RCAI
EventsEstablishment of RCAI

Major
Discoveries from
RCAI

Opening of RCAI in RIKEN
Yokohama InstituteTCR
microclusters

1st RCAI-JSI International
Symposium on Immunology

The zincsignal

1st RCAI International
Summer Program (RISP)

REGImmune, a spin-off from RCAI

Launch of PIDJ, Primary
Immunodeficiency Diseases
Network in Japan

α-C-GalCer
inhibits tumor
metastasis in
mice

Single molecule
imaging
microscopy

Establishment of RIKEN Jeffrey
Modell Diagnostic andResearch Center for Primary
Immunodeficiencies

1st Harvard Summer School at RCAI

Establishment of the RIKENTorii Joint Research Team forallergy vaccine development

Generation of
atopic mousemodel by ENU
mutagenesis

Bifidobacterium
protects from
E. coli infection

Establishment of the RIKEN Young
Chief Investigator Program

E4BP4 isresponsible
for IL-10
expression

Establishment of Medical I
mmunology World Initiative

Approval of NKT cell therapy forcancer by The Advanced Medical
Care Assessment System

Development ofan artificial
lymph node

IKKα is the key
player in IFN
induction of
innate immunity

15

5
25

2.

5
02
0.

25

0
0.

Interferon- A (ng/ml)

30

TLR7 ligand (SSRNA)(mg/ml)
Wildtype
IKK A KO

Np95
restoresDNA
methylation

K

PO

Th
Precursors

Helperinterplay

Runx

KillerAntagonistic
interplay
between ThPOKand Runx in thehelper/killer
lineage choice

Foxp3 + T cells
differentiate into
follicular B helper
T cells

Leukemicstem cells
in bonemarrow

Myeloid-basedmodel of T celldifferentiation

GP-2 is a
receptor formucosalantigens

Bcl11bregulates theformation of
T cells fromprogenitors

LAPTM5
negatively
regulates
TCR
signaling

Generation of
iPSC-derived
NKT cells

Modeling of
ERK signaling
networks that
determine cell
division ordifferentiation

CD169
macrophages
consumedead tumor
cells

In vivoimaging of B
cell migrationby two-photon
microscopy
ERK controls B cell development
and antigen production

Siglec-H controls cytokine production byplasmacytoid DCs

RIKEN
Research Center forAllergy and Immunology (RCAI)

Organization

Director
Masaru Taniguchi, M.D., Ph.D.

Senior Advisors
Kimishige Ishizaka, M.D., Ph.D.
Shizuo Akira, M.D., Ph.D.

International Advisory Council
Max D. Cooper, Chair, M.D.
Rudolf Aebersold, Ph.D.
Antonio Coutinho, M.D., Ph.D.
Alain Fischer, M.D., Ph.D.
Ronald N. Germain, M.D., Ph.D.
Paul W. Kincade, Ph.D.
Tadamitsu Kishimoto, M.D., Ph.D.
Bernard Malissen, Ph.D.
Ruslan Medzhitov, Ph.D.
Masayuki Miyasaka, M.D., Ph.D.
Hiromitsu Nakauchi, M.D., Ph.D.
William E. Paul, M.D.
Susan K. Pierce, Ph.D.
Klaus Rajewsky, M.D.
Ralph M. Steinman, M.D.
Kiyoshi Takatsu, Ph.D.
Dale Umetsu, M.D., Ph.D.
Arthur Weiss, M.D., Ph.D.

ii

GroupLab. for Cell Signaling Takashi Saito, Ph.D.
Lab. for Lymphocyte Differentiation Tomohiro Kurosaki, M.D., Ph.D.
Lab. for Immunogenomics Osamu Ohara, Ph.D.
Lab. for Developmental Genetics Haruhiko Koseki, M.D., Ph.D.
Lab. for Immune Regulation Masaru Taniguchi, M.D., Ph.D.
Lab. for Cytokine Signaling Toshio Hirano, M.D., Ph.D.
Lab. for Immunological Memory Toshitada Takemori, M.D., Ph.D.
Lab. for Transcriptional Regulation Ichiro Taniuchi, M.D., Ph.D.
Lab. for Human Disease Model Fumihiko Ishikawa, M.D., Ph.D.

Team
Lab. for Immune Diversity Ji-Yang Wang, Ph.D.
Lab. for Immunochaperones Hei-ichiro Udono, M.D., Ph.D.
Lab. for Mucosal Immunity Sidonia Fagarasan, M.D., Ph.D.
Lab. for Lymphocyte Development Hiroshi Kawamoto, M.D., Ph.D.
Lab. for Epithelial Immunobiology Hiroshi Ohno, M.D., Ph.D.
Lab. for Innate Cellular Immunity Masato Tanaka, M.D., Ph.D.
Lab. for Host Defense Tsuneyasu Kaisho, M.D., Ph.D.
Lab. for Dendritic Cell Immunobiology Katsuaki Sato, Ph.D.
Lab. for Infectious Immunity Satoshi Ishido, M.D., Ph.D.
Lab. for Immunogenetics Hisahiro Yoshida, M.D., Ph.D.

Deputy Directors

Lab. for Vaccine Design

Yasuyuki Ishii, Ph.D.

Lab. for Cellular Systems Modeling

Mariko Okada-Hatakeyama, Ph.D.

Takashi Saito, Ph.D.
Shigeo Koyasu, Ph.D.
Haruhiko Koseki, M.D., Ph.D.

Unit
Unit for Molecular Systems Immunology Makio Tokunaga, Ph.D.
Unit for Immune Homeostasis Shohei Hori, Ph.D.
Unit for Cellular Immunotherapy Shin-ichiro Fujii, M.D., Ph.D.

Research Coordinators

Unit for Immunoinformatics S. Sujatha Mohan, Ph.D.
Unit for Single Molecule Imaging Kumiko Sakata-Sogawa, Ph.D.

Toshitada Takemori, M.D., Ph.D.

Unit for Therapeutic Model Kanako Shimimzu, M.D., Ph.D.

Haruka Iwano, D.V.M., Ph.D.

Unit for Inflammatory Regulation Takashi Tanaka, M.D., Ph.D.

Peter Burrows, Ph.D.

Unit for Immunodynamics Takaharu Okada, Ph.D.

International Research Unit
Administrative
Coordination Office

Unit for Thymic Environment

Willem van Ewijk, Ph.D.

Unit for Immunoepigenetics Miguel Vidal, Ph.D.
Ichiro Taniuchi, M.D., Ph.D.

Unit for Immue Crosstalk Hilde Cheroutre, Ph.D.

iii

C o n t e n t s
10 years history of RCAI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

iOrganization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

iiContents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

iv

Director's Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

vi

Part 1Creation of New Paradigms

Medical Immunology World Initiative. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

2

Ensuring the persistence of immune memory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

5Preventing overreactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

6

Following an immune cell’s career path . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

7

CNS2 is a critical regulator of IL-4 production by follicular helper T cells. . . . . . . . . . . . . . . . . 

8

Keeping the immune system on track. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

9Reacting to changing circumstances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

10

PDLIM2 inhibits T helper 17 cell development and inflammation . . . . . . . . . . . . . . . . . . . . . . . 

11

Sorting out gut health. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

12

CIN85 drives B cell responses by linking the BCR to the NF-κB signaling pathway. . . . . . . . . 

13

Functional and developmental heterogeneity of invariant natural killer T cells. . . . . . . . . . . . . 

14Membrane-bound human SCF/KL promotes in vivo human hematopoietic engraftment andmyeloid differentiation in mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

15

Outstanding Contribution of the Year 2011. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

16Prize Winners 2011. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

20

Excellent Paper of the Year 2011. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

22Part 2

24

iPS technology development for immunological research and therapeutics . . . . . . . . . . . . . . 

26

The PIDJ Network develops a new genetic diagnosis method . . . . . . . . . . . . . . . . . . . . . . . . . 

28

NKT cell-targeted adjuvant cell therapy for cancer patients . . . . . . . . . . . . . . . . . . . . . . . . . . . 

30

Development of artificial adjuvant vector cells (aAVC) as a novel therapy . . . . . . . . . . . . . . . . 

30

Development of humanized mouse models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

31

Calpis-RIKEN Integrated Collaborative Research conducted in RCAI . . . . . . . . . . . . . . . . . . . 

32

RCAI Innovation Projects in Drug Discovery and Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

32

Part 3

Nurturing Young Scientists

Young Chief Investigator Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

34

YCI Laboratory for Bioenvironmental Epigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

35

YCI Laboratory for Mathematical Modeling of Immune System . . . . . . . . . . . . . . . . . . . . . . . . 

37

YCI Laboratory for Stem Cell Competency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

39

RIKEN Joint Graduate School Program International Program Associate. . . . . . . . . . . . . . . . . 

41

RIKEN Foreign Postdoctoral Researcher . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

42

RIKEN Special Postdoctoral Researcher (SPDR) Program. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

42

RIKEN’s Junior Research Associate (JRA ) Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

43

Adjunct Professorship Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

44Part 4iv

Technology and Innovation

RCAI and Torii collaborate for development of an allergy vaccine . . . . . . . . . . . . . . . . . . . . . . 

Collaborative Networks

Open Laboratory at RCAI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

46

Open Laboratory for Signal Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

47

Open Laboratory for Allergy and Mucosal Immune Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . 

49

University of Michigan-RCAI Joint Workshop. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

51

The 4th Joint RCAI-LIAI Workshop Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

53

RCAI-CGM collaboration is launched . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

53

RCAI 10th Anniversary Seminar Series. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

55

Multidisciplinary Research Projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

57

Student Exchange Programs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

57

International Research Collaboration Award . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

58

Part 5

Outreach Activities

RIKEN Yokohama Open Campus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

60

Immunology Workshop for High School Students . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

60

Lab visit by junior high school and high school students. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

61

Science Café "Regulation of the cell fate" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

61

Science Café "Is calico cat's pattern inheritable?" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

62

Science Café "Development of apollinosis vaccine and a novel cancer therapy" . . . . . . . . . . 

62Part 6

Laboratory Activities

Laboratory for Developmental Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

64

Laboratory for Lymphocyte Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

66

Laboratory for Transcriptional Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

68

Laboratory for Cell Signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

70

Research Unit for Single Molecule Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

72

Laboratory for Lymphocyte Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

74

Research Unit for Immunodynamics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

76

Research Unit for Molecular Systems Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

78

Laboratory for Epithelial Immunobiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

80

Laboratory for Mucosal Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

82

Laboratory for Immune Diversity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

84

Laboratory for Immunological Memory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

86

Laboratory for Host Defense. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

88

Laboratory for Infectious Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

90

Laboratory for Innate Cellular Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

92

Research Unit for Inflammatory Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

94

Research Unit for Therapeutic Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

96

Research Unit for Immune Homeostasis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

98

Laboratory for Immunochaperones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  100Laboratory for Immune Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  102Laboratory for Dendritic Cell Immunobiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  104Laboratory for Cytokine Signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  106Laboratory for Immunogenetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  108Laboratory for Vaccine Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  110Laboratory for Human Disease Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  112Research Unit for Cellular Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  114Laboratory for Immunogenomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  116Research Unit for Immunoinformatics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  118Laboratory for Cellular Systems Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  120Research Unit for Thymic Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  122Research Unit for Immunoepigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  124Research Unit for Immune Crosstalk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  126Central Facilities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  128Administrative Coorddination Office. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  132

Part 7

Data and Statistics

Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  134Invited Presentations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  138RCAI Seminars 2011. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  141Budget, Personnel and Patents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  143Cover and Section Heading Photo Legends. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  144v

Director'sReportAftermath of the Great East Japan Earthquake
After the devastating Great East Japan Earthquake on Mar. 11, 2011, RIKEN Research Center for Allergy and
Immunology (RCAI) launched a support program for immunology researchers, hoping to be of assistancein rebuilding the educational and research environment in the severely damaged Northeastern region. In
collaboration with the Japanese Society for Immunology (JSI), we provided support to 11 affected laboratories.
Because their individual circumstances varied so much, we provided need-based assistance with researchsupplies and biological samples (research equipment, cell lines, antibodies, mice, etc.), derivation of mice
to SPF status, and emergent support for research funding. We appreciate many offers from immunology
institutions abroad that kindly cooperated in these efforts: Max Planck Institute (Germany), the National
Institutes of Health (U.S.), INSERM (France), Jackson Laboratory (U.S.) and the La Jolla Institute for Allergy
and Immunology (U.S.).
Following the accident at the Fukushima Nuclear Power Plant, saving electricity became one of thebiggest challenges for the summer of 2011. The Ministry of Economy, Trade and Industry (METI) ordered
a 15% cut in the electricity usage compared to the last year with a penalty of up to 1 million yen / hour(on weekdays 9:00 a.m. - 8:00 p.m.). The consumption in RIKEN Yokohama Institute was monitored andreported every hour. At RCAI, air conditioners were set at 28 ℃, and people used stairs instead of elevators.
Some freezers and refrigerators were switched off, and many people shifted their working time, starting
earlier in the day. The summer was unusually quiet because of the cancellation of several events including
the Harvard Summer School at RCAI, the RCAI International Summer Program, RCAI-JSI International
Symposium on Immunology, Briefing of Joint Graduate School Programs, and the RCAI Retreat. Thanks tothe cooperation of every staff, we never exceeded the imposed limit.

Ralph Steinman's Vision Inspired Us to Create a New Program in Human
Immunology, the Medical Immunology World Initiative (MIWI)
Ralph Steinman, Professor of The Rockefeller University and a member of the RCAI Advisory Council,
received the Nobel Prize for Physiology and Medicine on October 3, 2011. However, just soon after thisviwonderful news, and to my great regret, we were informed that he had passed away on September 30. We
mourned his death and expressed our deep condolences to his family.
Ralph, who was famous as the discoverer of the dendritic cell, visited RCAI every year for the RCAI
Advisory Council Meeting. He always respected the ideas of the young scientists, offering positive andconstructive advice and suggestions with a very broad perspective. He repeatedly proposed a plan topromote internal collaborations to nurture young researchers by constructing a support system within thecenter. He was also a strong proponent of human immunology as a future direction for RCAI. Although he
was planning to visit us to attend the meeting in August 2011, the meeting had to be replaced with an email
review because of the cancellation caused by the earthquake. Instead of inviting the Advisory Committee
members to Yokohama, in June we asked them all to submit comments and an evaluation of the RCAI FuturePlan. Even though the document sent to the committee members consisted of more than 100 pages, Ralph
sent back his report within one week. He made valuable suggestions about how RCAI should approachhuman immunology in order to step into the next new phase and also to establish a system to nurture young
scientists.
Seriously taking his advice into consideration, in 2011 RCAI launched the Medical Immunology World
Initiative (MIWI) as a new human immunology consortium using humanized mice. Eight institutes are affiliatedwith MIWI: Immunology Fronteer Research Center (iFReC) Osaka University, NIH, IMSUT, two departmentsof Zurich University, INSERM/Necker Hospital, Pasteur Institute, Imperial College London, and RCAI. MIWI
is an international humanized mouse users' consortium and, as well, an integrative network for humanimmunology research in the next generation.
We also established the Young Chief Investigator Program as a tenure track system at RCAI. In thisprogram, we appoint researchers younger than 40 years old to positions guaranteed for 7 years, and provide
mentoring support by 3-4 senior PIs inside or outside RIKEN. In the 5th year, YCIs are reviewed by the
Program Committee for possible promotion and also for the relevance and significance of their research
projects to the future core research programs at RCAI. In 2011, we selected three researchers, whose
previous research fields were mathematics, neuroscience and epigenetics. The YCI program is also linked to
MIWI, with programs designed to nurture young scientists in interdisciplinary areas of research.
I strongly wish to establish this new and unique current of human immunology and the young investigator
program through MIWI and to develop it to realize Ralph Steinman's vision.

Research Achievements
Although we had many difficulties in 2011, RCAI researchers continued to make important research
discoveries. I would like to mention some of them.
The transcription factor Bcl6 was known to be important for germinal center B cells. Dr. Takaharu
Okada and colleagues used two photon imaging and reporter mice and found that the protein levels are very
important in dictating B cell behavior and also that Bcl6 is important for a special type of T helper cell, theT follicular helper (Kitano, et al. "Bcl6 Protein Expression Shapes Pre-Germinal Center B Cell Dynamics andFollicular Helper T Cell Heterogeneity", Immunity, Vol. 34, pp. 961–972, 2011).
IL-10 is an important anti-inflammatory cytokine and Dr. Masato Kubo and the team found that the E4
promoter-binding protein (E4BP4) transcription factor is responsible for driving the expression of IL-10 inmultiple types of immune cells (Motomura, et al. "The transcription factor E4BP4 regulates the productionof IL-10 and IL-13 in CD4+ T cells", Nature Immunology, Vol. 12, pp. 450-459, 2011).
The question of whether regulatory T cells can be “reprogrammed” to become CD4 T helper cells has
been very controversial in the field. Dr. Hori and colleagues showed that the real Tregs are not reprogrammable.
(Miyao, et al. "Plasticity of Foxp3+ T Cells Reflects Promiscuous Foxp3 Expression in Conventional T Cells
but Not Reprogramming of Regulatory T Cells", Immunity, Vol. 36, pp. 262-275, 2012).
Dr. Sato and the team used a clever combination of gene and cellular knockouts to reveal an importantrole for plasmacytoid dendritic cells in the regulation of inflammation and T cell immunity in vivo. (Takagi, etal. "Plasmacytoid Dendritic Cells Are Crucial for the Initiation of Inflammation and T Cell Immunity In Vivo",
Immunity, Vol. 35, pp. 958–971, 2011).
The minimal T cell receptor signaling unit is called a TCR microcluster, and these structures must moveand eventually coalesce by an unknown mechanism. Dr. Saito and colleagues discovered that this movement
is mediated by dynein, a ‘motor protein’ that shuttles cargos along the cytoskeleton (Hashimoto-Tane, et al.
"Dynein-Driven Transport of T Cell Receptor Microclusters Regulates Immune Synapse Formation and T Cell
Activation", Immunity, Vol. 34, pp. 919–931, 2011).

vii

Dr. Kubo and his team identified a cis-acting gene regulatory element in the Il-4 locus that is critical for
IL-4 production by T follicular helper cells, which are important in stimulating B cell antibody production andclearing viral and bacterial infections (Harada, et al. "The 3’ enhancer CNS2 is a crucial regulator in follicularhelper T cells of interleukin-4 mediated humoral immunity", Immunity, Vol. 36, pp. 188-200, 2012).
TH17 cells can induce a massive inflammatory response and also have essential roles in eliminating
microbial pathogens. Dr. Takashi Tanaka and collaborators discovered that the ubiquitin ligase PDLIM2
inhibits TH17 cell differentiation by degradation of the STAT3 transcription factor (Tanaka, et al. "PDLIM2
Inhibits T Helper 17 Cell Development and Granulomatous Inflammation Through Degradation of STAT3",
Science Signaling, Vol 4, ra85, 2011).
Dr. Hase and Dr. Ohno's team investigated the role of AP-1B–mediated protein sorting in the maintenanceof gastrointestinal immune homeostasis. AP-1B-deficient mice developed spontaneous chronic colitis andmoreover, AP-1B expression was reduced significantly in colonic epithelium samples from patients with
Crohn's disease (Takahashi, et al. "The Epithelia-Specific Membrane Trafficking Factor AP-1B Controls Gut
Immune Homeostasis in Mice", Gastroenterology, Vol. 141, pp. 621-632, 2011).
Dr. Kurosaki and his team discovered the importance of the CIN85 adaptor protein in BCR-mediatedsurvival, proliferation and antibody production due to its association with BLNK and participation in NF-κB
signaling (Kometani, et al. "CIN85 drives B cell responses by linking BCR signals to the canonical NF-kB
pathway", The Journal of Experimental Medicine, Vol. 208, pp.1447-1457, 2011).
Dr. Watarai and my team demonstrated that an important subset of iNKT cells, the IL-17RB+iNKT subset,
develops distinct from classical iNKT cell developmental stages in the thymus and play important roles in thepathogenesis of airway diseases (Watarai, et al "Development and Function of Invariant Natural Killer T Cells
Producing TH2- and TH17-Cytokines", PLoS Biology, Vol. 10, e1001255, 2012).
Dr. Ishikawa and his team created an improved humanized mouse that has significantly improved
engraftment of human hematopoietic cells (Takagi, et al. "Membrane-bound human SCF/KL promotes invivo human hematopoietic engraftment and myeloid differentiation", Blood, Vol. 12, pp. 2768-2777, 2012).
I would also like to emphasize here that several RCAI researchers have become professors, a fact that
underscores the vibrant and vital nature of RCAI; Dr. Kaisho became a professor of Osaka University, Dr.
Udono became a professor of Okayama University, Dr. Masato Tanaka became a professor of Tokyo Universityof Pharmacy and Life Sciences, and, recently, Dr. Kawamoto became a professor of Kyoto University, Dr.
Ishido became a professor of Showa Pharmaceutical University, Dr. Hoshino became a professor of Kagawa
University, and Dr. Hase became a professor of The Institute of Medical Science, the University of Tokyo.

Towards human immunology
Although fundamental research in immunology has always been a focal point and strength of our center,over the past years we have developed several programs with a translational component, for example avery large effort in primary immunodeficiency diseases. In March 2012, building on the work of the RCAI,RIKEN decided to establish a new research center for integrative medical science, starting in April, 2013.
Since its establishment in 2001, RCAI has continued to make new discoveries in immunology research andhas pioneered the creation of new paradigms. After these initial 12 years of research endeavor, RCAI will
open the door for the new direction to human immunology. I believe that our effort will continue to lead theworld's immunology.

Mar. 31, 2012

Masaru Taniguchi

viii

2011
Part 1Creation of New Paradigms

MIWI
Medical Immunology World Initiative is launchedR

CAI launched the Medical Immunology World Initiative ( MIWI )as a new human immunology program. MIWI is an international
humanized mouse users’ consortium and, as well, an integrative
network for human immunology research in the next generation.
Eight institutions are currently affiliated with MIWI: iFReC, NIH,
IMSUT, two departments of Zurich University, INSERM/Necker
Hospital, Pasteur Institute, Imperial College London, and RCAI.

The immune system is a critical component for

toward elucidating the role of the human immune

maintaining homeostasis of our body and also asystem in disease onset and progression.

key target for development of therapeutics and pre-The goal of MIWI is to use integrative immuno-

vention of numerous diseases. Understanding the

logical approaches to obtain fundamental knowledge

principles of the human immune system is therefore

about the human immune system and mechanisms ofof central importance to discover clinically useful bio-

disease development, and to discover the principles

markers as well as molecular and cellular targets forfor diagnosis and therapy of human diseases, partic-

therapies. However, our knowledge of the humanularly allergy, primary immunodeficiencies, influenza

immune system is still quite limited compared to what

and other viral infections, human cancer, and so on.

we know about the mouse and other model organtitative molecular data obtained from representative

To accomplish this goal RCAI proposed thefollowing 5 aims:

and well-defined human materials, and the lack of

1. Generate humanized mice with greater levels ofsuitable experimental strategies for functional dissec-

humanization of their immune and hematopoietic

tion of the human immune system.

microenvironments than are currently available

isms. This is mainly because of the shortage of quan-

Recent advances in bioinformatics and in technologies for genome and RNA sequencing, quantita-

above.

tive identification of proteins, metabolites and other

2. Accumulate tools to manipulate the reconstituted

small compounds have enabled us to collect an enor-

human immune system by depleting distinct

mous amount of digitalized data derived from humanimmune compartments with specific antibodies ormaterials. In fact, advances in human genomics havemodification of their functions by transduction ofallowed us to identify many genes that potentially may

human hematopoietic progenitor or other cells

contribute to the pathogenesis of human diseases.

with regulatory molecules, e.g. shRNA.

However, experimental strategies to mechanistically

3. Establish a database that integrates the accumu-

interpret these data are not sufficiently coordinated or

lated data from conventional studies on the humanrobust to understand the human immune system dur-

immune system and humanized mouse studies

ing pathogenic processes.

with the data collected from clinical samples.

To overcome these problems, it is necessary to

4. Utilize this integrated information to identify pivotal

establish an interdisciplinary biomedical researchmolecules in human immune cells as therapeutic

platform and an international consortium of multiple

targets and biomarkers in the different diseaseresearch groups that possess different experimental
expertise but share a central interest. For establishment and maintenance of such a consortium, it isthus critical to share comprehensive goals directed2

and use these mice to study the diseases listed

conditions.
5. Establish a system to nurture young researchers.

Figure:Scheme of MIWIA new platform for human immunology research is required. For this purpose, the Medical Immunology World Initiative (MIWI) as
the Human Immunology Consortium using Humanized Mice is being established. MIWI focuses on creation of the 3rd generationof humanized mice, integrative medical immunology, and an integrated database, as well as a mentoring system for nurturing young
investigators. The initiative involves RIKEN RCAI together with eight institutes; NIH for Systems Immunology (Dr. Ronald
Germain), Osaka University Immunology Frontier Research Center (iFReC) (Dr. Shizuo Akira) for immune imaging/dynamics,
Pasteur Institute (Dr. James Di Santo) and two centers at Zurich University (Dr. Markus Manz and Dr. Christian Munz) for the 3rd
generation of humanized mice with European type HLA genes and their application to establish human disease and therapy models,
INSERM/Necker Hospital (Dr. Alain Fischer) for human primary immunodeficiency diseases, the Institute of Medical Science
the University of Tokyo (IMSUT) (Dr. Yoshihiro Kawaoka) for pathogenic and nonpathogenic human influenza virus infection
models using humanized mice, and Imperial College London（Dr. Reiko Tanaka）for mathematical modeling and simulation.
Cooperation with these international institutes will complement those areas lacking in RCAI to effectively establish the system.

To realize the aims above, RCAI suggests thefollowing 5 plans:

any side effects.

1. Establishment of a new generation of humanizeddisease of interest, protocols and reagents will be

In order to assess functional

contributions of human immune cell types to themice and a user consortium

shared among the participating research groups.

There are several limitations with the present sec-

Although it is obviously likely that the humanized

ond generation of humanized mice because themice will only partly recapitulate human diseases,

immune microenvironments are entirely of mousethe comparative analysis of the accumulated data

origin.

In particular, the lack of human T cell-from patients and from the humanized mouseselecting HLA molecules and the species-speci-

models should ultimately allow us to understandficity of several cytokines and adhesion molecules

more precisely the differences between human

limit the efficient development of lymphocyte sub-and mouse immune systems as well as any advan-

sets and antigen recognition by human immune

tageous similarities between humanized mice andcells.

In this regard, a worldwide collaboration

the human system, both of which will have predic-

with research centers focusing on humanizedtive value for future analyses. However, this out-

mouse models will facilitate the investigation ofcome will occur only when a solid informational

human immunity and immunological diseases.

platform is established for this purpose.

2. Using humanized mice to understand the human

3. Integrative immunobiology and disease targets

immune system and immune responses

The immune system and immune responses will

Humanized mice will be integral components forbe systematically elucidated by using the quantita-

understanding the human immune system andtive measurements and mathematical/statistical

recapitulating disease onset and progression.

tools of systems biology. For this purpose, theThus, they are quite useful for vaccine/drug devel-

analysis of molecular and cellular behavior will be

opment programs and to examine the safety andhelpful to understand spatio-temporal dynamics ofefficacy of candidate vaccines and drugs in vivo,

immune cells in tissues and organisms.

particularly as to whether these agents effectively

studies will include quantitative analyses at theprotect against human disorders and if there are

single molecule and single cell level and in both

These

3

time and space ( systems dynamics ) , with the goal

talented young investigators who will lead humanof assembling such data into a computable

immunology in the next generation and accelerate

immune network.

In parallel, a pipeline will be

the establishment of interdisciplinary researchdeveloped to mine the data with bioinformatics

areas. To facilitate integrative biology and humantools and hopefully then construct useful disease

immunology research, the following directions inmodels that will allow us to obtain clues as to how

recruiting and developing young investigators will
be pursued: a ) researchers bridging biology andto eventually set up intervention strategies. Diseases and therapeutic targets will be 1) allergic
diseases, 2 ) primary immunodeficiencies, 3 ) influenza virus infection, 4 ) human tumor virus infection

informatics, b ) researchers who have clinical experience and understand both on human immunity

and immune control, 5 ) immune therapy and 6 )and diseases pathogenesis for successful clinicaltranslation in the future, c ) researchers innovating

normal immune and memory responses.

new technologies that can be applied to immunol-

4. Establishment of an integrated disease databaseogy and other biomedical sciences. The global

A disease-oriented database will be establishednetwork through MIWI will encourage young inves-by integrating immune cell data obtained directly

tigators to strengthen communication skills andfrom patient specimens or via analysis of the cor-foster collaboration among different fields of sci-

responding humanized mouse models. Human-ence.

ized mice are of particular value here because they
can provide us with human tissues/cells that are

MIWI will also coordinate with RIKEN Yokohama

not routinely available from patients. In this new
platform, the clinical information is integrated with

Institutes for whole genome sequencing, genomewide medical sciences ( GWAS ) and disease biomark-various lines of information obtained by laboratory

ers, and with QBiC ( Quantitative Biology Center) for

experiments, including genetic, proteomic andintegrative biology and the development of methods

cellular information obtained from various tissues

for mathematical modeling.

and immune cells of humanized mice.
5. Nurturing young investigators bridging immunology and other fields of science
One of the objectives for starting MIWI is to nurture

Dr. Shizuo Akira named the Senior Advisorand Dr. Shigeo Koyasu and Dr. Haruhiko
Koseki new Deputy Directors

Dr. Shizuo Akira

T

Dr. Shigeo Koyasu

wo professors, Dr. Shizuo Akira ( Osaka Uni-a new Deputy Director of RCAI, and Dr. Haruhikoversity ) and Dr. Shigeo Koyasu ( Keio Univer-

Koseki, Group Director of Laboratory for Develop-

sity ) joined the advisory and management boardsof Medical Immunology World Initiative ( MIWI ) . To

mental Genetics, was also promoted to a Deputy

take on the advisory task for MIWI, Dr. Akira

Dr. Takashi Saito, Dr. Shigeo Koyasu and Dr. Har-

became the new Senior Advisor of RCAI, in addi-

uhiko Koseki share the tasks of management oftion to the current advisor, Dr. Kimishige Ishizaka.

RCAI and MIWI.

For the management of MIWI, Dr. Koyasu became

4

Dr. Haruhiko Koseki

Director position.

Now three Deputy Directors,

Research highlights
Takaharu Okada

Ensuring thepersistenceof immune
memoryA gene regulatory
factor promotes
long-term protection
against infectious
threats by effecting
the maturation of avariety of immune
cells

Figure:S

Time-lapse photography at two-minute intervals shows the migration(white trail) of a Bcl6-expressing B cell (green) into the GC (blue) following
induction of an immune response (top). By comparison, B cells with inhibited
Bcl6 function fail to migrate to the GC (bottom). © 2011 Elsevier, Inc.

tructures within the lymph nodes known as germinal centers ( GCs ) help
the body to maintain long-term immune defense against foreign threats.

The GCs essentially act as sites where antibody-producing B cells undergo
a process of ‘evolution’ to generate higher-quality antibodies. This evolutionary mechanism is in turn supported by a class of T cells known as follicularhelper T ( Tfh ) cells.
Takaharu Okada and colleagues at the RIKEN Research Center forAllergy and Immunology in Yokohama recently tracked the process of B
cell development in mouse GCs by monitoring expression of Bcl6. This
protein facilitates B cell evolution by regulating expression of key developmental genes.
The researchers were surprised to note a broader scope of Bcl6 activity
than expected. “It became apparent that Bcl6 is important for Tfh cells as
well,” says Okada, “and I felt that we should track [the levels of] Bcl6 expression in both B and T cells at the same time.”
They quantified Bcl6 levels by generating transgenic mice in which a
DNA fragment encoding a fluorescent protein had been inserted into one
copy of the Bcl6 gene. Since this insertion partially disrupted Bcl6 function,
this reporter system enabled the researchers to examine the effects ofinhibiting this protein.
Following the induction of an immune response in the animals, Okada
and colleagues observed an increase of Bcl6 expression in antigen-specific
B cells within peripheral regions of the lymph node; these subsequently
migrated to the GC, where they continued to strongly express Bcl6 ( Figure ) .
This migration and maturation process is dependent upon interaction
with Bcl6-expressing helper T cells. All T cells expressing high levels of Bcl6

Masahiro Kitano ( left ) and Saya Moriyama (right )became Tfh cells, but many Tfh cells subsequently reduced Bcl6 production
to varying degrees. Okada was surprised that the dynamics of Bcl6
expression differed between B and T cells. “This suggests that they employ
different mechanisms for regulating expression of this important transcription factor,” he says.
Okada and his team also recorded observations suggesting that Tfh
cells expressing low levels of Bcl6 may give rise to ‘memory cells’, whichenable the immune system to react quickly to recurring threats. However,

Original Research Paper
Masahiro Kitano, Saya Moriyama, Yoshikazu Ando, Masaki
Hikida, Yasuo Mori, Tomohiro Kurosaki, and Takaharu
Okada. Bcl6 protein expression shapes pre-germinal
center B cell dynamics and follicular helper T cell
heterogeneity. Immunity Vol. 34, 961-972, 2011.

this will require further investigation.
Understanding how these various cells employ this shared regulatory
factor will also be a top priority moving forward. “We would like to learn themolecular mechanisms of Bcl6 expression in B and T cells by setting up
collaborations with experts in the field of gene and protein expression
control,” says Okada.

This
This article
article isis reproduced
reproduced fromfrom RIKEN
RIKEN RESEARCHRESEARCH with
with permission.
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5

Research highlights
Masato Kubo

Preventing
overreactions
Identification of thetranscription factor
that regulates aprotein that dampens
immune responses
could aid the fightagainst autoimmune
diseaseFigure:I

In T cells stained
blue (top left),the transcription
factor E4BP4(red) regulates
that production of
IL-13 (green) and
IL-10 (not shown).

nterleukin-10 ( IL-10 ) is an anti-inflammatory cytokine protein that reduces
immune responses and staves off autoimmune disease. Now, a researchteam led by Masato Kubo at the RIKEN Research Center for Allergy and
Immunology, has identified a transcription factor called E4 promoter-binding
protein ( E4BP4 ) that is responsible for driving the expression of IL-10 in multiple types of immune cells.
The researchers investigated E4BP4 because of a unique property of asubset of immune cells called T helper type 1 ( TH1) cells, which generally
enhance immune responses by secreting pro-inflammatory cytokines. However, under chronic stimulation with foreign antigens—that occur during
chronic infection—TH1 cells can also produce cytokines, such as IL-10 and
IL-13, which are normally made only by other immune-cell types. While theimmune system is fighting the infection, IL-13 modulates allergic responses,
and IL-10 prevents the immune system from attacking the body.
Kubo and colleagues compared genes expressed in TH1 cells with andwithout chronic antigen stimulation, and found that E4BP4 was expressed
only in instances of chronic antigen stimulation. When they expressed E4BP4
in TH1 cells that had not been chronically infected, it induced production of
IL-10 and IL-13 in conditions in which those cytokines would not normallyoccur ( Figure ) . E4BP4-deficient TH1 cells could not increase expression of
IL-10 and IL-13 after chronic antigen stimulation. The researchers found that
other T cell subsets also required E4BP4 to modulate the expression of IL-10,
but not IL-13.
Transcription factors can control the expression of genes by binding to a

Yasutaka Motomura

region on the genomic DNA called the promoter. Kubo and colleagues
observed that E4BP4 bound to the IL-13 promoter in TH1 cells that had beenchronically stimulated with antigen. No binding occurred with TH1 cells lacking chronic stimulation. Kubo explains, however, that: “E4BP4 seems to regulate the expression of IL-10 in a totally different way—by altering thechromosomal structure in the region of that gene.”
Mice lacking IL-10 can spontaneously develop intestinal autoimmune
disease. Interestingly, Kubo and his team found that E4BP4-deficient mice
produced lower levels of IL-10 than control mice, and showed some symp-

Original Research Paper
Yasutaka Motomura, Hiroshi Kitamura, Atsushi Hijikata,
Yuko Matsunaga, Koichiro Matsumoto, Hiromasa Inoue, Koji
Atarashi, Shohei Hori, Hiroshi Watarai, Jinfang Zhu, Masaru
Taniguchi & Masato Kubo. The transcription factor E4BP4
regulations the production of IL-10 and IL-13 in CD4+
T cells. Nature Immunology, Vol. 12, 450-459, 2011.

toms of gastrointestinal inflammation along with diarrhea. The mice lacking
E4BP4 also developed more severe symptoms of a neurological autoimmune
disease caused by exposure to brain antigens. E4BP4 is therefore a key factor in preventing the immune system from attacking the body’s own organs,and “induction of expression of E4BP4 may cure many types of autoimmune
inflammatory diseases,” says Kubo.

This
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articlearticleis isreproduced
reproduced
from
from
RIKEN
RIKEN
RESEARCHRESEARCH
withwithpermission.
permission.

6

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Research highlights
Shohei Hori

Figure:Following an
immune cell’scareer path
A protein ‘fingerprint’
used to identify
certain immune cells
is expressed more
broadly than firstthought, raising newquestions about how
these cells developT

The various classesof T cells are capableof responding topathogenic threats andrestraining the resulting
immune response to
avoid inflicting damage
on host tissues.(Source:
Dr. Triche, National
Cancer Institute, USA)he immune system produces diverse varieties of T cells ( Figure ) , suchas pathogen-destroying cytotoxic T cells and immune response-boosting helper T cells. Regulatory T ( Treg ) cells restrain these other cells andprevent the body from overreacting to threats or generating a dangerous
autoimmune response.
Treg cells are usually identified by expression of the transcriptional regulator protein Foxp3, but new work from a team led by Shohei Hori of the
RIKEN Research Center for Allergy and Immunology has demonstrated that
this is not a reliable signature.
Several groups have obtained data suggesting that Treg cells can essentially ‘change careers’, losing their Foxp3 expression and transforming into
other T cell types. However, findings from Hori and colleagues led them topropose an alternative ‘heterogeneity model’. “Our observations suggested
that these phenomena can be fully explained by a minor ‘uncommitted’population of Foxp3 + T cells without assuming reprogramming,” he says.
His group has now provided compelling evidence for this hypothesis byusing a labeling technique that allowed them to distinguish cells currently
expressing Foxp3 from those that are not, but which have expressed thisprotein in the past.
The researchers identified two groups of Foxp3-expressing cells that
responded differently to an immune stimulus. Most expressed this protein
stably and at high levels, and exhibited the functional characteristics ofTreg cells. A minority fraction displayed transient bursts of Foxp3 expression,
but ultimately developed into other T cell types. These ‘exFoxp3’ cells did
not appear to represent reprogrammed Treg cells, but rather a separate poolof T cells that only produce this protein sporadically.

Takahisa Miyao

Interestingly, his team also learned that some Treg cells do enter a state
where they stop expressing Foxp3, although they retain ‘memory’ of theiridentity as Treg cells. This is achieved via chemical modifications to the DNA
within the gene encoding Foxp3, and immune stimulation promptly leads torobust re-expression of this protein. “This should force people to reconsider
the popular but oversimplified view of Foxp3 as the master regulator ofTreg cells,” says Hori.
The events that determine this expression profile are therefore likely toprove more important in establishing cellular identity than the presence orOriginal Research Paper
Takahisa Miyao, Stefan Floess, Ruka Setoguchi,
Herve Luche, Hans Joerg Fehling, Herman Waldmann,
Jochen Huehn and Shohei Hori. Plasticity of Foxp3+
T cells reflects promiscuous Foxp3 expressionin conventional T cells but not reprogramming of
regulatory T cells. Immunity, 36, 161-163, 2012absence of Foxp3. “It has been speculated that the Treg lineage is determinedby a higher-order regulatory pathway upstream of Foxp3, but the nature ofthis system is unknown,” says Hori. In future work, he plans to partner withcolleagues at other RIKEN laboratories to investigate this question moreclosely.

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7

Research highlights
Masato Kubo

CNS2 isa criticalregulator of
IL-4 production
by follicularhelper T cells

Figure:U

TFH cells stimulate IgG1 and IgE antibody production

pon antigen stimulation, naïve helper T cells differentiate into various
types of effector helper T cells. Type 2 helper T ( Th2 ) cells are effector

helper T cells that secret the cytokine IL-4 and stimulate B cells to produce
IgG1 and IgE antibodies, thus mediating atopic and allergic diseases.
Follicular T ( Tfh ) cells are a recently defined subset of helper T cells thatare distinguishable from other helper T cells in two important ways: by theirlocation in the B cell follicles and by their function - to activate B cells andtrigger germinal center formation. This results in clearance of viral and bacterial infections through induction of potent humoral immunity. Like Th2 cells,
the Tfh cells also secrete IL-4 and stimulate antibody class switching by B
cells to produce IgG1 and IgE antibodies. Differences in the regulatory mechanisms and functions between Th2 and Tfh remained unknown.
To elucidate the mechanisms of IL-4 secretion by Th2 and Tfh cells,
Masato Kubo ( Open Laboratory for Signal Network) and his team generated
a series of mutant mice that genetically lack each cis-acting gene regulatory
element in the noncoding regions of the Il-4 locus. They found that micelacking CNS2, an enhancer located ~10 kb downstream of the Il-4 codingexons, had a reduced production of IgG1 and IgE. To understand the function of CNS2, they next generated CNS2 reporter mice, in which CNS2 actiYohsuke Haradavated cells could be detected by GFP expression. GFP positive cells werelocalized in B cell follicles and their gene expression pattern was similar to Tfh
cells. From these results, Kubo and the team discovered that CNS2 is thecell type-specific Il-4 enhancer in Tfh cells. They also analyzed the development of CNS2-active Tfh cells, and found that these cells developed fromnaïve T cells 3-5 days after immunization with antigen.
To test the impact of CNS2-deficiency on allergic diseases, they induced
bronchial asthma in CNS2 deficient mice. Asthma was observed in CNS2
deficient mice, indicating that development of asthmatic disease is mediatedby Th2 and not by Tfh cells.
"We revealed a unique role of CNS2 during the induction phase of Il-4

Original Research Paper
Harada, Y., Tanaka, S., Motomura, Y., Harada, Y., Ohno,
S., Ohno, S., Yanagi, Y., Inoue, H., Kubo, M.The 3'
enhancer CNS2 is a critical regulator of interleukin4-mediated humoral immunity in follicular helper
T cells. Immunity. 36 ( 2) :188-200. ( 2012 )gene expression in the Tfh cell lineage. Tfh cells are important in stimulating
B cell antibody production and clearing viral and bacterial infections.
production and the development of effective vaccines," said Kubo.

This article is reproduced from RIKEN RESEARCH with permission.

8

Our

discovery will be an important step toward the understanding of antibody

http://www.rikenresearch.riken.jp/eng/research/6968

Research highlights
Takashi Saito

Keeping theimmune systemon trackSpecialized motor
proteins help control
immune activationby physically haulingclusters of signaling
receptors to a central
site for eventual
disposal

Figure:S

Following activation, a TCR-MC (white arrow) travels along microtubulesof the cytoskeleton (green), making its way from the periphery to thecSMAC (time scale, seconds). © 2011 Elsevier Inc.

pecialized immune cells called T cells can recognize threats and induce
immune responses through T cell receptors ( TCRs ) , but these recep-

tors do not act alone. Multiple receptors gather together at the cell surface
to cooperatively switch on T cells. “The minimum unit for triggering T lymphocyte activation is known as the TCR microcluster [ TCR-MC ] ,” explains

Takashi Saito of the RIKEN Research Center for Allergy and Immunology.
“These are the key structure for T cells to recognize antigens and become
activated.”
At the interface between T cells and the antigen-presenting immune
cells that switch them on, TCR-MCs accumulate at a structure called thecentral supramolecular activation cluster ( cSMAC ) . Now, research from Saito
and colleagues has revealed unexpected insights into how this accumulation
occurs.
Saito and his team were the first to characterize TCR-MC function, butthey were uncertain how these clusters make their way from the periphery to
the core of the cSMAC. To understand this phenomenon, they performed aseries of experiments in which T cells were placed on an artificial lipid layerthat mimics the membrane of an antigen-presenting cell, allowing them tomicroscopically visualize activation-related events at the T cell surface.
Cellular structures are reinforced by protein fibers that form a network
called the cytoskeleton, and Saito and colleagues revealed that TCR-MC
movement is mediated by dynein, a ‘motor protein’ that shuttles cargos
along these fibers. “We knew lymphocyte activation was regulated throughAkiko Hashimoto-Tane ( left ) and Machie Sakuma (right)

the cytoskeleton,” he says. “But it was most surprising that TCR complexes
are physically associated with dynein and that their movement is mediatedby assembling with this complex.”
Upon TCR activation, the dynein-facilitated movement drags TCR-MCs
laterally along the surface of the membrane towards the cSMAC ( Figure ) , afunction previously unseen for this motor protein. Pharmacological inhibitionof dynein strongly impaired migration of TCR-MCs and undermined their

Original Research Paper
Akiko Hashimoto-Tane, Tadashi Yokosuka, Kumiko SakataSogawa, Machie Sakuma, Chitose Ishihara, Makio Tokunaga,
and Takashi Saito. Dynein-driven transport of T cell receptor
microclusters regulates immune synapse formation andT cell activation. Immunity, Vol. 34, 919-931, 2011.
Related paper
Tadashi Yokosuka, Kumiko Sakata-Sogawa, Wakana
Kobayashi, Michio Hiroshima, Akiko Hashimoto-Tane,
Makio Tokunaga, Michael L Dustin and Takashi Saito.
Newly generated T cell receptor microclusters initiate
and sustain T cell activation by recruitment of Zap70 and
SLP-76. Nature Immunology Vol. 6, 1253-1262, 2005.

assembly within the cSMAC, as did the selective reduction of a key subunitof the dynein complex. Intriguingly, the same treatments that impaired TCRMC migration also enhanced T cell activation. Saito and colleagues therefore
concluded that once these clusters reach the center of the cSMAC, they
become internalized within the cell and thereby taken out of action.
Saito hopes to exploit this effect by learning how the TCR-MC-dynein
complex is assembled. “It would be ideal if we had a specific inhibitor of thisassembly,” he says, “which could lead to stronger immune status withenhanced activation of T cells.”

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9

Research highlights
Katsuaki Sato

Reacting tochanging
circumstances
An analysis of asubset of immune
cells reveals how
these cells rally
defenses against
infection while
keeping potentiallyharmful inflammatory
reactions in check

Figure:T

In autoimmune conditions suchas the skin condition psoriasis,the immune system attacks
tissues in the body, with painfulconsequences for patients. ©
2012 iStockphoto/QUAYSIDE

cells represent a significant
component of the ‘muscle’ inthe immune system, promoting
aggressive action against perceived
threats or restraining fellow immune
cells from launching an unhealthy
autoimmune response ( Figure ) .
Dendritic cells ( DCs ) help to manage these cells, presenting bits of antigen to T cells in a context that allows
them to react appropriately.
As with everything in the immune system, however, the biological details
are considerably more complex. “DCs consist of heterogeneous subsets,
including conventional DCs ( cDCs ) and plasmacytoid DCs ( pDCs ) ,” explains

Katsuaki Sato of the RIKEN Research Center for Allergy and Immunology,“and the precise functional role of each DC subset in immune responses
remains unclear.” Sato is especially interested in pDCs, as the experimental
data obtained to date have done little to clarify their behavior within thebody.
Sato and his colleagues from Japan and France recently published adetailed analysis of pDC function, achieved by selectively obliterating this cell
population in mice. To do this, the team inserted a toxin gene into the geneencoding Siglec-H, a protein uniquely expressed by pDCs; by chemically
activating this fatal factor, the researchers could rapidly eliminate Siglec-Hproducing cells. As an added benefit, this insertion effectively knocked out
Siglec-H expression, revealing the functional contributions of this protein inotherwise normal pDCs.
pDCs express a protein called toll-like receptor 9 ( TLR9 ) , which responds
particularly to the presence of pathogens such as viruses and bacteria. The
researchers determined that pDCs generate various inflammatory signals in
response to TLR9 activation, but that the levels of these signals are normally
modulated by the inhibitory action of Siglec-H, which appears to be a key

Hideaki Takagi

regulatory molecule in these cells.
Their experiments confirmed a central role for pDCs in responding to
infection, driving both the inflammatory response pathway as well as the
production of pathogen-destroying cytotoxic T lymphocytes. However, pDCs
also appear to make an important contribution to the process of ‘peripheral
tolerance’, which holds the immune system in check and prevents it fromoverreacting to non-threatening antigens. Specifically, pDC signals inhibitedthe production of antigen-specific helper T cells, which activate other immune

Original Research Paper
Hideaki Takagi, Tomohiro Fukaya, Kawori Eizumi, YumikoSato, Kaori Sato, Azusa Shibazaki, Haruna Otsuka, Atsushi
Hijikata, Takashi Watanabe, Osamu Ohara, Tsuneyasu Kaisho,
Bernard Malissen, and Katsuaki Sato. Plasmacytoid dendritic
cells are crucial for the initiation of inflammation and T cell
immunity in vivo. Immunity, Vol. 35, 958-971, 2011

cells, and favored the formation of regulatory T cells, which help to restrain
the immune response.
These latter findings were somewhat surprising, and Sato hopes to delve
further into their implications for human health in future studies. “We have aplan to analyze the role of pDCs and their regulation in the control of autoimmune disease,” he says.

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Research highlights

Figure:Relative to wild-type mice (top), thelivers of PDLIM2-deficient animals(bottom) show considerably higher levelsof granuloma formation. Granulomas
are indicated by dark spots formed
following staining with hematoxylin
and eosin (scale bars, 100 µm).
© 2012 AAASThe T helper 17 ( TH17 ) cell belongs

Takashi Tanaka

PDLIM2
inhibits T
helper 17 cell
development
andinflammation

to a newly identified helper T cellsubset that is distinct from TH1 and TH2
cells. Whereas TH1 and TH 2 cells characteristically produce the cytokinesinterferon-γ and interleukin ( IL ) -4, respectively, TH17 cells secrete IL-17, IL-21,
and IL-22 as effector cytokines. TH17 cells can induce a massive inflammatory response and have essential roles in eliminating microbial pathogens.
However, TH17 cells are also potentially highly pathogenic, because excessive and prolonged activation of TH17 cells may cause human autoimmune
and inflammatory diseases, including rheumatoid arthritis and inflammatory
bowel diseases. TH17 cells are also critically involved in the development of
granulomatous inflammation, which suggests that controlling the development/function of TH17 cells will be an important strategy to prevent and treat
granulomatous diseases.
Although much is known about the differentiation of TH17 cells, little is
known about how these cells are inhibited. Takashi Tanaka ( Research Unit
for Inflammatory Regulation, RCAI ) in collaboration with Tadashi Matsuda( Hokkaido University ) discovered that the ubiquitin ligase PDLIM2 inhibits
TH17 cell differentiation.
To analyze TH17 induced inflammation, they used an established bacterial-induced inflammation model, Propionibacterium acnes-induced liver
granuloma. The granulomatous inflammation was significantly enhanced in
PDLIM2-deficient mice compared to wild-type mice ( Figure ) . T cells isolated
from PDLIM2 deficient mice produced two- to fivefold more IL-17, IL-21 andIL-22 than the control mice due to the enhanced differentiation of TH17 cells.
To clarify how PDLIM2 was involved in the regulation of TH17 cell differentiation, the researchers analyzed signaling molecules that interact withPDLIM2. Using coimmunoprecipitation and reporter assays, they found thatPDLIM2 interacts with STAT3 and terminates STAT3-mediated signaling.
Furthermore, they found that PDLIM2 mediates ubiquitination of STAT3, andthat the ubiquitinated STAT3 molecules are degraded through the proteasome pathway. Indeed, STAT3 was more stable in PDLIM2-deficient cells,and STAT3-dependent gene expression was substantially increased when
PDLIM2 was knocked-down by small interfering RNA ( siRNA ) .
STAT3 is a master regulator of the development of highly proinflamma-

Original Research Paper
Tanaka, T., Yamamoto, Y., Muromoto, R., Ikeda, O.,
Sekine, Y., Grusby, M.J., Kaisho, T. & Matsuda, T.
PDLIM2 inhibits T helper 17 cell development andgranulomatous inflammation through degradationof STAT3. Science Signaling 4, ra85 ( 2011) .
Related paper
Tanaka, T., Soriano, M.A. & Grusby, M.J. SLIM is a
nuclear ubiquitin E3 ligase that negatively regulates
STAT signaling. Immunity 22, 729-736 ( 2005) .

tory TH17 cells, and TH17 cells are critically involved in several inflammatory
and autoimmune diseases. Therefore, as a suppressor of STAT3, PDLIM2
may be a therapeutic target to prevent TH17 cell-mediated diseases. "Recent
studies suggest that TH1 and TH17 cell subsets are not mutually exclusive,
but cooperatively induce inflammatory responses," says Tanaka. "In thisstudy and in our previous work, we have demonstrated that PDLIM2 cannegatively regulate the development of both cell types, and thus represents
a useful new target for the treatment of human autoimmune and inflammatory diseases."This article is reproduced from RIKEN RESEARCH with permission.
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11

Research highlights
Hiroshi Ohno

Koji Hase

Sorting out
gut health
Gastrointestinal
inflammation isprevented by aprotein sorting factor
found in cells lining
the gut

Figure :T

AP-1B expression in gut epithelial cells prevents bacteria fromentering the gut and causing inflammatory bowel diseases.

he gastrointestinal tract is lined with intestinal epithelial cells ( IECs ) thatmaintain gut health by keeping bacteria and pro-inflammatory immune

cells from infiltrating gut tissues. Now, a team of researchers in Japan has
shown that a protein in these cells, which is responsible for sorting many
proteins to particular portions of the IEC surface, plays a key role in IEC
modulation of gut inflammation.
IECs are polarized cells, with a bottom surface that attaches to deeper
gut tissues, and a top surface that faces the inside of the gut, where it is
exposed to ingested food and gut-resident bacteria. Proteins that are created in the cell are sorted preferentially to either the top or the bottom portionof the IEC. For example, cytokine receptors are shuttled mainly to the bottomof IECs so they can respond to cytokines released by immune cells within
deeper gut tissues. Led by Koji Hase at the RIKEN Research Center forAllergy and Immunology, the researchers thought that disruption of proper
protein sorting could affect the ability of IECs to properly respond to theirenvironment.
To test their theory, the researchers generated mice lacking the μ1Bsubunit for a sorting protein called adaptor protein-1B ( AP-1B ) . These micedeveloped an inflammatory gut disease called colitis, in which large number
Daisuke Takahashi

of immune cells infiltrated the gut. Mice lacking AP-1B expressed fewer
antibacterial proteins, allowing bacteria to attack gut tissue ( Figure ) . Haseand colleagues showed that this bacterial entry enhanced immune cell
recruitment into the gut, because antibiotics could reduce the inflammationin these mice.
Cytokines such as interleukin-17 ( IL-17 ) are responsible for inducing antibacterial protein expression in IECs. However, the researchers found thatcells lacking AP-IB were unable to properly sort cytokine receptors, including
the IL-17 receptor, to the appropriate portion of the IEC membrane. Thissuggested that IECs may have failed to properly respond to IL-17 because its
receptors were in the wrong part of the cell.
When Hase and colleagues examined IECs in humans with an inflamma-

Original Research Paper
Daisuke Takahashi, Koji Hase, Shunsuke Kimura, Fubito
Nakatsu, Masumi Ohmae, Yasushi Mandai, Toru Sato,
Yasuhiro Date, Masashi Ebisawa, Tamotsu Kato, Yuuki
Obata, Shinji Fukuda, Yuki I. Kawamura, Taeko Dohi,
Tatsuro Katsuno, Osamu Yokosuka, Satoshi Waguri,
Hiroshi Ohno. The epithelia-specific membrane trafficking
factor AP-1B controls gut immune homeostasis inmice. Gastroenterology Vol. 141, 621-632, 2011.

tory bowel condition called Crohn’s disease, they found that expression ofthe μ1B subunit was reduced, and that one cytokine receptor seemed to sort
to the wrong portion of the IEC surface. “AP-1B-dependent protein sorting
therefore seems to control epithelial immune functions that keep the humangut healthy,” explains Hase. Enhancing the expression of μ1B could be apotential therapy for Crohn's disease, the team concludes.

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Research highlights
Tomohiro Kurosaki

CIN85 drives B
cell responses
by linking the
BCR to the
NF- κB signaling
pathway

Figure :W

Colocalization of CIN85 clusters and BCR

hen pathogens invade the body, B cells quickly respond to them byproducing antibodies. B cells can recognize the antigens on patho-

gens because they bind to receptors on their cell surface ( B cell receptor( BCR )) . Binding of antigen stimulates downstream signaling pathways that
promote activation, proliferation and differentiation of B cells to produce antibodies. Defects in these signaling pathways would lead to insufficient antibody production and immunodeficiency, whereas excess activation could
cause autoimmune diseases or B cell malignancies. Thus, elucidation of
BCR-mediated signaling pathways is essential for understanding the causeof such diseases and for the development of new therapies.
Dr. Tomohiro Kurosaki and his group ( Laboratory for Lymphocyte
Differentiation ) previously discovered that the adaptor protein BLNK is a key
molecule in BCR signaling and subsequent B cell responses. To further
understand the role of BLNK in BCR signaling, they decided to identify
proteins that bind to BLNK.
Using a yeast two-hybrid screen, they found that CIN85, another adaptor molecule, interacts with BLNK. Because CIN85 is expressed in manydifferent immune cell types, they generated a B cell-specific CIN85 knockout
mice to analyze its function in B cells. To determine its role in antibody production, they immunized the mice with a T cell independent type II ( TI-II )antigen. TI-II antigens are known to stimulate antibody production in theabsence of T cell help. The TI-II response was barely detectable in the CIN85
knockout mice, indicating the importance of CIN85 in T cell independent
antibody production.
To further dissect the proliferative signaling pathway involving CIN85,

Kohei Kometani (left) and Tadashi Yokosuka (right)

they gave various stimuli to CIN85 knockout B cells in vitro. The mutant B
cells exhibited defective survival after anti-IgM stimulation, demonstrating
CIN85 involvement in BCR-mediated proliferation and survival. Because
BCR stimulation induces multiple signaling pathways, they next decided toaddress which pathways are perturbed by the CIN85 knockout. They
identified a defect in the NF-κB signaling pathway in the mutant B cells andcould correct this defect in vivo by introduction of a constitutively active
form of IKK-β, an NF-κB activating enzyme, into the knockout mice. Thus,
they confirmed that CIN85 participates in the BCR-mediated NF-κB
signaling pathway.
In this study, the RCAI investigators discovered the importance of CIN85

Original Research Paper
Kometani, K., Yamada, T., Sasaki, Y., Yokosuka,T., Saito, T., Rajewsky, K., Ishiai, M., Hikida, M.
and Kurosaki, T. CIN85 drives B cell responses
by linking BCR signals to the canonical NF-κB
pathway. J Exp Med . 208 ( 7 ) : 1447- 57. 2011

in BCR-mediated survival, proliferation and antibody production due to its
association with BLNK and participation in NF-κB signaling. This finding
suggests that a functional defect in CIN85 would induce immunodeficiency,
or that hyperactivation of CIN85 could induce autoimmune diseases or cancer. Therefore, CIN85 may be a promising therapeutic target for regulationof B cell antibody production and control of immune diseases.

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13

Research highlights
Masaru TaniguchAllergy has become oneof the most prevalentdiseases in Japan, affecting
30%

of

the

population.

Atopic asthma is one of the

Functional anddevelopmental
heterogeneityof invariant
natural killer
T cells

more serious allergic diseases and it afflicts 300
million people worldwide,causing

250

deathsevery

numbersarethousand
year.

The

Figure:increasing;

Newly proposed model of NKT cell
development and function

childhood asthma is found in 6% of children these days, six times more thanin the 1980’s.
A research team lead by Masaru Taniguchi and Hiroshi Watarai ( Group
Director and Senior Researcher, respectively, at the Laboratory for Immune
Regulation ) had previously identified a subtype of Natural Killer T ( NKT ) cells
expressing IL-17 receptor B ( IL-17RB + ) that triggers the pathogenesis in airway hyper reactivity, an animal model for asthma. IL-17RB + NKT cells produce IL-4, IL-5, IL-13, IL-17A and IL-22, cytokines that cause airway
inflammation and damage bronchial mucosa. In further investigating thisNKT subtype, they recently discovered that there are distinct developmental
lineages and functions among NKT cells ( Figure ) .
It is known that NKT cells change their cell surface marker expression
patterns during development; NKT cells develop through Stage 1 ( CD44NK1.1- ) , Stage 2 ( CD44 + NK1.1- ) and Stage 3 ( CD44 + NK1.1+ ) in the thymus
and then move to the periphery. When the team analyzed the expression of
IL-17RB in mouse thymus, they found that only 10% of thymic NKT cells were
IL-17RB + , and most of them were in Stage 1 and 2. Surprisingly, these
IL-17RB + NKT cells did not require IL-15, which, until now, was thought to be
essential for NKT cell maturation. IL-17RB + NKT cells maturated from Stage
1 to Stage 2 and then, without changing to IL-17RB -, they moved to theperiphery. This observation suggested that IL-17RB + cells constitute a distinct NKT subtype, one which follows a different developmental pathway
from the IL-17RB - NKT cells. Based on cytokine production, two distinct
subpopulations of IL-17RB + NKT cells could be identified, IL-17RB + CD4 + andIL-17RB + CD4- NKT cells. IL-17RB + CD4 + NKT cells responded to IL-25 andproduced TH 2 cytokines ( IL-13, IL-4 ) , TH 9 cytokines ( IL-9, IL-10 ) and TH17
cytokines ( IL-17A, IL-22 ) , while IL-17RB + CD4- NKT cells responded to IL-23
and produced TH17 cytokines ( IL-17A, IL-22 ) . These cytokines are known toHiroshi Watarai

induce allergic reactions directly but also to stimulate other immune cells andcause inflammation.
Given this developmental heterogeneity, the team next analyzed in vivo
functions of IL-17RB + NKT cells. They found that the allergy prone BALB/cmouse strain had 3-4 fold more IL-17RB + NKT cells than the less allergic
C57BL /6 strain. Indeed, Il17rb knockout mice had a defective production of

Original Research Paper
Hiroshi Watarai, Etsuko Sekine-Kondo, Tomokuni Shigeura,
Yasutaka Motomura, Takuwa Yasuda, Rumi Satoh, Hisahiro
Yoshida, Masato Kubo, Hiroshi Kawamoto, Haruhiko
Koseki and Masaru Taniguchi. Development and functionof invariant natural killer T cells producing TH 2- andT H17-cytokines. PLoS Biol. Vol. 10, e1001255, 2012.
Related paper
Asuka Terashima, Hiroshi Watarai, Sayo Inoue, Etsuko Sekine,
Ryusuke Nakagawa, Koji Hase, Chiaki Iwamura, Hiroshi
Nakajima, Toshinori Nakayama, and Masaru Taniguchi.
A novel subset of mouse NKT cells bearing the IL-17
receptor B responds to IL-25 and contributes to airway
hyperreactivity. J. Exp. Med., Vol. 25, 2727-2733, 2008.

TH 2, TH 9, and TH17 cytokines, and when they were infected with respiratorysyncytial virus ( RSV ) , which causes bronchitis and pneumonia, Il17rb knockout mice did not develop any airway inflammation.
“IL-17RB + NKT cells were responsible for exacerbating the inflammation
caused by RSV infection. RSV infection is one of the major causes of pneumonia in asthmatic babies and children. Regulation of IL-17RB + NKT cells
would be a key for prevention of inflammation in childhood asthma,” said
Hiroshi Watarai.
This research was supported by Japan Science and Technology Agency’s
Precursory Research for Embryonic Science and Technology (PRESTO ).

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14

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Research highlights

HE staining

DAPI

hCD45

Tryptase staining

c-Kit

merge

Fumihiko Ishikawa

Membraneboundhuman SCF/KL
promotesin vivo humanhematopoietic
engraftment
and myeloid
differentiationin mice

Figure:T

Distribution of human mast cells in the stomach of a humanized mouse. The left
panel shows H&E (top) and mast cell tryptase (bottom) staining of humanized
hSCF Tg NSG mice. The right panel shows staining for human CD45 to identify
human hemopoietic cells, human c-kit to identify mast cells, and a merged image.
DAPI staining identifies nucleated cells in this section of mouse stomach.

he humanized mouse model system has served as a valuable tool toinvestigate human hematopoiesis, immunity, and diseases in vivo,

because biomedical research involving living humans is severely limited by
ethical and technical constraints. However, one of the major limitations of
the humanized mouse model is that the tissue microenvironments are primarily of mouse origin. The lack of an appropriate microenvironment contributes to the impaired development of human T-lymphoid and myeloid cells
in this system, thus limiting investigation of human hematopoiesis and immunity in the humanized mouse. To help overcome this problem, in collaboration with Dr. Leonard D. Shultz at the Jackson Laboratory, Dr. Fumihiko
Ishikawa and his team ( Laboratory for Human Disease Model ) had previously
reported the development of functional human T cells in NOD/SCID/IL2rgKO( NSG ) mice expressing human HLA class I, a molecule important for human
T cell selection/development and function in the mouse. Recently, they further modified this NSG mouse and successfully created an improved mouse( hSCF Tg NSG mouse ) that expresses a membrane bound form of humanstem cell factor ( SCF ) , which is critical for the maintenance of stem and progenitor cell activities. When this hSCF Tg NSG mouse was transplanted withhuman hematopoietic stem cell to generate a humanized mouse, chimerismof human hematopoietic cells was dramatically increased. The frequency ofhuman lymphoid and myeloid cells that developed in the bone marrow ofhSCF Tg NSG mice was significantly higher ( 97.1% and 49.7% respectively )compared with non-Tg NSG mice ( 63.1% and 26.2% respectively ) .
Among the leukocytes, the researchers identified human mast cells,
granulocytes and antigen-presenting cells. Notably, the greatest subfraction

Shinsuke Takagi

among engrafted human myeloid cells consisted of granulocytes including
neutrophils and mast cells. Both mature and immature human mast cells
were present in bone marrow, and human mast cells were identified not onlyin spleen and hematopoietic organs but also in lung, gastric tissue, andintestinal tissues of hSCF Tg NSG recipients.
In human hematopoiesis, SCF-cKit signaling is critical for the maintenance of stem and progenitor cell activities. When human mast cells fromgastric tissues of hSCF Tg recipients were examined, human cKit was present in these cells. The observed significant improvement in human hematopoietic cell chimerism could be attributed to preferential binding of human SCF
to human c-Kit in hematopoietic stem cells, resulting in efficient SCF-c-Kit

Original Research Paper
Takagi S, Saito Y, Hijikata A, Tanaka S, Watanabe T,
Hasegawa T, Mochizuki S, Kunisawa J, Kiyono H, Koseki
H, Ohara O, Saito T, Taniguchi S, Shultz LD, Ishikawa
F. Membrane-bound human SCF/KL promotes invivo human hematopoietic engraftment and myeloid
differentiation. Blood. 19 (12 ) :2768-77. 2012

signaling.
To further improve human hematopoietic cell development in the humanized mouse, it is clearly necessary to humanize the microenvironment. Thisis also required for in vivo investigation of the interactions between hematopoietic stem cells and their microenvironment. "The hSCF Tg NSG humanized
mice will facilitate investigation of in vivo differentiation, migration, function,and pathology of human mast cells," Dr. Ishikawa noted.

15

Outstanding
Contributionof the Year
2011

R

through lowering the intracellular concentration of free zinc ( Kitamura et al.,Nat Immunol, 2006 ) . To physiologically analyze the function of zinc, theygenerated knockout mice of various
zinc transporters. Using ZIP13 deficient mice, they found that ZIP13 isrequired for bone, teeth and connec-

IKEN RCAI Outstanding Contribution of the Year 2011
was given to three researchers, Toshio Hirano ( photo1) , Group Director of Laboratory for Cytokine Signaling,
Takaharu Okada ( photo 2 left) Leader of the Research Unit
for Immunodynamics and Tomokatsu Ikawa ( photo 3 left) ,Senior Researcher of the Laboratory for Lymphocyte Development. This award was established in 2006 to recognize
RCAI investigators who make outstanding contributions totive tissues development, and is involved in BMP/TGF-βsignaling, and that the same genetic defect causes EhlersDanlos syndrome in humans ( Fukada et al., PLoS ONE,2008 ) . Deletion of the ZIP14 gene resulted in growth retardation and dysregulation of G-protein coupled receptor signaling ( Hojyo et al., PLoS One, 2011) . They also reported thatthe zinc transporter ZnT5 is required for mast cell-mediateddelayed type allergic responses. ( Nishida et al., J Exp Med,2009 ) . In addition to the role of zinc

the Center.
Dr. Toshio Hirano received thetransporters,

Hirano’sgroup

found that zinc functions as a secondtion of Japanese and RIKEN’s Immu-messenger capable of converting an

nology research by the discovery of
IL-6, analysis of IL-6 receptor signal-

external signal into internal events( Yamasaki et al., J Cell Biol, 2007) .

ing, and further development of the

They named this the “Zinc wave”.

new area of zinc signaling. It is

Based on these results, Dr. Hirano

increasingly expected that immunolo-

proposed two types of zinc signal-

gists will elucidate disease mecha-

ings, “late zinc signaling” dependentnisms and therapeutics. Dr. Hirano’s

on changes in the transcription of Zn

discovery of IL-6 contributed greatly

transporter genes and “early zinc sig-

to our understanding of the pathogenic mechanisms of rheumatoid

Photo 1 : Dr. Toshio Hirano

arthritis and other autoimmune diseases ( Hirano, Proc. Jpn. Acd., Ser.
B, 2010 ) .

naling” which occurs without newtranscription (Fig. 1) (Hirano et al., Adv
Immunol, 2008 , Fukada et al., J. Biol.
Inorg. Chem, 2011).

Dr. Hirano had alreadyDr. Hirano also contributed to thebeen awarded the Crafoord Prizemanagement of RCAI as Deputy

from the King of Sweden and he also

Director.

received the Japan Prize last year forinternational symposium on immunol-“Discovery of interleukin-6 and its

ogy in collaboration with JSI, andapplicationdiseases.”

contributed to the establishment of a

These awards made impacts through-collaborative relationship between

out the world and encouraged RIKENRCAI and Osaka University.

researchers, especially the young

Hirano was the Dean of Graduate

scientists.

intreating

Dr. Hirano’s pioneeringresearch on zinc and zinc transport-

Photo 2 : Dr. Takaharu Okada (left) and
Dr. Masaru Taniguchi (right)

He organized the annual

Dr.

School of Medicine in Osaka University from 2008 till March 2011, and iners also contributed to the outstand-August of 2011 he became the Presi-

ing reputation of RIKEN’s science.

dent of Osaka University. In addition,

Dr. Hirano and his team discov-he has recently become an Executive

ered that zinc has roles in mediating
and controlling intracellular signaling

Member of the Council for Scienceand Technology Policy ( CSTP ) for the

events. In 2004, they found that theJapanese Cabinet. To avoid any

Zn

Slc39a6/Zip6/Liv1

appearance of conflict of interest, heis a STAT3 target gene and showed

decided to leave RCAI at the end ofthat it has a role in cell migration dur-

March 2012.

transportering early zebrafish development( Yamashita et al., Nature, 2004 ) . They
then found in dendritic cells that LPSinduced maturation is partly mediated

16

Dr.

award for his contribution to promo-

In his speech, Dr. Hirano recalled
the time he spent at RCAI. “I still
Photo 3 : Dr. Tomokatsu Ikawa (left) and
Dr. Masaru Taniguchi (right)

remember the day when Dr. Taniguchi was formally appointed as the16

in which Yellow Fluorescent Protein had been inserted into
one copy of the Bcl6 gene. Since this insertion partially disrupted Bcl6 function, this reporter system also enabled themto examine the effects of partial Bcl6 deficiency. Following
the induction of an immune response in the BCL6 reportermouse, Okada and colleagues observed an increase of Bcl6
expression in antigen-specific B cells within peripheral
regions of the lymph node; these cells subsequently migrated
to the GC, where they continued to express high levels of
Bcl6. This migration and maturation process was dependent
upon interaction with Bcl6-expressing helper T cells. All T
cells expressing high levels of Bcl6 became Tfh cells, but
Figure 1: Early Zn signaling and late Zn signaling. Intracellular Zn
signaling can be classified into at least two categories: early Zn
signaling, involving the Zn wave, which is directly induced by an
extracellular stimulus, and late Zn signaling, which is dependent
on a transcriptional change in Zn transporter expression

many Tfh cells subsequently reduced Bcl6 production to

Director of RCAI in June of 2001. Since then, under his
excellent leadership, everything began. This building wascompleted in October 2003. All people gathered here andstarted activities in April 2004. Then the time passed and weare now celebrating the 10th anniversary. During this 10year period, RCAI made major contributions to immunology,
not only in Japan but also in the world. RIKEN’s Second
Midterm Plan will end next year, and for the Third Midterm
Plan, a new initiative will start under a new director, focusing
on integrative biology. It is very sad for me that I have to leave
here formally in order to avoid any conflict of interests after
appointment to the CSTP. However, I think the passion ofthis Center, which has developed in the last 10 years, willremain in each of us forever.” Director Taniguchi congratulated Dr. Hirano on his decision to take on the most impor-

Figure 2: Two-photon imaging of the B cell entry into the pre-formed GC.
The top panels are representative images from 60 min recordings.
The bottom panels show the surface of GCs reconstructedfrom the CFP images and the trajectories of the cells.

tant burden for science in Japan, although it saddened himthat Dr. Hirano officially left RCAI.
Dr. Takaharu Okada was awarded for his contribution

varying degrees ( Fig. 2 ) . Based on these findings, the authors

for establishing “in vivo imaging of the dynamics of immune

suggest that Tfh cells expressing low levels of Bcl6

cells by two-photon laser scanning microscopy”.

may give rise to ‘memory cells’, which enable the immune
system to react quickly to recurring threats. ( Kitano et al.,This isbecoming a powerful new method in immunological studies,high level that has been reached by only a handful of groups

Immunity, 2011)
Dr. Okada said in his speech that he took the award asin the world. Recently, his lab further demonstrated theencouragement to move towards integrative biology. “Weand his lab has developed this technique to a remarkably

effectiveness of this technique by making important findings
regarding the dynamics of germinal center formation, thekey event in humoral immune responses. This technique is
expected to play an important role in RCAI’s future strategy
to promote integrative biology for understanding the mechanisms of inflammatory and immunological disease development.
Dr. Okada tracked the process of B cell development in
the mouse germinal center ( GC ) by monitoring expression of
Bcl6, a transcription factor that facilitates B cell evolution by
regulating expression of key developmental genes. In GCs,
antibody-producing B cells undergo differentiation to generate higher-quality antibodies, a process that is supported bya class of T cells known as follicular helper T ( Tfh ) cells. To
track the events in vivo, the team generated transgenic mice

Photo 4 : Dr. Takaharu Okada (left) and Dr. Gib
Bogle (right) at Dr. Bogle's office17

actually would like to do some modeling, but it will be reallycell lineage differentiation. In his lab, using E2A knockout

hard. It is necessary to incorporate many kinds of factors

progenitor cells, Dr. Ikawa found that these E2A knockout

like G-protein coupled receptors, various other receptors

hematopoietic progenitor cells have self-renewal activity withmultipotentcy ( Ikawa et al., Immunity, 2004 ) .

and also adhesion molecules, and it will also be hard tomodel that immune response in tissues.

This is a great

He joined Dr. Kawamoto’s lab in 2006 and started

opportunity RCAI has given me. In 2010, a group of research-

research on T cell lineage commitment. Their main interest

ers from New Zealand visited us and we had a joint work-

was how hematopoietic stem cells or early multipotent pro-shop with New Zealand and Chiba University. Two of theresearchers, Rod Dunbar ( Maurice Wilkinson Center) andgenitors commit to the T cell lineage in the prethymic or early

Gib Bogle ( Bioengineering Institute in University of Auckland )( photo 4 right ) , invited me for two weeks to launch the col-occurs in the thymic DN2 stage, so that in the earlier stages,

thymic stages. Dr. Masuda found that the commitment

laboration for modeling of immune responses.” Drs. Dunbar

MT progenitors still maintain other lineage potentials, suchas macrophage or NK cells ( Masuda et al., J Immunol, 2007;

and Bogle had developed an agent-based model for T cell

Wada et al., Nature, 2008 ) ( Fig. 3 ) . So the next question was

activation in a lymph node. Agent based models are compu-

how this commitment occurs. To address this question, Dr.

tational models for simulating action and interaction ofautonomous agents, and such models incorporate processes at a range of scales: cell receptor-ligand dynamics,
cell motility, TCR stimulation leading to T cell activation andproliferation, and cell trafficking. They are focusing on understanding of the immune response by integrating knowledge,
to build an in silico laboratory for biological experiments, andto provide a new tool for immunology research. “I think the
purpose of our modeling will be to try to extract the information that is hard to get through wet lab experiments, such as
lipid mediator micro-distribution; for example S1P or LPA,
those really important lipids for B cell and T cell dynamics. It
is really hard to get the real data, but how is it if we put X ina model and then are able to get some information like thatthrough the agent-based model of the B cell follicle? The
ultimate hope may be to make a virtual lymph node that

Figure 3: Intrathymic progenitors
become fully committed
to the T cell lineage
in the midst the DN2stage by terminating
myeloid potential

helps us to improve the design of the synthetic lymph node,
by providing this type of information,” said Dr. Okada.
Dr. Tomokatsu Ikawa was awarded for his contribution

Ikawa established a stromal cell-free culture system because

on the “elucidation of molecular mechanisms of T cell lineage

the only culture system to induce T cells was using stromalcommitment”.

Dr. Ikawa and colleagues established acells with Notch ligand. He tried various different culture

unique culture system that can induce development of
myeloid-T ( MT ) bipotent progenitors from multipotent

conditions and finally found out that reduction of the con-hematopoietic progenitors. Using this culture system, Dr.

T cell development without the need for any stromal cells.

Ikawa discovered Bcl11b as a master regulator for T lineage

This was the first example to show that T cell can be induced

commitment and also provided molecular evidence in sup-in stroma-free conditions. He also found that Bcl11b is anport of the “Myeloid-based model” in T cell development,

essential transcriptional factor in T cell lineage commitment.

which was discovered by Dr. Kawamoto at RCAI. This study

In Bcl11b knockout mice, T cell development was blocked at

centration of IL-7 in the middle of the culture period induces

has had a huge impact on the field of developmental immunology, and Dr. Ikawa has been recently selected to receive
the Minister of Education, Culture, Sports, Science andTechnology ( MEXT ) Prize for Young Investigators 2012.
Dr. Ikawa started his research career in 1997 when he
was a graduate student in Dr. Katsura’s laboratory in Kyoto
University. His first project was to understand how T or NK
lineage commitment occurs. He established a novel clonal
culture system to study T or NK commitment status, and healso showed that Id2 is essential for NK lineage commitment
from bipotent progenitors ( Ikawa et al., Proc Natl Acad Sci U
S A, 2001). After obtaining a Ph.D. degree, he moved toCornelis Murre’s lab at UCSD as a postdoc. Dr. Murre had
identified E2A, a famous transcription factor essential for B

18

Figure 4: DN2-determiantion step is a critical checkpoint of T
cell development, and Bcl11b is a key factor that drives T
cell lineage determination. The blockage of Bcl11b driven
checkpoint induces self-renewal of arrested progenitors

the DN2 stage, and these cells can be maintained in vitro

think about more integrative medical life sciences in the next

with self-renewal capacity. From these data, he showed that
Bcl11b is a key factor for T cell lineage determination ( Ikawa

third mid-term plan. This could not have been done without

et al., Science, 2010 ) ( Fig. 4 ) .

today’s awardees of the Outstanding Contribution of the

In his speech, he mentioned several ongoing projects.

RCAI’s achievements during the last 10 years, and I think
Year 2011 are also contributors to this new direction.

I

One was on transcriptional networks in T cell lineage com-believe RCAI will play a central role for RIKEN to make a new

mitment, in collaboration with the Omics Science Center

step forward towards the new direction,” said Mr. Okuma.

(OSC). Using the culture system he established together
with the CAGE ( Cap Analysis of Gene Expression ) system in

Mr. Okuma will resign his position at the end of March, 2012.

OSC, they are now working to draw transcriptional networks

Watanabe, Director of RIKEN Yokohama Institute Research

involved in T cell commitment.

Promotion Division .

Another one was on the

A large flower bouquet was given to him by Ms. Sonoko

maintenance of T cell progenitor fate by epigenetic factors,
in collaboration with Dr. Koseki of RCAI. Dr. Ikawa has beenable to reprogram T cells to B cells by inactivating epigenetic
factors. “I want to expand our knowledge of lineage commitment during lymphocyte development. I’d like to thank Dr.
Kawamoto for giving me the opportunity to work here in
RIKEN, probably the best institute in the world, and I hope tocontinue to contribute to the Center,” said Dr. Ikawa.
During the awards ceremony, Mr. Kenji Okuma, Directorof RIKEN Yokohama Institute ( Photo 5 ) , explained about the
new direction of the Center. RIKEN’s 5-year Mid-term Plan
will be finished in March next year, and RIKEN decided toestablish a new center focusing on integrative medical sciences. During the first five-year mid-term plan, RCAI initiatedits research efforts in immunology. The management sys-

Photo 5 : Mr. Kenji Okuma, Director of
RIKEN Yokohama Institutetem of RCAI was extraordinary and its advisory council system was unique.

In the second mid-term plan, RCAI

Dr. Taniguchi, RCAI’s Director said to the members ofstrengthened and expanded its efforts, and received excel-

RCAI, “I believe we’ve made extraordinary achievements

lent evaluations. However, after these 10 years, RIKEN is

and contributions to the immunology field during these 10

required to make qualitatively different research achieve-

years. This is the result of everyone’s tremendous continu-

ments and has to consider some new direction for the future.

ous efforts. I would like to thank all of you and we will never“I respect RCAI because it has been making tremendous

forget the efforts by all the RCAI members to cooperate for

efforts to rethink the direction of future immunology. Still

the Center. “

maintaining its central focus in immunology, RCAI tries toReferences :Fukada, T., Civic, N., Furuichi,
T., Shimoda, S., Mishima, K.,
Higashiyama, H., Idaira, Y., Asada, Y.,
Kitamura, H., Yamasaki, S., Hojyo, S.,
Nakayama, M., Ohara, O., Koseki, H.,
dos Santos, H. G., Bonafe, L., Ha-Vinh,
R., Zankl, A., Unger, S., Kraenzlin, M.
E., Beckmann, J. S., Saito, I., Rivolta,
C., Ikegawa, S., Superti-Furga, A., andHirano, T. (2008) The Zinc Transporter
SLC39A13/ZIP13 is Required forConnective Tissue Development; Its
Involvement in BMP/TGF-β Signaling
Pathways. PLoS ONE 3: e3642
Fukada T., Yamasaki, S., Nishida,K., Murakami, M., Hirano, T. (2011)
Zinc homeostasis and signaling in
health and diseases: Zinc signaling.
J Biol Inorg Chem 16, 1123-1134
Hirano, T. ( 2010) . Interleukin 6 inautoimmune and inflammatory
diseases: a personal memoir. Proc
Jpn Acd, Ser B 86, 717-730.
Hirano, T., Murakami, M., Fukada,T., Nishida, K., Yamasaki, S., andSuzuki, T. ( 2008) . Roles of zinc andzinc signaling in immunity: zinc as
an intracellular signaling molecule.
Adv Immunol 97, 149-176.

cultured E2A-deficient hematopoietic
progenitor cells are pluripotent.
Immunity 20, 349-360.

Hojyo, S., Fukada, T., Shimoda,
S., Ohashi, W., Bin, B.H., Koseki,
H., and Hirano, T. ( 2011) . The zinctransporter SLC39A14/ZIP14 controls
G-protein coupled receptor-mediatedsignaling required for systemic
growth. PLoS One 6, e18059.

Kitamura, H., Morikawa, H., Kamon,
H., Iguchi, M., Hojyo, S., Fukada, T.,
Yamashita, S., Kaisho, T., Akira, S.,
Murakami, M., et al. ( 2006) . Toll-likereceptor-mediated regulation of zinc
homeostasis influences dendritic cellfunction. Nat Immunol 7, 971-977.

Ikawa, T., Fujimoto, S., Kawamoto,H., Katsura, Y., and Yokota, Y.
( 2001) . Commitment to natural killer
cells requires the helix-loop-helix
inhibitor Id2. Proc Natl Acad
Sci U S A 98, 5164-5169.

Kitano, M., Moriyama, S., Ando, Y.,
Hikida, M., Mori, Y., Kurosaki, T.,
and Okada, T. ( 2011) . Bcl6 protein
expression shapes pre-germinal
center B cell dynamics andfollicular helper T cell heterogeneity.
Immunity 34, 961-972.

Ikawa, T., Hirose, S., Masuda,K., Kakugawa, K., Satoh, R.,
Shibano-Satoh, A., Kominami, R.,
Katsura, Y., and Kawamoto, H.
( 2010) . An essential developmental
checkpoint for production of the Tcell lineage. Science 329, 93-96.
Ikawa, T., Kawamoto, H., Wright, L.Y.,
and Murre, C. ( 2004) . Long-term

Masuda, K., Kakugawa, K., Nakayama,T., Minato, N., Katsura, Y., andKawamoto, H. ( 2007 ) . T cell lineage
determination precedes the initiationof TCR beta gene rearrangement.
J Immunol 179, 3699-3706.
Nishida, K., Hasegawa, A., Nakae,S., Oboki, K., Saito, H., Yamasaki,S., and Hirano, T. ( 2009 ) . Zinctransporter Znt5/Slc30a5 isrequired for the mast cell-mediateddelayed-type allergic reaction but
not the immediate-type reaction.
J Exp Med 206, 1351-1364.
Wada, H., Masuda, K., Satoh, R.,
Kakugawa, K., Ikawa, T., Katsura,Y., and Kawamoto, H. ( 2008 ) . Adult
T-cell progenitors retain myeloid
potential. Nature 452, 768-772.
Yamasaki, S., Sakata-Sogawa, K.,
Hasegawa, A., Suzuki, T., Kabu, K.,
Sato, E., Kurosaki, T., Yamashita,S., Tokunaga, M., Nishida, K.,
et al. ( 2007 ) . Zinc is a novel
intracellular second messenger.
J Cell Biol 177, 637-645.
Yamashita, S., Miyagi, C., Fukada,T., Kagara, N., Che, Y.S., and Hirano,T. ( 2004) . Zinc transporter LIVI
controls epithelial-mesenchymaltransition in zebrafish gastrulaorganizer. Nature 429, 298-302.

19

Prize Winners
2011
Makio Tokunaga ( Photo 1) , Leader of the Research
Unit for Molecular Systems Immunology received The
Commendation for Science and Technology from the
Minister of Education, Culture, Sports, Science and Technology, 2011. He won the Research Category Prize for his
studies on elucidation of molecular dynamics and interactions by the development of single molecular imaging
Photo 1 : Makio Tokunaga

microscopy. This prize is awarded to investigators whose
research is highly original and contributes to the science and technology of Japan.

Tomokatsu Ikawa ( Photo 2 ) , Researcher of the Laboratory for Lymphocyte Development, received the Research Encouragement Award of the Japanese Society for
Immunology for his research on transcriptional regulation of T cell/ B cell lineage commitment. He developed a unique cell culture system to analyze cell lineage commitments from hematopoietic progenitors. He found that Id2 is essential for NK lineage
commitment from T/NK bipotent progenitors, and that Notch signals are essential forearly T cell development. Recently, he showed that BCL11B is essential for T cell linPhoto 2 : Tomokatsu Ikawa

eage commitment. This award is given to young investigators, age 40 or younger, whohave performed distinguished immunological research.

Nyambayar Dashtsoodol ( Photo 3 ) , Researcher of the Laboratory for Immune Regulation, received the President of Mongolia'sPrize for the Distinguished Young Scientist of the Year 2011. He
was awarded for his study of the thymic development of NKT cells:
identification of CD4, CD8 double-negative NKT cell precursors.
This award is given to a young Mongolian scientist ( under the ageof 35 years ) whose research has greatly contributed to the advancement of science aimed for human benefit and representation of
Photo 3 : Nyambayar Dashtsoodol

Mongolia at the international level.

Toshiyuki Fukada ( Photo 4 ) , Researcher of the Laboratory for Cytokine Regulation,
received the Kanagawa Nambyo Study Foundation Award 2011 for elucidation of themechanisms of zinc-related diseases and received 400 thousand yen. This award isgiven to scientists working in Kanagawa Prefecture who contributed research advancements aimed at conquering intractable diseases.
RIKEN Research and Technology Incentive Awards 2011 were presented to two RCAI
researchers, Masahiro Kitano (Photo 5) and Atsushi Hijikata (Photo 6). Kitano,
Special Postdoctoral Researcher of the Research Unit for Immunodynamics, received
Photo 4 : Toshiyuki Fukada

the award for his study on in vivo imaging of immune cell dynamics by two-photon laser
scanning microscopy. Hijikata, a Research Associate of the Laboratory for Immuno-

Photo 5 : Masahiro Kitano

20

Photo 6 : Atsushi Hijikata

Photo 7 : Shinji Fukuda

genomics, received the award for the establishment of a high precision gene testing
method for mosaic autoinflammatory diseases. The RIKEN Research and Technology Incentive Award was established in FY2009 to recognize young researchers andtechnicians under age 40 who have contributed to furthering RIKEN’s ideals byachieving exemplary results in their research or research support activities. Kitano
also received the Medical Science Prize at RIKEN FY2011 SPDR & FPR Presentationof Research Results for his poster presentation, “Bcl6 Protein Expression Shapes
Pre-Germinal Center B Cell Dynamics and Follicular Helper T Cell Heterogeneity.”

Photo 8 : Shintaro Hojyo

Shinji Fukuda ( Photo 7) and Hiroshi Ohno, Laboratory for Epithelial Immunobiology, received the Bioengineering Research Paper Award, in collaboration with
Yasuhiro Date, Yumiko Nakanishi, Tamotsu Kato and Jun Kikuchi of RIKEN Plant Science Center in September 2011. They were awarded for the paper "New monitoring
approach for metabolic dynamics in microbial ecosystems using stable-isotopelabeling technologies", Vol. 110, pages 87-93, Journal of Bioscience and Bioengineering, 2010. In the paper, they reported a newly developed approach for monitoring
the metabolic dynamics in microbial ecosystems using a combination of DNA fingerprinting and metabolome analysis based on stable-isotope-labeling technologies.
This award is given to selected papers published in the Journal of Bioscience andPhoto 9 : Saya Moriyama

Bioengineering for their contribution to the advancement of bioengineering.

Shintaro Hojyo ( Photo 8 ) , Researcher of the Laboratory for Cytokine Regulation,
received The Best Poster Award at The 6th Annual Meeting of the Japan Transporter
Research Association. He was awarded for his poster presentation "Zinc Transporter,
SLC39A14/ZIP14, regulates the endocrine signals mediated by G protein-coupled
receptors ( GPCR ) "Saya Moriyama ( Photo 9 ) , Graduate Student of the Research Unit for Immunodynamics, received the Incitement Award at The 3rd Meeting of Signal Network Society
for her presentation, “BCL6 Protein Expression Shapes Pre-Germinal Center B Cell

Photo 10 : Kohei Kometani

Dynamics and Follicular Helper T Cell Heterogeneity.” She also received a Keystone
Symposia Scholarship to attend the meeting, "B Cells: New Insights into Normal versus Dysregulated Function ( D6 ) " held on Apr 12-17, 2011, in Whistler, Canada.

Kohei Kometani ( Photo 10 ) of the Laboratory for Lymphocyte Differentiation,
Takayuki Imanishi ( Photo 11) of the Laboratory for Cell Signaling, Rika Ouchida( Photo 12 ) of the Laboratory for Immune Diversity, Yuuki Obata ( Photo 13 ) ,Daisuke Takahashi ( Photo 14 ) and Shinji Fukuda ( Photo 7) of the Laboratory
for Epithelial Immunobiology received Tadamitsu Kishimoto International Travel Award
from the Japanese Society for Immunology to attend international immunology meet-

Photo 11 : Takayuki Imanishi

ings held overseas.

Photo 12 : Rika Ouchida

Photo 13 : Yuuki Obata

Photo 14 : Daisuke Takahashi

21

Excellent Paperof the Year2011

T

he RCAI Award for Excellent Paper was originally established in 2004 with donations from Dr. Masaru
Taniguchi and Dr. Toshio Hirano. The annual award aims to recognize excellent publications by RCAI
scientists. Although the funds were depleted by 2008, RCAI’s strategic committee decided that there was greatvalue in awarding excellent achievements by young researchers and encouraging their efforts, so they provided
the funding to continue this prize.
In 2011, 11 excellent papers were selected from 13 candidates for this award.

Masahiro Kitano, Saya Moriyama and
Takaharu Okada

Takashi Tanaka

Research Unit for Immunodynamics and Laboratory for“PDLIM2 inhibits T helper 17 cell development and granu-

Lymphocyte Differentiation

lomatous inflammation through degradation of STAT3”“Bcl6 protein expression shapes pre-germinal center B cell

Science Signaling, Vol 4, ra85, 2011

Research Unit for Inflammatory Regulation

dynamics and follicular helper T cell heterogeneity”
Immunity, Vol. 34, pp. 961- 972, 2011

Daisuke Takahashi, Koji Hase andHiroshi Ohno

Yasutaka Motomura and Masato Kubo

Laboratory for Bioenvironmental Epigenetics and Labora-Open Laboratory for Signal Network

tory for Epithelial Immunobiology“The transcription factor E4BP4 regulates the production of“The epithelia-specific membrane trafficking factor AP-1B+

IL-10 and IL-13 in CD4 T cells”

controls gut immune homeostasis in mice”

Nature Immunology, Vol. 12, pp. 450- 459, 2011

Gastroenterology, Vol. 141, pp. 621- 632, 2011

Takahisa Miyao and Shohei Hori

Kohei Kometani and Tomohiro Kurosaki

Research Unit for Immune Homeostasis

Laboratory for Lymphocyte Differentiation“Plasticity of Foxp3 + T cells reflects promiscuous Foxp3“CIN85 drives B cell responses by linking BCR signals to

expression in conventional T cells but not reprogramming

the canonical NF-κB pathway”

of regulatory T cells”

The Journal of Experimental Medicine, Vol. 208, pp.1447-1457,

Immunity, Vol. 36, pp. 262-275, 20122011

Hideaki Takagi and Katsuaki Sato

Hiroshi Watarai and Masaru Taniguchi

Laboratory for Dendritic Cell Immunobiology

Laboratory for Immune Regulation“Plasmacytoid dendritic cells are crucial for the initiation of“Development and function of invariant natural killer T cells

inflammation and T cell immunity in vivo”

producing TH2- and TH17-cytokines”

Immunity, Vol. 35, 958-971, 2011

PLoS Biology, Vol. 10, e1001255, 2012

Akiko Hashimoto-Tane and Takashi Saito

Shinsuke Takagi and Fumihiko Ishikawa

Laboratory for Cell Signaling

Laboratory for Human Disease Model“Dynein-driven transport of T cell receptor microclusters“Membrane-bound human SCF/KL promotes in vivo humanregulates immune synapse formation and T cell activation”

hematopoietic engraftment and myeloid differentiation”

Immunity, Vol. 34, 919-931, 2011

Blood, Vol. 119, pp. 2768-2777, 2012

Yohsuke Harada and Masato Kubo
Open Laboratory for Signal Network“The 3′enhancer CNS2 is a critical regulator of interleukin4-mediated humoral immunity in follicular helper T cells”
Immunity, Vol. 36, 188-200, 2012

22

2011
Part 2

Technology and Innovation

RCAI and Torii collaborate fordevelopment of an allergy vaccine

Japanese cedar ( Cryptomeria japonica ) pollinosis is a common allergy in Japan, with a prevalence estimated to be 26% based on a nationwide survey conducted in 2008. Impaired per-formance due to pollinosis and medications used for treating pollinosis is thought to be an
important cause of loss of concentration and productivity of patients in the workplace. Antigenspecific immunotherapy ( SIT ) is considered to be the only curative therapy for allergy. Vaccines
using allergoids and modified Cry j 1, a major allergen of Japanese cedar pollen, have beendeveloped and used for pre-clinical trials; however, none of them has been commercially available
for medical use either due to poor clinical outcomes of late stage clinical trials or the failure to finda cooperative pharmaceutical company to introduce the vaccines into the market. To fill the critical gap between basic research and later stage of drug development, RCAI offered a program
named ‘exchange zone’ where RCAI, universities, hospitals, and pharmaceutical companies work
together for drug development, including allergy vaccine development especially for Japanesecedar pollinosis. An exchange zone is based on the concept that sprinters in a relay race run
together in the exchange zone, where the baton is passed firmly and smoothly. Based on thisprogram, RIKEN and Torii Pharmaceutical Co., Ltd. set up joint research laboratory in RCAI in May
2010 and started research and development of a recombinant SIT vaccine, taking into consideration GLP toxicity tests and GMP. Our aims are to develop a recombinant SIT vaccine with highsafety, tolerability and efficacy, and to introduce the vaccine into the Japanese market.
Development of the recombinant SIT vaccine
The vaccine was originally developed in RCAI as a safer SIT vaccine, one that is less dangerous
in inducing adverse events, such as anaphylaxis, after administration. For the vaccine, recombinant technology is used to conjugate two major allergens from Japanese cedar pollen, namely
Cry j 1 and Cry j 2. The fusion protein is then further modified with polyethylene glycol ( PEG ) to
inhibit binding with immunoglobulin E ( IgE ) , the allergy-inducing antibody that is likely to pre-exist
in the target patient population. PEG is conjugated to the recombinant Cry j 1/Cry j 2 fusion
protein via a cysteine residue on the fusion protein. All cysteine residues except one in the fusion
protein were substituted with serine residues to control the number and location for conjugation
with PEG. The vaccine is expected to lose potential to bind human IgE, which may induce adverse
events after vaccine administration in vivo.
The stability and yield of the recombinant fusion protein were compared among several host/vector combinations including eukaryote and prokaryote. It is also necessary to develop a simple
method for the purification of the recombinant SIT vaccine expressed in prokaryotes or eukaryotes, taking into consideration cost-performance and purity. To date, they have set up a procedure to purify the recombinant protein followed by conjugation with PEG. It is now necessary to
optimize the procedure for large scale industrial production. They are also preparing monoclonal
antibodies specific to the vaccine and will develop vaccine-specific quantification systems such asa sandwich ELISA.

24

Therapeutic potential of the vaccine in a mouse model ofJapanese cedar pollinosis
Systemic injections of the vaccine prevent the increase of serum Cry j 1-specific IgE following
subsequent secondary or tertiary sensitization with native Cry j 1 in mice. In humans, the majorclinical symptoms of the pollinosis are local inflammation like conjunctivitis, rhinorrhea, and sneezing. They established a mouse model of the pollinosis by Cry j 1 sensitization resulting in infiltration of inflammatory cells such as eosinophills and mast cells into ocular and nasal mucosa. They
are assessing improvement in the ocular and nasal inflammation after treatment by local or systematic vaccination. They will then evaluate the therapeutic potential of the vaccine by comparing
the reduction of local inflammation or IgE production with various doses and frequencies of vaccination using the sensitized mouse and mouse model of pollinosis. To improve the therapeutic
effects of the vaccine, elucidation of the therapeutic mechanisms and identification of predictive
biomarkers are important issues. Therefore, they are searching for biomarkers to monitor therapeutic responses during or after the vaccination. They plan to evaluate the therapeutic effects ofthe vaccine in humans using the biomarkers identified in this translational research, after which
they will produce the vaccine as GMP grade.

Members of RIKEN-TORII Joint Research Team
Team Leader :Masaru Taniguchi

Senior Scientist :Takashi Fujimura

Visiting Scientists :Hiroyuki Miyazaki, Yasushi Okumura, Koji Fujinami, Masao Matsuda,
Ryosuke Ishikawa

Technical Staff :Kyounga Seo

Recent Publications :1. Fujimura T, Okamoto Y, Taniguchi M. Therapeutic effects and biomarkers in sublingual immunotherapy: a review. J. Allergy(Cairo) 2012: 381737. (2012)
2. Fujimura T, Yonekura S, Horiguti S, Taniguchi Y, Saito A, Yasueda H, Inamine A, Nakayama T, Takemori T, Taniguchi M, Sakaguchi
M, Okamoto Y. Increase of regulatory T cells and the ratio of specific IgE to total IgE are candidates for response monitoring or
prognostic biomarkers in two-year sublingual Immunotherapy ( SLIT ) for Japanese cedar pollinosis. Clin. Immunol.139: 65-74.
( 2011)
3. Fujimura T, Yonekura S, Taniguchi Y, Horiguti S, Saito A, Yasueda H, Nakayama T, Takemori T, Taniguchi M, Sakaguchi M,
Okamoto Y. The induced regulatory T cell level, defined as the proportion of IL10+Foxp3+ cells among CD25+CD4+ leukocytes,
is a potential therapeutic biomarker for sublingual immunotherapy: a preliminary report. Int. Arch. Allergy. Immunol.
153: 378-387. ( 2010 )4. Fujimura T and Okamoto Y. Antigen-specific immunotherapy against allergic rhinitis: the state of the art. Allergol. Int.
59: 21-31. ( 2010 )25

iPS technology development for
immunological research and therapeutics

Figure :I

Induction of antigen specific
CTL cells from CTL derived
iPS cells. MART1, melanoma
antigen recognized by T cells,
is a protein antigen expressedon the surface of melanocytes
and melanoma cells. Human
iPS colonies derived from
Mart1 antigen-specific CTL
were cut into small clumps
and plated on OP9 stromalcells. On day 13, cells were
harvested by tripsinizationand transferred to co-cultureswith OP9-DLL1 stromalcells. At day 35, DP cells weregenerated in vitro and mostof the DP cells were Mart1
tetramer positive T cells.

nduced pluripotent stem ( iPS ) cells possess tremendous therapeutic potential not only in thefield of regenerative medicine but also for immune therapy. RCAI has started an activity to apply

iPS technology for mouse and human immunology research and therapeutic development. The
core facility for iPS research is engaged in developing efficient protocols to reprogram various
lymphocytes and induce differentiation of iPS cells into lymphoid lineage cells. These studies arebeing done on a collaborative basis with individual research activities in RCAI, and this activity ispartly supported by CREST from JST.

Generation of iPSCs from mature lymphocytes and identification of genes that
resist reprogramming
Mature lymphocytes are believed to be more resistant than embryonic fibroblasts to the genetic
reprogramming needed to generate iPS cells. Since gene expression profiles and global epigenetic status are quite different between lymphocytes and fibroblasts, the iPS group hypothesized
the presence of genes in mature lymphocytes that are difficult to fully reprogram. To test thishypothesis, Daisuke Yamada and colleagues established 20, 2 and 5 independent iPSC clones
from mature B, T and NKT cells ( B-, T-, NKT-iPSC ) in mouse. All of them were qualified as authentic lymphocyte-derived iPSCs by analyzing the genomic configurations of immunoglobulin or T
cell receptor loci, gene expression profiles, and the ability to form teratomas and chimeric mice.

26

However, they found that only 7 of them were competent to differentiate into the germ cell lineage.
By comparing gene expression profiles between B-iPSCs and ESCs, they found that the Polycomb target genes in ESCs tend to be more repressed in B-iPSCs. This over-repression of Polycomb targets turned out to associate with over-loading with Polycomb repressive complex-1(PRC1). Interestingly, they did not observe such PRC1 over-loading in the few B-iPSCs that arecompetent for germline transmission. Consistent with these observations, B-iPSCs can more
efficiently differentiate upon conditional depletion of the PRC1 components Ring1A/B than ESCs.
These results suggest that Polycomb target genes are epigenetically unstable during reprogramming of B-lymphocytes and easily mis-reprogrammed. To test this possibility, the investigators
further analyzed local levels of Histone H3K9 trimethylation ( H3K9me3 ) , which are known to compete for the targets with Polycomb, and again found its over-loading at Polycomb target genes in
B-iPSCs that are incompetent for germline transmission. These results suggest that reprogramming of Polycomb target genes is a rather unstable process during induction of iPSCs. Thisobservation implies that local levels of PRC1 and H3K9me3 could be used as molecular markers
to predict pluripotency of iPSCs.
Generation of CD8 + singlepositive T cells in vitro from iPS cells derived fromhuman mature T cells
Induced pluripotent stem ( iPS ) cells are expected to be used as a source for production of various
types of cells to be used in regenerative medicine. In this study Kawamoto and colleagues established iPS cells from human mature T cells ( T-iPS cells ) , namely from total CD3 + cells or CD4-CD8 +( CD8SP ) cells of cord blood as well as of adult peripheral blood. These T-iPS cells were confirmed
to bear productively rearranged TCRβ chain genes. When co-cultured with OP9/DLL1 cells, T-iPS
cells efficiently generated TCRβ+ CD4 + CD8 + cells on day 40 of cultivation, in contrast to iPS cells
derived from human cord blood CD34 + cells. They further showed that T-iPS cells derived from
CD8SP cells formed substantial numbers of mature CD8SP cells on day 60 of the culture. The
present study thus provides a novel method for cloning and expansion of CD8SP cells with defined
antigen specificity, which can then be applied for cell therapy against cancer.

Members of iPS Group
Leaders :Haruhiko Koseki ( Group Director)
Hiroshi Kawamoto ( Team Leader)
Fumihiko Ishikawa ( Group Director)
Osamu Ohara (Group Director)

Senior Scientist :Hiroshi Watarai

Research Scientists :Daisuke Yamada
Kyoko Masuda

Technical Staff :Momoko Okoshi-Sato, Genta Kitahara, Masako Fujita,
Chieko Tezuka, Sakura Sakata, Yuko Nagata,
Mariko Tomizawa, Raul Eduardo Vizcardo Sakoda

Recent Publications :1. Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Va14-Ja18 TCRa gene.
Watarai H, Rybouchkin A, Hongo N, Nagata Y, Sakata S, Sekine E, Dashtsoodol N, Tashiro T, Fujii S, Shimizu K, Mori K, Masuda
K, Kawamoto H, Koseki H, and Taniguchi M. Blood 115:230-237 ( 2010 )2. Watarai H, Fujii S, Yamada D, Rybouchkin A, Sakata S, Nagata Y, Iida-Kobayashi M, Sekine-Kondo E, Shimizu K, Shozaki Y,
Sharif J, Matsuda M, Mochiduki S, Hasegawa T, Kitahara G, Endo TA, Toyoda T, Ohara O, Harigaya K, Koseki H, Taniguchi
M. Murine induced pluripotent stem cells can be derived from and differentiate into natural killer T cells. J Clin Invest.
120:2610-2618 ( 2010 )27

The PIDJ Network develops a new genetic
diagnosis methodT

he clinical archive for the Primary Immunodeficiency in Japan ( PIDJ ) is steadily growing. The
number of PIDJ entries was only 260 in 2008, but now exceeds 1500. Importantly, the data

entries are geographically widely distributed, deposited by more than 300 different hospitals, inaddition to RCAI's direct collaborators, the Japan PID study group supported by the Ministry of
Health, Labour, and Welfare. This outcome clearly indicates that the PIDJ network is establishing
firm roots in the clinician community in Japan.
As another important activity of PIDJ, RCAI, in collaboration with the Kazusa DNA ResearchInstitute, analyzed approximately 250 primary immunodeficiency ( PID ) samples during the 2011
fiscal year. In other words, the number of analyzed known and/or candidate PID genes exceeds
100 and the total number of the PID genes actually sequenced is more than 600 ( Figure 1) . Among
PIDs, there are three major disease entities that clinicians frequently submit for genetic testing
because they are difficult to accurately diagnose only from the symptoms; autoinflammatory diseases, mendelian susceptibility to mycobacterial disease, and common variable immunodeficiency. Because each of these diseases has multiple known causative genes, their genetic
analysis is highly labor-intensive and time-consuming. This fact has strongly motivated Dr. Ohara( RCAI's Laboratory for Immunogenomics and Kazusa DNA Research Institute ) to develop a more
efficient method of genetic testing than the conventional one based on amplification of genetic
regions of interest by polymerase chain reaction ( PCR ) followed by capillary DNA sequencing.
Massively parallel DNA sequencing ( MPS ) technology is an obvious method for this purpose.
However, a well-known caveat of MPS is the high frequency of sequencing errors, which is fine for
exploratory high throughput purposes, but which is a particularly serious problem when thesequence is used for conclusive genetic tests. To solve this problem, Ohara and his team tried toestablish an MPS-assisted statistically robust method for diagnosis of chronic infantile neurological cutaneous and articular syndrome ( CINCA ) , also known as neonatal-onset multisystem inflammatory disease ( NOMID ) . This disease is a dominantly inherited autoinflammatory disease that ischaracterized by neonatal onset and a triad of symptoms, including urticarial-like skin rash, neurological manifestations, and arthritis/arthropathy. CINCA is known to be caused by mutations in
the NLRP3 gene, which is a member of the Nod-like receptor family and a component of theinflamasome of the innate immune system. Dr. Ohara’s collaborators in Kyoto University previously
showed that there can be somatic mosaicism of NLRP3 gene mutations as a cause of CINCA/NOMID and that this explains the pathogenesis in about half of CINCA/NOMID patients who are
undiagnosed by a conventional direct PCR sequencing of the NLRP3 gene ( Saito et al., Blood,
2008, 111, 2132–41; Tanaka et al. Arthritis Rheum., 2011, 63, 3625–32). Because the occurrenceof somatically mutated cells is frequently
less than 50% in the presence of normal
cells, the diagnosis of this form of the disease requires higher accuracy than that for
diagnosis of conventional Mendelian diseases. The conventional approach for diagnosis of NLRP3 somatic mosaicism reliedon cloning of PCR products of exons of the
NLRP3 gene followed by capillary DNA
sequencing. Because somatic mosaicism
higher than 5% is usually considered diagnostic of the disease, more than 100 clones
have to be analyzed for each exon, which
inevitably imposes a heavy workload when
Figure 1: Statistics of PIDJ Genetic Tests

28

diagnosing somatic mosaicism. Thus, aproblem is how to avoid tedious cloning steps for diagnosis of NLRP3 somatic mosaicism whilestill maintaining high accuracy in the genetic test. The investigators succeeded in addressing thisdifficult issue by making error-occurrence reference maps for a gene to be analyzed by MSP ( Fig.
2: Izawa et al., DNA Res., 2012, 19, 143-52 ) . By comparing the error-occurrence map with actual
data, they could eventually discriminate sequencing errors from real genetic variations with statistical confidence. In fact, this method was successfully applied for diagnosis of CINCA/NOMID.
This approach is, as obvious in its principle, applicable to other conventional genetic tests, andhas the potential to greatly reduce the cost and labor of conventional genetic tests as well. Basedon the pipeline already developed for diagnosis of somatic mosaicism of the NLRP3 gene, development of an MPS-assisted genetic testing pipeline for autoinflammatory diseases, Mendelian
susceptibility to mycobacterial disease and common variable immunodeficiency is now underway.
The success rate of conventional genetic tests of known PID genes in PIDJ is 20-30% on
average. Thus, more than 500 clinical cases are currently accumulated in the PIDJ resource as
genetically unidentified. This resource is a great outcome of the PIDJ collaboration to date insearching for new PID-causing mutations. Because technical advances in DNA sequencing makeit possible to sequence all the exons in the human genome at a reasonable cost, the team has
actually started to perform a genome-wide search of disease causative genetic variations forsome of the unidentified PIDs. However, to understand the genotype-phenotype relationship ofPIDs, knowledge of basic immunology is indispensable. In particular, because the number of PID
patients is very limited in Japan and the PIDs are frequently sporadic, the functional analysis of thedetected genetic variation(s) eventually plays a key role in such an exploratory project for theidentification of PID genes. In addition, the role of bioinformatics becomes more and more criticalthan before because the amount of the information drastically increases. In this situation, it isstrong advantage that the PID collaboration network is composed of multidisciplinary researchers, including clinical PID specialists, genome scientists, and basic immunologists. By fully exploiting the PIDJ network, they will continue to tackle and try to clarify the mechanism through which
dysregulation of the homeostasis of the immune system takes place.

Figure 2: Example of an Error-Reference Map
The occurrence rates of sequencing errors (Insertion/Deletion in the upper panel,
Mismatches/Ambiguous calls in the lower panel) for exon 4 of the NLRP3 geneare shown in a strand-specific manner (indicated as “Reverse” or “Forward”).

29

NKT cell-targeted adjuvant
cell therapy forcancer patientsR

CAI has been developing a Phase I/IIa clinical study of the application of NKT cell-targeted
therapy for advanced lung cancer ( stage IIIB, IV or recurrence ) , in collaboration with Chiba

University Hospital ( Prof. Toshinori Nakayama, Prof. Takehiko Fujisawa and Associate Prof. Shinichiro Motohashi ) . α-GalCer-pulsed APCs ( 1x10 9 /m 2 PBMC-derived DCs ) were intravenously
administered four times. In this study, the MST (median survival time) of the responder group(defined by an increased number of IFN-γ producing NKT cells compared to the poor-respondergroup) was significantly longer than the poor-responder group (29.3 months versus 9.7 months).
Thus, the increased IFN-γ production by NKT cells upon α-GalCer stimulation showed a significant association with clinical outcome. These phase I/IIa trial results are encouraging and warrant
further evaluation of the survival benefit of this immunotherapy.
In September, 2011, this NKT cell-targeted therapy for lung cancer was approved by The
Advanced Medical Care Assessment System by the Japanese Ministry of Health, Labour andWelfare ( MHLW ) . The Advanced Medical Care Assessment System was introduced in Japan in1985 to ease the financial burden on patients who need state-of-the-art medical treatment that issafe and effective, but still not covered by health insurance because it is not yet approved under
the Pharmaceutical Affairs Law ( PAL ) . In response to recent rapid progress in medical technology
and the patients’ need to have safe and lower cost treatment with advanced technologies, thissystem was introduced to allow partial health insurance coverage, such as for clinical evaluations,medications and hospital stays. It is also the case that the collection of appropriate clinicalresearch data is facilitated by this system, and thus can then lead to approval of the advanced
medical technologies under the PAL.
After the approval, RIKEN and the National Hospital Organization ( NHO ) agreed to an integrative collaboration for NKT cell-targeted therapy and medical innovations in March, 2012. NHO
manages a Japanese network of 144 hospitals, including 53 thousands beds and a staff of 54
thousands. It supports high-quality clinical trials in the hospitals and facilitates collection of
appropriate clinical research data. They plan to start clinical trials of NKT cell-targeted therapy at
the Nagoya Center of the National Health Organization.

Development of artificial adjuvant vector cells(aAVC) as a novel therapyIn collaboration with Dr. Kakimi ( Tokyo Univ.) , Dr. Maeda ( Iwate Medical Univ.) and Dr. Mizuno( Yamaguchi Univ.) , RCAI's Drs. Fujii ( Research Unit for Cellular Immunotherapy ) , Shimizu( Research Unit for Immunotherapeutics ) and Ishii ( Laboratory for Vaccine Design ) have beendeveloping unique adjuvant vector cells that stimulate NKT cell activation.
These investigators previously demonstrated that allogeneic fibroblast cells loaded with
α-GalCer and transfected with antigen-encoding mRNA have a combination of effects; adjuvant
effects due to iNKT cell activation and the delivery of antigen to DCs in vivo ( Blood 2009 ) . Thesecells produced antigen protein and activated NK and iNKT cells. When injected into mice, they30elicited antigen-specific T cell responses and provided tumor protection. Thus, glycolipid-loaded,
mRNA-transfected allogeneic fibroblasts act as adjuvant vector cells ( aAVCs ) to promote iNKT
cell activation, leading to DC maturation and antigen-specific T cell immunity. In preclinical studies, they have set up canine studies to observe the safety profile and immune response. As was
the case in mice, administration of these aAVCs to dogs activates iNKT cells as well as elicits
antigen-specific T cell responses with no adverse events. This unique tool could prove clinicallybeneficial in the development of immunotherapies for malignant and infectious diseases.

Development of humanized mouse models

R

CAI's Laboratory for Human Disease Model ( Group Director: Fumihiko Ishikawa ) , Laboratory
for Developmental Genetics ( Group Director: Haruhiko Koseki ) and Laboratory for Immunog-

enomics ( Group Director: Osamu Ohara ) aim to develop the next generations of humanized micethat carry essential human genes, including MHC class I and II, cytokines and adhesion molecules.
Their goal is to develop humanized mice in which the immune responses to various pathogens
and environmental stresses will precisely recapitulate those seen in humans. These next generation mice will enable us to create useful models to better reconstitute human immune system andto recapitulate specific human diseases.
In 2010 in collaboration with The Jackson Laboratory, the team developed humanized micecarrying a human leukocyte antigen ( HLA ) . They showed that the expression of human genes isessential to fully recapitulate human lymphopoiesis in immunodeficient NOD/SCID/IL2rgKO ( NSG )mice. In the humanized mice that they created ( HLA class1 A02 transgenic NOD/SCID/IL2rgKO
mice ) , human cytotoxic T lymphocytes ( CD8 +T cells ) developed successfully and these human T
cells were functional in terms of cytokine production and cytotoxicity. They recognized EpsteinBarr virus ( EBV ) -infected cells and virus-associated peptides, and showed cytotoxicity in an HLArestricted manner. Moreover, this antiviral cytotoxic T lymphocyte response was inhibited by theaddition of an anti-HLA class I antibody ( Shultz et al., Proc Natl Acad Sci USA, 2010 ) .
In 2012, to humanize bone marrow microenvironment, they further modified this humanizedmouse model and created an NSG mouse that expressed human membrane-bound Kit ligand/stem cell factor ( hSCF Tg NSG mouse ) , a cytokine that plays an important role in hematopoiesis.
Transplantation of cord blood-derived human HSCs into the hSCF Tg NSG mouse resulted insignificantly higher long-term engraftment of human leukocytes in the bone marrow, spleen, andperipheral blood compared with non-hSCF transgenic NSG recipients. Humanization of themouse bone marrow microenvironment with hSCF has led to nearly 100% human hematopoietic
repopulation and efficient myeloid cell development. The frequency of human CD33 + myeloid cells
within the total human CD45 + population was significantly higher in the hSCF Tg NSG recipientscompared to the non-Tg NSG recipients and constituted the majority of human hematopoietic
cells. The reconstituted mice also have abundant human mast cells in spleen and gastrointestinal
mucosa. These findings demonstrate the essential role of membrane-bound SCF in human
myeloid cell development and, moreover, the new hSCF Tg NSG humanized mouse model may be
useful in studies of human allergy and innate immunity in vivo. ( Takagi et al., Blood, 2012 )These newly created humanized mouse models are expected to be essential tools for understanding the human immune system and recapitulating disease states. RCAI recently launched
the Medical Immunology World Initiative to facilitate a worldwide collaboration with research centers focusing on the investigation of human immunity and immunological diseases using humanized mice. The next generations of humanized mice will be reciprocally exchanged with othermembers of the research centers, thereby optimizing studies of human diseases and pioneering
human immunology research ( See Research Highlights section ) .

31

Calpis-RIKEN Integrated Collaborative Research
conducted in RCAII

n 2004, RIKEN started the Integrated Collaborative Research with Industry Program to promotejoint research focused on meeting industry needs that emphasizes industry initiatives to makeuse of RIKEN’s considerable and ever-growing research assets. The Gut Microbiology Research
Team was established under this program in RCAI in 2010 to conduct collaborative research
between Calpis Co. Ltd., a Japanese manufacturer of an uncarbonated milk and lactic acid-based
soft drink, and RCAI’s Laboratory for Epithelial Immunobiology. Dr. Naoyuki Yamamoto conducted
immunology research on probiotics at RCAI in collaboration with RCAI’s Hiroshi Ohno.

RCAI Innovation Projects in Drug
Discovery and MedicineIn order to conduct research and development of novel drugs and medical technologies,
RIKEN established the Program for Drug Discovery and Medical Technology Platforms ( DMP )in April 2010.

Since then, based on its guidelines, DMP has selected theme leaders, who

promote the projects for drug discovery and medical technology, focusing on areas of diseases
with low therapeutic satisfaction, orphan drugs and new concepts.

The theme leaders areresearchers at RIKEN, but DMP supports the project by assistance of the portfolio manager withexperience in drug discovery and by technical platforms established in DMP, linked to RIKEN
Research Centers.
Several RCAI researchers collaborate with DMP. Toshitada Takemori became a unit leader ofDrug Discovery Antibody Platform Unit of DMP, in order to develop monoclonal antibodies thatcan be used as therapeutic drugs for treatment of cancer and other diseases. The activity of the
Antibody Platform Unit is supported by RCAI Leaders Takashi Saito, Hiroshi Kawamoto and Fumihiko Ishikawa, who are experts in monoclonal antibody production, artificial lymph nodes, andhumanized mice, respectively.
Besides the Antibody Platform Unit, four other R&D projects were conducted under thecollaboration between RCAI and DMP. “NKT cell cancer therapy” ( Project Leader: Masaru Taniguchi ) , “Novel allergy therapy” ( Project Leader: Masaru Taniguchi ) , “Novel therapy for leukemia
targeting cancer stem cells” ( Theme Leader: Fumihiko Ishikawa ) and “CD8T therapeutic antibody
drugs” ( Theme Leader: Tsuneyasu Kaisho ) .
Considering this direction, RCAI launched a new program “Innovation Projects in Drug
Discovery and Medicine” in 2010. The aim of this program is to develop seeds that can becandidate projects for DMP. During 2011, two projects were conducted at RCAI.

Table : RCAI Innovation Projects in Drug Discovery and Medicine 201132

1.

Shin-ichiro Fujii“Development of artificial adjuvant vector cells”

2.

Takashi Tanaka“Development of a novel treatment for wounds using siRNA against PDLIM2”

2011
Part 3

Nurturing Young Scientists

Young Chief Investigator Program

Figure :R

Scheme of the Young Chief Investigator Program
The Young Chief Investigator (YCI) will run alaboratory independently in terms of funding andresearch. The laboratory will, however, share space,equipment and facilities with a host lab (RCAIResearch Group or Team). YCIs are supported bya mentor system. Three or four specialists (PIs)from the related fields inside or outside RCAI
will be the mentors and will provide guidancefor experimental design, preparation of papers
and presentations, promotion of international
visibility, and obtaining research funding.

CAI launched a new program, Young Chief Investigator

year. The committee will also discuss the relevance andProgram, in 2010 to provide a career path for young

value of the research project as part of the core research

investigators who conduct multidisciplinary research thatprojects at RCAI.

will bridge immunology with other research fields. In thisprogram, the selected Young Chief Investigator ( age below

sibility of extending for an additional 2 years after evaluation

40 ) will head an independent research laboratory but willby the Director and an internal committee. At that point, ahave an access to mentoring by multiple senior specialists in

Young Chief Investigator can leave RCAI to take a position at

related research fields, i.e. the leaders in various RIKEN Insti-

another institution or be promoted to another type of posi-tutes. Mentors provide guidance for experimental design,tion within RCAI.

There will be an initial 5-year appointment, with the pos-preparation of papers and presentations, promotion of inter-

Three researchers were selected for the Young Chief

national visibility, and obtaining research funding. The YCIInvestigator Program, Dr. Hayato Naka-Kaneda (stem cells
and aging reversal ), Dr. Shinji Nakaoka (development of inter-laboratory will also share space, equipment and facilities
with a host laboratory in RCAI ( Groups, Teams or Units )( Fig.) To consider necessary changes in the level of RCAI

face between integrative biology and mathematics ), and Dr.
Koji Hase (mucosal flora and epigenetic analysis ). Their men-

support for YCI, the YCI Program Committee meets twice a

tors and host laboratories are listed in Table.

Table: Young Chief Investigators and their mentors 2011
YCI lab

Mentors

Affiliation

Shinji Nakaoka“Laboratory for Mathematical
Modeling of Immune System”

Masahiro Ueda

Group Director, Laboratory for Cell Signaling Dynamics, RIKEN QBiC

Kenji Kabashima

Associate Professor, Department of Dermatology, Graduate School of Medicine, Kyoto University

Host Lab:
Osamu Ohara, RCAI

Osamu Ohara

Group Director, Laboratory for Immunogenomics, RIKEN RCAI

Masato Kubo

Professor, Research Institute for Biological Science, Tokyo University of Science
Senior Visiting Scientist, Open Lab. for Signal Network, RIKEN RCAI

Ronald Germain

National Institutes of Health, USA

Hayato Kaneda“Laboratory for Stem Cell
Competency”

Hitoshi Niwa

Team Leader, Laboratory for Pluripotent Stem Cell Studies, RIKEN CDB

Piero Carninci

Team Leader, Functional Genomics Technology Team, RIKEN OSC

Host Lab:
Ichiro Taniuchi, RCAI

Hiroshi Kawamoto

Team Leader, Laboratory for Lymphocyte Development, RIKEN RCAI

Haruhiko KosekiGroup Director, Laboratory for Developmental Genetics, RIKEN RCAIKoji Hase
Minoru Yoshida“Laboratory for
Bioenvironmental Epigenetics” Piero Carninci
Yoichi Shinkai
Host Lab:
Haruhiko Koseki, RCAI
Haruhiko Koseki

34

Chief Scientist, Chemical Genetics Laboratory, RIKEN ASITeam Leader, Functional Genomics Technology Team, RIKEN OSC
Chief Scientist, Cellular Memory Laboratory, RIKEN ASI
Group Director, Laboratory for Developmental Genetics, RIKEN RCAIYCI Laboratory for

Bioenvironmental
Epigenetics

Young Chief Investigator :Koji Hase
Student Trainee : Yukihiro Furusawa

Epigenetics is a mechanism that imposes a specific andphoid tissues, the largest collection of secondary lymphoid

heritable pattern of gene expression on the progeny oforgans, which are necessary for induction of mucosal IgA

differentiating cells, without affecting the base sequence of
the DNA. The major epigenetic mechanisms include DNA

responses. Certain enteric bacteria also facilitate differentiation of type17 helper T ( Th17) and regulatory T ( Treg ) cells,

methylation, chemical modifications of histone tails andboth of which are major T cell populations in the intestinal

higher order chromatin organization. Emerging evidencemucosa. In support of this concept, the development of

indicates the importance of epigenetic events in the development and proper functions of the immune system. Fur-

Th17 and Treg cells as well as IgA production are defective inmice housed in a germ-free isolator ( denoted as “germ-freethermore, it is assumed that transcriptional regulation via

mice” ) . These previous observations led to the notion that

epigenetic mechanisms may be influenced by environmental

host-microbe interactions establish immunological homeo-

factors, e.g., microbial products and cytokines. However,stasis in the gut, which further raises the important question:

identification of such environmental factors and defining their

how do commensal bacteria affect the host immune

impact on immunological homeostasis remain elusive goals.

system?

Humans harbor over 100 trillion bacteria in the distal
intestine. These commensal ( meaning ‘to share a dining
table’ in Latin ) bacteria have long been appreciated for the

Epigenetic modifications of host cells by intestinalcommensal bacteria

benefits they provide to the host, the most obvious being

We have so far conducted studies to understand thetheir capacity to metabolize indigestible food components tomaintenance of intestinal immune homeostasis and havesmall metabolites that are utilized as nutrients by host cells.

demonstrated the biological significance of intestinal M cells

Moreover, it is now clear that the presence of commensal

localized at gut-associated lymphoid tissues. During thebacteria contributes to shape the gut immune systemcourse of these studies, we noticed that the expression level

through promoting the development of gut-associated lym-of DNA methyltransferases is upregulated in the colonic35

Figure :Schematic of epigenetic regulationof intestinal immunity bycommensal bacteria. Intestinalcommensal bacteria actively produce
small molecule metabolites. These
metabolites may directly or indirectly
influence cell differentiation andfunction of immunocompetentcells such as T and B lymphocytes
via epigenetic mechanisms.

mucosa of mice maintained under conventional conditions compared to that in germ-freemice. This observation suggests that bacterial colonization may influence the DNA methylation status of host cells. Indeed, our methyl DNA immunoprecipitation-on-chip ( MeDIP-

Recent publications

chip ) assay revealed that the global DNA methylation level is substantially higher in colonic

1. Fukuda S, Toh H, Hase K, Oshima
K, Nakanishi Y, Yoshimura K, Tobe
T, Clarke JM, Topping DM, Suzuki T,
Taylor TD, Itoh K, Kikuchi J, Morita
H, Hattori M, Ohno H. Bifidobacteria
protect host from enteropathogenic
infection through production of
acetate. Nature 469, 543-7, (2011)

epithelium prepared from conventional mice compared to that from germ-free mice.
The enteric microflora constitute a potent bioreactor that controls several metabolic
functions. The principal metabolic functions include the fermentation of indigestible foodsubstances such as dietary fibers and resistant starch into simple sugars, absorbablenutrients, and short-chain fatty acids ( SCFAs ) . Notably, certain SCFAs have been reported
to exert histone deacetylase ( HDAC ) inhibitory activity, at least in vitro. Based on theseobservations, we hypothesize that intestinal microbiota may secure mucosal immune
homeostasis via epigenetic mechanisms by the action of metabolites that they produce( see Fig. )Goal of the study: Identification of the missing link
between commensal bacteria and the development of gut immune system
This newly launched laboratory aims to explore the epigenetic regulation of the immune
system by intestinal microbiota, and also to elucidate the biological significance of thisepigenetic regulation in gut immunity. To simplify the host-bacteria interactions, I will
employ germ-free mice and various gnotobiotic mice that will be colonized with one or only
a few bacterial strains. Mucosal immunocompetent cells from these mice will be subjected
to a genome-wide analysis of epigenetic modification ( i.e. DNA methylation and histone tailtrimethylation such as H3K4me3, H3K9me3 and H3K27me3 ) . Furthermore, transcriptome
analysis of the host cells as well as metabolic profiling of fecal samples will be performed.
Bioinformatic analysis will be applied to define positive correlations between epigenetic
changes in host cells and specific metabolites. Thus, identification of a key metabolite ( s )that facilitates epigenetic changes in host cells is currently underway.

36

2. Takahashi D, Hase K, Kimura S,
Nakatsu F, Ohmae M, Mandai Y,
Sato T, Date Y, Ebisawa M, Kato
T, Obata Y, Fukuda S, Kawamura
Y. I, Dohi T, Katsuno T, Yokosuka
O, Waguri S and Ohno H. The
Epithelia-specific membranetrafficking factor AP-1B controls
gut immune homeostasis
in mice. Gastroenterology
141, 621-32 (2011)
3. Hase K, Kawano K, Nochi T,
Pontes G.S., Fukuda S, Ebisawa
M, Kadokura K, Tobe T, Fujimura
Y, Kawano S, Yabashi A, Waguri
S, Nakato G, Kimura S, Murakami
T, Iimura M, Hamura K, Fukuoka
S-I, Lowe AW, Itoh K, Kiyono H and
Ohno H. Uptake via Glycoprotein
2 of FimH+ bacteria by M cells
initiates mucosal immune response.
Nature 462, 226-230 (2009)
4. Hase K, Kimura S, Takatsu H,
Ohmae M, Kawano S, Kitamura
H, Ito M, Watarai H, Hazelett CC,
Yeaman C and Ohno H. M-Sec
promotes membrane nanotube
formation by interacting with Ral
and the exocyst complex. Nat.
Cell Biol. 11, 1427-32 (2009)YCI Laboratory forMathematical
Modeling of
Immune System
Young Chief Investigator :Shinji NakaokaT

he skin tissue is a first line of host defense that acts as

immune cell dynamics in the skin tissue, and (iv) Develop-

barrier and sensor for invading pathogens. Atopic der-ment of supportive theory for multi-scale mathematicalmatitis is a common skin disease that could occur as a con-modeling -- in silico representation of immunological events

sequence of loss of skin barrier function, followed byand rules.

extensive allergic/inflammatory immune responses. Despite
and mechanisms that would mediate chronic allergic immune

Construction of resource/information-base foratopic dermatitis

responses in the skin tissue remain unknown. Dysregulated

Current existing subfields in atopic dermatitis study include

immune responses in the skin tissue of atopic dermatitis

various disciplines such as skin biology, immunology, neu-

patients are typically observed at heterogeneous spatio-

roendocrinology and psychology. Encouragement of multi-

temporal scales. Spatio-temporal multiscaleness and par-disciplinary collaborations over different subfields of atopic

ticipation of multiple types of immune cells are key issues todermatitis study holds the promise of bringing comprehen-be elucidated for obtaining a comprehensive view of the pro-sive understanding to this disease. Construction of agression of atopic dermatitis.

resource/information-base for atopic dermatitis is an essen-

extensive basic and clinical research, definitive components

The main goal of the research in my laboratory is to con-tial step to facilitate multidisciplinary study by offering vari-struct multi-scale mathematical models for atopic dermatitis,

ous types of structured knowledge. Although existing

psoriasis vulgaris and other eczemas. The entire researchversatile database and computational tools developed inplan consists of the following four essential building blocks:( i ) Construction of a resource/information-base for atopic

bioinformatics enable us to obtain relevant information about

dermatitis, ( ii ) Exploration of intracellular gene expression
dynamics of skin and immune cells, ( iii ) Quantitative study ofknowledge in subfields is required to mediate interdisciplin-

atopic dermatitis, integration of existing vast yet dispersed
ary collaborations. The main purpose of this project is to

37

A
C

B

Th2
Th1
DC

Th2

Th17

B

Figure :A schematic representationof the four building
blocks for multi-scalemathematical modelingof immune responses.

construct an integrated knowledge-base for atopic dermatitis. As a first step, a referencedatabase system for atopic dermatitis is now under construction.
Exploration of intracellular gene expression dynamics of skin andimmune cells
The first step to clarify the onset of atopic dermatitis is to define basic characteristics of theinitial allergic and inflammatory reactions. Exploration of differences in gene expression
profiles and progression of allergic inflammation processes between normal and atopic
dermatitis mice should provide informative guides to identify definitive components andmechanisms that mediate chronic and allergic immune responses. In this project, several
atopic dermatitis mouse models are used to investigate gene expression dynamics of skinand immune cells. The main purpose of this project is to investigate the role of the JAK/STAT signaling pathway: how differences in signal transduction would result in diverse
outcomes. The project is currently in progress.
Quantitative study on immune cell dynamics in the skin tissue
The first step to clarify the progression ( post-initial phase ) of atopic dermatitis is tounderstand the dynamic interplay among skin and immune cells. Newly developed measurement devices offer quantitative, multivariate, high-throughput and time-series data.
Mathematical modeling and statistical/information-theoretic analyses are essential toextract qualitative insights from this kind of data. The main purpose of this project is toinvestigate immune cell dynamics in the skin tissue under tight collaboration with experiments on the basis of quantitative research design. The theoretical goal of this project is to
extract essential control mechanisms that are dysregulated during the progression of
atopic dermatitis. The project is now being developed.
Development of supportive theory for multi-scale mathematical modeling
In general, the whole process of immune responses is achieved under complex dependencies in terms of a wide range of spatio-temporal interactions and participation of multiple
types of immune cells. Any mathematical models that describe process and dynamics ofimmune responses should appropriately reflect such complex dependencies. However, alack of theoretical guidance to describe complex immune responses in silico hampers
construction of multiscale mathematical models. The main purpose of this project is todevelop a supportive theory for representing events and rules of immune responses insilico. Our primary aim is to extract in silico-translatable rules underlying allergic ( Th2 )immune reactions from the existing literature. The project is currently in progress.

38

Recent publications
1. Katsuyama C., Nakaoka S.,
Takeuchi Y., Tago K., Hayatsu
M., Kato K., Complementarycooperation between two syntrophic
bacteria in pesticide degradation, J.
Theor. Biol. 256 644-654 ( 2009 ) .
2. Nakaoka S., Wang W., Takeuchi
Y., Effect of parental care andaggregation on population
dynamics, J. Theor. Biol.
260 161-171 ( 2009 ) .
3. Iwami S., Nakaoka S., TakeuchiY., Miura Y., Miura T., Immune
impairment thresholds in HIV
infection, Immunol. Letters123 149 -154 ( 2009 ) .
4. Diekmann O., Gyllenberg M., Metz
J.A.J., Nakaoka S. and de Roos A.,
Daphnia revisited: local stability andbifurcation theory for physiologicallystructured population models
explained by way of an example, J.
Math. Biol. 61 277-318 ( 2010 ) .
5. Nakaoka S., Aihara K., Mathematical
study on kinetics of hematopoietic
stem cells -- theoretical conditions
for successful transplantation --,J. Biol. Dyn. IFirst, 1–19 ( 2011) .

YCI Laboratory for

Stem Cell
Competency

Young Chief Investigator :Hayato Kaneda
Technical staff : Shiho NakamuraAge-related disruption of tissue homeostasis induces

Competence regulation of neural stem cells

reduction of regenerative ability, resulting in ageassociated pathologies and a lower quality of life ( QOL ) .

We have been investigating the molecular mechanisms governing the differentiation of neural stem cells ( NSCs ) using an

Japan has already become an ageing society and the demo-

in vitro neurosphere culture system that we had originallygraphic trends predict a doubling of the world population
over 65 years of age within the next 30 years. However, wedeveloped. In this system, NSCs are induced from embryonicstem cells (ESCs ) through embryoid body (EB ) formation andknow little about the mechanisms of ageing and even muchselectively amplified as neurospheres ( Fig. 1). NSCs haveless about how to counteract it. A tight balance between

multipotency to differentiate into neurons and glial cells. Neu-

cellular proliferation and cell death maintains tissue homeostasis. In the former case, somatic stem cells ( SSCs ) playrogenesis largely precedes gliogenesis during central nervous system (CNS ) development in vertebrates and thisimportant roles by supplying tissue-specific cells over theneurogenesis-to-gliogenesis switching requires the temporal

lifespan of the animal. SSCs are generally defined by multi-

identity transition of NSCs. This transition can also be

potency and the ability to self-renew. However, recent stud-observed in our culture system as it recapitulates in vivo

ies have demonstrated that SSCs themselves undergomouse CNS development. We previously identified Coup-tfI

ageing, changing their functions with age. Their dysfunction
and decreased regenerative capacity can cause physiologi-

and II (Coup-tfs) as critical molecular switches for the neurogenic-to-gliogenic transition of NSCs ( Naka H. et al. Nature

cal deficiencies, e.g., inefficient muscle repair, reduced bone

Neuroscience 2008 ). Their knockdown caused sustained

mass, neurodegenerative diseases, and dysregulation ofhematopoiesis. Therefore, we predict that the restoration ofgeneration of the early-born neurons and loss of gliogenesis( Fig. 2). Remarkably, Coup-tfs neither repress neurogenesis

SSCs functions towards those in young healthy individuals

nor promote gliogenesis. Instead, we found that they are only

would contribute to recovery of tissue homeostasis andinvolved in the temporal “competence change” of NSCs toimprovements in our health.

permit the transition of their developmental status by changing their responsiveness to extrinsic gliogenic signals ( Fig.3 ).

39

in vivo Neural development

Blastocyst
Inner cell mass

Speciﬁcation of
three germ layers

Neurogenic
neural stem cells

Gliogenic
neural stem cells

in vitro Neurosphere culture system

ES cells

Embryoid body (EB)p2

passage 0 (p0)neurospherep1
neurosphere

Early-born neuron

Late-born neuron
Glia

p3

COUP-TFs knockdown

Figure 1: In vitro neurosphere culture system
NSCs are induced from ESCs as neurospheres. Coup-tfs knockdown caused
sustained generation of early-born neurons and loss of gliogenesis.

Classical model
Sequential expression of cell fate speciﬁc transcription factors

Figure 2: Alteration of responsiveness to gliogenic cytokines
Coup-tfs knockdown neurospheres continue to produce
neurons even in the p2 stage and resist the inductionof gliogenesis by gliogenic cytokines, LIF and BMP2.
Tbr1

ER81

Brn2

GFAP

Neurogenic signal

Neurogenic signal

Gliogenic signal

Gliogenic signal

maintenance ofan undiﬀerentiated state
self-renewal

activation of gliogenic genes

COUP-TFI/II
Competence change
Proposed model
E10

E12

E14

E16

E18

Figure 3: Model for the temporal identity transitionof NSCs that incorporates the concept
of competence regulation
Cytogenesis is integration of outputs frommultiple dynamic regulatory programs in stem
cells. Expression of COUP-TFI/II triggers NSC
competence change. Responsiveness to the gliogenic
signals is altered and the NSCs acquire gliogenic
competence. At that point, gliogenic signals induce
gliogenesis only from the gliogenic NSCs.

after birth

Stem cell ageing
Recently, we could further reveal the molecular mechanisms underlying the NSC competence change. The results led us to hypothesize that competence change, which isreflected by changes in responsiveness to extrinsic signals, is a common and fundamental
molecular mechanism among various SSCs in the regulation of their properties. Thus, weare trying to expand our discovery made in NSC development to the regulation of a varietyof SSC regulations and are now focusing on stem cell ageing.
Among various SSCs, stem cell ageing is best studied in hematopoietic stem cells( HSCs ) . Ageing induces expansion of the HSC population, but its capacity for long-term
blood cell reconstitution becomes reduced and also its differentiation potency becomes
biased towards myelopoiesis at the expense of lymphocyte production. Numerous cellintrinsic and extrinsic mechanisms connecting stem cell ageing and age-associatedpathologies have been identified as HSC ageing modulators. They include DNA damage
accumulation, oxidative stress, chromatin dysregulation, induction of senescence-associated proteins and ageing of niche cells, for example. However, no critical molecular regulators of stem cell ageing have been identified. Thus, we are trying to understand thecontributions of competence regulation to stem cell ageing.

40

Recent publications
1. Sato T., Shimazaki T., Naka H.,
Fukami S., Satoh Y., Okano H.,
Lax I., Schlessinger J., Gotoh N.
FRS2alpha regulates Erk levels tocontrol a self-renewal target Hes1
and proliferation of FGF-responsive
neural stem/progenitor cells. StemCells 28, 1661-1673 ( 2010) .
2. Naka-Kaneda H., ShimazakiT., Okano H. Neurogenesis
to gliogenesis switching.
Experimental Medicine (Jikken
Igaku) 28, 815-822 ( 2010) .
3. Tao O., Shimazaki T., Okada Y.,
Naka H., Kohda K., Yuzaki M.,
Mizusawa H., Okano H. Efficient
generation of mature cerebellar
Purkinje cells from mouseembryonic stem cells. J. Neurosci.
Res. 88, 234-247 ( 2010) .

RIKEN Joint Graduate School Program
International Program Associate

R

CAI accepted eight international students as RIKEN International Program Associates (IPA).
Under this IPA program, RCAI lab heads host international students from collaborating

graduate schools and supervise their Ph.D. program as Joint Supervisors. The students receive
a daily living allowance and housing costs for up to a maximum of three years.

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The IPA students who studied at RCAI in 2011 were
Li Yingqian( Nanjing University, China) studied in the Laboratory for Immune Diversity ( Photo 1)
Sebastian Nieke( Tübingen Univ., Germany ) studied in the Laboratory for Transcriptional Regulation ( Photo 2)
Li Shuyin( China Agriculture University, China ) studied in the Laboratory for Immune Diversity ( Photo 3 )Yue Ren( Jilin University, China ) studied in the Laboratory for Immune Regulation ( Photo 4 )Mohamed El Sherif Gadelhaq Gadelhaq Badr
( Tokyo Medical and Dental University) from Edypt studied in the Laboratory for Cell Signaling ( Photo 5 )Zhang Yanfei( Peking University, China ) studied in the Laboratory for Immune Diversity ( Photo 6 )Joo Ann Ewe( Universiti Sains Malaysia, Malaysia ) studied in the Laboratory for Epithelial Immunobiology ( Photo 7)
Huey Shi Lye( Universiti Sains Malaysia, Malaysia ) studied in the Laboratory for Epithelial Immunobiology ( Photo 8 )41

RIKEN Foreign Postdoctoral Researcher

T

he RIKEN Foreign Postdoctoral Researcher ( FPR ) program offers aspiring
young foreign researchers with creative ideas and who show promise of

becoming internationally active in the future the opportunity to pursue innovative
research at RIKEN under the direction of a RIKEN laboratory head. The FPR
program is one of RIKEN’s initiatives to open up its facilities and resources to theworld and create a stimulating research environment that will place RIKEN at theforefront of global science and technology.
In 2011, Jafar Sharif ( Photo ) studied in the Laboratory for Developmental
Genetics as a RIKEN FPR.

RIKEN Special Postdoctoral Researcher (SPDR) Program

R

IKEN’s program for Special Postdoctoral Researchers was instituted to provide young and creative
scientists the opportunity to be involved in autonomous and independent research in line with RIKEN

objectives and research fields. The positions are competitive, but if selected, researchers receive salaries
and research budgets (1 million yen) from RIKEN and they are able to conduct their research at one of its

laboratories.
This year, nine postdocs conducted their research at RCAI through the SPDR program.

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Shinsuke Ito ( Laboratory for Developmental Genetics ) ( Photo 1)
Mizuho Kajikawa ( Laboratory for Infectious Immunity ) ( Photo 2)
Takashi Kanaya ( Laboratory for Epithelial Immunobiology) ( Photo 3)
Shimpei Kawamoto ( Laboratory for Mucosal Immunity) ( Photo 4)
Masahiro Kitano ( Research Unit for Immunodynamics ) ( Photo 5)
09

Yoshitaka Shirasaki ( Laboratory for Immunogenomics ) ( Photo 6)
Yuki Horisawa-Takada ( Laboratory for Developmental Genetics ) ( Photo 7)
Hirokazu Tanaka ( Laboratory for Transcriptional Regulation ) ( Photo 8)
Nayuta Yakushiji ( Laboratory for Developmental Genetics ) ( Photo 9)

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RIKEN’s Junior Research Associate ( JRA ) ProgramT

he Junior Research Associate program was launched in 1996 to encourage young scientists withfresh ideas and youthful enthusiasm to collaborate with, and learn from, senior scientists withyears of experience. This program provides part-time positions at RIKEN for young researchersenrolled in university Ph.D. programs. The JRA program serves the dual purpose of fostering thedevelopment of these young scientists while also energizing RIKEN with their innovative thinking.
This year, eleven JRA students studied in RCAI.

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Yuki Aoki ( Laboratory for Human Disease Model ) ( Photo 1)
Misao Hanazato ( Laboratory for Epithelial Immunobiology) ( Photo 2)
Rikiya Ishikawa ( Laboratory for Infectious Immunity ) ( Photo 3)
Toshi Jinnohara-Yuyama ( Laboratory for Epithelial Immunobiology ) ( Photo 4)
Chie Kano ( Laboratory for Immune Diversity ) ( Photo 5)
Tamotsu Kato ( Laboratory for Epithelial Immunobiology ) ( Photo 6 )Nanako Shimura ( Laboratory for Immunogenomics ) ( Photo 7 )Masanaka Sugiyama ( Research Unit for Inflammatory Regulation) (Photo 8)
Shinsuke Takagi ( Laboratory for Human Disease Model ) ( Photo 9)
Hideaki Takagi ( Laboratory for Dendritic Cell Immunobiology ) ( Photo 10 )Yuuhou Najima ( Laboratory for Human Disease Model ) ( Photo 11)43Adjunct Professorship Programs

R

CAI accepts graduate students through the mechanism of adjunct professorships at various
Japanese universities. RCAI collaborates with and accepts graduate students from 10 domestic

university graduate schools. There are now a total of 25 adjunct professors/associate professors in
RCAI. Seventy-eight students studied at RCAI in 2011.

Table: Adjunct professorship programs
Graduate Program

Affiliated RCAI Investigator

Graduate School of Frontier Bioscience,
Osaka University

Tomohiro Kurosaki ( Professor )Ichiro Taniuchi ( Visiting Professor )Keigo Nishida ( Visiting Associate Professor )Graduate School of Medicine,
Osaka University

Takashi Saito ( Visiting Professor )Department of Immunology, Graduate School of Medicine,
Chiba University

Takashi Saito ( Visiting Professor )Toshiyuki Fukada ( Visiting Associate Professor )Haruhiko Koseki ( Visiting Professor )Hiroshi Ohno ( Visiting Professor )Shin-ichiro Fujii ( Visiting Associate Professor )Yasuyuki Ishii ( Visiting Associate Professor )Fumihiko Ishikawa ( Visiting Associate Professor )Graduate School of Pharmaceutical Sciences,
Chiba University
School of Biomedical Science,
Tokyo Medical and Dental University

Osamu Ohara ( Visiting Professor )Takashi Saito ( Visiting Professor )Tomohiro Kurosaki ( Visiting Professor )Masato Kubo ( Visiting Professor )Mariko Okada ( Visiting Professor )Sidonia Fagarasan ( Visiting Associate Professor )International Graduate School of Arts and Sciences,
Yokohama City University

Hiroshi Ohno ( Visiting Professor )Tsuneyasu Kaisho ( Visiting Professor )Satoshi Ishido ( Visiting Associate Professor )Research Institute of Biological Sciences,
Tokyo University of Science

Masato Kubo ( Professor )Osamu Ohara ( Visiting Professor )Shohei Hori ( Visiting Associate Professor )Tadashi Yokosuka ( Visiting Associate Professor )Department of Computational Biology,
Graduate School of Frontier Sciences,
The University of Tokyo

44

Mariko Okada ( Visiting Associate Professor )Graduate School of Medicine, Kyoto University

Fumihiko Ishikawa( Visiting Associate Professor )Graduate School of Medicine, Kobe University

Masaru Taniguchi( Adjunct Lecturer )2011Part 4

Collaborative Networks

Open Laboratory at RCAI

R

CAI’s

Open

Laboratoryfororatory space and staff in RCAI andory ) , and Dr. Taeko Dohi’s ( National
Center for Global Health and Medicine ) project “Intestinal flora andAllergy Research was estab-

have joined various internal researchlished in 2009 to provide a frame-

meetings and discussions to pro-work for researchers from externalmote extensive research communi-

institutes, universities or hospitals

cation with RCAI investigators.

epigenomic imprinting” was hosted
by Dr. Hiroshi Ohno ( Laboratory for
Epithelial Immunobiology ) .

that would allow them access to the

Two other projects were also

Center’s resources and the opportu-

conducted at RCAI under the Allergy

In addition to allergy research,nity for collaborative allergy research

RCAI also established an Openprojects.

Open Laboratory system. Dr. Kenji
Matsumoto ( National Research Insti-

In 2011, two researchers, Drs.
Masato Kubo ( Open Laboratory fortute for Child Health and Development) and Dr. Naoki Shimojo’s ( Chiba

where multidisciplinary researchers

Signal Network ) and Dr. Noriko M
Tsuji ( Open Laboratory for Allergy

University ) project “Cohort study ofcomplexallergy in infants and the generation

mathematicaland Mucosal Immune Tolerance ) ,of a humanized mouse model” wasapproaches.

were promoted to Senior Visiting

hosted by Dr. Toshitada Takemori( Laboratory for Immunological Mem-

Scientists. They have their own lab-

Laboratory for Systems Biology,
can interact at RCAI to model the
immuneandsystemusing

computational

Open Laboratory for Allergy Research
Masato Kubo (Research Institute for Biological Science, Tokyo University of Science)“Laboratory for Signal Network”
Noriko M Tsuji (Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology ( AIST ))“Laboratory for Allergy and Mucosal Immune Tolerance”
Kenji Matsumoto (National Research Institute for Child Health and Development) andNaoki Shimojo (Chiba University)“Cohort study of allergy in infants and the generation of a humanized mouse model”
Taeko Dohi (National Center for Global Health and Medicine)“Intestinal flora and epigenomic imprinting”

Open Laboratory for Systems Biology
Kazuyuki Aihara (Institute of Industrial Science, The University of Tokyo)
Tatsuji Hara (Graduate School of Information Science and Technology, The University of Tokyo)
Yoshiharu Yamamoto (Graduate School of Education, The University of Tokyo)
Kenko Uchida (Graduate School of Advanced Science and Engineering, Waseda University)
Jun-ichi Imura (Graduate School of Information Science and Engineering, Tokyo Institute of Technology)
Koji Tsumura (Graduate School of Information, Science and Technology, The University of Tokyo)
Hidenori Kimura (RIKEN BSI-TOYOTA Collaboration Center)
Shinji Nakaoka (Graduate School of Mathematical Sciences, The University of Tokyo)46

Open Laboratory for Signal Network

Figure :T

Expression of E4BP4 regulates IL-10 expression in several T cell subsets.
E4BP4 controls the plasticity of IL-13 production by Th1 cells
following chronic antigen stimulation and functions as a master
regulator for IL-10 expression by Th1, Th2, Treg and NKT cells.

cells play a central role in the effector and regulatory

helper T cell subsets, innate type immune cells andfunctions in immunological surveillance, and aberra-

innate lymphoid cells.

tions in these functions can lead to various immunological disorders. T helper 1 ( TH1) cells secrete interleukin-2
response to intracellular pathogens and viruses. TH2

E4bp4, a mammalian basic leucine zipper( bZIP) transcriptional factor, is a critical regulator of IL-10 and IL-13 production in CD4 T cells

cells, which are mainly protective against extracellularInterleukin-10 ( IL-10 ) is an anti-inflammatory cytokine

pathogens but also account for allergic immunethat downregulates immune responses and staves off

responses, produce IL-4, IL-5, IL-6, IL-10 and IL-13. These

autoimmune disease. This cytokine is secreted mainly bycytokines secreted by the different effector helper T cells

TH2 cells but also by TH1 cells during chronic infections.

play a critical role in controlling the outcome of immuno-

We observed plasticity of IL-10 and IL-13 expression fol-

logical surveillance. The helper T cell subsets differenti-

lowing chronic TH1 stimulation and found that IL-10 andate from common precursor cells, the naïve T cells, andIL-13 are regulated by the basic leucine zipper type of

this differentiation is accompanied by acquiring thetranscriptional factor, E4BP4/NFIL-3. TH1 cells lacking(IL-2), IFN-γ and TNF-α during the cellular immune

capacity to produce certain types of cytokines. Recent

E4bp4 had attenuated IL-10 and IL-13 expression follow-

studies suggest that TH2 cytokines that are tightly asso-

ing chronic antigen stimulation and, consistently, forced

ciated with allergic immune responses are also expressed

expression of E4bp4 initiated IL-10 and IL-13 expression

by innate type immune cells, such as mast cells andin conventional TH1 cells. On the other hand, TH2 cells

basophils, as well as by type II innate lymphoid cells,lacking E4bp4 had a marked impairment in IL-10 produc-

including natural helper cells and neocytes. These innatetion with no effect on IL-13. Moreover, Treg and NKT cells

cell-derived as well as TH2-derived cytokines play aderived from mice lacking E4bp4 had a reduction in IL-10

critical role in eliciting allergic disease. The overriding

expression.

goal of our laboratory is to understand the molecularTranscription factors can control the expression of

mechanisms underlying cytokine gene expression in

genes by binding to a specific region of the enhancer

47

and the promoter.

Mas-DTR mice resulted in deletion of both MCs andWe observed thatbasophils. Using these mice, we found that MCs andE4BP4 bound tobasophils played distinctive roles in the early and chronic

the Il13 promoter
in TH1 cells thatphases of the IgE-mediated allergic response. Basophils

had been chroni-

production induced by systemic antigen immunization.

callystimulated

However, basophil deletion partly impaired IL-4 produc-with antigen, but not in TH1 cells that had not beention by activated T cells from Trichinella infected mice.

chronically stimulated. However, E4BP4 regulates theWe also identified heterogeneity between TSLP-elicitedexpression of IL-10 in an epigenetic manner by altering

versus IL-3-elicited basophils. In humans, TSLP expres-were dispensable for asthmatic responses, and for IgE

the chromosomal structure in the region of the Il10 gene.

sion is associated with asthma, atopic dermatitis andThese results indicate that E4BP4 has multiple functions

food allergies, and studies in murine systems demon-in controlling the plasticity of IL-13 in Th1 cells and in

strated that TSLP promotes TH2 cytokine-mediatedfunctioning as a master regulator for IL-10 expression in

immunity and inflammation. We demonstrated that TSLP

Th1, Th2, Treg and NKT cells. Interestingly, E4BP4-defi-

induces TH2 cytokines by eliciting basophil responses.

cient mice, which produced lower levels of IL-10 than

Furthermore, in Mas-TRECK mice, contact hyper-

control mice, also showed some symptoms of gastroin-sensitivity (CHS) was attenuated when MCs were

testinal inflammation along with diarrhea. Therefore,

depleted during the sensitization phase. We examined

E4BP4 is also a key transcriptional factor in preventing

the role of mast cells in CHS and in FITC-induced cuta-

autoimmune inflammation.

neous DC migration and found that both maturation andmigration of skin DCs were abrogated by MC depletion.

Role of basophils and mast cells in allergic
responses and helminth infections.

CHS was attenuated when MCs were depleted during
the sensitization phase. Consistent with these findings,

We established a diphtheria toxin ( DT )-based condi-

co-culture with bone marrow-derived MCs enhanced

tional deletion system using Il4 enhancers specific foreither mast cells ( MCs ) or basophils to drive the expres-the maturation and chemotaxis of BMDCs in vitro. Thesesion of the DT receptor ( DTR ) ( Mas-DTR and Bas-DTR
mice ) . While diphtheria toxin treatment of Bas-DTR miceand enhance DC functions. This interaction might be

resulted in specific deletion of basophils, treatment of

CHS.

results suggest that MCs interact with DCs in the skinessential for establishing the sensitization phase of

Leader :Masato Kubo

(Research Institute for Biological Science, Tokyo University of Science)

Recent Publications :1. Tanaka, S., Motomura, Y., Suzuki, Y., Yagi, R., Inoue, H., Miyatake, S., Kubo, M. The enhancer HS2 critically regulates GATA-3-mediated Il4transcription in TH2 cells. Nat Immunol. 12, 77-85, 2011
2. Sofi, M. H., Qiao Y., Ansel, K. M., Kubo, M., Chang, C-H. Induction and maintenance of IL-4 expression are regulated differently by the
3’enhancer in CD4 T cells. J Immunol. 186, 2792-9. 2011
3. Motomura, Y., Kitamura, H., Hijikata, A., Matsunaga,Y., Matsumoto, K., Inoue, H., Atarashi, K., Hori. S., Watarai, H., Zhu, J., Taniguchi, M., andKubo, M. The transcription factor E4BP4 regulates the production of IL -10 and IL-13 in CD4+ T cells. Nat Immunol. 12, 450-459, 2011
4. Siracusa, M. C., Saenz, S. A., Hill, D. A., Kim, B. S. Headley, M. B., Doering, T. A., Wherry EJ., Jessup, H. K., Siegel, L. A., Kambayashi, T., Dudek,
E. C., Kubo, M., Cianferoni, A., Spergel, J. M., Ziegler, S. F., Comeau, M. R., and Artis, D.; TSLP promotes interleukin-3-independent basophil
hematopoiesis and type 2 inflammation. Nature 477 , 229-233, 2011.
5. Otsuka, A., Kubo, M., Honda, T., Egawa, G., Nakajima, S., Tanizaki, H., Kim, B., Matsuoka, S., Watanabe, T., Nakae, S., Miyachi, Y., Kabashima,
K. Requirement of interaction between mast cells and skin dendritic cells to establish contact hypersensitivity. Plos One 6 , e25538. 201148

Open Laboratory for Allergy andMucosal Immune Tolerance (AMIT)

To accommodate a vast antigenic exposure fromunwanted inflammation and stabilizing its homeostasis isfood components as well as from commensal bac-

an ideal approach for the fundamental treatment of

teria, the gut has evolved a naturally anti-inflammatory

inflammatory disorders such as allergy. To this end,

environment. Under normal physiological conditions, theinduction of Ag-specific oral tolerance is an attractive

GALT develops mechanisms to tolerate, or exist in asolution. Promising strategies for induction of Ag-specificsymbiotic relationship with, a large, normally innocuous

regulatory T cells might include usage of soluble pep-

community of gut microbes. In order to maintain thistides, tolerogenic adjuvants and/or cytokines, as well as

tolerant state, the immune system of the gut must exhibit

the direct application of Ag via the mucosal route.

“dominant tolerance” and possess the ability to generatea regulatory immune response that can even control

The AMIT open lab will elucidate:

systemic immunity. Oral tolerance is therefore a major(1) the mechanism for generation and functional matu-

physiological mechanism to maintain intestinal and sys-

ration of intestinal Ag-specific Treg and their sup-temic immune homeostasis. It is also a promising method

pressionfor controlling both regional and systemic allergic inflam-

responses.

mation.

mechanism

inallergic

inflammatory(2) how suppression mechanisms that originate in the

Defects in the induction of oral tolerance against
egg proteins, for instance, lead to a food allergy. Therefore co-opting the immune system towards avoiding

intestine dominantly regulate systemic immunity,(3)

the mechanism of rush specific oral tolerance
induction (SOTI), a feasible clinical treatment method

Figure 1: Oral tolerance: its suppression mechanism originates in the intestineand dominantly regulates systemic immunity, thus it is essential for
maintenance of immune homeostasis. It is also a promising methodof regulating both regional and systemic allergic inflammation.

49

for food allergy, byallergy model mice. These experimental models will be

developing an ani-utilized to elucidate how gut-induced Tregs suppress

mal model.

allergic inflammation and related effector cells such as

The AMIT open

mast cells. Since oral tolerance is the most physiologicallab has establishedprototypea model system for

response, further understanding of antigen presenting

type I allergy (ana-

of

the

Ag-specifictolerogenic

immunecells and regulatory T cells in the gut should lead tophylaxis), regular and rush SOTI in sensitized mice, andeffective

confirmed prolonged effects of the SOTI protocol in

dysfunction.

therapeuticstrategies

targetingimmuneFigure 2 : Induction of regulatory T cells in the intestine
Induced Treg (iTreg) that develops in GALT is “TGF-β-producing iTreg”,“Foxp3+ iTreg”, and “IL-10-producing iTreg” whose differentiation factors
are TGF-β, TGF-β plus retinoic acid, and IL-10, respectively. Althougheach group of Treg has the ability to inhibit Th1 immune responses andinflammation, their exact suppressive mechanisms are largely unknown.
(Tsuji NM & Kosaka A, Trends Immunol (2008) 29: 532-540)

Leader :Noriko M Tsuji( Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology ( AIST ))Recent Publications :1. Kawashima T, Hayashi K, Kosaka A, Kawashima M, Igarashi T, Tsutsui H, Tsuji NM, Nishimura I, Hayashi T, Obata A. Lactobacillus plantarumstrain YU from fermented foods activates Th1 and protective immune responses. Int Immunopharmacol. 11( 12) :2017-24. 2011
2. Yan H, Kakuta S, Nishihara M, Sugi M, Adachi Y, Ohno N, Iwakura Y, Tsuji NM. Kjellmaniella crassifolia Miyabe ( Gagome) extract
modulates intestinal and systemic immune responses. Biosci Biotechnol Biochem. 75( 11) :2178-83. 2011
3. Jeon SG, Kayama H, Ueda Y, Takahashi T, Asahara T, Tsuji H, Tsuji NM, Kiyono H, Ma JS, Kusu T, Okumura R, Hara H, Yoshida H, Yamamoto M,
Nomoto K, Takeda K. Probiotic Bifidobacterium breve induces IL-10-producing Tr1 cells in the colon. PLoS Pathog. 2012 8( 5) :e1002714. 2012
4. Faria AM, Mucida D, McCafferty DM, Tsuji NM, Verhasselt V. Tolerance and inflammation at the gut mucosa. Clin Dev Immunol. 2012:738475. 201250

University of Michigan-RCAI Joint Workshop
December 1-2, 2011

F

ive immunologists from University of Michigan visited RCAI for the first University of Michigan-RCAI Joint Immunology Workshop held on December 1-2,

2011. This workshop was originally proposed by the University of Michigan in orderto launch collaborative relationships with RCAI researchers. In the first session "Tcell Signaling", Takashi Saito ( RCAI ) introduced his recent discovery of TCR signaling regulation by PD-1 microclusters. Hei-ichiro Udono ( RCAI, photo 1) discussed
the role of HSP90 in antigen cross-presentation, and Malini Raghavan ( U of M,
photo 2 ) discussed the functions of calreticulin in MHC class I folding. The session
on "Mucosal Immunity" led to a hot discussion on commensal bacteria. Gabriel
Nunez ( U of M, photo 3 ) , Naohiro Inohara ( U of M, photo 4 ) , Sidonia Fagarasan( RCAI ) , Hiroshi Ohno ( RCAI ) and Hilde Cheroutre ( LIAI and RCAI, photo 5 ) foundsome differences in commensal bacteria in the same mouse strains, but it was difficult to explain the reasons. The last session of the first day was "Clinical Immunology". Sophie Paczesny ( U of M, photo 6 ) described her studies to identify
biomarkers for GVHD, Shin-ichiro Fujii ( RCAI ) explained his new allogeneic immunotherapy using cells loaded with NKT cell ligand, and Fumihiko Ishikawa ( RCAI ) introduced his anti-leukemic stem cell ( LSC ) therapeutic development using thehumanized mouse model. The second day started with a "Lymphocyte Development" session. Wes Dunnick ( U of M, photo 7) talked about class switch recombination regulated by chromosomal translocations, Hiroshi Kawamoto ( RCAI ) describeda lineage check point during the development of T cells, and Ichiro Taniuchi ( RCAI )and Hilde Cheroutre ( LIAI and RCAI ) discussed Thpok regulation of T cell fate. In theafternoon of the second day, each researcher from the University of Michigan held
individual discussions with RCAI researchers with common interests. By the end of
the workshop, all participants agreed to the continuation of communications anddiscussions for collaborations, and the organizers, Wes Dunnick and Haruhiko
Koseki ( Photo, 8 ) promised to make efforts for the next joint workshop, hopefully inMichigan.

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Table: Program
Thursday, December 1
Opening remarks

Masaru Taniguchi, RCAI and Wesley Dunnick, University of Michigan

Session 1 : T cell Signaling

Chair : Hiroshi Ohno

Takashi Saito (RCAI )Imaging of T cell activation regulation: -Negative regulation of TCR signaling by PD-1
microcluster.

Hei-ichiro Udono (RCAI )Dendritic cells require HSP90 as a cytosolic translocator of extracellular antigen for crosspresentation

Malini Raghavan (University of Michigan )Functions of calreticulin in MHC class I folding and antigen presentation

Session 2 : Inflammation

Chair : Naohiro Inohara

Takashi Tanaka (RCAI )HSP70 is essential for PDLIM2-mediated termination of NF-κB signaling

Masato Tanaka (RCAI )Immune regulation by dead cell clearance

Session 3 : Mucosal Immunity

Chairs : Masato Tanaka & Hilde Cheroutre

Gabriel Nunez (University of Michigan )Role of Nod-like receptors and IL-1 signaling in intestinal immunity and host defense against
enteric pathogens

Naohiro Inohara (University of Michigan )The interaction between host immunity and commensals: A view from the bacterial side

Sidonia Fagarasan (RCAI )Vitamin A-dependent transcriptional activation of NFATc1 is critical for the development andsurvival of B1 cells

Hiroshi Ohno (RCAI )The epithelia-specific membrane trafficking factor AP-1B secures gut immune homeostasis inmice

Session 4 : Clinical Immunology

Chairs : Malini Raghavan & Takashi Tanaka

Sophie Paczesny (University of Michigan)

Discovery and validation of graft-versus-host disease biomarkers

Fumihiko Ishikawa (RCAI)

Understanding human immunity & diseases through a creation of humanized mouse model.

Shin-ichiro Fujii (RCAI)

Dendritic cell (DC) targeting immunotherapy by antigen mRNA transfected, allogeneic humancells loaded with NKT cell ligand as artificial adjuvant vector cells (aAVCs)

Friday, December 2

52

Session 5 : Lymphocyte Development

Chairs : Takashi Saito & Wesley Dunnick

Wesley Dunnick (University of Michigan )Regulating class switch recombination and chromosomal translocations

Hiroshi Kawamoto (RCAI )Lineage restriction process from hematopoietic stem cells to T cell progenitors: An essential
developmental checkpoint for production of the T cell lineage.

Ichiro Taniuchi (RCAI )Mechanisms of helper versus cytotoxic lineage choice

Hilde Cheroutre ( LIAI / RCAI )Silencing of Thpok drives plasticity of CD4 effector T cells in vivo

The 4th Joint RCAI-LIAI Workshop ProgramT

he 4th Joint RCAI-LIAI workshop

sented their recent data. Over thement of treatments and cures forwas jointly organized by the Lacourse of the workshop, the partici-

immune system disorders. Past col-Jolla Institute for Allergy & Immunol-pants actively discussed these top-

laborations resulted in the creationogy (LIAI) and RIKEN at LIAI on Feb-

ics, and this opportunity facilitatedof a new research unit (by Hilderuary 7-8. Nearly 100 participants,

further collaboration between RCAI

Cheroutre) in RCAI in 2011.

including Masaru Taniguchi, Directorand LIAI researchers.

2011 workshop also resulted in a few

of RIKEN RCAI, and Mitchell Kronen-

The

Both RCAI and LIAI were estab-

new collaborations. Future collabo-

Chieflished with the goals of making

rations between the two research

Scientific Officer, attended the work-

breakthroughs in our understanding

institutes will help in achieving their

shop.

of the immune system and improving

common goals.

berg,

LIAI

President

andThis workshop addressed cur-human health through the develop-

rent progress in six major areas, Th& Treg cells, Signaling in the immune
system, Disease regulation, Antigen
recognition by T cells & antibodies,
and T cell development & memory.
Eighteen leading scientists in thesefields from the two institutes pre-RCAI-CGM collaboration is launched;
toward integrative medical sciences

R

IKEN RCAI and RIKEN Center
for Genomic Medicine ( CGM )with clinical data accumulated atlaunched a collaboration to link theirof how to integrate these similar but

two research fields; RCAI's mecha-

different research fields, a kick-off

nistic research using mice and otherexperimental models and CGM's

meeting was held on Aug 26, 2011
( photo 1-8 ) . Researchers from bothresearch on human disease suscep-

centers introduced their themes,

tibility genes.

approaches

In this collaborative

scheme for the analysis of common
human

immunological

CGM. To discuss the practicalities

andunpublishedresults to promote discussions.

disorders,

At the end of the meeting, theRCAI will work closely with CGM andresearchers agreed to focus on fiveplans to collect and integrate thedifferent themes and each working

data from genetically modified micegroup will start concrete collabora-

53

tions; 1) Rheumatoid arthritis andautoimmune diseases, 2 ) Vasculitis

in humans, and CGM researchers

about variations of HLA and dis-suggested their candidate genes foreases. For the second seminar on

and vascular diseases, 3 ) Allergy, 4 )Colitis and 5 ) Pharmacogenetics,

functional analysis using geneticallymodified mice. In addition to theseFeb. 21, Dr. Shigeharu Wakana( RIKEN BioResource Center ) was

QTL and other diseases.

discussions and analysis, a series ofinvited to talk about the International

During the fall and winter ofjoint seminars was started based on

Mouse2011, each working group continued

requests from researchers. For thetheir standardized analysis and pipe-

discussions and started collabora-

first one, held on Jan 24, Dr. Katsushi Tokunaga ( The University oflines. The RCAI-CGM collaboration

Tokyo, photo 9 ) and Dr. Yuta Kochi( CGM, photo 10 ) were invited to talkreach toward the goal of establishingtive analysis. Based on their mouse
experiments, RCAI researchers suggested candidate genes for diseases

54

01 Naoyuki Kamatani ( CGM )02 Michiaki Kubo ( CGM )05 Masaaki Murakami(Osaka Univ.)

06 Haruhiko Koseki ( RCAI )07 Toshitada Takemori ( RCAI )08 Kazuhiko Yamamoto (CGM )Phenotyping

Consortium,

will continue to expand in order tointegrative medical sciences.

03 Masaru Taniguci ( RCAI )04 Toshihiro Tanaka ( CGM )09 Katsushi Tokunaga( Univ. Tokyo )10 Yuta Kochi (CGM )RCAI 10th Anniversary Seminar SeriesIn 2011, RCAI held a series of guest seminars to enhance communication with outside researchers, because theCenter's major external events had been canceled after the Great East Japan Earthquake. A total of 35 active

researchers ( 23 from Japan and 12 from abroad ) , especially young ones, were selected and invited from various
fields.
Table : RCAI 10th Anniversary Seminar Series
Date

Speaker

Affiliation

Country

Title

2011.7. 29

Masahiro
Yamamoto

Department of Microbiology and
Immunology, Graduate School of Medicine,
Osaka University

Japan

Exploration of Host-Parasite Interface
Possibly Reveals a New Aspect of
Immunology

2011.8.2

Yumi
Matsuzaki

Keio University, School of Medicine, Centerof Integrated Medical Research

Japan

Donor mesenchymal stem cells trigger
chronic graft-versus-host disease following
minor antigen mismatched bone marrowtransplantation

2011.8.4

RichardJones

The University of Chicago

USA

Proteinomic analysis of cell signaling
networks with micro-western arrays

2011.8.9

Jun Seita

Institute for Stem Cell Biology &Regenerative Medicine, and Department of
Pathology, Stanford University School of
Medicine

USA

Systems Approach to Hematopoietic Stem/
Progenitor Cell Biology

2011.8.30

Taro
Kawai

Immunology Frontier Research Center,
Osaka University

Japan

Signaling pathways activated by innate
receptors and their roles in host defense

2011.9.6

Kiichiro
Yano

Cardiovascular-Metabolic Laboratories,
Daiichi-Sankyo Co., LtdJapan

Role of Crosstalk between Adipocytes andEndothelium in Sepsis

2011.9.13

KazuyoMoro

Keio University School of Medicine,
Department of Microbiology and
Immunology

Japan

Natural helper cells play a critical role in IL33-dependent eosinophilia in the lung.

2011.9.29

TomoyaKatakai

Department of Molecular Genetics, Instituteof Biomedical Science, Kansai Medical
University

Japan

High-speed interstitial T cell migration in
lymph node involves LFA-1-dependent and-independent mechanisms controlled bystromal cell network

2011.10.7

Taro
Fukao

Max-Planck Institute of Immunobiology andEpigenetics

Germany

MicroRNA Regulation of Energy
Metabolism in Mammalian Immune System

2011.10.11

J. Rodrigo
Mora

Massachusetts General Hospital & Harvard
Medical School

USA

Retinoic acid and gut-homing T cells in
intestinal immune homeostasis

2011.10.13

Takahiro
Maeda

Department of Medicine, Harvard Medical
School, Brigham and Women's HospitalUSA

LRF Transcription Factor Maintains HSC
Homeostasis by Preventing LymphoidPrimed LT-HSCs from Excessive
Differentiation

2011.10.19

Shinobu
Saijo

Medical Mycology Research Center, Chiba
University

Japan

C-type lectins: their roles in the hostdefense against fungal infection

2011.10.25

AtsushiMizoguchi

Harvard Medical School, Massachusetts
General Hospital

USA

Regulatory B cells, lessons from the modelof IBD2011.11.2

TaishinAkiyamaThe Institute of Medical Science, The
University of Tokyo

Japan

Molecular mechanisms required for thymic
self-tolerance

2011.11.16

MakotoArita

Graduate School of Pharmaceutical
Sciences, The University of TokyoJapanMediator Lipidomics in Inflammation
Research

2011.11.21

Takeshi
Egawa

Department of Pathology and Immunology
Washington University School of Medicine

USA

Transcription factors in cytotoxic T cell
development

Taro Fukao

J. Rodrigo Mora

Shinobu Saijo

Makoto Arita

Hidehiro Fukuyama

55

2011.12.8

Hidehiro
Fukuyama

Institute of Molecular and Cellular Biology,
CNRS

FranceDrosophila models to study chronic
inflammation

2011.12.8

KosukeYusa

Wellcome Trust Sanger InstituteJapan

Targeted gene correction of α1-antitrypsin
deficiency in induced pluripotent stem cells

2011.12.12

Takeshi
Nitta

National Center for Global Health andMedicineJapan

Revisiting ‘Thymic Nurse Cell’: Uniquelympho-epithelial complexes in the thymic
cortex2011.12.16

KohsukeImai

Department of Community Pediatrics,
Perinatal and Maternal Medicine
Tokyo Medical and Dental University

Japan

Primary immunodeficiency in Japan

2011.12.22

Kenya
Honda

Department of Immunology, Graduate
School of Medicine, The University of Tokyo

Japan

Intestinal microbiota shapes the immune
system2011.12.26

Takashi
Yamamura

Department of Immunology, National
Institute of Neuroscience, NCNP

Japan

Central nervous system autoimmunity andgut flora2012.1.19

Matthias
HebrokUCSF Diabetes Center

USA

Deciphering pancreas development anddiseases2012.1.25

Mark
Shlomchik

Yale University

USA

Germinal Center Selection and theDevelopment of Memory B and Plasma
Cells2012.1.31

TakakoHirataKyoto University Graduate School of
Medicine, Oxford University

Japan

The role of the ERM protein moesin in
lymphocyte homeostasis

2012.2.13

Rob Klose

Department of Biochemistry, University of
OxfordUK

Interpreting the CpG island signal

2012.2.14

Tomohiro
Sawa

Department of Microbiology, Graduate
School of Medical Sciences, Kumamoto
University

Japan

Signaling functions of reactive oxygen
species and electrophiles: regulatory
mechanisms and implications in chronic
inflammation-associated diseases

2012.2.20

Sudhir
Gupta

University of California, Irvine

USA

Paradox of Immunodeficiency andInflammation & Autoimmunity in Human
Aging

2012.2.20

Jun
Kunisawa

Division of Mucosal Immunology, Instituteof Medical Science, The University of TokyoJapan

Lipid, vitamin, and nucleotide in theregulation of gut immunity

2012.2.23

Rudi
Balling

Director, Luxembourg Centre for systems
Biomedicine (LCSB), University of
Luxembourg

Germany

A systems biology approach to Parkinson´sdisease: Research at the new Luxembourg
Centre of Systems Biomedicine

2012.2.23

Yoko
HamazakiDepartment of Immunology and Cell
Biology, Graduate School of Medicine,
Kyoto University

Japan

Identification of claudin-expressing
medullary thymic epithelial stem cells thatmaintain the functional medulla during life

2012.2.28

Kumar
SelvarajooInstitute for Advanced Biosciences, Keio
University

Japan

Unraveling governing principles from
immune cell dynamics

2012.2.28

Yoshinori
Fukui

Medical Institute of Bioregulation, Kyushu
University

Japan

Immune regulatory functions of DOCK
family proteins in health and disease2012.3.8

Yoshiko
Mito

Massachusetts General Hospital/Harvard
Medical School

USA

Allele-specific profiling of genomewide epigenetic marks and sequencepolymorphisms

2012.3.23

Toshiyuki
Takai

Institute of Development, Aging andCancer, Tohoku University

Japan

Regulatory effects of IgG on B cellfunctions

Kosuke Yusa

56

Kenya Honda

Masahiro Yamamoto

Jun Seita

Kazuyo MoroMultidisciplinary Research ProjectsW

ith the aim of developing new paradigms in immunology, RCAI launched a program in 2009 to support alimited number of multidisciplinary collaborative projects led by RCAI researchers. The program provides

5-10 million JPY/year to each collaborative research project for up to five years. In FY2011, eight projects wereselected to receive this support.

Table : Awardees of RCAI Multidisciplinary Research Projects 2011
Year
2009-

Name
Hiroshi Ohno

Project Title
Genomic, transcriptomic and metabolomic
analysis of human ulcerous colitis

Collaborators
Mamoru Watanabe ( Tokyo Medical and Dental Univ.)
Masahira Hattori (The Univ. of Tokyo)
Jun Kikuchi (RIKEN Plant Science Center)

2009-

Creation of artificial immune cells

Harukazu Suzuki
Jun Kawai
Yoshihide Hayashizaki
Carsten O. Daub
Piero Carninci (RIKEN Omics Science Center)200920092009-

Hiroshi Kawamoto
Haruhiko Koseki
Fumihiko Ishikawa
Masato Tanaka
Ichiro Taniuchi
Osamu Ohara
Toshitada Takemori
Masaru Taniguchi
Satoshi Ishido
Hisaaki Shinohara
Ichiro Taniuchi

Yuji Sugita (RIKEN Advanced Science Institute)Mariko Hatakeyama (RIKEN Advanced Science Institute)
Atsushi Mochizuki (RIKEN Advanced Science Institute)

2010-

Tsuneyasu Kaisho

2010-

Haruhiko Koseki

2010-

Takashi Saito

Modeling of antigen presentation
Modeling of NF-κB activation signals
Regulation of gene expressions for thymic
development
Generation of antibodies against chemokine
receptor XCR1 and elucidation of its crystal
structure
Elucidation of the lymphocyte differentiation
mediated by alternative splicing
Elucidation of the structure and function of
the immune receptor complex

Shigeyuki Yokoyama
Tomomi Someya(RIKEN Systems and Structural Biology Center)
Yoshinori Naoe
(National Center for Geriatrics and Gerontology)
Shigeyuki Yokoyama
Mikako Shirouzu (RIKEN Systems and Structural Biology
Center)Student Exchange ProgramsI

n 2011, RCAI's International Summer Program ( RISP ) started twoceled and RCAI could not accept

student exchange programs withthree students to Germany. Hisashi

immunology schools in Germany;

Wada, a master course student in

Spring School held by Deutsche
Gesellschaft für Immunologie ( DGFI )the Laboratory for Transcriptionaland Summer School held by Zentrum
für Infektioinsbiologie und Immunität( ZIBI ) . Although RISP 2011 was can-students from Germany, RCAI sent

Regulation, participated in DGFI
Spring School in Ettal ( photo 1) .

photo 1 : Monastery in Ettal where the
DGFI Spring School was heldLaboratory for Epithelial Immunobi-"I can never forget the experience.

ology participated in the ZIBI Summer School held in Berlin ( photo 2 ) .

In a small lecture room in a monas-"There were 40 participants from 16

tery, sixty students andcountries in the world. Although thelecturers from all overcourse was targeting mainly toEurope had so much

master course students and was a

discussions, even until

little too basic for me, I enjoyedmidnight," said Wada.

hands-on experiments using nema-

Two postdocs, Shimpei

tode," said Fukuda. RCAI is planning

Kawamoto, Laboratory

to accept students from Germany infor Mucosal Immunity

RISP 2012.

andShinji

Fukuda,photo 2 : Group photo of ZIBI Summer School57

International Research Collaboration AwardT

he RCAI International Research

Since the program began in

Kawamoto’s work on thymic pro-Collaboration Award is a uniqueresearchers

2004, 14 projects have been funded( Table ) and the collaborations have

genitor cells resulted in three papers
in Development ( 2006 ) and Mol.

outside of Japan in setting up semi-

resulted in several important papers.

Immunol. ( 2009 and 2010 ) . Drs. Bix

independent research units withinDrs. Vidal and Koseki’s collaborativeand Kubo published their work on

the laboratory of their collaboration

project on Ring1 genes resulted in 5
papers, Dev. Cell ( 2004 ) , Develop-

the IL4 repressor in Nat. Immunol( 2009 ) , and Drs. Ellemeier and Tani-

ment ( 2006 ) , Nat. Cell. Biol. ( 2007) ,Mol. Cell. Biol. ( 2008 ) and Develop-

uchi published their studies on CD8

ment ( 2008 ) . Drs. Dustin and Saito

Sci (2011) and on CD4/CD8 lineage
choice in Nat. Immunol. ( 2010 ) .

programsupporting

partner at the Center. The programprovides up to 10 million JPY/year toeach collaborative research projectfor up to three years. ( Until 2007, theprogram awarded 15 million JPY/
year but the amount was reduced in2008.)

published their work on T cell microclusters in Nat. Immunol. ( 2005 ) and
Immunity ( 2006 ) .

gene regulation in Proc. Natl. Acad.

Drs. Ewijk andTable : Awardees of RCAI International Collaboration Award Program

58

Year

Host Lab.

Title of ResearchAwardee20042006

Takashi
Saito

Analysis of dynamism and function of immunologicalsynapse using planar membrane and knock-in T cells

Dr. Michael DUSTIN
New York University School of Medicine

20042006

Hiroshi
Kawamoto

Regulatory role of lymphoid progenitors during
development of thymic microenvironments

Dr. Willem van EWIJK
Leiden University Medical Center

20042006

Haruhiko
Koseki

Genomic and functional analysis of the role of the
Polycomb Ring1 genes in B-cell development

Dr. Miguel VIDAL
Centro de Investigaciones Biologicas, CSIC

20042005

MasaruTaniguchi

Role of NKT cells in TSLP-mediated allergic
inflammation

Dr. Steven ZIEGLER
Benaroya Research Institute at Virginia Mason
Medical Center

20042006

Ji-Yang
Wang

Expression and function of FcRY-a novel Fc receptorrelated gene expressed in B cells

Dr. Peter BURROWS
University of Alabama at Birmingham

20052007

IchiroTaniuchiStudy of T cell differentiation mediated by regulated
expression of CD8 genes

Dr. Wilfried ELLEMEIER Institute of Immunology,
Medical University Vienna
Dr. Hilde CHEROUTRE La Jolla Institute for Allergy
and Immunology

20052007

Masato
Kubo

Understanding genetic regulation of interleukin 4
production by a CD4(+) T cell-intrinsic mechanism.

Dr. Mark BIX
University of Washington, Seattle, Washington

20052006

Yasuyuki
Ishii

Gene-array analysis and proteomics of Th2 tolerance

Dr. Yun-Cai LIU
La Jolla Institute for Allergy and Immunology

20052007

Osami
Kanagawa

Visualization of STAT protein in the cytokine mediatedsignaling at a single molecular level.

Dr. Kenneth MURPHY
Howard Hughes Medical InstituteWashington University School of Medicine

20052007Tomohiro
KurosakiRole of signaling molecules in B cell synapse
formation and its maintenanceDr. Facundo BATISTA
Cancer Research UK London

20062008

MasatoTanakaIdentification of Novel Necrotic Molecules from
Necrotic Hepatocytes and Examination of Its Effecton the Inflammatory Response

Dr. Sunhwa KIM and Dr. Michael KARIN
Department of Pharmacology, Univ. of California, San
Diego, USA

20072008

TakeshiWatanabe

A study on the spleen and lymph nodes mesenchymal
cells that participate in the assembly of artificial
secondary lymphoid organs

Dr. Andrea BRENDOLAN
Cornell University Medical Center, Department of Celland Developmental Biology

20072009

IchiroTaniuchiUnderstanding of tumor suppressive mechanismof Runx complexes against leukemia andgastrointestinal cancer

Dr. Motomi OSATO and Dr. Yoshiaki ITO
Institute of Molecular and Cell Biology, National
University of Singapore

20072009

Sidonia
Fagarasan

Nuclear reprogramming of terminally differentiatedplasma cells to study the specific role of IgA
in mucosal and systemic immunity and B cell
development

Dr. Stefano CASOLAIFOM-The FIRC Institute of Molecular Oncology
Foundation, Milano, Italy

2010-

MarikoOkadaHatakeyama

Proteomics based-quantitative analysis of signaltranscriptional network

Dr. Boris KHOLODENKO
University College Dublin, Ireland
Dr. Richard JONES
University of Chicago, USA

2011-

IchiroTaniuchiUnderstanding and Engineering of Dendritic
Epidermal T Cells (DETC) development

Dr. Florent GINHOUXSingapore Immunology Network (SIgN), Singapore

2011Part 5

Outreach Activities

RIKEN Yokohama
Open Campus

Immunology
Workshop for HighSchool Students

Oct 8, 2011
1

2

November 1, 2011
1

T

2

he RIKEN Yokohama Institute Open Campus washeld on Oct. 8, 2011. There were 1,900 visitors

ranging in age from children to retired folks.
On Oct. 3, only 5 days before the event, there wasan announcement of this year's Nobel Prize for Physiol-

S

eventeen students of Kanagawa Sohgoh HighSchool visited RCAI on November 1, 2011.

The

Bruce A. Beutler, Jules A. Hoffman and Ralph Stein-workshop started with lectures by Dr. Ishikawa and Dr.
Kawamoto. In his lecture, Dr. Ishikawa ( photo 1) told ofman. There was great excitement that Dr. Steinman, an

his own experiences in high school, medical school,

RCAI Advisory Council member, had received the prizeand hospitals in Japan and the US; what he thoughtfor his discovery of the dendritic cell, but it soon turned

and why he chose to be a leukemia researcher. The

to great sorrow after we learned that he had passed
away three days previously. RCAI's Hiroshi Kawamoto,young students felt empathy with his story and hisstrong passion for leukemia research ( photo 2 ) . "I amtogether with Shinichiro Fujii, Tsuneyasu Kaisho andvery impressed by his message, 'being pure' to myself.

Takashi Tanaka prepared posters, leaflets and a model

I can think more positively now about my future," oneof the dendritic cell to introduce the discoveries of thestudent said. "I was almost giving up my dream to go toNobel laureates: activation of innate immunity and the

medical school, but I will rethink about it. This was arole of the dendritic cell in adaptive immunity.

valuable opportunity to listen to someone from a clinicalogy and Medicine, given to three immunologists, Drs.

Twenty RCAI teams exhibited posters or movies.

field." Then, Dr. Kawamoto explained some very basic

The highlight of the Open Campus was hands-on learn-

immunology using his original cartoons, and took theing. Kawamoto and the team members arranged anexperiment to isolate CD4 + CD8 + T cells from mice
( photo 1) . "For this rather precise experiment, we canstudents to his own laboratory for hands-on practice( photo 3 ) . With the help of his laboratory members,

only accept 30 people who are high school students ortern of lymphocytes from spleen and thymus using

above. Still, I think it offers various experiences in one

FACS. "It was my first time to do a dissection. It looked

opportunity; a brief immunology lecture, mouse dissec-

the same as the anatomy figures in textbooks," one stu-tion, sophisticated cell sorting, laboratory atmosphere,

dent said. "I liked the cartoons. They were helpful tocommunication with researchers... participants can feel

understand the principles of immunology. I wish ourwhat we actually do everyday," said Kawamoto.

biology classes were like this," another said. The highstudents dissected mice and compared CD4/CD8 pat-

Yoshida and his team prepared stained samples ofschool teacher said this kind of opportunity is valuable

fetal mice and newborn mice at various developmental

for young students and he hoped RIKEN and RCAI

stages (photo 2). Two elementary school girls had lined

would continue to organize the immunology workshop

up in front of the microscopes before the samples

for high school students.

weren’t even ready. "We were waiting for this for a year.
When we came last year, they were amazing, so wecame back to take photos," they said.
This annual event, originally planned in July, wasrescheduled due to the urgent need to save electricity
this summer because of the massive East Japan Earthquake. When the summer heat passed by and the government lifted its electricity saving policy on September
9, various events were begun in many places. "ThisOpen Campus ultimately overlapped with a Sports Festival of the local school, but still many people visited us.
We have to appreciate that people maintain their interest in our science," a RIKEN staff member commented.

60

3

Lab visit by juniorhigh school and highschool students

Science Café

December 16, 2011

March 3, 2012

"Regulation of thecell fate"

1

2

O

n March 3, 2012, Ichiro Taniuchi, Group Director ofLaboratory for Transcriptional Regulation, talked

at the Science Café held at Kanagawa Prefectural
Kawasaki Library. Among 170 applications, 50 partici-Tpants were selected by a random drawing. Taniuchi's
wenty-six students of Shinagawa Joshi Gakuin, atalk started with various sensory organs. We are famil-

women’s junior and senior high school, visited

iar with the eye, skin, nose or ear, but "What about

RIKEN Yokohama Institute.

Ms. Hanae Fujimoto, a

immunity? Where is it? What does it sense?" Dr. Tani-technical scientist in the FACS laboratory explained theuchi explained various immune cells, how they sense

mechanisms of the cell sorter and demonstrated thepathogenic organisms, memorize and prevent. Then helymphocyte isolation process (photo 1), and then three

explained how immune cells are developed.

female RCAI researchers, Drs. Kumiko Sakata-Sogawa(photo 2), Mari Hoshino (photo 3) and Reiko Onishi

various illustrations, he talked about T cell selection inthymus, where functional selection ( positive selection )(photo 4) took 3-4 students each for laboratory visits

and autoreactive selection ( negative selection ) occur.

and discussions. Taking students to their own laborato-

Finally he talked about transcriptional regulation of cellries, they explained what kind of work immunology

fate during their development. Using Yoda in Star Wars

researchers actually do, what the environment of ato illustrate the concept, he explained how a master

research institute is like, why they decided to become atranscription factor, ThPOK, regulates the cell fate deci-

researcher, and women’s work-life balance. “I am inter-sion to become a helper or a killer T cell. "My image of

ested in biology, so it was a good opportunity to visit a

researchers was a kind of isolated people, but I am sur-research institute, although I still don’t have a clear

prised that he was frank but respectful to anyone. He

vision for the future,” one high school student said.

was trying to explain easily to any of the questions," aUsingparticipant said. "There were many good questions
from the participants. Still so many questions exist in3

the world that even researchers cannot answer. I hope
this can be one of the occasions that general people
start to find and think of scientific questions in theireveryday life," said Dr. Taniuchi.

461

Science Café

Science Café

"Is calico cat's patterninheritable?""Development of apollinosis vaccine anda novel cancer therapy"

March 14, 2012

March 24, 2012

D

r. Taniguchi, Director of RCAI and Group Directorof the Laboratory for Immune Regulation, talked at

Science Café held at Yokohama City Library on MarchO

24. His talk consisted of two parts, development of an March 14, Dr. Haruhiko Koseki, Group Director

pollinosis vaccine and development of a novel cancerof the Laboratory for Developmental Genetics,

therapy. As many as 1/3 of the Japanese population

talked at RIKEN Yokohama Science Café held at Yoko-

suffers from allergy, and because March is the cedarhama City Central Library. Thirty-four people, including

pollinosis season in Japan, various people from teen-

two junior high school students attended the event.

agers to retired folks aged over 70 participated in the

The talk started with the genetics of calico cats. In cats,

event. Dr. Taniguchi started his talk from an epidemio-

each of the genes encoding orange fur or black fur islogic perspective of pollinosis. It is said that multiple

linked with X chromosome. Only when the cat has two

factors such as hygienic environment, use of antibiotics,

X chromosomes (XX), one with the orange fur gene andsmog emission, diesel fumes, house dust, etc. correlate

the other with the black fur gene, the cat becomes cal-with induction of allergy. Then he explained immuno-

ico by mosaicism.

Thus, 99.9% of calico cats are

logical mechanisms and the discovery of IgE by Dr.

female, Dr. Koseki explained. Then, will the pattern be

Kimishige Ishizaka. The vaccine being developed at

the same if the genes are identical? Dr. Koseki intro-

RCAI is designed to promote the suppression of Th2

duced a result of the cloning of a calico cat. Because

and B cells by activation of NKT and Th1 cells. He

one of the X chromosomes was already inactivated inemphasized the importance of safety for the develop-

the somatic cell used for cloning, the fur color turned

ment of allergy vaccines. In the second half, he talked

out to be different; the cloned cat was a brown tabby.

about NKT cell targeted therapy for lung cancer patients,

This kind of functionally relevant modification of theand generation of NKT cells from iPS cells for therapeu-

genome is called epigenetics. "Definitely, the experi-

tic purposes.

ence you had in your life, starving or life habit for exam-

audience were at a surprisingly high level. One manple, is imprinted into your genome, and it can be

mentioned a recent newspaper article on Histamine

inherited," said Dr. Koseki. After his talk, participants

Releasing Factor by Dr. Kawakami of LIAI and thewrote down questions on Post-it notes and Dr. Koseki

structural analysis of a histamine receptor by Dr. Iwata

answered them. "I like cats and that was why I cameof Kyoto University. He asked if these results would be

here, but realized that genomic and epigenetic systems

useful for the development of therapies. "I am impressed

control the cat's fur coat pattern," a participant said.

by the audience. They must have been studying a lot,

During the event, questions from thealthough they might not be researchers," said Dr.
Taniguchi.

62

2011
Part 6

Laboratory Activities

Laboratory for

Developmental
Genetics

Group Director :Haruhiko Koseki
Senior Scientist :Kyôichi Isono

Research Scientists : Yûichi Fujimura, Mitsuhiro Endô, Osamu Masui, Takashi
Kondô, Yixin Dong, Kit Wan Ma, Nayuta Yakushiji (SPDR), Yuki
Takada (SPDR), Shinsuke Ito (SPDR), Jafar Sharif (FPR)
Technical Staff :Tamie Endo, Naoko Ônaga, Rie Suzuki, Kayoko Katsuyama,
Yôko Koseki, Kaoru Kondô, Mami Kumon, Fuyuko Kezuka,

Raul Eduardo Vizcardo Sakoda

T

he Developmental Genetics Research Group fulfills a

repression remains not fully understood. We could show that

dual role within RCAI. A large proportion of the man-high levels of H2AK119u1 deposition occur at a subset of

power and financial resources of the group is devoted to thetarget loci that are critical for ESC maintenance. We further

maintenance of a high-standard mouse facility. Through thedemonstrated that H2AK119u1 deposition occurs at theseAnimal Core Facility, the group is also responsible for thetargets in PRC2-deficient ESCs, albeit at lower levels, sug-

generation of knock-out and transgenic animals for the vari-

gesting the presence of H3K27me3-independent compen-

ous research laboratories at the center. At the same time,satory mechanisms for recruitment of PRC1 function. Finally,

the laboratory is pursuing a research program to elucidate

we showed that the H2A ubiquitination activity of PRC1 is

the molecular mechanisms underlying organ development

essential for target gene repression and ESC maintenanceand stem cell functions. Particular emphasis has been put

and that distinct PRC1 functions mediate chromatin com-

on epigenetic regulation mediated by combinatorial actionsof Polycomb group ( PcG ) gene products and DNA methyla-

paction and contribute to silencing activity, most notably attion mechanisms in development and stem cell functions.

PRC1 utilizes these diverse sensing and effector mecha-

Hox loci. Based on these results, we have proposed thatnisms, which provide a means to maintain a repressive

The role of epigenetic regulators during
development and differentiation

state that is robust yet highly responsive to developmental
cues during ES cell self-renewal and differentiation.

Two distinct Polycomb complexes, PRC1 and PRC2, col-Although mechanistic aspects of PcG proteins in ESCs

laborate to maintain epigenetic repression of key developmental loci in embryonic stem cells ( ESCs ) . PRC1 and PRC2

are being elucidated, it is still unknown how PcG proteins

have histone modifying activities, catalyzing mono-ubiquitination of histone H2A ( H2AK119u1) and trimethylation of H3

onic tissues or differentiating cells. To tackle this issue, welysine 27 ( H3K27me3 ) respectively. However, the interplay of

Ring1A and/or Ring1B, which are essential E3 ligases forthese modifications and their contribution to Polycomb

H2A monoubiquitination, in a limb bud-specific manner. In

regulate differentiation and patterning processes in embrytested the role of PRC1 on limb organogenesis by depleting

Recent publications
1. Harada M, Murakami H, Okawa A, Okimoto N, Hiraoka
S,Nakahara T, Akasaka R, Shiraishi YI, Futatsugi N,
Mizutani-Koseki Y, Kuroiwa A, Shirouzu M, Yokoyama S,
Taiji M, Iseki S, Ornitz DM, Koseki H. FGF9 monomerdimer equilibrium regulates extracellular matrix affinity
and tissue diffusion. Nat Genet. 41, 289-98 (2009)64

2. Watarai H, Fujii S, Yamada D, Rybouchkin A, Sakata S,
Nagata Y, Iida-Kobayashi M, Sekine-Kondo E, Shimizu K,
Shozaki Y, Sharif J, Matsuda M, Mochiduki S, Hasegawa
T, Kitahara G, Endo TA, Toyoda T, Ohara O, Harigaya K,
Koseki H, Taniguchi M. Murine induced pluripotent stem
cells can be derived from and differentiate into naturalkiller T cells. J Clin Invest.120, 2610-2618 ( 2010)

3. Li X, Isono KI, Yamada D, Endo TA, Endoh M, Shinga
J, Mizutani-Koseki Y, Otte AP, Casanova M, Kitamura
H, Kamijo T, Sharif J, Ohara O, Toyada T, Bernstein
BE, Brockdorff N, Koseki H. Mammalian Polycomblike
Pcl2/Mtf2 is a novel regulatory component of
PRC2 that can differentially modulate Polycomb
activity at both the Hox gene cluster and at Cdkn2a
genes. Mol Cell Biol. 31, 351-364 ( 2011)

Figure:PcG bodies in the Hoxb1 regions. PcG bodies(green) are immunostained in a speckled pattern.
The Hoxb1 loci (red) are detected by DNA-FISH.

Ring1B single KO mice, we found shortening of the radius

Phc2 modulates the structural organization of PcG com-and ulna, suggesting that PRC1 defects predominantly affectplexes to enable robust yet reversible PcG-mediated repres-distal limb development. Ring1A /B double KO mice exhib-

sion during development. Since Hox gene clusters areited severe limb truncation. To identify target genes for PRC1,

unusual in retaining a special genomic configuration, we

we used microarray analysis for Ring1A /B double KO limbs

went on to test whether PRC1 also uses this mechanism atand ChIP-Chip analysis for binding of Ring1B in developing

solitary target genes. Intriguingly, by re-examining ChIP-seq

limb buds. We found the involvement of PRC1 in repressing

data for Ring1B distribution in ESCs, we found that PRC1 isthe expression of transcription factors that demarcate the

distributed not only to promoter regions but also to genomic

proximal region of the limb in the distal regions. Interestingly,

regions surrounding stop codons of repressed target genes.

the expression of these genes is also under the control of

We hypothesized that promoter regions are associated withretinoic acid (RA) signals suggesting functional antagonism

3’ regions of their respective targets. We focused on the

between RA signals and PRC1 for specification of the distal

Meis2 gene, which occupies a large genomic segment

region of limb buds. Consistent with this hypothesis, RA sig-

spanning around 200Kb in mammals, and which has a com-nificantly affected Ring1B binding to these target genes andexaggerated distal limb defects in Ring1B single KO mice.

plex expression patterns during development. We used 3C( chromatin conformation capture ) and FISH and immuno-

We thus propose a role for PRC1 in mediating RA signals to

FISH histological sections to analyze the interaction of thegenerate the distal part of limbs.

promoter and the 3’-PcG binding sequence or midbrain specific enhancer region, which was used as a reference. We

Regulation of large scale chromatin structures by
epigenetic regulators

found that the 3’-PcG binding sequence locates in close

Accumulating evidence documents a role for PcG proteins in

Meis2 and both regions associate with a single PcG body.

regulating higher order chromatin structures, but the mecha-

By using Ring1A/B dKO mice, we could also show that thisproximity to the promoter within cells that do not express

nisms and impact of such structures on transcriptional regu-

interaction is dependent on PRC1. Here we saw no associa-

lation remain obscure. In this study, we identified PcG bodies

tion of midbrain-specific enhancer with the promoter region.

in mouse primary fibroblasts as distinct foci at which PRC1

By contrast, the midbrain-specific enhancer comes close to

and H3K27me3 are colocalized and canonical PcG target
genes are condensed ( Figure ) . We found that PcG bodythe promoter in the midbrain region, in which Meis2 isformation requires Phc2-SAM polymerization, which criti-

not. These results suggested that PRC1-mediated regulation

cally contributes to condensation and repression of PcG

of higher chromatin structures is also used to repress soli-

target genes. We further show that Phc2-SAM polymeriza-

tary target genes, likely by using another PRC1 bindingtion limits the dynamic nature of PRC1, and thereby promotes

sequence that frequently localizes around stop codons.

strongly expressed, while the 3’-PcG binding sequence does

stable association of PRC1 with PcG target genes. Our findings suggest a novel model by which SAM polymerization of

4. Takada Y, Naruse C, Costa Y, Shirakawa T,
Tachibana M, Sharif J, Kezuka-Shiotani F, Kakiuchi
D, Masumoto H, Shinkai Y, Ohbo K, Peters AH,
Turner JM, Asano M, Koseki H. HP1γ links
histone methylation marks to meiotic synapsis inmice. Development 138, 4207-17 ( 2011)

5. Sharif J, Endoh M, Koseki H. Epigenetic
memory meets G2/M: to remember or toforget? Dev Cell. 20, 5-6 ( 2011)

65

Laboratory for

Lymphocyte
Development

Team Leader :Hiroshi Kawamoto
Research Scientists : Tomokatsu Ikawa, Kiyokazu Kakugawa, Kyoko Masuda
Research Associate : Rumi SatohT

Technical Staff :Asako Shibano-Satoh, Chiho Matsuura,
Ryosuke Yashi, Midori Kawauchi

Student Trainees :Raul Sakoda

Visiting Scientists :Nagahiro Minato, Yoshimoto Katsura,
Toshio Kitamura, Takeshi Watanabemechanisms that regulate cell fate decisions in the pro-enous, ( ii ) cells are normal ( unlike immortalized cell lines ) ,( iii ) time course analyses can be performed, ( iv ) a large

cess of lineage restriction from multipotent hematopoietic

number of cells are available, making it possible to analyze

he major aim of our team is to elucidate the molecular

stem cells to unipotent progenitors. A series of studies fromgenome-wide gene expression profiles and epigenetic sta-

our laboratory on early hematopoiesis have led to a funda-

tus and to do biochemical studies. Another potentially very

mental redefinition of lymphoid progenitors as well as theimportant advantage is that these systems may become

ontogeny and phylogeny of T- and B-cell development. We

applicable in clinical settings as a method to expand humanthus have proposed our new model of hematopoiesis, thehematopoietic progenitors.

myeloid-based model, in which myeloid potential is retained

We recently applied one of such systems for the study

along the specification pathways towards erythroid, T, and Bof the mechanisms of the T cell lineage determination step,

cell lineages.

which is where T cell progenitors have terminated all non-T
lineage potential and are fully committed to T cell lineage.

Development of an arrest-restart operation systemof cell-differentiation capable of manipulating
lineage commitment of hematopoietic progenitors

We have previously determined that the early intrathymic T

The ideal experimental system for studies on the mechanisms of lineage commitment and differentiation would be

lineage determination step takes place at the midst of theso-called double negative ( DN ) 2 stage of intrathymic T cell

one in which commitment and differentiation can be syn-development ( Masuda et al. J. Immunol. 2007 ) . We recently

chronously controlled. We succeeded in establishing several

found that when murine hematopoietic progenitors weresuch systems in which the arrest and restart of development

cultured on immobilized Notch ligand DLL4 protein in theof progenitors can be induced at a stage prior to the criticalpresence of a cocktail of cytokines including interleukin-7,

step of early T cell or B cell development. The merits of thesesystems in terms of basic research are ( i ) cells are homog-

progenitors just prior to the T cell lineage determination step

cell progenitors retain myeloid potential after terminating B
cell potential ( Wada et al, Nature, 2008 ) , and that the T cellunderwent developmental arrest and the arrested progeni-

Recent publications
1. Ikawa, T, S Hirose, K Masuda, K Kakugawa, R Satoh, A
Shibano-Satoh, R Kominami, Y Katsura, H Kawamoto.
An essential developmental checkpoint for productionof the T cell lineage. Science. 329: 93-96, ( 2010 ).

66

2. Kawamoto H, T Ikawa, K Masuda. H Wada, Y Katsura.
A map for lineage restriction of progenitors during
hematopoiesis: the essence of the myeloid-basedmodel. Immunol Reviews. 238: 23-36, ( 2010) .

3. Moro K., T. Yamada, M. Tanabe, T. Takeuchi, T.
Ikawa, H. Kawamoto, J-I. Furusawa, M. Ohtani,
H. Fujii, and S. Koyasu. Innate production of Th2
cytokines by adipose tissue-associated c-Kit+Sca-1+
lymphocytes. Nature 463 : 540-544, ( 2010) .

Figure :Arrest-restart operation system
for the study of the T celllineage specification step
A. Induction of the developmentallyarrested cells at the multipotent
progenitor stage. LKS cells fromfetal liver of EBF1-/- mice were
cultured under B cell inducing
conditions. Cells were arrested
at the B220+CD19- prepro-B
cell stage, which is thought torepresent progenitors retaining
T, B and myeloid potential.
B. Induction of the T cell lineage
specification step. When EBF1-/progenitors were transferred tothe stromal cells expressing Notch
ligand DLL4, the cells restartdifferentiation towards T cells.

tors entered a cycle of self-renewal. Reducing the concentration of interleukin-7 in the cultures promoted T cell lineage

These early events occur at the so-called double negative( DN ) 1 stage. Such DN1 progenitors then begin to express

determination. A similar arrest and self-renewal of progeni-

CD25 to enter the DN2 stage, just synchronizing with thetors was observed in thymocytes of mice deficient in the

upregulation of Bcl11b.

transcription factor Bcl11b. This study thus identifies the ear-

Therefore, in the DN1 stage, two important events takeplace: namely i ) termination of B cell potential, ii ) expression

liest checkpoint during T cell development and shows that it
is Bcl11b-dependent ( Ikawa et al, Science, 2010 ) .

of Bcl11b. The step for the termination of B cell potential can

The culture system established in the above study

alternatively be described as the “T cell lineage specifica-

allows control of the T-lineage determination step and will betion” step. To recapitulate this process, we used EBF1-defi-a useful method for further study on the mechanisms ofcient progenitors that represent multipotent progenitors
retaining T, B and myeloid potential ( Figure A ) . These multi-

T cell lineage determination.

potent progenitors were transferred to stromal cells that

Dissection of the earliest stage of intrathymic
T cell development

highly express Notch ligand DLL4. We found that the trans-

We have recently developed another arrest-restart operation

ferred cells subsequently differentiate towards the T cell lineage in a very synchronous manner, generating CD25 + DN2

culture system in which we can reproduce the earliest intra-

cells in four days. Therefore, the first two to three days ofthymic T cell developmental process. It is known that in theculture appear to represent the DN1 stage. We decided toperform a very detailed time course analysis ( 0h, 1h, 2h,

fetus the thymus seeding progenitors are mostly specified to
the T cell lineage but still retain residual B cell potential( Kawamoto et al. Immunol. Rev., 2010 ) . Such B cell potential

4h…..and so on ) and the cells will be included in the FAN-

is immediately terminated just after the progenitors encoun-

Center to analyze genome-wide gene expression profiles.

ter the thymic environment where Notch ligand is abundantly

We hope to dissect the precise features of the DN1 stage byexpressed, whereas myeloid potential is retained for a while.

using this approach.

TOM5 project conducted by the RIKEN Omics Science

4. Kakugawa K., T. Yasuda, I. Miura, A. Kobayashi, H.
Fukiage, R. Satoh, M. Matsuda, H. Koseki, S. Wakana,H. Kawamoto, H. Yoshida. A novel gene essential
for the development of single positive thymocytes.
Mol. Cell. Biol. 29:5128-5135, ( 2009) .

67

Laboratory forTranscriptional Regulation

Group Director :Ichiro Taniuchi
Research Scientists : Taku Naito

Mari Tenno
Hirokazu Tanaka (SPDR)
Wooseok Seo (JSPS)O

Technical Staff :Sawako Muroi
Chizuko Miyamoto
Risa Chihara
Mayu Okoshi
Mika Ikegaya

Student Trainees :Sebastian Nieke (IPA)
Hisashi Wada

ne of the major questions in developmental biology issary to induce cytotoxic fate in MHC class I-restricted cells.

how the fate of progenitor cells differentiating into

We have also shown that the Runx/Cbfβ transcription factor

opposing lineages is determined. Even as we learn more

complexes are essential to activate two distinct silencers

about cell fate determination, other questions arise, namely

embedded on either the ThPOK or the Cd4 locus. We are

how genetic programming after lineage specification func-

expanding our findings to understand how the activity oftions to establish cell identity and then how cell identity, once

these silencers is regulated at the molecular level and how

established, is maintained in differentiated cells. Research in
my laboratory is directed toward understanding ( a ) how pre-ment.

Runx complexes are involved in immune system develop-

cursor cells sense external or intrinsic stimuli and turn on a
genetic program for regulating fate decision and (b) how a

Mechanism regulating ThPOK gene expression

lineage-specific gene expression pattern is established dur-

Both CD4/CD8 co-receptor expression and the specificity of

ing a commitment process to become fully differentiated

TCRs to MHC molecules correlate well with the outcome ofcells. We are addressing these questions by studying lym-

lineage decision by CD4 + CD8 + DP thymocytes. Cells

phocyte development. In particular, we have been studying
the transcriptional regulation of lineage choice by CD4 + CD8 +double-positive ( DP ) thymocytes differentiating into either

expressing MHC class I-restricted TCR differentiate into thecytotoxic-lineage and terminate Cd4 gene expression,

CD4 helper- or CD8 cytotoxic-lineage T cells. Expression

whereas cells expressing class II-restricted TCR give rise to
the CD4 + helper-lineage and loose Cd8 gene expression.

of a functional ThPOK transcription factor is essential for

However, it remains unclear how differences in TCR signals

development of helper-lineage cells. Our previous study has
identified a transcriptional silencer ( the ThPOK silencer) inare sensed and integrated into developmental programming

the ThPOK locus, and has shown that the ThPOK silencer ishelper lineage-specific expression of the ThPOK gene isessential to restrict ThPOK gene expression in cells express-

regulated by the activity of the ThPOK silencer, we suppose

ing MHC class II-restricted TCR, thereby making it neces-that the ThPOK silencer would serve as nuclear sensor and++in the cell nucleus of post-selection thymocytes. Given that

Recent publications
1. Setoguchi R., Tachibana M., Naoe Y., Muroi
S., Akiyama K., Tezuka C., Okuda T., Taniuchi
I. Repression of the Transcription Factor ThPOK by Runx Complexes in Cytotoxic T Cell
Development. Science 319, 816-19 (2008)

68

2. Muroi S., Naoe Y., Miyamoto C., Akiyama K., Ikawa
T., Masuda K., Kawamoto H., Taniuchi I. Cascading
suppression of transcriptional silencers by ThPOK seals
helper T cell fate. Nat. Immunol. 9, 1113-21 ( 2008)

3. Sakaguchi S., Hombauer M., Bilic I., Naoe Y.,
Schebesta A., Taniuchi I. Ellmeier W. The zinc finger
protein MAZR is part of the transcription factor
network controlling CD4/CD8 cell fate decision of
DP thymocytes. Nat. Immunol.11, 42-48 ( 2010).

DP thymocytes

MAZR
Bcl11bRunx
thymicenhancer

K27

Me3

ATG TAA

H3

ThPOK
silencer

Ia

Ib

proximalenhancer

II

III

II

III

ClassII TCR signaled thymocytes

MAZR Me3
Bcl11bThPOK
K4
Gata3 Runx H3

Ia

Gata3
Ib

Figure 1: Model for ThPOK gene regulation. Expression of the ThPOK geneduring thymocyte development is regulated by combined activationand inactivation of the two enhancers and the silencer. Binding ofRunx, MAZR and Bcl11b is essential to exert silencer activity, which
includes, at a minimum, deposition of repressive epigenetic marks
such as H3 K27 tri-methylation (H3K27me3). Gata3 functions as an
upstream factor for ThPOK expression by activating enhancers thatremove H3K27me3 and impose active epigenetic marks, H3K4me3.

Figure 2: Roles of Runx1/Cbfβ in regulating
early B cell development. Runx1/
Cbfβ complexes play essential roles in
specification to the B lymphocyte lineage
in part by epigenetically activating
the Ebf1 gene. LMPPs=Lymphoidprimed multipotent progenitorsconverter of TCR signals. Therefore it is crucial to define thehelper-lineage T cells has evolved and imprinted on ourmolecule(s) or mechanism that acts as a switch to reverse

genome.

the ThPOK silencer activity for inducing ThPOK expression
recently we have isolated novel proteins that bind to theRole of Runx complexes in early B lymphocyte
development.

ThPOK silencer and are doing functional characterization

Since the precise roles of Runx complexes in early B lym-

using mouse genetics.

phocyte differentiation remain elusive, we examined mouseupon engagement of class II-restricted TCRs. To this end,

In addition to the reversal of the ThPOK silencer, activa-

strains in which the Runx1, Runx3 or Cbfβ gene is inactivatedtion of positive regulatory elements, such as enhancers andin early B lineage progenitors by an mb1-cre transgene. We

the promoter, is required for efficient induction of ThPOK

found that loss of Runx1, but not Runx3, function causes a

expression. Previously, we have identified a proximalenhancer that is necessary to maintain ThPOK expression atsevere developmental block during early B lymphopoiesis,
resulting in the lack of IgM + B cells and VH to DJ H recombina-

the later developmental stage. Recently we have mapped

tion. Observing reduced expression of core transcription

another enhancer at the 5’ end of the gene, in close proxim-

factors regulating early B cell development, such as E2A,

ity to the silencer. This enhancer, designated as a thymic

Ebf1 and Pax5, in B cell precursors lacking Runx1, we per-enhancer, is necessary for efficient induction of ThPOK

formed ChIP-on chip approaches and detected binding of

expression in freshly selected thymocytes, in part via erasing

Runx complexes to the Ebf1 proximal promoter, where

pre-existing repressive epigenetic marks induced by the

repressive histone marks, H3K27-tri-methylation, are accu-

silencer. Although these enhancers and the silencer are all
T-lineage specific, there is another enhancer ( s ) that allows

mulated in Runx1-deficient B cell progenitors. Interestingly,

low level ThPOK expression in all mature lymphocytes. Thus
appropriate kinetic ThPOK expression, which is key indeficient progenitors restored not only development of B220 +cells that underwent VH to DJ H rearrangement but alsochoosing the correct developmental pathway, is orches-

expression of B-lineage signature genes including the Ebf1

trated by the sophisticated interaction between these T cellspecific regulatory regions ( Figure 1) . Further studies on thegene itself. Thus Runx1/Cbfβ complexes are essential tomolecular control of these regulatory elements as well astion of the Ebf1 gene. Our results place the Runx1/Cbfβ

epigenetic regulation at the ThPOK locus will advance ourcomplex in the right position in the transcription factor network that governs specification to the B-lineage ( Figure 2 ) .

understanding of how TCR signals are converted into fateretroviral transduction of Ebf1, but not Pax5, into Runx1-

initiate B-lineage specification, in part via epigenetic activa-

determination programs and how the system that generates

4. Seo W., Ikawa, T., Kawamoto H., Taniuchi I.
Runx1-Cbfβ facilitates early B lymphocyte
development by regulating expression of Ebf1
gene. J. Exp. Med. 209, 1255-62 ( 2012) .

5. Taniuchi I and Ellmeier W. Transcriptional andEpigenetic Regulation of CD4/CD8 Lineage
Choice. Adv. Immunol. 110, 71-110 ( 2011)

69

Laboratory for

Cell Signaling
Group Director :Takashi Saito
Tadashi Yokosuka

Senior Scientist :Research Scientists : Takayuki Imanishi, Reiko Onishi, Yasuo Shikamoto, 			
Arata Takeuchi, Akiko Hashimoto-Tane, Shin-ichi Tsukumo		Visiting Scientists :Hiroshi Ike, Ikuo Ishige, Yasutaka Wakabayashi, Sho Yamasaki

Technical Staff :				

Wakana Kobayashi, Machie Sakuma, Masako Takamatsu,
Akiko Takumi, Midori Unno

Student Trainees :Mohamed El Sherif Gadelhaq Gadlehaq Badr (IPA), 			
Minoru Sawaguchi (JRA)Central Facility FACS Laboratory :Hanae Fujimoto, Yukiko Hachiman

Central Facility Confocal Laboratory :		Yasutaka Wakabayashi (Leica)				
Ikuo Ishige (BM Equipment)

Central Facility Monoclonal Laboratory : Tomomi Aoyama, Kazuyo Uchida (Program for Drug 					Tdiscovery and Medical technology platform)

cells mediate central roles in immune regulation and are

the single-cell level by using a combination of imaging tech-

responsible for immune defense against pathogens andcancer. Because of their critical function in immune regula-niques with model planar bilayers and total internal reflection
fluorescence microscopy ( TIRF ) . We have identified TCR-

tion, impairment in the T cell activation process and function

microclusters ( MCs ) as the signalsome to assemble signal-

results in immune diseases. The group aims to determine

ing molecules and induce activation signals, which thenthe molecular mechanisms of activation, differentiation andtranslocate into the center of the IS and form the cSMAC.

homeostasis of T cells in order to modulate T cell activation/

We then analyzed the dynamic features of co-stimulation

function in immune disorders from the signal transduction

signals by the positive co-stimulatory receptor CD28 and its

perspective. Particularly in studies of T cell activation, thenegative counterpart CTLA-4 and their relationship withgroup has been using real-time imaging analysis to elucidate

TCR-MCs. CD28 is initially co-localized with TCR-MCs andthe dynamic regulation of the signaling complex and signal

then accumulates into the cSMAC together with PKCθ fortransduction of related downstream pathways. These stud-sustained co-stimulation. Later on, CTLA-4 also accumu-

ies also include the roles of co-stimulation signals, innate-

lates in the same area of the cSMAC as CD28, where it inhib-

related signals and cytoskeletal regulation. We also analyze

its activation by competing with CD28 for ligand-binding.

regulation at later phases of T cell activation for cell migra-

Furthermore, we analyzed the dynamic regulation of inhibi-tion, functional differentiation, and establishment of periph-

tory signals by PD-1. Unlike CTLA-4 accumulation in theeral effector functions and tolerance.

cSMAC, PD-1 accumulates in the TCR-MCs and mediates
inhibition of proximal signals within TCR-MCs by recruiting

Dynamic regulation of T cell activation andcostimulation

the phosphatase SHP-2. Thus, we have clarified the spatio-

We have studied the dynamic movement of signaling mole-lation and how these signals exhibit quantitatively fine-tuned

cules in the process of the formation of the immunologicalsynapse ( IS ) and T cell activation upon antigen recognition at

regulation.

temporal dynamic regulation of positive/negative co-stimu-

Recent publications
1. Hashimoto-Tane A., Yokosuka T., Sakata-Sogawa
K., Sakuma M., Ishihara C., Tokunaga M., Saito T.
Dynein-driven transport of T cell receptor microclusters
regulates immune synapse formation and T cell
activation. Immunity 34, 919-931( 2011)70

2. Kong K-F., Yokosuka T., Canonigo-Balancio A
J., Isakov N., Saito T., Altman, A. A motif in theV3 domain of the kinase PKC-θ determines
its localization in the immunological synapse
and functions in T cells via association with
CD28. Nat. Immunol. 12, 1105-12 ( 2011)

3. Yokosuka T., Kobayashi W., Takamatsu M., SakataSogawa K., Zeng H., Hashimoto-Tane A., Yagita H.,
Tokunaga M., Saito T. Spatiotemporal basis of CTLA-4
costimulatory molecule-mediated negative regulationof T cell activation. Immunity 33, 326-339 ( 2010)

Figure 1 : Dynein-driven translocation of TCR-microclusters along microtubules regulates immune synapse formation and T cell
activation. AND-TCR transgenic T cells expressing GFP-tubulin and CD3ζ-Halo were stimulated on a planar bilayer. Real
time imaging analysis revealed that TCR-microclusters move along microtubules and ultimately form the cSMAC. Dynein andthe components of the dynein complex accumulated within TCR microclusters and moved together. Cells in which dynein or
microtubules were disrupted failed to generate the cSMAC and this resulted in enhancement of T cell activation due to the failure
to degrade the TCR-microclusters, leading to sustained activation signals such as phosphorylation in the microclusters.

Figure 2 : Model for dynein-mediated translocation of TCR
microclusters and T cell activation regulation. TCRmicroclusters are generated at the periphery upon antigen
recognition by the TCR. They move towards the center ofthe engaged region, initially by actin-retrograde flow, andthis is followed by translocation along microtubules in a
dynein-dependent fashion. This pathway is mediated by
the association between the TCR complex and the dynein
complex upon antigen stimulation. Strong but not weakstimulation induced the translocation of microclusters,
thus suggesting that dynein-driven translocation of
TCR-microclusters regulates T cell activation.

Cytoskeletal regulation of T cell activation

Regulation of T cell responses by innate signals

TCR-MCs generated at the periphery of the IS move to theWe have analyzed the interconnected activity of signaling

center to form the cSMAC. This translocation of TCR-MCs

molecules of the innate and acquired immune systems. After

thus regulates TCR activation by reducing the number of

we identified the critical roles of IRAK4 in T cell activation, wefunctional TCR-MCs through their degradation at the cSMAC.

analyzed the function of TLRs and RLRs expressed in T

We analyzed the mechanism of TCR-MC translocation andcells. Initial analysis revealed that TLR2/3/9 mediate co-found that the minus-end microtubule-associated motor

stimulatory functions whereas TLR2 directly activates Th1

protein dynein and its complex are co-localized with TCR-

cells to induce cytokine production independently of TCR

MCs and co-precipitate with the TCR complex. We havestimulation. Whereas T cell co-stimulation through TLR2 isshown that TCR-MCs are translocated along microtubules

MyD88-dependent, surprisingly, the co-stimulation by theto form the cSMAC. Depletion of the dynein complex or dis-ligand of TLR3/9 was found to be independent of MyD88/

ruption of microtubule network formation resulted in

TRIF. We found that for the optimal T cell co-stimulatory

enhanced T cell signals and activation. These data indicate

activity, nucleic acids have to be aggregated and this appears

that signal strength regulates the assembly of the TCR-

to be mediated by an as yet unknown novel T cell-specificdynein complex and its translocation to form the cSMAC.

sensor of nucleic acids, whose identity is currently underinvestigation.

4. Hashimoto-Tane A., Yokosuka T., Ishihara C., Sakuma
M., Kobayashi W. and Saito T. TCR-microclusters criticalfor T-cell activation are formed independently of lipid
raft clustering. Mol. Cel. Biol. 30, 3421-3429 ( 2010)

5. Takeuchi A., Itoh Y., Takumi A., Ishihara C., Arase N.,
Yokosuka T., Koseki H., Yamasaki S., Takai Y., Miyoshi J.,
Ogasawara K., and Saito T. CRTAM confers late-stage
activation of CD8+ T cells to regulate retention within
lymph node. J. Immunol. 183, 4220-4228 ( 2009)71

Research Unit forSingle Molecule
Imaging

Unit Leader :Kumiko Sakata-SogawaThe goal of our laboratory is the development of new

Visualization of cell signaling

microscopy systems for elucidation of immunological

Cells are activated by stimuli that usually come from the out-

responses. We have been focusing on the study of signalingside of the cells. The stimulation detected at the cell mem-processes in immune cells using the technique of single mol-brane is transmitted by different signaling proteins to theecule imaging and quantification. Single molecule approaches

nucleus, where the transcription of target genes are initiatedenable us to capture transient intermediates and heteroge-or terminated depending on the response of the cell to theneous behavior, thus avoiding ensemble averaging. This

stimulation. We are applying our single molecule analysis

capability is powerful for elucidating mechanisms of cellularmethod to the understanding of complex signaling pathways

functions: which molecule interacts with what, when, where,by visualization and quantification of the interaction of signal-and how it works in living cells. Thus fluorescence imaging

ing proteins. For quantitative analysis of these interactions, itand quantitative analysis of single molecules are valuable

is important to observe multiple molecules simultaneously. It

methods to study the individual behavior of biological sys-is also necessary to prepare many different cell lines harbor-tems. For this purpose we developed a novel type of fluorescence microscopy ( HILO ) for use in single cell/single

ing target genes for high throughput data collection. To thismolecule studies. One of the main technical challenges inend, we have established methods to obtain isogenic celllines expressing two fluorescence tagged ( GFP- or RFP )single molecule microscopy is to obtain a high signal to noise

fusion proteins at a homogenous and low level. In addition toratio in order to capture the subtle fluorescent signal of single

this method, we utilize fluorescence-labeled antibodies spe-molecules. HILO has an advantage for observations insidecific for physiological protein modifications such as phos-

cells with the high signal to noise ratio of 7.6. Our single mol-phorylation, methylation or acetylation. Together withecule microscope system has been optimized to allow
observation of immunological responses from the cell mem-quantum dot technology and fluorescence-dye labeledprotein by the “double-click” technique ( Figure ) , we realize

brane to the nucleus by introducing a computer system for

multi-color

control of optical devices.

analysis.

singlemolecule

imagingandquantitative

Recent publications
1. Hashimoto-Tane A. ,Yokosuka T., Sakata-Sogawa
K., Sakuma M., Ishihara C., Tokunaga M., Saito T.:
Dynein-driven transport of T cell receptor microclusters
regulates immune synapse formation and T cell
activation.Immunity 34, 919-931 ( 2011)72

2. Yokosuka T., Kobayashi W., Takamatsu M., SakataSogawa K., Zeng H., Hashimoto-Tane A., Yagita
H., Tokunaga M., Saito T.: Spatiotemporal basisof CTLA-4-mediated negative regulation of T-cell
activation. Immunity 33, 326-339 ( 2010 )3. Miletic AV., Graham DB. Sakata-Sogawa K.,
Hiroshima M., Hamann HJ., Cemerski S., KloeppelT., Billadearu DD., Kanagawa O., Tokunaga M. andSwat, W: Vav Links the T Cell Antigen Receptor
to the Actin Cytoskelton and T Cell Activation
Independently of Intrinsic Guanine Nucleotide
Exchange Activity. PLoS One 4, e6599 ( 2009 )Figure:Introduction of Cy3-labeled calmodulin in LAT-GFP expressing Jurkat cells.
Calmodulin was labeled with a single molecule of Cy3 in vitro and introduced into LAT-GFP Jurkat
cells by electroporation. Co-localization of LAT and calmodulin is indicated by yellow fluorescence.
(Collaboration with Dr. Nobuhiro Hayashi at the Tokyo Institute of Technology) Bar: 10 µm.

Spatio-temporal regulation of NF-kB inactivation
by PDLIM2

regulation of NF-κB activation ( Tanaka T et al. Nat. Immunol.,8, 584-591 2007) .

NF-κB is an important transcription factor that activates

Aiming to elucidate PDLIM2 regulatory mechanisms, we

expression of inflammatory genes in response to stimulation.
In resting cells, NF-κB binds IκBα, which keeps it in an inac-

established dual gene expressing cell lines of GFP- and
RFP-fusion proteins of PDLIM2 and NF-κB ( p65 subunit ) .

tive state sequestered in the cytoplasm. Stimulation leads toUsing single molecule fluorescence microscopy, we were

phosphorylation of Ser residues on IκBα and its subsequent

able to visualize the dynamics of PDLIM2 function. In resting

ubiqutination and degradation by the proteasome. As acells, both proteins localized in the cytoplasm, but upon

result of IκBα degradation, liberated NF-κB translocates tothe nucleus and activates expression of target genes includ-stimulation, they translocated to the nucleus and colocalized. A PDLIM2 mutant ( LIM ) that lacks the ubiquitin ligase

ing the IκBα gene itself. The nascent IkBα binds to nuclear

functional domain also translocated to the nucleus but did

NF-κB and exports it back into the cytoplasm, resulting in a

not colocalize with p65 in stimulated cells. These results

negative feedback loop. In addition to this canonical path-demonstrate that the LIM domain is relevant to the control ofway, another protein, PDLIM2, was found by RCAI investiga-

NF-κB localization, suggesting that this domain is responsi-

tors to function as an E3 ubiquitin ligase responsible for theble for regulation of the transcriptional activity of NF-κB.

73

Laboratory for

Lymphocyte Differentiation

Group Director :Tomohiro Kurosaki
Research Scientists : Yuuich Aiba

Kohei KometaniMemory antibody responses are typically seen to

Visiting Scientists :Yoshihiro Baba
Wataru Ise
Takeshi Inoue
Masanori Matsumoto
Rinako Nakagawa

Technical Staff :Yoko Fujii
Shiori Maeda
Miwako Tochigi

Student Trainee :Saya MoriyamaTencounter antigens in vivo, almost all the B cells induce AID

cell-dependent antigens and are characterized by theat the transcriptional level. Thus, we can genetically label

rapid production of high-titers of high-affinity antigen-specificthe antigen-experienced B cells by using AID expression( Figure 1) .

antibody. The previous hallmark of T cell-dependent memory
B cells is their expression of somatically mutated, isotypeswitched B cell antigen receptors, features that are mainly
generated in germinal centers (GCs). Indeed, classical studies have focused on isotype-switched memory B cells

Employing this system, we have isolated two subsets of
IgM type memory B cells ( IgM + IgD + and IgM + IgD- ) and shownthat the IgM + IgD + memory B cells undergo somatic hypermutation, whereas the IgM + IgD- cells do not. These observa-

toring antigen-experienced B cells have revealed the exis-

tions suggest that IgM type memory B cells are generated
through GC-dependent ( IgM + IgD + ) and independent

tence

cells,( IgM + IgD- ) pathways. When the IgM + IgD + or IgM + IgD- mem-demonstrating the considerable heterogeneity of memory B

ory B cells are adoptively transferred together with activatedcells. As a first step to understand how such cellular andT cells, both types of memory B cells re-initiate the GC reac-possibly functional heterogeneity is generated and integrated
during humoral memory responses, our laboratory has now

tion. Given that IgG type memory B cells have the propensity
to differentiate into plasma cells ( described below ) , these

focused on clarifying the mechanisms underlying the robust-

results suggest that IgM type memory B cells have charac-

ness of memory antibody responses.

teristics that are distinct from IgG type memory B cells.

Existence of two types of IgM memory B cells
Fate mapping-methods have allowed us to monitor both unswitched ( IgM-type ) and switched ( IgG-type ) memory

Contribution of transcription factors to efficientdifferentiation of IgG type memory B cells into
plasma cells

B cells after injection of antigen into mice. When B cells

Although robust antibody responses of IgG type memory B(mainly the IgG isotype). However, recent advances in moniof

un-switched

IgM

type

memoryB

Recent publications
1. Yasuda T., Kometani, K., Takahashi, N., Imai,
Y., Aiba, Y. and Kurosaki, T. Erk kinases control
plasma cell differentiation by regulating expressionof Blimp-1. Sci. Signal. 4, ra25 ( 2011)74

2. Matsumoto, M., Fujii, Y., Baba, A., Hikida, M., Kurosaki,
T. and Baba, Y. The calcium sensors STIM1 and
STIM2 control B cell regulatory function throughIL-10 production. Immunity 34, 703-714 ( 2011)

3. Limnander, A., Depeille, P., Freedman, T.S., Liou, J.,
Leitges, M., Kurosaki, T., Roose, J.P. and Weiss, A.
Stim1, PKCδ and RasGRP proteins set a threshold for
pro-apoptotic Erk signaling during B cell development.
Nat. Immunol. 12, 425-433. ( 2011)

Figure 1: Fate mapping methods for
antigen-experienced B cells
In AID-Cre Tg mice, the Cre geneis controlled by the promoter of the
AID gene. This Cre gene is expressedin antigen-experienced B cells, since
Aid is expressed in activated B cells
after the exposure to the antigens. By
crossing AID-Cre Tg mice with ROSAloxP-RFP mice, antigen-experienced
GC B cells and memory B cells but notnaive B cells were labeled with RFP.

Figure 2: Preferential differentiation to plasma
cells by IgG-type memory B cells
IgG-type memory B cells express less Pax5 andBach2 genes compared to IgM naive B cells.
Pax5 and Bach2 suppress Blimp1 expression,
which is the essential transcription factor forplasma cell differentiation. This led to the modelthat IgG-type memory B cells can differentiate
to plasma cells preferentially and quickly by
reducing Pax5 and Bach2 expression.

cells have been well appreciated, the cellular and molecularspecific IgG1 type cloned mice, allowing us to obtain anti-

basis underlying this phenomenon has been unclear. It has

gen-non-experienced IgG1 B cells. By using these B cells,

long been suspected that inherent differences in the struc-we have demonstrated that antigen-experienced, but not
non-experienced, IgG1+ B cells rapidly differentiated into

ture of membrane IgM and IgD on naive B cells versus membrane IgG on memory B cells account for the robust
secondary antibody responses ( BCR-intrinsic model ) .

plasma cells. Furthermore, the heightened differentiation

Both membrane IgM and IgD have short, three amino

was associated with low amounts of the transcription factor

acid cytoplasmic tails that seem not to play a direct role in

Bach2, since enforced expression of Bach2 in IgG1 memory

BCR signaling. By contrast, all membrane IgG subclasses

B cells reduced this enhanced differentiation capability.

have unique cytoplasmic domain structures of 28 residues

Based on these results, we favor a model in which reorgani-that are highly conserved between species. Thus, in addition

zation of transcription factors takes place during generation

to BCR signaling executed by the common Igα/Igβ heterodi-of IgG1 type memory B cells after primary antigen exposure,mers, the IgG type BCRs could exert additional BCR signaling functions via the cytoplasmic domains of membrane IgG,

and this altered transcription factor profile is critical for rapid
responsiveness of memory B cells ( BCR-extrinsic model )thereby contributing to the robust secondary responses.

( Figure 2 ) .

capability of antigen-experienced IgG type memory B cells

To directly test this model, we have established NP-

4. Kometani, K., Yamada, T., Sasaki, Y., Yokosuka,T., Saito, T., Rajewsky, K., Ishiai, M., Hikida, M.
and Kurosaki, T. CIN85 drives B cell responses
by linking BCR signals to the canonical NF-κB
pathway. J. Exp. Med. 208, 1447-1457 ( 2011)

5. Kitano, M., Moriyama, S., Ando, Y., Hikida,M., Mori, Y., Kurosaki, T, and Okada, T. Bcl6
Protein Expression Shapes Pre-Germinal Center
B Cell Dynamics and Follicular Helper T Cell
Heterogeneity. Immunity 34, 961-72 ( 2011)75

Research Unit for

Immunodynamics

Unit Leader :Takaharu Okada
Research Scientists : Masahiro Kitano (SPDR )Yoshikazu Ando
Technical Staff :T

Noriko Takahashi

he goal of our research is to understand the mecha-( GC ) reactions. A subpopulation of Tfh cells that has physi-

nisms regulating cell migration and interactions in thecal access to the GC is believed to be responsible for con-tissues that shape immune responses. For this purpose wetrolling GC reactions. However, little has been known about

use real time imaging, in particular two-photon microscopy,

mechanisms that regulate the dynamics of GC Tfh cells. We

to analyze in vivo cellular migration and interactions. This

have performed two-photon imaging analysis of antigen-microscopy method, which was introduced recently to thespecific Tfh cells within GC-containing B cell follicles. By

field of immunology, has been revealing striking dynamics ofsimultaneously visualizing GC B cell clusters, we have been

immune cells in the lymphoid organs, underlining the importance of this approach to resolve the complexity of theable to analyze the migration of Tfh cells into the GC area
and the surrounding follicular mantle ( FM ) . The analysis

immune system. Last year, we were focusing on clarifying

clearly showed that Tfh cells in the GC and those in the FM

dynamics of antigen-engaged B cells during the formation ofgerminal centers ( GCs ) , a process that ultimately leads totended to be retained in the respective regions, suggesting

long-term, high affinity antibody production. This year, we

localization. Our previous imaging analysis of Tfh cells har-have started new projects to understand the mechanisms

boring hypomorphic mutations in the gene encoding Bcl6,

controlling the dynamics of two different T cell types, follicular helper T ( Tfh ) cells and cytotoxic T cells, which play piv-

which is an essential transcription factor for Tfh cells, sug-otal roles in humoral and cellular immunity, respectively.

impaired than that of FM Tfh cells by the partial deficiency of
Bcl6 ( Figure ) . This observation led us to perform gene

Dynamics of GC Tfh cells.

expression analysis of Tfh cells with various surface pheno-

Tfh cells are essential for initiation and maintenance oftypes and Bcl6 genotypes to search for genes differentiallyT-dependent B cell responses, particularly germinal center

expressed between GC Tfh cells and FM Tfh cells. We havedifferential expression of molecules responsible for theirgested that formation of GC Tfh cells was more severely

Recent publications
1. Baumjohann D, Okada T, Ansel KM. Cutting
Edge: Distinct waves of BCL6 expression
during T follicular helper cell development.
J. Immunol. 187, 2089-2092 ( 2011)76

2. Kitano M, Moriyama S, Ando Y, Hikida M, Mori Y,
Kurosaki T, Okada T. Bcl6 protein expression shapes
pre-germinal center B cell dynamics and follicular helper
T cell heterogeneity. Immunity 34, 961-972 ( 2011)

3. Tanizaki H, Egawa G, Inaba K, Honda T, Nakajima
S, Moniaga CS, Otsuka A, Ishizaki T, Tomura M,
Watanabe T, Miyachi Y, Narumiya S, Okada T,
Kabashima K. Rho-mDia1 pathway is required
for adhesion, migration, and T-cell stimulation in
dedritic cells. Blood 116, 5875-5884 ( 2010)

Figure:Bcl6 expression and function in GC Tfh cells.
The left image shows a lymph node section stained for antigen-specific helper T cells (adoptively transferred, T cell
receptor transgenic CD4+ T cells), IgD, and Bcl6. The GC region is demarcated by the cluster of Bcl6-expressing
GC B cells, which are surrounded by IgD positive B cells mostly in the FM. Many of antigen-specific helper T
cells in the GC also express Bcl6. The image on the right shows a 105 μm z-projection image of a GC-containing
follicle in an intact lymph node. This two-photon image is a part of a time-lapse imaging data set that shows
impaired GC accessibility of Tfh cells harboring hypomorphic mutations in the Bcl6 gene (Bcl6yfp/yfp) comparedto Bcl6+/+ Tfh cells. The GC and FM regions are demarcated based on the density of polyclonal B cells.

identified several candidate genes responsible for localiza-

DCs, which consist of numerous subsets with distinct phys-tion of these Tfh cell subsets. Preliminary results using gene-iological roles. Because of these limitations, there remains

targeted animals have begun to show a role for one of thesemuch to be learned about interaction dynamics between

candidate molecules in the localization of GC Tfh cells.

cytotoxic T cells and native DC subsets in response to vari-

Cellular interaction dynamics during cytotoxic T
cell differentiation.

ous forms of antigen. In collaboration with Dr. Kaisho’sgroup ( Lab for Host Defense, RIKEN RCAI; and Immunology
Frontier Research Center, Osaka University ) , we have devel-

Cytotoxic T cells are known to be generated through interac-

oped novel methods that allow visualization by two-photon

tions of antigen-specific CD8 + T cells with dendritic cells( DCs ) . Interaction dynamics between CD8 + T cells and DCs

microscopy of specific DC subsets. These methods will pro-have been visualized by two-photon microscopy. However,subsets in generation and maintenance of cytotoxic T cells

the previous imaging studies utilized either in vitro differenti-

with effector and/or memory function.

vide a basis to study spatiotemporal roles for different DC

ated DCs or reagents that would visualize all conventional

4. Okada T. Two-photon microscopy analysisof leukocyte trafficking and motility. Semin.
Immunopathol. 32 : 215-225 ( 2010)

5. Katagiri K., Katakai T., Ebisuno Y., Ueda Y.,
Okada T., Kinashi T.. Mst1 controls lymphocyte
trafficking and interstitial motility within lymph
nodes. EMBO J. 28, 1319-1331 ( 2009)77

Research Unit for

Molecular Systems Immunology

Unit Leader :Makio Tokunaga
Student Trainees : Yuma Ito

Jun Takimoto
Katsuo Ichinomiya
Masahiro Shimozawa
Ryuta Okada
Naomichi Inaba
Satoshi Ikeda
Hiroshi OyamaOur laboratory has developed technologies that allow

for single-molecule imaging at cell surfaces, but cannot be

immune responses and signaling processes to be visu-

used for molecular imaging inside cells.

alized at the single-molecule level. Single molecule imaging

To overcome this limitation, we have devised a newcoupled with the ability to simultaneously visualize several
different proteins in cells has enabled the quantification of

approach, called highly inclined and laminated optical sheet( HILO) microscopy for single molecule imaging inside cells

molecular dynamics, interactions, and kinetics. Based on( Fig. 1a ) . We have achieved notable success in increasingthese three-dimensional and temporal parameters, we

the signal/background ratio by inclining the illumination

examine numerical modeling and computer simulations ofbeam and by minimizing the illumination area. The incident

cell functions. Using the combination of single molecule

laser beam is highly inclined by a large refraction, and isquantification and “in silico” modeling, we aim to open up

laminated as a thin optical sheet at the specimen side.

new frontiers for understanding immune cells as molecular

In HILO microscopy, this thin optical sheet is used forsystems.

illumination.

Single Molecule Imaging and Molecular
Quantification in Cells

ized single molecules of the Sp1 transcriptional factor Sp1 ina living cell ( Fig. 1b) . Clear point-like images of single Sp1

We have demonstrated that clear visualization of single mol-molecules were obtained without the need for deconvolution

ecules in cells enables their molecular quantification. Clear

to remove out-of-focus haze. The fluorescence intensity ofsingle-molecule visualization was achieved using TIRF and

the Sp1 point images corresponded well to the theory of

HILO microscopy. The main technical challenge of single-

image formation. Further, there was much less photobleach-molecule fluorescence imaging is increasing the signal/

ing than in conventional confocal microscopy because of the

background ratio. We have been involved in the development of total internal reflection fluorescence ( TIRF ) micros-

lower intensity and non-focused nature of the illumination.

copy, a light-microscopic technique. TIRF is now widely used

clear images. The background intensity of images depends

To evaluate the HILO microscopy technique, we visual-

Reduction of the background intensity of images yields

Recent publications
1. Hashimoto-Tane A. ,Yokosuka T., Sakata-Sogawa
K., Sakuma M., Ishihara C., Tokunaga M., Saito T.:
Dynein-driven transport of T cell receptor microclusters
regulates immune synapse formation and T cell
activation.Immunity 34, 919-931 ( 2011)78

2. Shiina N., Tokunaga M.: RNA Granule Protein
140 ( RNG140): A Paralog of RNG105
Localized to Distinct RNA Granules in Neuronal
Dendrites in the Adult Vertebrate Brain. J.
Biol. Chem. 285, 24260-24269 ( 2010 ) .

3. Yokosuka T., Kobayashi W., Takamatsu
M., Sakata-Sogawa K., Zeng H., Yagita H.,
Tokunaga M., Saito T.: Spatiotemporal basis of
CTLA-4-mediated negative regulation of T-cell
activation. Immunity 33, 326-339 ( 2010 ) .

a
a

b

b

Figure 1: Molecular imaging enables one to visualize and quantify molecular
dynamics, interactions, and kinetics in cells for molecularsystems biology. (a) HILO microscopy for molecular imaging in cells.
Illumination by a highly-inclined and thin beam increases image intensityand decreases background intensity, yielding a signal/background ratio up
to about eightfold greater than that of epi-illumination. A high ratio yielded
clear single-molecule images and three-dimensional images. (b) To evaluate
the HILO microscopy technique, we visualized single molecules of the Sp1transcription factor in a living cell. Molecular dynamics and interactions can bequantified by image analysis using these single molecule images. Bar, 5 µm.

Figure 2: The combination of single molecule quantification and“in silico” modeling opens new approaches for developing
molecular systems biology. (a) Automatic tracking of
single molecules of the Sp1 transcription factor visualized
in a living cell. Molecular dynamics and interactions canbe quantified by image analysis using these single molecule
images. Bar, 5 µm. (b) Aiming at understanding immune
cells as molecular systems, we plan to construct “in silico” cell
models based on single-molecule quantification. Bidirectional
research is essential to reconstruct cell functions in silico;
research from molecules to systems by single molecule
analysis, and feedback research from systems to molecules.

on the volume of the illuminated region in specimens. To

cargo through nuclear pores in cells as bright spots on theexamine the thickness of the illuminated region, we obtained

nuclear envelope. Molecular interactions with the assembled

intensity profiles of illumination along the z-direction, that is,

NPC were quantified by single molecule analysis. Retention

the depth direction in specimens. We devised a method totimes, the number of associated molecules, the dissociation

decrease the illumination thickness by narrowing the illumi-

constant, and stoichiometry of import were all determined.

nation area using a field stop. Notably, we have achieved the
reduction of the illumination thickness to less than 10 µm ( for“In silico” Modeling and Simulation

example, 7 µm of thickness with a field stop for 20µm-diameter illumination ) .

As shown above, molecular interactions with the assembled

Further, we evaluated the signal/background ratio ofunderstand the molecular mechanism of nuclear import, aimages in HILO. Inclination of the illumination beam increases

numerical model of import was constructed using these

intensities of the fluorescence images up to 2.8-fold com-

kinetic parameters. Computer simulation was carried out

pared with epi-illumination. The 2.8-fold increase is in excel-

based on the model with two types of binding sites. The

lent agreement with the theory. By contrast, the backgroundsimulation fit very well with both the results of single-mole-intensity is substantially decreased by illumination inclina-

cule experiments and the molecular kinetic features in cells.

NPC were quantified by single molecule analysis. In order totion. As the background is composed of out-of-focus images,

We are now expanding the simulation studies into

the decrease is explained by the reduction of the illuminatedwhole-cell simulation of single lymphocytes based on single
molecule imaging and quantification ( Fig. 2 ) . Direct com-

range. As a result, illumination inclination increased the ratioof image to background ( signal/background ) up toparison with molecular imaging is indispensable for the

3.1-3.5-fold. Reduction of the illumination diameter further

simulation, since the values of the parameters have a hugedecreased the background intensity. Consequently, reduc-number of degrees of freedom.

tion of the diameter increased the ratio of signal to back-We demonstrated that clear visualization of single mol-ground up to 2.2-2.9-fold. Overall, the HILO illumination

ecules in cells enabled accurate quantification. The combi-

microscopy approach notably increased the signal/back-nation of single molecule quantification and “in silico”

ground ratio up to 7.6-fold.

reconstructions of cell functions opens new approaches for

To explore potential new uses of this technology, we
performed quantitative analysis of nuclear import to demon-

developing molecular system biology in immunology andother fields.

strate its application to kinetic studies. We could visualize
single molecules of GFP-importin β mediating the import of

4. Fukagawa, A., Hiroshima, M., Sakane, I., Tokunaga
M.: Stochastic emergence of multiple intermediates
detected by single-molecule quasi-static mechanicalunfolding of protein. BIOPHYSICS, 5, 25-35 (2009 ).

5. Tokunaga, M., Imamoto, N., Sakata-Sogawa, K.: Highly
inclined thin illumination enables clear single-molecule
imaging in cells. Nat. Methods, 5, 159 -161 ( 2008 ) .

79

Laboratory for

Epithelial
Immunobiology
Team Leader :Hiroshi Ohno
Research Scientists : Shinji Fukuda, Gaku Nakato, Takashi Kanaya (SPDR)
Research Associate : Daisuke Takahashi

T

Technical Staff :Yumi Chiba, Yumiko Fujimura, Kumiko Nakai,
Masumi Ohmae, Sayuri Sakakibara,
Chikako Uetake, Ayako Yamashita

Student Trainees :Toshi Jinnohara (JRA), Kazunori Kadokura,
Tamotsu Kato (JRA), Hideaki Shima (JSPS),
Misato Hanazato (JRA ), Akemi Fujiwara, Keiko Kato,
Yuuki Kitahara, Yuuki Obata, Takao Sato,
Ai Takahashi, Yoshiko Usami, Shunji Yamada

he mucosal epithelium that lines the inner surfaces ofUnderstanding of M-cell differentiation steps

the body, especially within the intestine, is exposed to a

M cells are atypical epithelial cells specialized for uptake ofwide range of antigens, including food-derived macromole-luminal antigens, including the phagocytosis of viruses andcules and microorganisms as well as numerous commensal

bacteria, to deliver them to dendritic cells that accumulate

bacteria, collectively called the intestinal microbiota. Appro-

beneath the FAE. More than three decades after their initialpriate recognition of these antigens is vital for maintaining

description, however, the molecular mechanisms of M-cell

immune homeostasis. Epithelial cells that overlay the gutassociated lymphoid tissue ( GALT ) , such as Peyer’s patchesdifferentiation are not well understood. Recently, Dr. Ifor I.

( PPs ) and isolated lymphoid follicles, are distinct fromstromal cells beneath the FAE is essential for the induction ofabsorptive epithelial cells of the villi and are termed follicleassociated epithelium ( FAE ) . The FAE contains a specialized

M cells. They have further shown that systemically adminis-

subset of epithelial cells, the M cells, which are thought toM-cell differentiation in the small intestinal villi. In collabora-

play a pivotal role in immune surveillance by delivering lumi-tion with Dr. Williams, we set up an in vivo M-cell differentia-

nal microorganisms to the underlying lymphoid cells via

tion assay to trace the expression kinetics of several M-celltranscytosis. One of the primary aims of our laboratory is tomarkers we have identified during RANKL-induced ectopic

understand the mechanisms that underlie the differentiation

M-cell differentiation. We found that Marcksl1 and Annexin

and function of FAE and M cells. Our research team is also

V were expressed as early as 1 day after RANKL injection,

investigating the interaction of commensal microbiota with

whereas CCL9 appeared on day 2, and that GP2 expression

the intestinal epithelium and its influence on mucosal andwas not detected until day 3. This same sequence of expres-systemic immunity. These studies may lead to the develop-

sion of the M-cell markers was seen not only in this relatively

ment of new and more efficient mucosal vaccination proto-

artificial model of RANKL-induced ectopic M-cell differentia-

cols/drug delivery systems as well as functional foods/

tion, but also in PP M cells during normal M-cell differentia-

preventive medicine based on host-microbiota interactions.

tion in mouse ontogeny; Marcksl1 was already observed on

Williams’ group identified that RANKL expressed by thetered exogenous recombinant RANKL induces ectopic

Recent publications
1. Fukuda S., Toh H., Hase K., Oshima K., NakanishiY., Yoshimura K., Tobe T., Clarke J. M., Topping D.
L., Suzuki T., Taylor T. D., Itoh K., Kikuchi J., Morita
H., Hattori M., Ohno H. Bifidobacteria can protectfrom enteropathogenic infection through productionof acetate. Nature 469, 543-547 ( 2011)

80

2. Takahashi D., Hase K., Kimura S., Nakatsu F.,
Ohmae M., Mandai Y., Sato T., Date Y., Ebisawa M.,
Kato T., Obata Y., Fukuda S., Kawamura Y., Dohi
T., Katsuno T., Tokosuka O., Waguri S., Ohno H.
The epithelia-specific membrane trafficking factor
AP-1B controls gut immune homeostasis in mice.
Gastroenterology 141, 621- 632 ( 2011)

3. Prakash T., Oshima K., Morita H., Fukuda S., Imaoka
A., Kumar N., Sharma V.K., Kim S.W., Takahashi
M., Saitou N., Taylor T.D., Ohno H., Umesaki,Y., Hattori, M. Complete genome sequences of
rat and mouse segmented filamentous bacteria,
a potent inducer of th17 cell differentiation.
Cell Host Microbe, 10, 273-284 ( 2011)

Stage 1 M cellStage 2 M cell
FAE

VillusStage 3
GP2

Stage 3 M cell
(Mature M cell)Stage 2
CCL9, Sgne-1, M-Sec
Stage 1
Marcksl-1, Annexin V

RANKL
Crypt

Transit amplifying cell
Stem cell

Figure :Schematic diagram of M-cell differentiation
Transit-amplifying cells, the immatureintestinal epithelial cells in the crypt, candifferentiate into the M-cell lineage following
stimulation with RANKL and begin to
express M-cell markers. Expression of thesemarkers occurs in a temporally distinct
pattern; Marcksl1 and Annexin V appear
within 1 day after RANKL treatment, CCL9,
Sgne-1 and M-Sec follow, and GP2 is finallyexpressed by morphologically mature M cells.

embryonic day 18, CCL9 in the day 2 neonate, and GP2 wasertheless, the molecular mechanisms underlying these ben-

finally detected 7 days after birth. These observations indi-

eficial effects have barely been elucidated. To address thiscate that the process of M-cell differentiation can be practi-

important but complex question, we have developed a com-

cally divided into a series of distinct stages defined by theexpression profile of M-cell markers, and that in vivo RANKL

prehensive ‘multi-omics’ approach, where exhaustive analyses, including ( meta ) genomics, ( meta ) transcriptomics andtreatment is a powerful experimental tool for tracing the indi-metabolomics, are combined. We employed a simplified

vidual steps of M-cell differentiation. Taking advantage ofthis strategy, we are now investigating M-cell specific tran-model of lethal infection with enterohaemorrhagic Escherichia coli O157:H7 ( O157) of mice associated with certain

scription factors expressed early during RANKL-induced

bifidobacterial strains to prove that our multi-omics approachM-cell differentiation, since they should regulate and serve
as a key to elucidating the molecular mechanisms of M-cellis useful for analyzing host-microbial interactions. We
showed that genes encoding ATP-binding-cassette ( ABC ) -differentiation.

type carbohydrate transporters present in certain bifidobacteria contribute to protecting mice against death induced by

Elucidation of host-intestinal microbiota
interactions important for host immunity andbiological defense mechanisms

O157. The bacteria that possess these transporters were

Inside our gut dwell a very large number of bacteria. Thisbifidobacteria exerts its action on the colonic epithelium byintestinal microbiota impacts on both human physiology andinducing anti-inflammatory and/or anti-apoptotic effects,

pathology. Certain of the commensal microbiota, such as

which prevent translocation of the O157 Shiga toxin from thethose belonging to the bacterial genus Bifidobacterium, havegut lumen into the blood. We therefore propose that acetate

beneficial effects on our health. Among the most distinctive

produced by protective bifidobacteria improves intestinal

benefits of these bacteria are the modulation of host defense

defense mediated by epithelial cells and thereby protects the

responses and protection against infectious diseases. Nev-

host against lethal infection.

4. Hase K., Kawano K., Nochi T., Pontes G. S., Fukuda
S., Ebisawa M., Kadokura K., Tobe T., Fujimura, Y.,
Kawano S., Nakato G., Kimura S., Murakami T., Iimura
M., Hamura K., Fukuoka S. I., Lowe A. W., Waguri S.,
Itoh K, Kiyono H., Ohno H. Uptake via Glycoprotein
2 of FimH+ bacteria by M cells initiates mucosal
immune response. Nature 462, 226-230 ( 2009)able to produce acetate from fructose. Our data strongly
suggest that the acetate produced in large amounts by these

5. Hase K., Kimura S., Takatsu H., Ohmae M., Kawano S.,
Kitamura H., Ito M., Watarai H., Hazelet C. C., Yeaman
C., Ohno H. M-Sec promotes membrane nanotube
formation by interacting with Ral and the exocyst
complex. Nat. Cell Biol. 11, 1427-1432 ( 2009)81

Laboratory for

Mucosal Immunity

Team Leader:Sidonia Fagarasan
Research Scientists : Tran Huy Thinh						Shimpei Kawamoto
Michio Miyajima
Duncan Sutherland (JSPS)
Research Associate : Mikako Maruya

ATechnical Staff :Yasuko Doi
Yumi Tsutsui

Visiting Scientist :Hongyan Qin (Chinese Fellowship)

the establishment of mutualistic and symbiotic relation-

cells. The germinal center ( GC ) T cells, generally known as T
follicular helper ( TFH ) cells, express high amounts of PD-1.

ships that have contributed to the development of ourPD-1-deficiency leads to species-specific, antibody-medi-

immune system and maintenance of homeostasis. In spite ofated autoimmune diseases. Interestingly, the incidence of

having available a wealth of immune sensing and effector

disease in PD-1-deficient mice varies among mouse colo-mechanisms capable of triggering inflammation in response

nies, depends on AID and is nil in germ-free conditions.

to microbial intrusion, we can live together with our body’s

These observations suggest that autoreactive antibodies inbacteria without any adverse effects. This is made possible

PD-1-deficient mice may arise after AID-induced genetic

by a continuous dialog between bacteria and host cells thatalterations in GCs, which are driven by stimulation from thegenerates finely tuned signaling programs ensuring a state

gut microflora. Indeed, we found that PD-1 regulates the gutof ‘hypo-responsiveness’ against dietary antigens and com-microbiota. The total numbers of ‘healthy’ bacteria such as

mensal bacteria. At the same time and somewhat paradoxically, these signaling programs generate a state of active

Bifidobacterium and Bacteroides were undetectable ormarkedly reduced in PD-1-/- mice. By contrast, bacteria of

readiness that allows efficient and prompt immune responses

the Enterobacteriaceae family, which are minor representa-

against pathogens.

tives in the small intestine of WT mice, were significantly

daptive coevolution of mammals and bacteria has led to

Our long-term goal is to understand the host-microbial

increased in PD-1-/- mice. These results obtained by 16S

relationship in the gut and to apply this knowledge for healthrRNA gene pyrosequencing of cecal contents are shown inand disease. Current vaccine strategies and immune thera-

Figure 1A. At the Phylum level, PD-1-/- mice had increased

pies could be vastly improved through further advances inpercentages of Firmicutes and Proteobacteria and amucosal immunology.

decreased frequency of the Bacteroidetes compared with
WT mice. Importantly, the skewed microbiota in the gut

PD-1 regulates bacterial communities in the gut
PD-1 is a co-inhibitory receptor expressed on activated T

induces hyperactivation of the systemic immune system in
PD-1-/- mice. Indeed, along with T cell and GC B cell hyper-

Recent publications
1. Kawamoto S., Tran T.H., Maruya M., Suzuki K., Doi
Y., Yumi T., Kato M.L., Fagarasan S. The inhibitory
receptor PD-1 regulates IgA selection and bacterial
composition in the gut. Science 336 , 485-9 (2012) .

82

2. Wei M., Shinkura R., Doi Y., Maruya M., FagarasanS., Honjo T. Mice carrying a knock-in mutation of
Aicda resulting in a defect in somatic hypermutation
have impaired gut homeostasis and compromised
mucosal defense. Nat Immunol. 12, 264-70 ( 2011) .

3. Maruya M., Suzuki K., Fujimoto H., Miyajima M.,
Kanagawa O., Wakayama T., Fagarasan S. Vitamin
A-dependent transcriptional activation of the nuclear
factor of activated T cells c1 ( NFATc1) is critical
for the development and survival of B1 cells. Proc
Natl Acad Sci U S A. 108 , 722-7 ( 2011) .

Figure 2: Enlarged germinal centers and excessive number
of TFH cells in PPs of PD-1-/- mice. Representative
sections of the PPs stained as indicated to reveal
the structure and characteristics of GCs.

Figure 1: Skewed gut microbiota and a defective
mucosal barrier in PD-1-deficient mice.
A. Phylogenetic classification of 16S rRNA frequencies
in the cecal contents from WT and PD-1-/- mice.
B. Anti-commensal serum IgGs in PD-1-/- mice. Lysed
commensal bacteria from cecal contents of SPF WT mice
were used as the target antigens in a Western blot, using
serum from WT and PD-1-/- mice with or without antibiotic
treatment. Note the decrease of commensal specificserum IgG in PD-1-/- mice after antibiotic treatment.

plasia in PPs and peripheral lymph nodes, we found that
serum from PD-1-/- mice contained antibodies specific forcomponents of commensal bacteria, indicating a breach in
the normal mucosal-systemic compartmentalization ( Figure
1B ) . Importantly, administration of broad spectrum antibiotics led to normalization of the phenotype of PD-1-/- mice. On
the basis of our results, we think that the skewed gut microbial communities that result from the dysregulated selectionof IgAs ( see below ) , drive the expansion of auto-reactive B
and T cells and production of auto-antibodies. This is the
subject for future studies.

Figure 3: Bacteria shielding by IgAs in the gut.
A. Pictures showing in green IgA plasma cells in lamina propria orsecretory IgAs coating bacteria in the intestinal lumen. Blue stains
the DNA of eukaryotic and prokaryotic cells. Note the absence of
IgAs and the SFB attached directly to the epithelium in AID-/- mice.
B. Representative sections of the small intestine stained asindicated to reveal IgA plasma cells in the lamina propria and thefrequency of IgA-coated bacteria as determined by flow cytometry.

PD-1 regulates selection of IgA in germinal centersof Peyer’s patches

critical for the containment of the gut commensal microflora,

Intestinal IgA production occurs via both T helper cell-

partly through bacterial shielding, as shown in Figure 3A. In

dependent and independent pathways. Diversification of the
IgA repertoire by somatic hypermutation ( SHM ) , however,

the absence of IgA coating, such as in AID-/- mice, wetakes place mostly in specialized microenvironments called
germinal centers ( GC ) , in which B cell interaction with TFH

The production of IgA by plasma cells in the gut isobserved expansion of segmented filamentous bacteria( SFB ) that attach to the epithelial cells. In PD-1-deficient
mice the IgAs, even though produced in large amounts bycells induces the expression of activation-induced cytidine
deaminase ( AID ) . PD-1-deficiency generates an excess

normal numbers of IgA plasma cells sitting in the lamina pro-number of TFH cells ( Figure 2 ) with altered phenotypes ( less
IL-21, more IFN-γ production ) , resulting in dysregulated

Figure 3B. Our results indicate that PD-1 plays a critical roleselection of IgA precursor cells in the GCs of Peyer’s patches.

maintenance of intact mucosal barrier. Ongoing experiments
aim at understanding the nature of TFH cells in PD-1-/- mice

In short, a large number of IgA B cells, selected and nonselected, receive TFH help and differentiate into IgA plasmablasts and plasma cells.

4. Suzuki K., Maruya M., Kawamoto S., Sitnik K.,
Kitamura H., Agace W.W., Fagarasan S. The sensingof environmental stimuli by follicular dendritic
cells promotes immunoglobulin A generationin the gut. Immunity 33 , 71- 83 ( 2010 ) .

pria, have reduced bacteria-binding capacity as shown inin regulation of antibody diversification required for theand how they impact on selection of IgA-producing cells in
the gut.

5. Tsuji M., Komatsu N., Kawamoto S., Suzuki K.,
Kanagawa O., Honjo T., Hori S., Fagarasan S.
Preferential generation of follicular B helper
T ( TFH ) cells from Foxp3+ T cells in gut Peyer’s
patches. Science 323, 1488-1492 ( 2009 ) .

83

Laboratory for

Immune Diversity

Team Leader :Ji-Yang Wang
Research Scientists : Rika Ouchida			Yohei Kawai

A

Student Trainees :Chie Kano			Shuyin Li (IPA)
Yanfei Zhang (IPA )Technical Staff :Hiromi Morintibodies are crucial effectors for host defense medi-

databases, and have identified several uncharacterized

ated by neutralization and opsonization of pathogens

and by activation of the complement system. Antibodies are

genes that are exclusively expressed in B cells, including thegerminal center ( GC ) subset of B cells and other subsets.

also important regulators of immune responses, acting

We have established knockout mice for these genes andthrough binding to the Fc receptors expressed by various

obtained some interesting preliminary results. Deficiency of

immune cells. Insufficient or excess production of antibodies

an ELL-associated molecule resulted in increased survival of

can lead to immunodeficiencies or autoimmune diseases,
respectively. The goal of our current research is to under-

the GC B cells and enlarged GC after immunization with aprotein antigen ( Figure 1) . Further analysis of these and addi-

stand how B cell activation and differentiation are positively

tional mice deficient in the other B cell-specific genes should

and negatively regulated to allow appropriate production ofallow us to uncover new pathways that regulate various

diversified antibodies of different classes and to achieve

aspects of B cell activation, differentiation and antibody pro-effective humoral immunity.

duction.

Uncover new pathways that regulate B cell activation and differentiation through analysis of mice
deficient in B cell-specific genes

Explore the mechanism of A/T mutations in
GC B cells

With the tools provided by completion of human and mouse
genome projects, the availability of microarray data, and theHigh-affinity antibodies are central to humoral immunity.
Somatic hypermutation ( SHM ) of Ig genes in GC B cells is antechnical advancement in generation of gene-manipulated

essential process for generating high-affinity B cells. SHM isinitiated by the activation-induced cytidine deaminase ( AID) ,mice, we have taken a molecular genetic approach to study

which is thought to convert cytosine ( C ) to uracil ( U ) andB cell activation and differentiation. We performed compre-

generate U:G lesions on DNA. Consistent with its substratehensive searches of the RCAI RefDIC and public microarrayspecificity, ectopic expression of AID in fibroblasts induces

Recent publications
1. Kano, C., Hanaoka, F. and Wang, J.-Y. Analysis of mice
deficient in both REV1 catalytic activity and POLH
reveals an unexpected role for POLH in the generationof C to G and G to C transversions during Ig gene
hypermutation. Int. Immunol., in press ( 2012).

84

2. Imai, T., Kato, Y., Kajiwara, C., Mizukami, S.,
Ishige, I., Ichiyanagi, T., Hikida, M., Wang, J.-Y.
and Udono, H. Hsp90 contributes to cytosolictranslocation of extracellular antigen for crosspresentation by dendritic cells. Proc. Natl.
Acad. Sci. USA 108: 16363 -16368 ( 2011) .

3. Li, Y., Gao, X. and Wang, J.-Y. Comparison of two
POLQ mutants reveals that a polymerase-inactive
POLQ retains significant function in tolerance
to etoposide and γ-irradiation in mouse B
cells. Genes Cells 16: 973-983 (2011).

Figure 1 : Enhanced GC formation inmice deficient in an ELLassociated molecule. Threepairs of WT and KO mice were
immunized with 100 μg of
NP-chicken gamma globulin inalum and two weeks later spleen
sections were stained with peanut
agglutinin (PNA) to detect
GCs. Representative staining
from each mouse is shown.

Figure 2 : B cells express two types ofFc receptors with opposing
functions. FcγRIIB is known
to inhibit B cell activation upon
IgG/antigen binding. We foundthat FcμR is a positive regulatorof B cell activation. Therefore, B
cell activation can be positively
and negatively regulated by IgM
and IgG, respectively. It remains
to be investigated whether FcμR
can directly interact with the
IgM B cell antigen receptor(BCR) and provide a survival
and proliferative signal.

the mutations in Ig genes of GC B cells are induced at non-

Elucidate the function of the IgM Fc receptor in the
humoral immune response

targeted A / T bases. We found that induction of A / T muta-

The existence of a receptor for IgM ( FcμR) was first sug-

mutations predominantly at C/G bases. Remarkably, half oftions is dependent on the GC B cell environment but

gested 40 years ago, but the gene encoding FcμR was iden-

independent of the target gene. Experiments are in progresstified only recently. FcμR is expressed exclusively in B cells into address how AID-triggered U:G lesions lead to mutations

mice and in B and T cells in humans. In collaboration withat A / T bases in GC B cells but not fibroblasts.

the Laboratory for Epithelial Immunobiology of RCAI and the
University of Alabama at Birmingham, we found that FcμR
positively regulates B cell activation and the humoral immune
response ( Figure 2 ) .

4. Kano, C., Ouchida, R., Kokubo, T. and Wang J.-Y.
Rapid cell division contributes to efficient inductionof A / T mutations during Ig gene hypermutation.
Mol. Immunol. 48, 1993-1999 ( 2011).

5. Ouchida, R., Kurosaki, T. and Wang, J.-Y. A role
for lysosomal-associated protein transmembrane
5 in the negative regulation of surface BCRlevels and B cell activation. J. Immunol. 185:294-301 (2010) . ( Featured in “In This Issue” )85

Laboratory for

Immunological
MemoryGroup Director :Toshitada Takemori

M

Research Scientist :Kaji Tomohiro

Technical staff :Akiko Sugimoto			Mitsue Hanami			Natsumi Yoneda
Natsuki Kobayashi
Sakiko Nagaoka

Visiting Scientists :Yoshimasa Takahashi
Masaki Hikida

Student Trainees:

Naoka ItohHisashi Tanida

emory B cells are antigen ( Ag ) experienced cells in aestablishes a long-lived IgG1 memory compartment inresting state, have a unique morphology and pheno-

which, over time, the non-mutated cells are graduallytype and acquire several intrinsic properties that differ from

replaced by mutated GC B cell progeny. Non-mutated mem-other B cell stages, such as longevity and rapid response to

ory B cells are capable of inducing an IgG1 secondary

antigen reexposure by differentiating into plasma cells. How-

response and attain functional maturation to elicit a more

ever, how and when memory B cells are established in therobust response, which correlates with specific changes in

immune system and the mechanisms underlying their main-

gene expression. Thus, our results demonstrate that thetenance and prompt differentiation remain obscure. Our aim

IgG1 memory B cells are generated by two distinct pathways

is to understand the origin and developmental pathways ofand that the memory compartment undergoes qualitative

memory B cells and the regulatory networks responsible forchanges after its initial establishment early in the immune
response ( submitted ) .

the acquisition of memory B cell properties and functions.
Furthermore, we wish to understand the dynamics of humanThus, B cell memory sustains two classes of antibody

B cells under physiological and pathological conditions incommon variable immunodeficiency ( CVID ) , which is char-repertoire, VH genes with germline sequence and thoseaccumulating somatic hypermutations ( SHM ) as a conse-

acterized by a reduced number of memory B cells in thequence of selection in the GC. We find that IgG1 memory B

peripheral blood.
While it is known that mutated, high-affinity memory B

cells respond to antigen reexposure and accumulate mutations in their rearranged VH genes through the GC reaction,

cells originate in GCs, the memory compartment consists ofregardless of whether they express unmutated or mutated orboth mutated and non-mutated cells and little is known

low-affinity or high-affinity VH genes. In the secondary

about the cell population dynamics generating this hetero-

response, unmutated B cells generated a large number ofgeneity. Our studies indicate that within the first week postimmunization, IgG1+ non-mutated memory B cells develop

siblings that accumulated mutations resulting in high-affinity

with T-cell help, prior to GC formation. This population

results support the view that memory B cells can reestablish

amino acid substitutions for the reexposed antigen. These

Recent publications
1. Fujii H, Ato M, Takahashi Y, Otake K, Hashimoto S, Kaji
T, Tsunetsugu-Yokota Y, Fujita M, Adachi A, Nakayama
T, Taniguchi M, Koyasu S, Takemori T. HIV-1 Nef impairs
multiple T-cell functions in antigen-specific immune
response in mice. Int Immunol 2011 23 ( 7 ) :433-41.

86

2. Fujimura T, Yonekura S, Horiguchi S, Taniguchi
Y, Saito A, Yasueda H, Inamine A, Nakayama T,
Takemori T, Taniguchi M, Sakaguchi M, Okamoto
Y. Increase of regulatory T cells and the ratio of
specific IgE to total IgE are candidates for responsemonitoring or prognostic biomarkers in 2-year

sublingual immunotherapy ( SLIT ) for Japanese cedar
pollinosis. Clin Immunol 2011 139 ( 1) :65 -74.
3. Fujimura T, Yonekura S, Taniguchi Y, Horiguchi S, Saito
A, Yasueda H, Nakayama T, Takemori T, Taniguchi M,
Sakaguchi M, Okamoto Y. The Induced Regulatory T

Figure:Both unmutated and mutated memory B cells are required for protection against pathogens. After
infection several B cells that cross-react with the initiating pathogen are selected and differentiate intomemory cells in GCs or prior to GC formation. GC-dependent memory cells express high-affinity antibodies
for the initiating pathogen as a consequence of SHM and selection, whereas GC-independent memory
cells sustain a cross-reactive Ab repertoire. Upon reinfection with a variant of the initiating pathogen thatemerges due to mutations accumulating during the spreading infection, unmutated memory B cells easily
recognize the variant by cross-reactivity and undergo adaptation to the variant epitope as a consequenceof subsequent SHM and selection, which takes place in the preformed GCs after affinity maturation.

the antibody repertoire that is adapted to the reexposed

memory cells and plasma cells. By contrast, development of

antigen. The primary infection caused production of long-

early memory B cells and plasma cells does not require theterm and high-affinity antibodies against the initiating patho-

help of Tfh, indicating that this B cell response is regulated

gens, a mechanism that is critical for protection againstwith the help of different T cell subsets. After antigen encoun-

reinfection with the same pathogen. The generation of newter, CD4 T cells begin to express BCL6 at the border of thehigh-affinity somatic mutants in the memory compartment,

B- and T-cell area after interaction with dendritic cells. Thisin addition to the preexisting antibodies, are advantageous

is followed by their development into Tfh cells upon interac-

for the system to rapidly and efficiently exclude the initiatingtion with B cells, which takes outside and inside of GCs.

pathogen. Of importance, the germ line encoded antibody

Thus our results support the view that during the process ofrepertoire in the memory compartment flexibly adapts to the

CD4 T cell differentiation, B cells interact with T cells andvariants of invading pathogens by cross-reactivity and pro-differentiate into early memory B cells, which localize to thevides a large amount of high-affinity antibodies via SHM.

B cell follicle. We are currently analyzing the features of the

These could be useful as a first-line of defense against com-

non-Tfh cells required for memory B cell generation early

mon pathogens.

after immunization by using CD4 T cells with conditional

It is known that CXCR5 high /PD-1high T follicular helper( Tfh ) cells play an important role in GC development. Con-by deletion of Bcl6 in CD4 T cells, which has prompted us tosistently, our analysis suggests that depletion of Tfh signifi-

also analyze how memory T cell development is regulated.

deletion of Bcl6. CD4 memory T cell development is blocked

cantly affects the generation and maintenance of high-affinity

Cell Level, Defined as the Proportion of IL-10Foxp3 (+)
Cells among CD25 (+) CD4 (+) Leukocytes, Is a Potential
Therapeutic Biomarker for Sublingual Immunotherapy.
Int Arch Allergy Immunol 2010;153 ( 4) :378-87.

4. Kishi Y, Aiba Y, Higuchi T, Furukawa K, Tokuhisa T,
Takemori T, Tsubata T. Augmented antibody responsewith premature germinal center regression in CD40Ltransgenic mice. J Immunol 2010 185 ( 1) :211-9.

5. Hikida M, Casola S, Takahashi N, Kaji T, Takemori T,
Rajewsky K, Kurosaki T. PLC-gamma2 is essential
for formation and maintenance of memory B
cells. J Exp Med 2009 206 ( 3) :681-9.

87

Laboratory for

Host Defense

Team Leader :Tsuneyasu Kaisho
Research Scientists : Katsuaki Hoshino

		 Hiroaki Hemmi

S

uccessful host defense in vertebrates requires bothinnate and adaptive immunity. The innate system func-

tions as a pathogen sensor and is involved in the initial effort
to eradicate an infection. Furthermore, innate immunity also

Clarifying the molecular mechanisms for type I IFN
production by plasmacytoid dendritic cells.
DCs sense nucleic acid adjuvants and produce type I interferon ( IFN ) in a subset-dependent manner. Among nucleic

contributes to the establishment of adaptive immunity. Dendritic cells ( DCs ) are antigen presenting cells criticallyacid sensors, TLR7 and TLR9 are peculiar in that they recog-

involved in a sequence of these immune responses. DCs

Accumulating evidence suggests that TLR7/9-induced typesense various pathogen-derived molecular components and

I IFN production play important roles in the pathogenesis ofexert their immunostimulatory functions by producing inflam-autoimmune disorders such as SLE.

nize not only pathogen- but also host-derived nucleic acids.

matory cytokines or upregulating expression of costimula-

The plasmacytoid dendritic cell ( pDC ) is notable for its

tory molecules. Those pathogen components are called

ability to produce large amounts of type I IFNs, both IFN-α

immune adjuvants based on these DC activating abilities.

and IFN-β, in response to signaling through nucleic acid sen-

Immune adjuvants are recognized by various types of pat-sors such as TLR7 and TLR9. We have clarified that a serine
threonine kinase, IκB kinase α ( IKKα ) , is critical for type I IFN

tern recognition receptors including Toll-like receptors( TLRs ) . Identification of new types of immune adjuvants andproduction from TLR7/9-stimulated pDC. IKKα interacts withcharacterization of the mechanisms by which they activate

and phosphorylates a transcription factor, IRF-7, which isDCs should contribute to development of novel immuno-

critical for type I IFN induction. Critical involvement of IKKα

regulatory maneuvers. We are attempting to clarify how DCs

in human pDC was verified with an IKK inhibitor, BAY11-7082.

are activated through pattern recognition receptors and to

The inhibitor suppressed IFN-α, but not TNF, production byobtain critical information for manipulating the immune

TLR7- or TLR9-stimulated human pDC at less than 1 µM.

response effectively. Various immune adjuvants including

Nuclear translocation of IRF-7 was also impaired by the

TLR ligands and gene targeted mice are important tools forinhibitor. Thus, the IKK-IRF-7 axis should also play a criticalthis purpose.

role in human pDC.

Recent publications
1. K. Hoshino , I. Sasaki , T. Sugiyama , T. Yano , C.
Yamazaki , T. Yasui , H. Kikutani, T. Kaisho. Cutting
edge: Critical role of IκB Kinase α in TLR7/9-Induced
type I interferon production by conventional dendritic
cells. J. Immunol 184:3341-3345 ( 2010) .

88

2. K. Gotoh, Y. Tanaka, A. Nishikimi, R. Nakamura, H.
Yamada, N. Maeda, T. Ishikawa, K. Hoshino, T. Uruno, Q.
Cao, S. Higashi, Y. Kawaguchi, M. Enjoji, R. Takayanagi,
T. Kaisho, Y. Yoshikai and Y. Fukui. Selective control
of type I IFN induction by the Rac activator DOCK2
during TLR-mediated plasmacytoid dendritic cell
activation. J. Exp. Med. 207:721-730 ( 2010) .

3. C. Yamazaki, R. Miyamoto, K. Hoshino, Y. Fukuda, I.
Sasaki, M. Saito, H. Ishiguchia, T. Yano, T. Sugiyama,
H. Hemmi, T. Tanaka, E. Hamada, T. Hirashima, R.
Amakawa, S. Fukuharab, S. Nomura, T. Ito, T. Kaisho,2010. Conservation of a chemokine system, XCR1 andits ligand, XCL1, between human and mice. Biochem.
Biophys. Res. Commun. 397:756 -761 ( 2010).

Figure :Type I interferon production
by a TLR7/9-stimulated
plasmacytoid dendritic cell.
Plasmacytoid dendritic cells(pDC) belong to a DC subset thatis distinguished by the ability to
produce large amounts of type I
interferon upon TLR7/9 signaling.
TLR7/9 can respond not only
to virus- but also host-derived
nucleic acids and pDC-derived
type I IFNs play important roles
in both antiviral immunity andpathogenesis of autoimmune diseases.
In addition to the IKKα-IRF-7
axis, pDC-specific molecules arealso involved in this pDC-specificsignal transduction pathway.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl-

cells and to crosspresent antigens to generate cytotoxic

coenzyme A ( HMG-CoA ) reductase and commonly used forCD8 + T cell responses, thereby playing key roles in anti-viraltreatment of hypercholesterolemia. Beneficial effects ofor anti-tumor immunity. TLR3 signaling can augment

statins have been reported in autoimmune diseases, but

crosspresentation activity of CD8α+ DC. The CD8α+ cDC

the underlying molecular mechanisms are still unclear. We

also has a high capacity to produce proinflammatory

have found that statins can inhibit IFN-α production andcytokines in response to signaling by various TLRs. How-

nuclear translocation of IRF-7 in TLR7- or TLR9-stimulated

ever, it remains unknown whether or how the CD8α+ cDC ishuman pDC.

involved in a variety of immune responses or inflammatory

IRF-7 expression is constitutively high in pDC andconditions.

critical for pDC-specific functions, e.g. the ability to produce

We have found that a chemokine receptor, Xcr1, islarge amounts of type I IFNs. However, IRF-7 is abundantly
account for pDC-specific functions. Among pDC-specificexclusively expressed by CD8α+ cDC. Expression of human
Xcr1 was specific to BDCA3 + DCs, which are a human counterpart of murine CD8α+ cDC ( BBRC 397:756, 2010 ) . By

genes, we have found a transcription factor that canusing a knock-in strategy into the Xcr1 locus, we have gener-transactivate the type I IFN promoter in synergy with IRF-7(Figure ) .

ated gene-manipulated mice in which CD8α+ cDC can be

induced also in other DC subsets and IRF-7 alone cannot

traced or deleted. We plan to clarify the in vivo behavior andbiological roles of CD8α+ cDCs in various immune responses

Clarifying the regulatory mechanisms for a TLR3
expressing DC subset

and inflammatory conditions. Imaging analysis is ongoing incollaboration with Dr. Takaharu Okada ( Research Unit for

TLR3 is a sensor for dsRNA and specifically expressed on

Immunodynamics ) .

CD8α+ DC among murine splenic DC subsets. The CD8α+
DC is distinguished by its high activity to incorporate dead4. H. Amuro, T. Ito, R. Miyamoto, H. Sugimoto, Y. Torii, Y.
Son, N. Nakamichi, C. Yamazaki, K. Hoshino, T. Kaisho,Y. Ozaki, M. Inaba, R. Amakawa, S. Fukuhara. 2010.
Statins, inhibitors of 3-hydroxy-3-methylglutarylcoenzyme A reductase, function as inhibitors of cellularand molecular components involved in type I interferon
production. Arthritis Rheum. 62:2073-2085 ( 2010) .

5. R. Miyamoto, T. Ito, S. Nomura, R. Amakawa, H. Amuro,Y. Katashiba, M. Ogata, N. Murakami, K. Shimamoto,C. Yamazaki, K. Hoshino, T. Kaisho, S. Fukuhara. 2010.
Inhibitor of IkappaB kinase activity, BAY11-7082,
interferes with the interferon regulatory factor 7 nuclear
translocation and type I IFN production by plasmacytoid
dendritic cells. Arthritis Res Therap. 12:R87 ( 2010) .

89

Laboratory for

Infectious Immunity

Team Leader :Satoshi Ishido
Research Scientists : Mari Ohmura-Hoshino		

Eiji Goto
Mizuho Kajikawa			
Pai-Chi Li
Student Trainee :Rikiya Ishikawa

Technical Staff :		

Masami Kawasumi-Aoki
Mari Yoshida-Mito
Naoko Tachibana

		
		

M

HC class II and CD86 are critical molecules for initia-

genetically modified mice.

tion of immunity and but are also related to immune

diseases, therefore, it is important to understand how these

MARCH-I belongs to a family of membrane-bound E3
ubiquitin ligases, referred to as MIR ( modulator of immune

molecules are regulated in vivo. In this vein, we discovered

recognition ) . The MIR family consists of viral MIRs and theiran E3 ubiquitin ligase referred to as MARCH-I. The expression level of MHC class II-peptide complexes ( pMHC II ) ismammalian homologues including MARCH-I. Importantly,

regulated through MARCH-I-mediated ubiquitination of thebrane molecules. Viral MIRs encoded by human herpesvirus

MHC II β chain. In the steady state, MARCH-I is expressed
mainly in the antigen-presenting cells ( e.g. DCs, B cells and8 were shown to down-regulate MHC class I and CD86, sug-

macrophages ) . In the case of DCs, pMHC II is continuously

evasion strategy. Therefore, from the clinical perspective, itubiquitinated by MARCH-I when the cells are in an immature

is important to understand the molecular basis of MIR sub-state; however, this ubiquitination is quickly terminated after

strate recognition. However, it is still difficult to uncover how

DC maturation as a result of downregulation of MARCH-I.

MIRs recognize their substrates in detail because of the dif-Thus, loss of pMHC II ubiquitination and downregulation of

ficulty in preparing MIR proteins. To address this issue, we

MARCH-I are thought to be an important step in the DC

are employing an integrative approach for viral MIRs.

the different MIR family members recognize different mem-gesting that these MIRs are part of the herpesvirus immune

maturation process. Since the stabilization of pMHC II is
MARCH-I is expected to be a critical process for initiation ofRegulatory T cells induce DC apoptosis by loss ofpMHC II in the steady state

immunity and maintenance of tolerance. At present, we are

Downregulation of MARCH-I and loss of MHC II ubiquitina-

intensively examining this issue by generating suitabletion are DC maturation processes; however, the biologicalincreased by loss of pMHC II ubiquitination, regulation of

Recent publications
1. Baravalle, G., Park, H., McSweeney, M., OhmuraHoshino, M., Matsuki, Y., Ishido, S. and Shin,
JS. Ubiquitination of CD86 Is a Key Mechanism
in Regulating Antigen Presentation by Dendritic
Cells. J. Immunol. 187, 2966-73 (2011)90

2. Tze, LE., Horikawa, K., Domaschenz, H., Howard,DR., Roots, CM., Rigby, RJ., Way, DA., OhmuraHoshino, M., Ishido, S., Andoniou, CE., Degli-Esposti,
MA. and Goodnow, CC. CD83 increases MHC II
and CD86 on dendritic cells by opposing IL-10driven MARCH1-mediated ubiquitination anddegradation. J. Exp. Med. 208 , 149-65 (2011)

3. Goto, E., Yamanaka, Y., Ishikawa, A., Aoki-Kawasumi,M., Mito-Yoshida, M., Ohmura-Hoshino, M., Matsuki, Y.,
Kajikawa, M., Hirano, H., and Ishido, S. Contribution of
K11-linked ubiquitination to MIR2-mediated MHC class
I internalization. J Biol Chem. 285 , 35311- 9 (2010)

Figure :Induction of DC apoptosis by natural Tregs through stabilization of pMHC II
In immature DCs, MHC class II-peptide complexes (pMHC II) are continuously degraded as a result of MARCH-I-mediatedubiquitination (left). During the process of maturation, the function of MARCH-I is inhibited and pMHC II is stabilized
through loss of ubiquitination. Stabilized pMHC II is effectively recognized by natural Tregs, and the mature DCs undergo
apoptosis through interaction between Fas and FasL. This regulation might be critical for prevention of autoimmunity.

consequences of these events in vivo are unknown. To
address this point, we have generated genetically modified

Inter-transmembrane region of viral MIR2 contributes to CD86 downregulation

mice in which the March1 gene can be conditionally deleted
by administration of 4-hydroxytamoxifen ( OHT ) . After dele-

Human herpesvirus 8 ( HHV8 ) , a human tumor virus, encodestion of MARCH-I, caspase 8-dependent apoptosis wasobserved in splenic DCs. Given that caspase 8 is involved inand MIR2 downregulate the surface expression of major
histocompatibility complex class I ( MHC I ) molecules throughFas or TNF-mediated apoptosis, the contribution of theseubiquitin-mediated endocytosis followed by lysosomal deg-

pathways was examined by using blocking antibodies. This

radation. Since MIR2 additionally downregulates a costimu-

experiment showed the importance of Fas-FasL-mediatedlatory molecule CD86 and an integrin ligand CD54, it issignals. By adoptive transfer experiments of BMDCs and thethought to be a more important molecule for the immune

analysis of an MHC II KI in which the ubiquitination site wasevasion than MIR1. To address the molecular basis of sub-mutated, stabilization of pMHC II on the cDCs as well as

strate specificity of MIR2, we examined which regions ofappropriate in vivo microenvironments were shown to be

B7-2 and MIR2 are required for MIR2-mediated B7-2 down-

required for DC apoptosis. Importantly, deletion of CD25
CD4 T cells prevented DC apoptosis. Finally, adoptively

regulation. Experiments using domain-swapping chimeras

transferred FoxP3 + CD4 T cells induced apoptosis in BMDCs
whose MHC II was stabilized by loss of ubiquitination. Thesealso between MIR1 and MIR2 demonstrated a significant
contribution of the juxtamembrane ( JM ) region of B7-2 andresults suggest that in the steady state, maturation might

the inter-transmembrane ( ITM ) region of MIR2 to MIR2-

lead to the suicide of DCs in an antigen-dependent manner.

mediated suppression. Computational simulation and muta-

Given that DCs must be tightly regulated to prevent exces-

genesis analyses suggest that Phe119 and Ser120 of thesive immunity, loss of pMHC II should be critical for mainte-

MIR2 ITM and Asp244 in the B7-2 JM region contribute tonance of tolerance in the steady state. Right now, we are

the recognition of B7-2 by MIR2. This finding sheds new

studying how MARCH-I is regulated in the steady state toinsight into the molecular basis of substrate recognition by

test our hypothesis.

MIR family members, and suggests a novel tactic for drug+

two MIRs, MIR 1 and 2, to evade host immunity. Both MIR1

between human B7-2 and CD8α, a non-MIR2 substrate, anddesign to treat HHV8-related cancer.

4. Walseng, E., Furuta, K., Bosch, B., Weih, KA.,
Matsuki, Y., Bakke, O., Ishido, S., and Roche, PA.
Ubiquitination Regulates MHC Class II-Peptide Complex
Retention and Degradation in Dendritic Cells. Proc.
Natl. Acad. Sci. U S A. 107 , 20465-70 (2010)

5. Ohmura-Hoshino, M., Matsuki, Y., Mito-Yoshida,M., Goto, E., Aoki-Kawasumi, M., Nakayama, M.,
Ohara, O., and Ishido, S. Cutting edge: Requirementof MARCH-I-mediated MHC II ubiquitination
for the maintenance of conventional dendritic
cell. J. Immunol. 183 , 6893-7 (2009)91

Laboratory for

Innate Cellular Immunity

Team Leader:

Masato Tanaka
Research Scientists : Kenichi Asano

Qiu Chunhong
Gen Nishitai

PTechnical Staff :Minako Aihara
Miyuki Suzuki

Student Trainees :Kazunori Karasawa
Misa Monya
Kazuyuki Okada
Yuya Anai
Masatoshi Sakuma

hagocytes, such as macrophages and dendritic cells( DCs ) , play important roles as a first-line defense for thetionally conserved physiologic process vital to the elimina-

immune system and exhibit a number of activities. At the site

place during tissue injury and inflammation, and phagocytesof bacterial infection, macrophages rapidly recognize invad-

rapidly recognize these cell corpses for removal. The corpse

ing bacteria via a wide variety of surface receptors that bindclearance is executed so rapidly by phagocytes that it iscomponents found on bacterial surfaces. Phagocytes also

extremely difficult to find dying/dead cells that are not asso-

have the ability to recognize invading microorganisms

ciated with phagocytes even in the thymus, where a large

through pattern-recognition receptors such as toll-likereceptors ( TLRs ) . TLRs recognize microbial components,

number of thymocytes die by apoptosis under physiological

known as pathogen-associated molecular patterns, includ-

clearance of dead cells prevents the release of potentiallying LPS and nucleic acids. Through the recognition by these

toxic or immunogenic intracellular materials from the cell

receptors, phagocytes can distinguish microorganisms,

corpses. Therefore, the prompt elimination of dying cells andresultingantipathogenic

damaged resident cells is thought to be required for the

responses including the production of inflammatory cytok-

maintenance of tissue integrity, the resolution of inflamma-

ines and chemokines. These factors induce inflammation,

tion, and normal tissue repair.

indistinctandappropriatetion of unnecessary cells. Apoptotic cell death also takes

conditions because they have failed selection. This rapid

and the inflammatory response plays a critical role in the

The Laboratory for Innate Cellular Immunity is investi-

effective clearance of invading microorganisms and thegating the molecular mechanisms for recognition andinduction of appropriate adaptive immunity.

phagocytosis of dying cells, and the pathological relevanceOn the other hand, phagocytes also have the ability torecognize dead “self” cells. Apoptotic cell death is an evolu-of impaired phagocytosis to inflammatory disorders including autoimmune diseases.

Recent publications
1. Asano K., Nabeyama A., Miyake Y., Qiu C.H.,
Kurita A., Tomura M., Kanagawa O., Fujii S.I., andTanaka M. CD169-positive Macrophages Dominate
Antitumor Immunity by Crosspresenting Dead CellAssociated Antigens. Immunity 34,85-95 (2011)92

2. Chow A., Lucas D., Hidalgo A., Mendez-Ferrer
S., Hashimoto D., Scheiermann C., Battista M.,
Leboeuf M., Prophete C., Rooijen N., Tanaka M.,
Merad M., and Frenette P. Bone Marrow CD169+
Macrophages Promote the Retention of Hematopietic
Stem and Progenitor Cells in the Mesenchymal
Stem Cell Niche. J Exp Med 208, 261-71 ( 2011)

3. Nabeyama A., Kurita A., Asano K., Miyake Y., YasudaT., Miura I., Nishitai G., Arakawa S., Shimizu S.,
Wakana S., Yoshida H., and Tanaka M. xCT Deficiency
Accelerates Chemically Induced Tumorigenesis. Proc
Natl Acad Sci U S A 107, 6436-6441 ( 2010)

On the contrary, subcutaneously injected apoptotic
cells are often immunogenic and researchers have taken
advantage of immunogenic tumor corpses for tumor vaccination. These findings suggest that apoptotic cells in the
periphery are cleared and processed in a different way fromblood-borne apoptotic cells in spleen. Antigen presentationin peripheral LNs is thought to be coordinately executed bymigratory DCs from peripheral tissues and LN-resident
APCs. However, little is known about the role of different
APCs in the clearance of dead cells and in the presentationof dead cell-associated antigens in peripheral tissues.
We recently documented how tumor cell-associatedantigens derived from dead tumor cells are crosspresented
in the draining LNs. We concluded that macrophages that
reside in the LN sinus take up dead tumor cells and directly
crossprime CTLs. Mice lacking sinus macrophages at thetime of dead tumor cell vaccination failed to induce antitumor immunity. These findings are expected to improve ourunderstanding of how to optimally generate and activate
tumor- specific T cell immunity in response to tumor cell
Figure :CD169-positive macrophages(Green) in the lymph nodesinus engulf subcutaneously injected dead tumor cells(red).

death.

CD169-positive macrophages as first-line
defenders
The LN sinus is a highway that links afferent and efferent

Critical Roles of Sinus Macrophages in Anti-tumor
Immunity

lymph and is believed to be a filtering zone for lymph-borne

Phagocytes, such as macrophages and DCs, swiftly phago-

lymphatic fluid, make initial contact with CD169 + mac-

cytose apoptotic cell corpses by recognizing moleculesrophages that serve as sentinels located along the length of

exposed only on the surface of the corpses. Defects in thisthe LN sinus. In the case of spleen, CD169 + marginal metal-

process result in autoimmune disorders, indicating thatlophilic macrophages as well as marginal zone macrophages

apoptotic cell clearance by phagocytes essentially contrib-

are located between the lymphoid compartment and theutes to the maintenance of self-tolerance under physiologi-

scavenging red pulp compartment. These macrophages arecal conditions. Consistent with these findings, the intravenous

well equipped to constantly screen the blood for foreign par-injection of apoptotic cells induced antigen-specific immu-

ticles and organisms, as well as aberrant molecular debris
and dying cells. We can detect the CD169 + macrophagenosuppression or tolerance to cell associated proteins. For

molecules. Large particles, including cellular antigens in thesuch tolerance induction, both rapid corpse clearance bysplenic macrophages in the marginal zone ( MZ ) and also

subpopulation in the boundary border between the outerselective phagocytosis and subsequent antigen presenta-

that this macrophage subpopulation plays a critical role as ation by the splenic DC subpopulation located in the MZ are

sentinel in each organ, and we are studying the physiologicalrequired.

and pathological roles of these macrophages.

4. Qiu C. H., Miyake Y., Kaise H., Kitamura H.,
Ohara O., and Tanaka M. Novel Subset of CD8α+
Dendritic Cells Localized in the Marginal Zone
is Responsible for Tolerance to Cell-associated
Antigens. J. Immunol. 182, 4127-4136 ( 2009)environment and the tissue in several organs. We speculate

5. Tanaka M., Miyake Y., and Asano K. Maintenanceof Self-tolerance by Apoptotic Cell Clearance.
Front. Biosci. 13, 6043-6049 ( 2008)93

Research Unit for

Inflammatory Regulation

Unit Leader :Takashi Tanaka
Technical Staff :Emiri Haga

Student Trainees : Masanaka Sugiyama

Takeshi Hirashima
Rumiko Ono		T

he inflammatory response is an important host defense
mechanism to sense and eliminate invading microbial

now focuses on the role of PDLIM2 ( PDZ and LIM-domain
protein-2 ) and related LIM proteins in the negative regulation

pathogens. Dendritc cells first detect pathogens by theirpathogen sensors ( e.g. Toll-like receptors [ TLR ]) . TLR sig-of inflammatory responses.

naling then leads to the activation of the transcription factor

PDLIM2 inhibits granulomatous inflammation

NF-κB, which enters the nucleus and induces the expres-

Granuloma formation is an important host defense mecha-sion of a series of inflammation-related genes, including

nism against intracellular bacteria. However, uncontrolled

those encoding proinflammatory cytokines such as interleukin-6 ( IL-6 ) and IL-12. These inflammatory responses thengranulomatous responses cause tissue damage and impair

direct T-helper ( Th ) lymphocyte differentiation into distinct

eases, such as Crohn’s disease and sarcoidosis. Recent

effector T cell subsets, such as Th1, Th2 and Th17, to com-studies have implicated exaggerated Th17 cell-mediatedbat various pathogens. These initially helpful inflammatory

responses in the development of granulomatous inflamma-

responses must be terminated at the appropriate time point,tion, suggesting that controlling Th17 cell development will

however, otherwise excessive responses can damage normal tissue and may cause autoimmune or allergic diseases.

be an important strategy for preventing and treating granulomatous diseases. PDLIM2, also known as SLIM ( STAT-

Our research goal is to identify key regulators of inflamma-

interacting protein ) , is a nuclear protein that contains bothtion-related signal transduction pathways and to clarify the

PDZ and LIM domains and belongs to a large family of LIM

molecular

ment of new therapeutic tools to control the exaggerated

proteins. PDLIM2 was originally isolated as a nuclear ubiquitin E3 ligase for the STAT4 transcription factor in CD4 + T
cells, suppressing Th1 cell differentiation ( Tanaka T et al,

inflammation seen in certain human diseases. Our research

Immunity, 2005 ) . We have recently shown that PDLIM2

mechanisms

forregulating

inflammatory

responses. These studies should contribute to the develop-

normal organ function in several human autoimmune dis-

Recent publications
1. Tanaka, T., Yamamoto, Y., Muromoto, R., Ikeda, O.,
Sekine, Y., Grusby, M., J., Kaisho, T., Matsuda, T.
PDLIM2 inhibits T Helper 17 cell development andgranulomatous inflammation through degradationof STAT3. Sci. Signal. 4(202), ra85 (2011)94

2. Yan, P., Fu, J., Qu, Z., Li, S., Tanaka, T., Grusby,
M.J., Xiao, G. PDLIM2 suppresses HTLV-I
Tax-mediated tumorigenesis by targeting
Tax into the nuclear matrix for proteasomal
degradation. Blood 113, 4370-4380 ( 2009)

3. Kaisho, T. and Tanaka, T. Turning NF-κB and IRFs on and
off in DCs. Trends Immunol., 29, 329-336 ( 2008 )Figure 1: Enhanced P. acnes-induced
liver granuloma formationin PDLIM2-deficient mice.
Hematoxylin and eosin stained
liver sections on day 7 after
intraperitoneal injection of heatkilled P. acnes. Scale bars, 100 µm.

Figure 2: PDLIM2 and PDLIM4 regulate
Th17 cell-mediated inflammatory
responses through different
mechanisms. PDLIM2 interacts with
STAT3 and promotes its ubiquitin/
proteasome-dependent degradation in
the nucleus, whereas PDLIM4 binds to
STAT3, recruits PTP and enhances its
dephosphorylation in the cytoplasm.

negatively regulates Th17 development and granulomatous

proteins differentially regulate inflammatory responses. STAT

responses by acting as a nuclear ubiquitin E3 ligase target-transcription factors are critical for T-helper cells to differen-

ing STAT3, the transcription factor critical for commitment to
the Th17 lineage ( Tanaka T et al, Sci. Signal., 2011) . PDLIM2

tiate into particular lineages of effector T cell subsets. We

promoted polyubiquitination and proteasome-dependent

member, is a negative regulator of STAT-mediated signaling.

degradation of STAT3, thereby disrupting STAT3-mediated

In contrast to PDLIM2, PDLIM4 is located in the cytoplasm.

gene activation. PDLIM2 deficiency resulted in an increase in

PDLIM4 bound to STAT3, STAT4 and STAT6, and suppressed

nuclear STAT3 and enhanced Th17 cell differentiation. There

activation of target genes. Interestingly, PDLIM4 did not pro-

was also exacerbated liver granuloma formation induced bymote polyubiquitination and degradation of these STAT mol-

Propionibacterium acnes, a gram-positive intracellular bac-ecules, but instead inhibited the phosphorylation of tyrosine

terium that has been suggested to be involved in the patho-

residues essential for cytokine-induced STAT activation. We

genesis of human sarcoidosis. Our findings delineate an

further demonstrated that PDLIM4 bound to and recruitedessential role of PDLIM2 in negatively regulating Th17-medi-

PTP-BL, a protein tyrosine phosphatase, and facilitatedated inflammatory responses and provide a potential thera-

dephosphorylation of STAT proteins. PDLIM4-deficiency inCD4+ T cells resulted in augmented tyrosine phosphorylation

peutic target for autoimmune diseases.

recently found that PDLIM4, another LIM protein familyof these STAT proteins and consequently enhanced Th1, Th2

The role of LIM proteins in the regulation of
inflammatory responses

and Th17 cell differentiation. Our findings delineate an essen-

The LIM protein family is classified into three groups andeffector T cell differentiation through a mechanism distinct

PDLIM2 belongs to Group 3. About 10 proteins are included

from PDLIM2. Collectively, LIM proteins may be a new family

in Group 3, but their functions in the immune system remainof adaptors that can negatively regulate signal transduction

unclear. We are in the process of clarifying how these LIM

pathways in the immune system.

4. Tanaka, T. Grusby, M.J. and Kaisho, T. PDLIM2-mediatedtermination of transcription factor NF-κB activation
by intranuclear sequestration and degradation of the
p65 subunit. Nat. Immunol. 8, 584-591 ( 2007 )tial role for PDLIM4 in negatively regulating STAT-mediated

5. Nakahira, M., Tanaka, T., Robson, B.E., Mizgerd, J.P.
and Grusby, M.J. Regulation of Signal Transducer andActivator of Transcription Signaling by the Tyrosine
Phosphatase PTP-BL. Immunity 26, 163-176 ( 2007)95

Research Unit for

Therapeutic Model

Unit Leader :Kanako Shimizu
Technical staff : Miki AsakuraT

he goal of our laboratory is to develop immunothera-

Unit ) under the support and training program for translational

peutic models for cancer. We have been focusing on the
biological role of dendritic cells ( DCs ) in vivo as a link betweenof screening the iNKT cell ligand-loading capacity of antigen

innate and adaptive immunity. NKT cells have unique immu-

presenting cells. This approach demonstrated that in vivo

noregulatory features that include the ability to rapidly pro-murine NKT cell responses can be used to quantitate theduce large quantities of cytokines. We have attempted to

α-GalCer loading capacity of human APCs.

research of Japan.

Second, we have established a method

develop an approach for inducing adaptive immunity basedon the adjuvant effect of NKT cell ligands and using in vivo
DC maturation, which we have found to be more effective
than ex vivo manipulation of DCs. We have recently established an immunotherapeutic strategy using adjuvant vector
cells ( AVCs ) , which are glycolipid-loaded, allogeneic fibroblasts that can be induced to express virtually any antigen by

Antigen mRNA-transfected, allogeneic fibroblasts
loaded with NKT cell ligand confer antitumor
immunity.
A collaboration with Drs. Fujii ( Cellular Immunotherapy Unit)
and Ishii ( Vaccine Design Team ) in RIKEN RCAI, Dr. Mizuno( Yamaguchi Univ.) , Dr. Kakimi ( Tokyo Univ.) and Dr. Maeda

transfecting them with antigen-encoding mRNA. These
AVCs enhance both innate ( NKT and NK cells ) as well as( Iwate Medical Univ.)

adaptive ( T cells ) immunity. We are now focusing on the

α-GalCer-loaded tumor (tumor/Gal ) cells could generatemechanism of DC maturation. Also, to prepare for the launch

innate and adaptive immunity through the maturation of DCsof clinical studies, we have been performing preclinical studies in collaboration with Dr. Fujii ( Cellular Immunotherapy

in a tumor specific manner. In the current study, instead of

We previously demonstrated that the administration oftumor cells, we used allogeneic fibroblast cells loaded with

Recent publications
1. Fujii S., Shimizu K. ( 2011) DC-based immunotherapy
targeting NKT cells. In Terabe M and Berzofsky
JA (eds), Natural killer T cells: Balancing theregulation of tumor immunity. Springer New
York Dordrecht Heidelberg London, 95-110.

96

2. Shimizu K., Asakura M., Fujii S. Prolonged antitumor
NK cell reactivity elicited by CXCL-10-expressing
dendritic cells licensed by CD40L+CD4+ memory
T cells. J. Immunol. 186, 5927-37 ( 2011)

3. Shimizu K, Hidaka M, Bickham K, Moriwaki
M, Fujimoto K, Fujii S. Human leukemic cells
loaded with α-GalCer activate murine NKT cells
in situ. Int J Hematol 92, 152-60 ( 2010)

Figure :Human DC/Gal activate primary
NKT cells from mice of different
genetic backgrounds
Murine iNKT cell activation following
i.v. injection of human DC/Gal was
evaluated in C57BL/6 and BALB/cmice. Two days after injection, spleencells were collected and the numberof CD1d/Gal-dimer+CD19- cells
was assessed by flow cytometry.
This method should be helpful forevaluation of human antigen presenting
cells loaded with iNKT cell ligand.

α-GalCer and transfected with antigen-encoding mRNA as

stimulation with antigen presenting cells ( APCs ) loaded withadjuvant vector cells (AVCs), thus combining the adjuvant

the NKT cell ligand, α-galactosylceramide ( α-GalCer) . Wheneffects of iNKT cell activation with delivery of an antigen of

we investigated whether human APCs loaded with α-GalCer

choice to DCs in vivo. As preclinical studies, we have begun

have the ability to activate NKT cells in mice, we found thatmurine and canine experiments to evaluate the safety profile

the human DCs stimulated the secretion of IFN-γ by acti-

and immune responses. These aAVCs administered to micevated murine NKT cells when injected into C57BL/6 mice.

and dogs activate invariant NKT (iNKT) cells as well as elicit

Furthermore, the number of transferred hDC/Gal cells cor-

antigen-specific T cell responses with no adverse events. By

related with the number of recovered IFN-γ-producing spleen

harnessing the innate immune system and generating ancells, indicating that the capacity of APCs to load α-GalCer

adaptive immune response to a variety of antigens, thiscan be measured by IFN-γ release in an ELISPOT assay.

unique tool could prove clinically beneficial in the develop-

Finally, α-GalCer-loaded human leukemic cells, both cell

ment of immunotherapies for malignant and infectious dis-lines and primary leukemic cells, injected into C57BL /6 miceeases.

also had the capacity to stimulate murine NKT cells in vivo.
These results indicate that in vivo murine NKT cell responses

Human leukemic cells loaded with
α-galactosylceramide (α-GalCer) activate murine
iNKT cells in situ -a method to quantitate theα-GalCer loading capacity of human APCs.

can be used to quantitate the α-GalCer loading capacity ofhuman APCs. This method could be utilized to develop
future immunotherapies in which NKT cells are targeted for
activation.

Invariant NKT cells (i NKT ) cells become activated after

4. Fujii S, Motohashi S, Shimizu K, Nakayama T, Yoshiga
Y, Taniguchi M. Adjuvant activity mediated by iNKT
cells. Semin Immunol 22, 97-102 ( 2010)

5. Fujii S, Goto A, Shimizu K. Antigen mRNA-transfected,
allogeneic fibroblasts loaded with NKT-cell ligand confer
antitumor immunity. Blood 113, 4262-72 ( 2009 )97Research Unit for

Immune Homeostasis

Unit leader :Shohei Hori
Research Scientists : Norihito Hayatsu

		 Maria Encarnita
		 Mariotti-Ferrandiz
Visiting Scientists :		 Ruka Setoguchi

		 Masashi Tachibana
Technical Staff :		 Takahisa Miyao

		 Guanying Wang
		 Kuniko KamiyaT

he ultimate goal of our laboratory is to understand thecells respond to extrinsic signals and deal with environmen-

basic rules of immunological tolerance and homeosta-

tal changes to ensure the robustness of self-tolerance andsis. While the clonal selection theory has predicted thehomeostasis.

importance of cell intrinsic mechanisms such as clonal deletion and anergy, their significance in naturally acquired self-

Plasticity of Foxp3 + T cells: the controversy resolved

tolerance still remains elusive. Over the last two decades,

The emerging notion of environment-induced Treg cell repro-

evidence has accumulated that natural self-tolerance may

gramming remains highly controversial. We and others havebe rather dominant and based on cell-extrinsic regulation ofpreviously shown that at least some of Foxp3 + T cells candifferentiate into Foxp3 - helper T ( Th ) cells in response toautoaggressive lymphocytes by other functional classes oflymphocytes, in particular by regulatory T ( Treg ) cells exhib-

environmental changes such as lymphopenia or inflamma-

iting suppressive functions. Our studies have demonstrated

tion, whereas other recent reports argue against the plastic-that a subset of Treg cells characterized by expression of the

ity phenomena. We have recently proposed that thistranscription factor Foxp3 plays an essential role in thecontroversy can be resolved by considering the “heteroge-

establishment and maintenance of natural self-tolerance andimmune homeostasis, particularly at the environmental

neity model” of plasticity, which hypothesizes that theobserved plasticity of Foxp3 + T cells does not reflect lineage

interfaces.

reprogramming of Treg cells but a minor population of

In order to understand the principles of immunologicaluncommitted Foxp3 + T cells. By genetic fate mapping or

tolerance and homeostasis, it is crucial to understand how

adoptive transfers, we have addressed the nature of suchthe immune system robustly maintains tolerance to “self” yetuncommitted Foxp3 + T cells and identified them as a minor

allows for aggressive responses toward “non-self” in the

population of non-regulatory cells exhibiting promiscuous

face of diverse and unpredictable perturbations from theand transient Foxp3 expression. Such cells give rise tointernal as well as external environments. To address thisquestion, we are currently investigating how Foxp3 + Treg

Foxp3 - Th cells and selectively accumulate in inflammatory
cytokine milieus or in lymphopenic environments including

Recent publications
1. Miyao T., Floess S., Setoguchi R., Luche H., Fehling
H.J., Waldmann H., Huehn J., Hori S. Plasticity of
Foxp3+ T cells reflects promiscuous Foxp3 expressionin conventional T cells but not reprogramming of
regulatory T cells. Immunity 36, 262-275 (2012)98

2. Hori S: Stability of regulatory T-cell lineage.
Adv Immunol. 112, 1-24 ( 2011)

3. Hori S: Regulatory T cell plasticity: beyond thecontroversies. Trends Immunol. 32, 295-300 ( 2011)

Figure :A model of Treg cell lineage commitment. During thymic or peripheral Treg cell differentiation, TCR and othersignals lead to Foxp3 up-regulation. (The RFP reporter visualizes Foxp3+ cells and their progeny, independently ofcontinuous or transient Foxp3 expression, see Miyao et al. Immunity for details.) However, not all Foxp3+ T cells
are committed to the Treg cell fate, because some others transiently up-regulate Foxp3 without acquiring a Treg cell
phenotype and function. The latter population eventually loses Foxp3 expression and differentiates into Foxp3- (exFoxp3)
Th cells, while Treg cells eventually undergo complete demethylation of the TSDR to stabilize Foxp3 expression.
Nevertheless, Treg cells may transiently lose Foxp3 expression in environments where TCR and/or IL-2 signals arelimited but retain an epigenetic memory of Foxp3 expression and suppressive function (“latent” Treg cells). Thus, the
TSDR appears to act as a “memory module” that ensures the committed state of Treg cells in a changing environment.
The initial signals that lead to the “imprinting” of a stable Treg cell phenotype remain to be uncovered however.

those in early ontogeny. By contrast, Treg cells did nottion strategies and knock-in mutagenesis in mice. Seren-

undergo reprogramming under those conditions irrespec-

dipitously, we have identified one hypomorphic mutation thattively of their thymic or peripheral origins. Moreover, although“latent” Treg cells retain phenotypic memory and robustly

had no effect on the in vitro suppressive activity, thymic differentiation, or global gene expression profile of Foxp3 + T
cells. This mutation, however, led to impaired fitness of

re-express Foxp3 and suppressive function upon activation.

CCR7lo “tissue-seeking” Foxp3 + T cells. Accordingly, we

The stable differentiated state of Treg cells was associatedhave found that mutant Foxp3 + T cells are much less com-with complete DNA demethylation of a conserved non-coding region termed Treg-specific demethylation region ( TSDR )petitive than wild-type Foxp3 + T cells in peripheral tissues,

within the Foxp3 locus. This study established that Treg cells

differed from tissue to tissue, suggesting an environmental

constitute a stable cell lineage, whose committed state in acontrol of Treg cell fitness. Importantly, the impaired fitnesschanging environment is ensured by an epigenetic mecha-

of mutant Foxp3 + T cells was associated with reduced

nism, irrespective of ongoing Foxp3 expression. Our next

expression of one transcription factor and was corrected byfocus is to discover the signals that lead to irreversible

its retroviral transduction. We suggest that fitness of Treg

commitment to the Treg cell fate.

cells in tissue environments is controlled by both genetic anda few Treg cells transiently lose Foxp3 expression, such

although the extent of the reduction of mutant Foxp3 + T cells

environmental factors and represents an essential determi-

Genetic and environmental control of Treg cell
fitness in peripheral tissues

nant of self-tolerance and immune homeostasis. We are
currently investigating i ) the molecular nature of such

In an attempt to understand the molecular mechanisms byenvironmental factors; ii ) how such extrinsic cues are trans-

which Foxp3 controls Treg cell differentiation and function,
we have addressed whether and how Foxp3 gene mutations

lated into Foxp3-dependent intracellular control mechanisms, and iii ) how “fit” Treg cells in turn ensure the fitness ofthat had been identified in human IPEX impinge on Treg cell

peripheral tissues.

development and function, using retroviral gene transduc-

4. Hori, S. Developmental plasticity of Foxp3+ regulatory
T cells. Curr Opin Immunol. 22, 575-582 ( 2010 )5. Komatsu N., Mariotti-Ferrandiz M.E., Wang Y., MalissenB., Waldmann H., Hori S. Heterogeneity of natural
Foxp3+ T cells: a committed regulatory T cell lineage andan uncommitted minor population retaining plasticity.
Proc Natl Acad Sci USA. 106, 1903-1908 ( 2009 )99

Laboratory for

Immunochaperones

Team Leader :Heiichirô Udono
Technical Staff :Chiaki Kajiwara						Takashi Matsuura						Yu KatoStudent Trainee : Shizuha Uda

C

D8 + T cells recognize naturally processed peptides in

efficiently detect cell surface hsp90 on DCs and mac-the context of MHC class I molecules. During viralrophages. Since hsp90 has no transmembrane domain, it

infection or malignant transformation, intracellular changes
in non-APC ( antigen presenting cells ) must be reported tomust be associated with unknown molecules for anchorage

CD8 + T cells, which are indispensable in fighting virusinfected cells and cancer cells. However, these abnormal

logical significance of cell surface hsp90 in the context of theto the cell surface. We are currently examining the physioimmune response.

cells cannot act by themselves as APC to prime T cells,
although they express tumor or viral antigens on their cellsurface. In addition, many viruses cannot infect professional
APC, such as dendritic cells ( DCs ) , and thus the APC cannot
present such viral antigens directly to CD8 + T cells. Therefore, DCs must internalize neighboring tumor or infected
cells, digest them, and then present antigen peptides to

The role of hsp90 in antigen cross-presentation.
In general, the main functions of HSPs are ( i ) facilitate protein folding/refolding and prevent aggregation of newly synthesized proteins on the ribosome; ( ii ) maintain the normal
function of mature proteins; ( iii ) target misfolded and/or

CD8 + T cells in the context of MHC class I molecules. Thisdamaged mature proteins for degradation by the proteasome; ( iv ) transport proteins between distinct intracellularpathway is called cross-presentation and is believed to becompartments such as the cytosol and mitochondria. Pro-

specific to DCs and not other APC types such as mac-teins targeted to the proteasome undergo degradation inrophages or B cells. We have evidence that molecular chaperones such as hsp90 play important roles in this antigenorder to recycle amino acids and the byproducts of this process ( degradation products, peptides ) are utilized as antigen

cross-presentation. One of our research goals is to identify

peptides, mainly presented by MHC class I molecules. Thus,

the mechanisms of cross-presentation, especially focusing

proteasomal degradation is important at the end of the lifeon the role of the molecular chaperone hsp90. During thecycle of all proteins to prevent waste of raw materials and isstudy of cross-presentation, we developed a mAb that canused by the immune system to alert T cells of any alterations

Recent publications
1. Mizukami S, Kajiwara C, Tanaka M, Kaisho T, Udono H.
Differential MyD88/IRAK4 requirements for crosspriming and tumor rejection induced by hsp70-model
Ag fusion protein. Cancer Sci. 103, 851-859, 2012.

100

2. Imai T, Kato Y, Kajiwara C, Mizukami S, Ishige I,
Ichiyanagi T, Hikida M, Wang JY, Udono H. HSP90
contributes to cytosolic translocation of extracellularantigen for cross-presentation by dendritic cells.
Proc Natl Acad Sci USA. 108, 16363-16368, 2011.

3. Ichiyanagi T, Imai T, Kajiwara C, Mizukami S, Nakai A,
Nakayama T, Udono H. Essential role of endogenous
HSP90 of dendritic cells in antigen cross-presentation.
J Immunol. 185, 2693-2700, 2010.

Figure:Models for cross-presentation of extracellularantigen by DCs after its translocation into thecytosol by HSP90. DCs take up extracellularantigen through the endosome/phagosome
pathway. Cytosolic HSP90 binds to therelatively unfolded antigen as it emerges acrossthe endosomal membranes through a putativetranslocon (Sec61 complex?) and promotes
its translocation into the cytosol. Once theextracellular antigen appears in the cytosol it
can be processed by the conventional MHC
I pathway: degraded by the proteasome into
short peptides that are loaded onto MHC I
molecules in the ER. CD8+ T cells then recognize
the MHC class I-epitope complex on the cellsurface. In model 2, the proteasome is attachedon the cytosolic face of the ER-phagosome andimmediately degrades translocated antigen into
short peptides. The peptides then re-enter the
ER-phagosome from which it was translocated.

in the intracellular antigenic milieu. Each step towards prote-cess that was blocked by free 6H8 mAb, indicating that theolysis by the proteasome is regulated, at least in part, bycross presentation truly depends on cell surface HSP90.

HSPs.

Alexa-labeled 6H8 mAb accumulated mainly on CD8 + and to

Regarding how HSPs are involved in cross-presentation,a lesser extent on CD8 - DCs in vivo one hour after intrave-

recently

retro-

nous injection. Administration of less than a microgram of atranslocation of proteins from the endosome ( PNAS 2011) .

6H8-OVA conjugate induced vigorous proliferation of adop-

Cross-presentation and -priming were decreased in both
HSP90α-null-DCs and -mice. CD8α+ DC apoptosis mediatedtively transferred OT-I CD8 + T cells, a response that did notoccur in DC-deficient mice or TAP ( transporter associatedwedemonstrated

that

HSPs

directby translocation of exogenous cytochrome c to the cytosolwith antigen processing ) 1-deficient mice. Furthermore,

was also eliminated in HSP90α-null mice. Ag translocation

6H8-OVA induced CTL in naïve mice protected them from

into the cytosol was diminished in HSP90α-null DCs and inlung metastasis with melanoma MO5 cells expressing OVA.

DCs treated with an HSP90 inhibitor. Ag within purified pha-Thus, targeting cell surface HSP90 by 6H8 coupled antigens

gosomes was released in an HSP90-dependent manner.
Our results demonstrate that cytosolic HSP90 pulls endo-

of interest enables cross-priming but not cross-tolerance ofantigen-specific CD8 + T cells, raising the possibility of asomal Ag out into the cytosol during cross-presentation.

novel antibody-based vaccine strategy for induction ofcytotoxic T cell immunity.

The role of cell surface hsp90 on dendritic cells.

In addition, we are currently investigating more funda-

With a novel mAb, 6H8, we found that HSP90 is alsomental aspects of this process – the physiological signifi-

expressed on the surface of bone marrow derived dendritic
cells ( BMDCs ) , macrophages and splenic DCs after heatcance of cell surface hsp90 and the mechanism by which

killed Propionibacterium acnes injection. The 6H8 mAbrophages. A full answer to these questions may open an

bound to DCs was rapidly internalized. Incubation of 6H8
coupled with OVA ( 6H8-OVA ) with DCs resulted in efficientavenue to a novel role of the molecular chaperone hsp90 in

hsp90 is localized on the cell surface of DCs and mac-immunity.

cross-presentation of the dominant OVA 257-264 epitope, a pro-

4. Ohkusu-Tsukada K, Toda M, Udono H, Kawakami Y,
Takahashi K. Targeted inhibition of the p38 MAP kinaseof IL-10-secreting CD25- T regulatory cells in cancer
immunotherapy. Eur J Immunol. 40,1011-1021, 2010.

5. Udono H, Ichiyanagi T, Mizukami S, Imai T. Heat
Shock Proteins in Antigen Trafficking -Implications
on Antigen Presentation to T cells- Intl J
Hyperthermia. 25, 617- 625, 2009.

101

Laboratory for

Immune Regulation
Group Director :Masaru Taniguchi
Senior Scientist :Hiroshi Watarai

Research Scientists : Takuya Tashiro

		
		
		

Michishige Harada
Diana Eissens		
Masao Shiozaki

Visiting Scientist :Nyambayar Dashtsoodol

Technical Staff :		

Sakura Sakata
Etsuko Sekine-Kondo		( until Aug 2011)

		

Yuko Nagata
Tomokuni Shigeura

Student Trainee :Yue Ren (IPA)

Intern : 				( from Jan 2012)

		

N

Sabrina Hamouche 		KT cells act as innate immune cells but also bridge the

A Phase I/IIa clinical trial has been carried out on 17 patients

innate and acquired immune systems. The nature ofwith advanced lung cancer. 60% of patients with high IFN-γ
production had a median survival time ( MST ) of 31.9 months,

the NKT cell response is dictated by the initial cytokine environment: interaction with IL-10-producing cells induces
negative regulatory TH 2/regulatory T cell-type NKT cells,

and the disease was stable with only with a primary treat-

while that with IL-12-producing cells results in pro-inflammatory TH1-type responses. A prime example of one such func-

molecular target drugs, where the MST was 10 months( p=0.0015 ) . These results are encouraging and warrant fur-tion is adjuvant activity: NKT cells augment anti-tumor

ther evaluation of the survival benefit of this immunotherapy.

responses by their production of IFN-γ, which acts on NK

For the next step, we have applied in 2011 for thecells to eliminate MHC negative target tumor cells and also

advanced medical care assessment system established inon CD8 cytotoxic T cells to kill MHC positive tumor cells.
Thus, upon administration of α-GalCer-pulsed DCs, both2008 by the Japanese Ministry of Health, Labor and Welfare( MHLW ) , so that the patients’ immunotherapy treatment will

MHC negative and positive tumor cells can be effectively

be covered in part by health insurance. The system waseliminated. Based on these findings, we have developed

established because advanced medical technologies using

NKT cell-targeted adjuvant cell therapies with strong antitu-

medical devices or pharmaceuticals, such as immune cell

mor activity in humans.

therapy, that are not yet approved under the Pharmaceutical
Affairs Law ( PAL ) , are not covered by health insurance in

A phase I-II clinical study of α-galactosylceramide(α-GalCer) -pulsed IL-2/GM-CSF-cultured peripheral
blood mononuclear cells in patients with advanced
and recurrent non-small cell lung cancer.

Japan. However in response to recent rapid progress inment. This is significantly more effective than treatment withmedical technology and the patients’ need to have safe andlower cost treatment with such technologies, this system
was introduced to allow health insurance payments. It is also

Recent publications
1. Watarai H, Sekine-Kondo E, Shigeura T, Motomura
Y, Yasuda T, Satoh R, Yoshida H, Kubo M, Kawamoto
H, Koseki H, Taniguchi M. Development and Functionof Invariant Natural Killer T cells Producing TH2- andTH17-cytokines. PLoS Biol. 10, e1001255 ( 2012) .

102

2. Motomura Y, Kitamura H, Hijikata A, Matsunaga Y,
Matsumoto K, Inoue H, Atarashi K, Hori S, Watarai H,
Zhu J, Taniguchi M, Kubo M. E4bp4, a mammalianbasic leucine zipper transcriptinal factor, is a criticalregulator of IL-10 and IL-13 production by CD4
T cells. Nat Immunol. 12, 450-459 ( 2011) .

3. Watarai H, Fujii SI, Yamada D, Rybouchkin A, Sakata S,
Nagata Y, Iida-Kobayashi M, Sekine-Kondo E, Shimizu K,
Shozaki Y, Sharif J, Matsuda M, Mochiduki S, Hasegawa
T, Kitahara G, Endo T, Toyoda T, Ohara O, Harigaya KI,
Koseki H, Taniguchi M. Murine induced pluripotent stem
cells can be derived from and differentiate into naturalkiller T cells. J Clin Invest. 120, 2610-2618 ( 2010).

Figure :the case that the collection of appropriate clinical researchdata is facilitated by this system, and thus can then lead toapproval of the advanced medical technologies under the
PAL. The NKT cell-targeted therapy was recently ( October,2011) approved by the government to use the advanced

The NKT cell subsets. NKT cells canbe divided into four subsets based on
the expression of IL-17RB and CD4.
The newly identified IL-17RB+ CD4- andIL-17RB+ CD4+ NKT cells respond to
IL-23 and IL-25, respectively, triggering
inflammation by the production of
TH2/9/17 cytokines, especially in the lung.

adapted for allogeneic NKT cell-targeted therapy in humans( Patent application ) .

Development and function of NKT cells producing
TH 2- and TH17-cytokines.
NKT cells are heterogeneous in terms of the expression of
CD4 and the IL-17 receptor B ( IL-17RB ) , a receptor for IL-25

medical care assessment system.

Induced pluripotent stem cells (iPSCs ) -derived NKT
cells are transplantable without acute graft-vshost disease ( GvHD ) .

pathway and precise function of these NKT cell subtypes

NKT cells show antitumor activity when activated to produce

mic CD44 +/- NK1.1- population and develop normally even in

Th1 cytokines by DCs loaded with α-GalCer. However, mostthe absence of IL-15, which is required for maturation andcancer patients do not have sufficient numbers of NKT cells

homeostasis of IL-17RB - NKT cells producing IFN-γ. These

to induce an effective immune response in this context, indi-

results suggest that NKT cells contain at least two subsets,cating a need for a source of NKT cells that could be used to

IL-17RB + and IL-17RB -. The IL-17RB + NKT cells can be further

supplement the endogenous cell population. iPSCs holddivided into two subtypes on the basis of CD4 expression,

tremendous potential for cell-replacement therapy, and we
successfully derived iPSCs both from embryonic fibroblasts

both in the thymus and in the periphery. CD4 + IL-17RB + NKT
cells produce TH 2 ( IL-13 ) , TH 9 ( IL-9 and IL-10 ) and TH17 ( IL-17A

from mice harboring functional NKT cell-specific rearranged

and IL-22 ) cytokines in response to IL-25 in an E4BP4-

T cell receptor loci in the germline and from splenic NKT

dependent fashion, whereas CD4- IL-17RB + NKT cells are aretinoic acid receptor-related orphan receptor ( ROR ) γt + sub-cells from adult B6 mice. These iPSCs could be differenti-

and a key factor in TH 2 immunity. However, the development
remain unknown. IL-17RB + NKT cells are present in the thy-

ated into NKT cells in vitro and secreted large amounts of theTH1 cytokine IFN-γ. Importantly, iPSC-derived NKT cells

set producing TH17 cytokines in an E4BP4-independent

recapitulated the known adjuvant effects of natural NKT cells

are abundant in the lung in the steady state and mediate thepathogenesis in virus-induced airway hyperreactivity ( AHR ) .

and suppressed tumor growth in vivo. Furthermore, iPSCderived B6 NKT cells, in contrast to B6 CD4 helper T cells,

fashion upon stimulation with IL-23. The IL-17RB + NKT cells

did not induce acute GvHD when transferred into T/B/NKT-In this study we demonstrated that the development of IL17RB + NKT cell subsets is distinct from the classical NKT

deficient Rag1-knockout BALB/c mice. These studies dem-

cell developmental stages in the thymus and that these IL-

onstrate the feasibility of expanding functionally competent

17RB + NKT cells play important roles in the pathogenesis of

NKT cells via an iPSC phase, an approach that may be

airway diseases.

4. Matsuoka N, Itoh T, Watarai H, Sekine-Kondo
E, Nagata N, Okamoto K, Mera T, Yamamoto H,
Yamada S, Maruyama I, Taniguchi M, Yasunami
Y. High-mobility group box 1 is involved in theinitial events of early loss of transplanted islets inmice. J Clin Invest. 120, 735-743 ( 2010).

5. Watarai H, Rybouchkin A, Hongo N, Nagata Y, Sakata
S, Sekine E, Dashtsoodol N, Tashiro T, Fujii SI, Shimizu
K, Mori K, Masuda K, Kawamoto H, Koseki H, Taniguchi
M. Generation of functional NKT cells in vitro fromembryonic stem cells bearing rearranged invariant
Vα14-Jα18 TCRα gene. Blood 115, 230-237 ( 2010) .

6. Motohashi S, Nagato K, Kunii N, Yamamoto H, Yamasaki
K, Okita K, Hanaoka H, Shimizu N, Suzuki M, Yoshino
I, Taniguchi M, Fujisawa T, Nakayama T. A phase
I-II study of alpha-galactosylceramide-pulsed IL-2/
GM-CSF-cultured peripheral blood mononuclear cells
in patients with advanced and recurrent non-small celllung cancer. J Immunol. 182, 2492-2501 (2009) .

103

Laboratory for

Dendritic Cell
Immunobiology

Team Leader :Katsuaki Sato
Technical Staff : Kaori Sato

Yumiko Sato​
Azusa Shibazaki
Haruna Otsuka
Student Trainee : Hideaki Takagi (JRA)D

endritic cells ( DCs ) are essential antigen-presenting
cells ( APCs ) that initiate primary immune responses.

periphery, a function of likely importance in self-tolerance as

DCs are heterogeneous, but the two major subsets are conventional DCs ( cDCs ) and plasmacytoid DCs ( pDCs ) , which

the precise functional role of each DC subset in immune

are distinguishable by surface and intracellular phenotypic

DC subsets in the immune system in vivo and to identify themarkers, immunologic function, and anatomic distribution.
Immature DCs ( iDCs ) serve as sentinels, recognizing themolecular basis for the regulation of their function.

presence of invading pathogens or virus-infected cells

Crucial roles of B7-H1 and B7-DC expressed on
mesenteric lymph node dendritic cells in thegeneration of antigen-specific CD4 + Foxp3 +regulatory T cells in the establishment of oral
tolerance

through various pattern-recognition receptors. Subsequently, they become mature DCs ( mDCs ) with up-regulated
expression of MHC and costimulatory molecules under
inflammatory conditions. The mDCs then migrate via thewell as immune disorders and transplant rejection. However,
responses remains unclear. Our goal is to clarify the role ofafferent lymphatics into the T-cell areas of secondary lym-

Oral tolerance, which generates systemic tolerance to fed

phoid tissues, where they prime rare antigen-specific naive T
cells for differentiation into effector T ( Teff ) cells, including T
helper type ( TH ) 1 cells, TH 2 cells, and TH17 cells, depending

antigens, is a key feature of intestinal immunity. However, theon environmental cues. DCs thereby play a crucial role inof costimulatory molecules in the establishment of oral toler-linking innate and adaptive immunity. Conversely, iDCs areance. Deficiencies of B7-H1 and B7-DC abrogated oral tolerance, accompanied by enhanced antigen-specific CD4 +T

also crucial for the induction of immunological tolerance
under steady-state conditions. The tolerogenic mechanisms
include recessive tolerance mediated by clonal deletion andanergy as well as dominant tolerance involving active immune
suppression by CD4 + Foxp3 + regulatory T ( Treg ) cells in themolecular mechanism mediating oral tolerance remains
unclear. In this study, we examined the role of the B7 familycell-responses and IgG1 production. Mesenteric lymph node
( MLN ) dendritic cells ( DCs ) express higher levels of B7-H1
and B7-DC than systemic DCs, whereas they have similarlevels of CD80, CD86, and B7-H2. Owing to their dominant

Recent publications
1. Takagi H., Fukaya T., Eizumi K., Sato Y., Sato K.,
Shibazaki A., Otsuka H., Hijikata A., WatanabeT., Ohara O., Kaisho T., Malissen B., Sato, K.
Crucial role of plasmacytoid dendritic cells
in the initiation of inflammation and T cell
immunity in vivo. Immunity 35, 1-14 ( 2011)104

2. Fukaya T., Takagi H., Sato Y., Sato K., Eizumi K.,
Taya H., Shin T., Chen L., Dong C., Azuma M., Yagita
H., Malissen B., Sato K. Crucial roles of B7-H1
and B7-DC expressed on mesenteric lymph nodedendritic cells in the generation of antigen-specificCD4+Foxp3+regulatory T cells in the establishmentof oral tolerance. Blood 116, 2266-2276 ( 2010)

3. Fukaya T., Takagi H., Taya H., Sato K. DCs in
immune tolerance in steady-state conditions.
Methods Mol. Biol. 677, 113 -126 ( 2010 )Figure 1: Proposed model for the role of MLN DCs in
the establishment of oral tolerance.
In MLNs, CD103+ DCs migrating from the lamina
propria (LP) after sampling ingested antigens present
them to naïve (n)CD4+ T cells to generate CD4+Foxp3+
iTreg cells mediated through B7-H1 and B7-DC as well as
TGF-β and retinoic acid (RA). These CD4+Foxp3+ iTreg
cells dampen antigen-specific CD4+ Teff cell-responses
and that leads to the establishment of oral tolerance.

expression of B7-H1 and B7-DC, MLN DCs enhanced theantigen-specific generation of CD4 + Foxp3 + inducible regulatory T ( iTreg ) cells from CD4 + Foxp3 - T cells rather than CD4 +Figure 2: Hypothetical model for the role of Siglec-H in
the control of cytokine production by pDCs.
Siglec-H impairs the TLR9-MyD88-mediated activationof the signaling cascades for NF-κB and IKK-α in
pDCs after the recognition of self-DNA from hostdamaged cells or CpG DNA from bacteria and virus,
and that leads to the inhibition of the productionof type I IFN and proinflammatory cytokines.

ethylated CpG DNA through endosomal toll-like receptors( TLRs ) . Sialic acid binding Ig-like lectin ( Siglec ) -H is exclusively expressed on murine pDCs and is unique among

effector T ( Teff ) cells relative to systemic DCs. Furthermore,

Siglec proteins in that it associates with the ITAM-bearing

the antigen-specific conversion of CD4 Foxp3 T cells into
CD4 + Foxp3 + iTreg cells occurred to a greater extent in MLNs

adaptor molecule, DAP12. To understand how Siglec-H

compared to other peripheral lymphoid organs during oral

deficient mice. We made three fundamental observations

tolerance under steady-state conditions. Such a conversion

about the role of Siglec-H: 1. By regulating a MyD88-medi-

was much more dependent on B7-H1 and B7-DC than onated downstream signaling pathway, it controls the thresholdother B7 family members, and it was severely impaired underof responsiveness to TLR ligand for the production of

inflammatory conditions. In conclusion, our findings suggest

type I IFN and proinflammatory cytokines. 2. By controllingthat B7-H1 and B7-DC expressed on MLN DCs are essential

MHC class II expression it regulates the ability of pDCs toprime antigen-specific CD4 + T cells for the generation of
CD4 + Foxp3 + inducible regulatory T ( iTreg ) cells and CD4 ++-for establishing oral tolerance through the de novo generation of antigen-specific CD4 + Foxp3 + iTreg cells.

might regulate the function of pDCs, we generated Siglec-H-

effector T ( Teff ) cells. 3. By regulating cross-presentation of

A critical role of Siglec-H in the regulation of
inflammation and T cell immunity by plasmacytoid
dendritic cells in vivosoluble and viral antigens it is required for the capacity ofpDCs to prime antigen-specific CD8 + T cells for the generation of cytotoxic T lymphocytes ( CTLs ) . Thus, our findings

Plasmacytoid dendritic cells ( pDCs ) are specialized immune

suggest that Siglec-H plays a critical role in the function ofcells that are capable of producing large amounts of type I
interferon ( IFN ) after sensing single-stranded RNA or unm-

pDCs for the regulation of inflammation and T cell immunity

4. Sato K., Eizumi K., Fukaya T., Fujita S., Sato Y.,
Takagi H., Yamamoto M., Yamashita N., Hijikata A.,
Kitamura H., Ohara O., Yamasaki S., Saito T., SatoK. Naturally occurring regulatory dendritic cells
regulate murine cutaneous chronic graft-versushost disease. Blood 113, 4780-4789 ( 2009 )in vivo.

5. Candolfi M., Yagiz K., Foulad D., Alzadeh G.E.,
Tesarfreund M., Muhammad A.K., Puntel M., Kroeger
K.M., Liu C., Lee S., Curtin J.F., King G.D., Lerner J.,
Sato K., Mineharu Y., Xiong W., Lowenstein P.R., Castro
M.G. Release of HMGB1 in response to proapoptotic
glioma killing strategies: efficacy and neurotoxicity.
Clin. Cancer Res. 15, 4401-4414 ( 2009 )105

Laboratory for

Cytokine Signaling
Group Director :Toshio Hirano
Senior Scientists :Toshiyuki Fukada
Keigo Nishida

Research Scientists : Satoru Yamasaki

Wakana Ohashi
Shintaro Hojyo

D

Technical Staff :Ayumi Ito
Masami Kawamura
Mayumi Hara

Student Trainee :Tomohiro Miyai

espite their increasing prevalence in developed coun-

Slc39a6 ( Kitamura et al., Nature Immunology. 2006 ) ,tries, the molecular mechanisms leading to allergy andsuggesting involvement of Zn in MHC class II cell surface

autoimmune diseases remain poorly understood. The ulti-

expression through regulating endocytosis and membranemate goal of the Cytokine Signaling Research Group is totrafficking. In order to understand the role and function ofhelp elucidate the underlying molecular and immunologicalZn and Zn transporters in vivo, we have generated micemechanisms of allergy and autoimmune diseases from thedeficient in the Slc39/Zip family of zinc transporters. We
found that Slc39a13/Zip13 knockout mice ( Slc39a13-KO )viewpoint of signal transduction within the immune system.
We have shown that heavy metal cations such as Zinc ( Zn )might act as intracellular signaling molecules, i.e., molecules

show changes in connective tissues reminiscent of thehuman disease Ehlers-Danlos syndrome ( EDS ) , in which

whose intracellular status is altered in response to an extra-

there are defects in the maturation of osteoblasts, chondro-

cellular stimulus, and that are capable of transducing theextracellular stimulus into an intracellular signaling event. Zn

cytes, odontoblasts, and fibroblasts. Impairment of bonemorphogenic protein ( BMP ) and TGF-β signaling was

is known to be important in the immune system, although its

observed in the corresponding tissues and cells. Homozy-precise roles and mechanisms have not been resolved.

gosity for a SLC39A13 loss of function mutation was identi-

Therefore, we are focusing our attention on the largely

fied in sibs affected with a unique variant of EDS that

unknown universe of signaling through Zn transporter pro-

recapitulates the phenotype observed in Slc39a13-KO mice.

teins, to reveal the role of Zn in immune and other physiolog-

Hence, our results have revealed a crucial role of SLC39A13

ical systems.

in connective tissue development at least in part due to its
involvement in BMP/TGF- β signaling pathways ( Fukada etRole of Zn and its transporters in immune andnon-immune tissues

al, PLoS ONE, 2008 ) . We have found that the human ZIP13
protein forms homo-dimers ( Bin et al, J. Biol. Chem, 2011) ,We found that the Zn transporter Slc39a6/Zip6/Liv1 is aand its further characterization by crystallography has been

STAT3 target gene and showed that it has a role in cell migration during early zebrafish development. ( Yamashita et al.,under investigation. In addition, we have investigated the role

Nature. 2004 ) , and that LPS-induced maturation of DCs is

Slc39a14-KO mice showed progressive systemic growth

partly mediated through lowering the intracellular concentra-

defects, and impaired signaling of G-protein-coupled receptors ( GPCR ) in the growth plate, pituitary grand, and livertion of free Zn by down regulating Zn transporters, includingof Slc39a14 by generating genetically deficient mice. The

Recent publications
1. Bin, B-H., T. Fukada, T. Hosaka, S. Yamasaki, W.
Ohashi, S. Hojyo, T. Miyai, K. Nishida, S. Yokoyama
and T. Hirano. Biochemical characterization of humanZIP13 protein: a homo-dimerized zinc transporter
involved in the Spondylocheiro dysplastic Ehlers-Danlos
syndrome. J. Biol. Chem. 286, 40255-40265 ( 2011)

106

2. Nishida K., S. Yamasaki, A. Hasegawa, A. Iwamatsu, H.
Koseki, and T. Hirano. Gab2, via PI-3K, Regulates ARF1
in FcεRI-Mediated Granule Translocation and Mast Cell
Degranulation. J. Immunol. 187, 932-941 ( 2011)

3. Murakami, M*., Y. Okuyama*, H. Ogura*, S. Asano,
Y. Arima, M. Tsuruoka, M. Harada, M. Kanamoto,Y. Sawa, Y. Iwakura, K. Takatsu, D. Kamimura, T.
Hirano. ( *equal contribution) Local microbleeding
facilitates IL-6– and IL-17–dependent arthritis in theabsence of tissue antigen recognition by activatedT cells. J. Exp. Med. 208, 103-114 ( 2011)

Figure 1: Schematic model of the regulation of GPCR-mediatedsignaling by Slc39a14. Slc39a14 regulates basal cAMPlevels by suppressing PDE activity, which enhances the
GPCR-cAMP-CREB pathway to control systemic growth.

Figure 2: Model of the Fc epsilon RI-mediated Fyn/Gab2/
PI-3K/ARF1 signaling pathway involved in granuletranslocation and mast cell degranulation.
Fc epsilon RI activates the Src family kinase Fyn, which
phosphorylates/activates Gab2, which then associates with theSH2 domain of PI-3K. PI-3K controls granule translocation,
degranulation, and anaphylaxis responses via ARF1 activation.

was observed. We found that Slc39a14-mediated Zn trans-

mast cells in allergy, inflammation, and autoimmune diseases

port plays a role in the control of GPCR-cAMP response
element-binding protein ( CREB ) signaling via regulating

are being investigated. We have dissected the degranulationcAMP levels by suppressing phosphodiesterase ( PDE )activity ( Figure 1, Hojyo et al, PLoS ONE, 2011) , suggesting a

microtubule polymerization and granule translocation to the

crucial role of Slc39a14 in control of mammalian systemic

ond, the granules fuse with the plasma membrane in a well-

growth.

characterized calcium-dependent manner. Furthermore, we

Recently, we showed that Zn suppresses Th17-mediatedautoimmune diseases by inhibiting the development of Th17

showed that the Fyn/Gab2/RhoA, but not the Lyn/SLP-76

cells via attenuating STAT3 activation. In mice injected withpendent microtubule-dependent pathway. At present, we

type II collagen to induce arthritis, Zn treatment inhibitedare especially focused on clarifying the molecular mecha-

Th17 cell development. IL-6-mediated activation of STAT3
and in vitro Th17 cell development were all suppressed by Zn.

nisms

Importantly, Zn binding changed the α-helical secondary

granule translocation and degranulation, we establishedstructure of STAT3, disrupting the association of STAT3 with

Gab2 knock-in mice that express Gab2 mutated in either the

JAK2 kinase and with a phospho-peptide that included a

PI-3K- or SHP-2-binding sites. We found that Gab2 knock-in

STAT3-binding motif from the IL-6 signal transducer gp130.

mice that express Gab2 mutated in either of these bindingThus, we conclude that Zn suppresses STAT3 activation,
which is a critical step for Th17 development ( Kitabayashi etal, International Immunology, 2010 ) .

sites had defective mast cell-mediated immediate-type aller-

Together these results support the idea that Zn has

ing site was involved in Fc epsilon RI-dependent ARF1 acti-

roles for mediating and controlling intracellular signaling

vation and granule translocation to the plasma membrane in

events. We propose the term “Late Zn signaling” for this typethe degranulation process, further dissecting the signalsof Zn signaling, in contrast to “Early Zn signaling”. The former

required for degranulation. Finally, we revealed the molecular

is dependent on changes in the transcription of Zn transporter genes, whereas the latter is not ( Hirano et al, Adv.

framework of the Fyn/Gab2/PI-3K-dependent ARF1-medi-

Immunol., 2008 ) .

process of mast cells. First, Fc epsilon RI stimulation triggers
plasma membrane in a calcium-independent manner. Sec-signaling pathways play a critical role in the calcium-inde-ofcalcium-independent

microtubule-dependent

granule translocation. In order to clarify how Gab2 mediates

gic responses, but not delayed-type responses. We also
showed that the PI-3K-binding site but not the SHP-2-bind-ated granule translocation and mast cell degranulation( Fig. 2 ) . Thus, these findings provided us with potential
therapeutic targets.

Molecular Mechanisms of Mast Cell degranulation
Immunological and molecular mechanisms of the role of

4. The Zinc Transporter SLC39A14/ZIP14 Controls
G-Protein Coupled Receptor-Mediated Signaling
Required for Systemic Growth. Hojyo, S.*, T. Fukada*,
S. Shimoda, W. Ohashi, B-H. Bin, H. Koseki, T. Hirano.
PLoS ONE 6 (3) : e18059 (2011) ( *Equally contribution)

5. Kitabayashi C., T. Fukada, M. Kanamoto1, W. Ohashi,
S. Hojyo, T. Atsumi, N. Ueda, I. Azuma, H. Hirota,
M. Murakami, and T. Hirano. Zinc suppresses
Th17 development via inhibition of STAT3
activation. Int. Immunol. 22, 375-386 ( 2010)

6. Nishida K*., A. Hasegawa*, S. Nakae, K. Oboki,
H. Saito, S. Yamasaki, and T. Hirano. ( *equal
contribution) Zinc transporter Znt5/Slc30a5 isrequired for the mast cell-mediated delayed-typeallergic reaction but not the immediate-type
reaction. J. Exp. Med. 206, 1351-1364 ( 2009 )107

Laboratory for

Immunogenetics

Team Leader :Hisahiro Yoshida
Research Scientist : Takuwa Yasuda
Technical Staff :T

he main activity of our team is the screening of a large
ethylnitrosourea ( ENU ) mutant mouse panel and is acollaborative effort with the RIKEN Genomic Sciences

Hitomi Kodera
Akimi Banno
Ayako Kobayashi
Mikiko Sato
Takeyuki Goto

3,000 sites throughout the entire genome, resulting inapproximately 100 sites in protein coding regions per firstgeneration ( G1) mutant mouse.

Center. An important goal of this project for RCAI is theWe have analyzed the mutant phenotypes by patho-

development of novel mouse models for various immuno-

logical, cytological and molecular biological evaluations oflogical disorders, notably allergic and autoimmune disorders.

affected tissues, lymphoid organs and blood cells.

Since ENU mutagenesis introduces approximately 3,000

The mutant loci responsible for any abnormal pheno-

point mutations in a genome, we can expect 100 coding

types identified in candidate lines are mapped by backcross-region mutations in one pedigree. In parallel, we are screen-

ing mutant individuals with the C3H/HeJ strain for geneing the mutant mice under environmental bias, using a

detection by single-nucleotide polymorphism analysis. Can-variety of approaches including immunization with allergens

didate gene mapping has been done in collaboration withand adjuvants to identify allergy modifier genes.

the Phenome Informatic Team and Mouse Mutation Resource
Exploration Team in the GSC, RIKEN

ENU mutant panel study
In order to identify the genetic basis for immune disorders,Allergic disease model mutant mouse model

we are screening a pool of mutant mice generated by ran-

Using this approach, we identified and established a mutant

dom

randommouse line with phenotypic features reminiscent of a typicalsingle-base pair changes in genomic DNA at approximately

human allergic disease. The ear skin became thicker and red

chemicalmutagenesis.

ENU

induces

Recent publications
1. Sugiyama M, Nakato G, Jinnohara T, Akiba H,
Okumura K, Ohno H and Yoshida, H Expression
pattern changes and function of RANKL during
mouse lymph node microarchitecture development.
Int Immunol. 24, 369-378 (2012)108

2. Tachibana M, Tenno M, Tezuka C, Sugiyama M, Yoshida
H, Taniuchi I. Runx1/Cbfβ2 complexes are required
for lymphoid tissue inducer cell differentiation at twodevelopmental stages. J Immunol. 186 ,1450-7 (2011)

3. Matsushima Y, Kikkawa Y, Takada T, Matsuoka K, Seki
Y, Yoshida H, Minegishi Y, Karasuyama H, Yonekawa H.
An Atopic Dermatitis-Like Skin Disease with HyperIgE–Emia Develops in Mice Carrying a Spontaneous
Recessive Point Mutation in the Traf3ip2 (Act1/
CIKS ) Gene. J Immunol. 185,2340-9 ( 2010 )Figure 1: An atopic dermatitis-like mutant
mouse was established from the
ENU screen.
The diagram illustrates the
stepwise progression of AD
disease in the mutant animals.

Figure 2: Summary of four years of
ENU mutant screening.
In total, approximately 8,000 mutant
mice from 80 genomes have been
screened and 145 phenotypes
related to immunity or allergy
have been identified. The images
in the bottom right show the 3
dimensional renderings of the lungsof WT and mutant mice after OVA
immunization and challenge.

and, as the mice aged, they started to scratch the ear skin orand established more than 145 mutant lines with immune orface. Finally, they developed chronic facial and ear skinblood disease phenotypes. In keeping with our expectation,

inflammation and increased levels of not only Th2 serum

more than 50 lines had phenotypes related to allergic dis-immunoglobulins but also Th1 serum immunoglobulins. Thiseases. From these mutant lines, we are now analyzing a

stepwise development of symptoms and other findings aremutant line with features of experimental allergic asthma,compatible with the criteria for human infantile atopic dermatitis ( AD ) . We have mapped the phenotype-causing point

rhinitis and conjunctivitis by OVA immunization.

mutation to a distinct gene and directly confirmed this cau-pendent mutant loci to distinct regions, and nine of themsality by genetic manipulation to introduce the same muta-

have been identified to be point mutations in independenttion into the gene of wild type mice. We have clarified thegenes. Along with allergic disease model mutant screening,disease onset mechanism and succeeded in disease onset
prevention based on the results of our studies ( manuscript

we have identified and characterized a few other interestingunder submission ) . We are now further analyzing the step-

listed in the publications section. One is a good model forwise progression of this AD-like disease in the mutant mousethe human neurodegenerative disease infantile neuroaxonal
dystrophy ( INAD ) ( Wada et al., 2009 ) , and another is a pointin combination with various gene-manipulated mice to better
understand the allergic disease progression in skin and in
the immune system.
By ENU recessive mutant screening, we have identified

4. Wada H., Yasuda T., Miura I., Watabe K., Sawa C.,
Kamijuku H., Kojo S., Taniguchi M., Nishino I., WakanaS., Yoshida H., Seino K. Establishment of an improved
mouse model for infantile neuroaxonal dystrophy thatshows early disease onset and bears a point mutationin Pla2g6. Am J Pathol. 175, 2257-63 ( 2009 )As of December 2011, we have mapped thirty-two inde-

phenotypes in mutant lines with/without allergic defects asmutation in the Themis gene, which we found plays animportant role in thymocyte development ( Kakugawa et al.,2009 ) .

5. Kakugawa K., Yasuda T., Miura I., Kobayashi A.,
Fukiage H., Satoh R., Matsuda M., Koseki H.,
Wakana S., Kawamoto H., Yoshida H. A novel geneessential for the development of single positive
thymocytes. Mol Cell Biol. 29 , 5128-35. ( 2009)109

Laboratory for

Vaccine Design

Team Leader :Yasuyuki Ishii
Research Scientists : Hidetoshi Akimoto

Satoshi Komaniwa
Emi FukudaIn the course of research and development for cedar pollen
allergy vaccines, we had found that a liposomal form ofVisiting Scientists :Omar Duramad
Kenichi Masuda

Technical Staff :Haruka Katagiri
Risa Nozawa

antigen-specific phenomenon could be reproduced by the
adoptive transfer of CD4 + CD25 +T cells derived from mice

α-GalCer, but not an aqueous one, could predominantly

treated with the liposomal α-GalCer encapsulated OVA. We

enhance the immunoregulatory functions of invariant Natural
Killer T ( iNKT ) cells, such as IL-10 production. Then, we

are now trying to elucidate the precise mechanism of antigen-specific IgE suppression by the CD4 + CD25 +Treg cells.

attempted to suppress IgE antibody formation in a mouse
In the case of primary IgE antibody formation, a systemic

Mechanism for generation of CD4 + CD25 + regulatory
T cells by liposomal α-GalCer -antigen

injection of liposomal α-GalCer prior to the immunization

Last fiscal year, we found that rhodamine-labeled liposomal

with alum-absorbed antigen resulted in the remarkableα-GalCer was incorporated into splenic B220 + CD1d high cells,

suppression of not only IgE but also IgG antibody. However,

but not B220 + CD1d low cells, and much more than into CD11b +the secondary antibody response of fully antigen-sensitizedmacrophages or CD11c + dendritic cells. Immunohistochemi-

mice after antigen challenge was not suppressed by evencal observation clearly showed that rhodamine-positive dots

repeated systemic injection of liposomal α-GalCer. Since itcorresponding to B220 + CD1d high cells were localized at theallergy model by the administration of liposomal α-GalCer.

had been suggested that regulatory T cells such as Foxp3-

marginal zone area in spleen and that co-localization of iNKT

positive naturally occurring Treg cells or IL-10-producing Tr1

cells after the rhodamine-liposome injection could becells might be involved in the suppression of the secondary

detected in the mice transferred NKT-ES-V cells in whichantibody response, we tested whether the liposomal

IRES-Venus was inserted downstream of the TCR Cα stop

α-GalCer encapsulating an antigenic polypeptide would
generate antigen-specific Treg cells. We demonstrated thatcodon in NKT-ES cells (1). The expression of CXCL16, aligand for CXCR6, on rhodamine-positive B220 + CD1d highthe systemic administration of liposomal α-GalCer encapsu-

cells and CD11c + cells was analyzed since CXCR6 is a major

lated OVA could induce suppression of the secondary anti-

chemokine receptor on iNKT cells. The result of cell surface

OVA IgE antibody response in OVA-immunized mice but not

staining with anti-CXCL16 mAb showed that the expressionthat in cedar antigen Cry j 2-immunized mice. Moreover, thislevel of CXCL16 on B220 + CD1d high cells and CD11c + cells

Recent publications
1. Duramad O, Laysang A, Li J, Ishii Y and Namikawa
R, Pharmacologic Expansion of Donor-Derived,
Naturally Occurring CD4 (+) Foxp3 (+) Regulatory
T Cells Reduces Acute Graft-versus-Host Disease
Lethality Without Abrogating the Graft-versusLeukemia Effect in Murine Models. Biol Blood
Marrow Transplant 17 , 1154-1168, 2011110

2. Okayama T, Matsuno Y, Yasuda N, Tsukui T, Suzuta
Y, Koyanagi M, Sakaguchi M, Ishii Y, Olivry T, Masuda
K. Establishment of a quantitative ELISA for themeasurement of allergen-specific IgE in dogs using
anti-IgE antibody cross-reactive to mouse and dog IgE.
Vet Immunol Immunopathol. 139, 99-106, 2011

3. Ishii Y, Motohashi S, Shimizu K, Nakayama
T, Taniguchi M, and Fujii S, Applicationof NKT cells in immunotherapy, Curr
Immunol. Rev. 6, 109 -115, 2010

response in mice treated with liposomal α-GalCer
encapsulated antigen. In future studies, we willdefine the characteristics, such as Foxp3, IL-10
and/or IL-21, of CD4 + CD25 +Treg cells expanded byin vivo treatment with liposomal α-GalCer encapsulated OVA.
1) Watarai et al. ( 2010 ) Blood 115 ( 2 ) :230-237.

Establishment of humanized cedar pollen
allergy mice
Based on the RCAI mission, we have already produced two types of allergy vaccines for Japanesecedar pollinosis. One is a polyethylene glycol ( PEG ) modified recombinant fusion protein of Cry j 1 andCry j 2, which are major cedar pollen antigens, theother is the same fusion protein encapsulated in
Figure :Correlation between human CD19-positive B cell expansion andhuman lgE production in NOD/SCID/ll2rγKO mice
infused with PBMCs from allergic volunteers.

α-GalCer liposomes. Though both vaccine projects
are still in the pre-clinical study phase, new systems
for prediction of human efficacy and safety areneeded before going into clinical studies. We

was quite low in unmanipulated mice, but was augmented

started a program for the establishment of humanized cedar

after rhodamine-liposome injection, suggesting the involve-

pollen allergy mice, in which there would be production ofment of the CXCR6/CXCL16 system. To test whether CXCR6/

human IgE antibody specific for cedar antigens and a type I

CXCL16 was involved in the interaction of the iNKT cells with
the B220 + CD1d low cells, neutralizing antibody against

allergic reaction including anaphylaxis following antigen

CXCL16 and rhodamine-labeled liposomes were co-admin-als with the highest CAP-RAST value were selected for aistered into the NKT-Venus cloned mice. Immunohistochem-transfer experiment. Their peripheral blood mononuclear

challenge. Among cedar pollinosis volunteers, three individu-

ical analysis showed that iNKT cells failed to take up

cells were infused into NOD/SCID/Il2rgKO mice, which wereresidence in the marginal zone area. To determine the sig-

simultaneously immunized with the cedar antigen Cry j 1.

nificance of the interaction of iNKT cells with marginal zone
B220 + CD1d high cells, cytokine expression of splenic iNKTAfter ten days, the recipient mice were boosted by intraperi-cells after the injection of liposomal α-GalCer with or without

titers in sera were measured by ELISA 21 days after the firstanti-CXCL16 neutralizing mAb was analyzed by Q-PCR. The
result showed that IL-10 and IL-21mRNA expression among

immunization. In mice that had received peripheral bloodmononuclear cells of one volunteer ( #015 ) , both total human

toneal injection with antigen. Total IgE and anti-Cry j 1-IgE

the tested cytokines was remarkably reduced in iNKT cells

IgE and anti-Cry j 1-IgE antibody were detected even though

recovered from the mice treated with anti-CXCL16 neutraliz-

their levels were much lower than that in the blood of thising mAb, suggesting that IL-10 and IL-21 production by iNKT

volunteer. No IgE was detected in recipients of cells from theother two volunteers ( #002 & #008 ) . As shown in the figure,cells might be indispensable for the interaction with the liposomal α-GalCer-pulsed B220 + CD1d high cells. We are now
speculating that this IL-10 production could play a role in thehuman CD19-positive cells were not observed in the spleenof the non-IgE + mice but were found in the high IgE mice,

expansion of CD4 + CD25 +Treg cells, while IL-21 production

suggesting that the some sort of treatment to maintain sur-could be involved in the inhibition of IgE class switching. In

vival and induce the IgE class switch of human CD19-positive

our previous studies, IL-10 expression by α-GalCer-pulsed

B cells in NOD/SCID/Il2rgKO recipient mice will be essential.

B220 + CD1d high cells after their interaction with iNKT cells

We are now examining the effect of infusing antigen-pulsed

was much higher than that of the iNKT cells, and this influ-

peripheral blood mononuclear cells, with or without human

enced the expansion of Foxp3 + CD4 + CD25 +Treg cells.

IL-4, into NOD/SCID/Il2rgKO mice. We hope to find condi-

Although IL-21 production by iNKT cells may partly explain

tions to extend the survival of the human B cells in the trans-

the IgE suppression mechanism, it may not explain the sup-ferred mice for the purpose of long term observation ofpression of the antigen-specific secondary IgE antibody

vaccine safety.

111

Laboratory for

Human Disease Models

Group Director :Fumihiko Ishikawa
Senior Scientist : Yoriko Saito
Technical Staff :Nahoko Suzuki
Mariko Tomizawa-Murasawa
Akiko Sone
Hiroshi Kajita
Ikuko OgaharaStudent Trainees : Yuki Aoki (JRA)Shinsuke Takagi (JRA)Yuho Najima (JRA)S

ince 2006, our lab has been focusing on the investiga-

to explore the possibility of drug development in order totion of normal and diseased human hematopoiesis andeliminate chemotherapy-resistant AML stem cells. Finally, in

immunity through creation of humanized mouse models.

collaboration with other laboratories within RCAI, we intend

In this fiscal year, we have created a humanized mouseto analyze hematopoietic development from human iPS cells

model with expression of membrane-bound human Kit

in vitro and in vivo.

ligand/stem cell factor. Humanization of the mouse bonemarrow microenvironment has led to nearly 100% humanhematopoietic repopulation and efficient myeloid develop-

Creation of hSCF transgenic humanizedmouse model

mast cells in spleen and gastrointestinal mucosa, which may

We have aimed to improve lymphoid-skewed differentiationof transplanted human hematopoietic stem cells ( HSCs ) inenable us to apply this new humanized mouse model to thethe bone marrow of the humanized mouse. Since inefficientarea of allergy research.

ment. The reconstituted mice also have abundant human

In addition to the creation ofhuman myeloid development could be attributed to theimmune-compromised mice with a humanized microenvi-

mouse microenvironment not fully supporting differentiationronment, we have aimed to create in vivo models for humandiseases such as leukemia, primary immunodeficiency, andand maturation of human myeloid lineage cells, we createdmembrane-bound form of human Kit ligand ( KL ) /stem cellvirus infection. In the leukemia study, we found distinct

factor ( SCF ) transgenic mice in collaboration with Dr. Leon-mechanisms of leukemogenesis between adult myeloid leukemia ( AML ) and infant lymphoid leukemia and have started

ard D. Shultz at The Jackson Laboratory. Transplantation ofcord blood-derived human HSCs resulted in significantly

Recent publications
1. Takagi S, Saito Y, Hijikata A, Tanaka S, Watanabe T,
Hasegawa T, Mochizuki S, Kunisawa J, Kiyono H, Koseki
H, Ohara O, Saito T, Taniguchi S, Shultz LD, Ishikawa
F. Membrane-bound human SCF/KL promotes invivo human hematopoietic engraftment and myeloid
differentiation. Blood, 119, 2768-2777, 2012112

2. Shultz LD, Saito Y, Najima Y, Tanaka S, Ochi T,
Tomizawa M, Doi T, Sone A, Suzuki N, Fujiwara H,
Yasukawa M, Ishikawa F. Generation of functional
human T-cell subsets with HLA-restricted immune
responses in HLA class I expressing NOD/SCID/
IL2rγ null humanized mice. Proc. Natl. Acad.
Sci. U.S.A. 107, 13022-13027, 2010

3. Saito Y, Uchida N, Tanaka S, Suzuki N, TomizawaMurasawa M, Sone A, Najima Y, Takagi S, Aoki Y, Wake A,
Taniguchi S, Shultz LD, Ishikawa F. Induction of cell cycle
entry eliminates human leukemia stem cells in a mousemodel of AML. Nat. Biotech. 28, 275-280, 2010.

Figure :Creating a novel NSG strain with ahumanized BM microenvironment.
(A) To humanize the bone marrow
microenvironment, we created a newstrain of NSG mice expressing humanmembrane-bound stem cell factor.
Transplantation of human cord bloodderived HSCs resulted in higher levelsof human hematopoietic engraftment
in hSCF TG NSG recipients comparedwith non-TG NSG controls. (B,C) In particular, the developmentof human myeloid lineage cells
became more efficient in the human
SCF-expressing humanized mice.

higher long-term engraftment of human leukocytes in the

IFN-γ production by humanized mouse-derived CD8 + T cells

bone marrow, spleen, and peripheral blood of hSCF Tg NSG

in an HLA-restricted manner. In addition to the HLA-restricted

recipients compared with those of non-transgenic NSG

human CTL response against virus-derived antigens, werecipients. In the bone marrow, the frequency of humanCD33 + myeloid cells within the total human CD45 + popula-

have sought to evaluate whether the HLA-class I transgenic

tion was significantly higher in the hSCF Tg NSG recipients

human immune therapy against malignancies. To this end,

than in the non-Tg NSG recipients and constituted the major-

we chose as a target antigen WT1, a molecule that we andity of human hematopoietic cells. With expression of humanothers have previously reported as being expressed in a

SCF in the recipient microenvironment, human mast cells

wide variety of tumors and tumor stem cells,. We immunizedefficiently developed in the bone marrow, spleen, and gas-

HLA class I transgenic humanized mice with WT1 peptide

trointestinal tract of the hSCF Tg NSG recipients. Taken

and TLR adjuvant or with WT1 peptide-loaded dendritic cells

together, the data from our new in vivo humanized mousethree times, with 10-day intervals between injections. Com-model demonstrate the essential role of membrane-boundpared with the human CTL response against EBV-associatedSCF in human myeloid development, and moreover theantigens such as LMP1 or EBNA1, the human HLA-restricted

hSCF Tg NSG humanized mice may be useful in studying

immune response against WT1 peptide was weaker but was
readily detectable. Human CD8 + T cells harvested from HLA

human allergic responses and innate immunity in vivo.

humanized mouse can be utilized as an in vivo model forclass I TG humanized mouse spleen and lymph nodes wereDevelopment of an immune-therapy model using
HLA class I transgenic humanized miceable to secrete IFNγ in response to WT-1 expressing cells.

Last year, we tried to overcome one of the major limitations

We are currently trying to evaluate cytotoxic activity ofhumanized mouse-derived CD8 + T cells against WT1-of the current humanized mouse models, the lack of an HLA-

expressing AML stem cells in vitro and in vivo. Through thisrestricted human immune response, through creation of HLA

analysis, we hope to address whether immune-therapy

class I transgenic humanized mice. When we infected theseagainst WT1 could be a successful therapeutic strategy tar-

humanized mice with EBV, we consistently detected
tetramer+ CD8 + T cells in the recipient lymphoid organs, andgeting chemotherapy-resistant AML stem cells.

4. Saito Y, Kitamura H, Hijikata A, Tomizawa-Murasawa M,
Tanaka S, Takagi S, Uchida N, Suzuki N, Sone A, Najima
Y, Ozawa H, Wake A, Taniguchi S, Shultz LD, Ohara
O, Ishikawa F. Identification of therapeutic targets forquiescent, chemotherapy-resistant human leukemia
stem cells. Sci. Transl. Med. 2, 17ra9, 2010.

113

Research Unit for

Cellular
Immunotherapy

Unit Leader :Shin-ichiro Fujii
Technical staff : Hidetoshi Sugahara

Jun Shinga
Student Trainee : Yusuke SatoH

uman Vα24 + NKT cells bearing an invariant Vα24Jα18

mizu, ( Therapeutic Model Research Unit) and with theantigen receptor can be activated by a specific ligand,support and training program for translational research ofα-GalCer, in a CD1d-dependent manner. We previously

Japan.

showed that circulating Vα24 + NKT cells present in lung can-developing a joint clinical study with Chiba University using
α-galactosylceramide ( α-GalCer ) -pulsed autologous den-

Increased number of NKT cells in the lung after
α-GalCer-loaded dendritic cell (DC/Gal ) therapy intranslational research (A collaboration with Drs.
Nakayama and Motohashi in Chiba University and Dr.
Taniguchi in RCAI)

dritic cells ( DCs ) . Based on our initial analyses, the immuno-

We have been developing a Phase I/IIa clinical study of thelogical and clinical results of the phase I/IIa trials areapplication of NKT cell therapy for advanced non-small celllung cancer ( NSCLC ) patients refractory to standard treat-cer patients were functional. Therefore, to evaluate theimmunological and clinical responses to NKT cell therapy inadvanced non-small cell lung cancer patients we have beenencouraging.
immunity by focusing on the link between innate and adap-

ment as a post second line therapy ( stage IIIB, IV or recurrence). α-GalCer-pulsed APCs ( PBMC-derived DCs ) weretive immunity. Using a mouse model, we successfully devel-

intravenously administered four times. In this study, we dem-

oped a strategy for the induction of antigen-specific T cell

onstrated that the increased IFN-γ production by NKT cells

We have also studied the role of DCs in situ for tumor

responses using tumor-associated antigen expressing,upon α-GalCer stimulation was significantly associated with

α-GalCer-loaded allogeneic fibroblasts to stimulate in situ

clinical outcome. Also, we detected a larger number of NKT

DC maturation. Based on these preclinical findings, we arecells capable of producing IFN-γ in the lung tumor rather

attempting to launch clinical studies of such human artificial
adjuvant vector cells ( aAVCs ) , in collaboration with Dr. Shi-

than peripheral blood. To develop this therapy, we havestarted to use a bronchoscope for trans-bronchial injection

Recent publications
1. Fujii S., Shimizu K.( 2011) DC-based immunotherapy
targeting NKT cells. In Terabe M and Berzofsky
JA (eds), Natural killer T cells: Balancing theregulation of tumor immunity. Springer New
York Dordrecht Heidelberg London, 95 -110.

114

2. Shimizu K., Asakura M., Fujii S. Prolonged antitumor
NK cell reactivity elicited by CXCL-10-expressing
dendritic cells licensed by CD40L+CD4+ memory
T cells. J. Immunol. 186, 5927-37( 2011)

3. Asano K., Nabeyama A., Miyake Y., Qiu CH., Kurita
A., Tomura M., Kanagawa O., Fujii S., Tanaka M.
CD169-Positive Macrophages Dominate Antitumor
Immunity by Crosspresenting Dead Cell-Associated
Antigens. Immunity 34, 85-95 ( 2011)

Figure:Efficacy of aAVCs for induction of Innate Immunity and Adaptive immunity
C57BL/6 mice were immunized with CD1dhi-NIH3T3/Gal-ova, Rae-1ε-NIH3T3-ova,
Rae-1γ-NIH3T3-ova, Mult1-NIH3T3-ova or CD70-NIH3T3-ova. Spleen cells fromdifferent groups of immunized mice were collected one week after immunization. CD8+T
cells were positively selected from spleens in these mice and cocultured with OVA 257-264
peptide-pulsed CD11c+ cells for 36 hours. The number of IFN-γ secretion from CD8+
T cells (/5x105 cells) in response to OVA 257-264 peptide was evaluated by ELISPOT.

of DC/Gal, that is, via the bronchial instead of intravenous

antigen-specific T cell responses and provided tumor pro-route.

tection. Thus, glycolipid-loaded, mRNA-transfected allogeneic fibroblasts act as adjuvant vector cells ( aAVCs ) to

Since this study includes a novel technique, westarted it as new phase I study to confirm the safety profile.

promote iNKT cell activation, leading to DC maturation and T

Preclinical study with antigen mRNA-transfected,
allogeneic fibroblasts loaded with NKT cell ligand(A collaboration with Drs. Shimizu (Therapeutic Unit )and Ishii (Vaccine Design Team ) in RIKEN RCAI, Dr.
Mizuno (Yamaguchi Univ. ) , Dr. Kakimi (Tokyo Univ. ) and
Dr. Maeda ( Iwate Medical Univ. )versus ligands of NK cells, the number of IFN- γ-producing T
cells was much higher in mice given CD1d hi-NIH3T3/Gal-ova
than in other groups ( Figure ) . As preclinical studies, we have

We previously utilized allogeneic fibroblast cells loaded withimmune response. These aAVCs administered to dogs acti-

α-GalCer and transfected with antigen-encoding mRNA,

vate iNKT cells as well as elicit antigen-specific T cellthus combining the adjuvant effects of iNKT cell activation
with delivery of antigen to DCs in vivo ( Blood 2009 ) . We

responses with no adverse events. This unique tool could

found that these cells produce antigen protein and activate

therapies for malignant and infectious diseases.

cell immunity. When we compared the magnitude of T cell
responses after priming with the iNKT cell ligand α-GalCer

set up canine studies to observe the safety profile andprove clinically beneficial in the development of immuno-

NK and iNKT cells. When injected into mice, they elicited

4. Watarai H, Fujii S, Yamada D, Rybouchkin A, Sakata S,
Nagata Y, Iida-Kobayashi M, Sekine-Kondo E, Shimizu K,
Shozaki Y, Sharif J, Matsuda M, Mochiduki S, Hasegawa
T, Kitahara G, Endo TA, Toyoda T, Ohara O, Harigaya K,
Koseki H, Taniguchi M. Murine induced pluripotent stem
cells can be derived from and differentiate into naturalkiller T cells. J. Clin. Invest. 120, 2610-2618 ( 2010 )5. Fujii S, Goto A, Shimizu K. Antigen mRNA-transfected,
allogeneic fibroblasts loaded with NKT-cell ligand confer
antitumor immunity. Blood 113, 4262-72 ( 2009 )115

Laboratory for

Immunogenomics

Group Director :Osamu Ohara
Research Scientists : Takashi Watanabe, Yoko Kuroki, Yoshitaka Shirasaki (SPDR)
Research associates : Atsushi Hijikata, Mai Yamagishi

TTechnical Staff :Nobutake Suzuki, Atsuo Kobayashi, Tomoko Hasegawa, 		Keiko Takahashi, Naomi Inagaki, Ritsuko Ozawa,
Fumie Yokoyama (temporary employee),
Noriko Utsumi (temporary employee),
Emi Abe (temporary employee),Nobuyuki Goto (temporary employee)

Student Trainees :Nanako Shimura (JRA),Asahi Nakahara, Masayuki Ishii

he very basic mission of the immunogenomics grouphas been to function as a “Gateway” to genomics for

immunologists. For this mission, we established a three-

Development of a highly accurate method to detect
low-frequency mosaicisms in the NLRP3 gene using
next generation DNA sequencing

pronged approach to our research activities since our group
first launched: ( 1) central support activities; ( 2 ) strategic and

NGS results in a great reduction in the cost of DNA sequenc-

collaborative research activities; and ( 3 ) exploratory researchmosaicism with low allele frequencies. However, one of theactivities aimed at new technology development. New inmajor concerns with NGS is how to discriminate real changes

2011, we have introduced two next-generation DNA sequencers ( Roche GS Junior and Illumina HiSeq1000 ) to make itfrom sequencing errors. We tackled this issue in collabora-

possible to exploit recent advances in DNA sequencing
technology at RCAI. Next-generation DNA sequencing ( NGS )method of choice for practical genetic tests in clinical situa-

ing and offers new applications including detecting somatiction with clinicians in Kyoto University to make NGS thetions.

will drastically change the genomic approaches that will now

Chronic infantile neurological cutaneous and articular

become practical for use by immunologists, and the current
technology is just the beginning of the change. In parallel, we

syndrome ( CINCA ) , also known as neonatal-onset multisystem inflammatory disease ( NOMID ) , is a dominantly-inherited

have also placed a significant emphasis on development ofsystemic autoinflammatory disease caused by a heterozy-

new technology to ultimately enhance strategic and collab-

gous germline gain-of-function mutation in the NLRP3 gene.

orative research activities as well as the central support

The recent finding of a high incidence of NLRP3 somatic

activities. Our current emphasis is on the development ofmosaicism in apparently mutation-negative CINCA/NOMID

technology for single-cell analyses and for efficient genetic

patients suggests that a rapid diagnosis of somatic NLRP3

testing using next-generation DNA sequencing technology.

mosaicism is crucial to ensure proper treatment. However,

Examples of our recent achievements along these directions

conventional DNA sequencing approaches require large

are described below.

investments of time, cost, and labor, making it difficult toroutinely diagnose low-level somatic NLRP3 mosaicism bythis approach. To address this problem, we used NGS to

Recent publications
1. Detection of Base Substitution-Type Somatic Mosaicism
of the NLRP3 Gene with >99.9% Statistical Conﬁdenceby Massively Parallel Sequencing: Izawa K, Hijikata
A, Tanaka N, Kawai T, Saito KM, Goldbach-Mansky
R, Aksentijevich I, Yasumi T, Nakahata T, Heike T,
Nishikomori R, Ohara O. DNA Res. 2012 in press.

116

2. XPS and NEXAFS studies of VUV/O ( 3 ) -treatedaromatic polyurea and its application to microchip
electrophoresis: Shinohara H, Nakahara A, Kitagawa
F, Takahashi Y, Otsuka K, Shoji S, Ohara O, Mizuno
J. IET Nanobiotechnol. 2011 5 ( 4 ) :136.

3. Plasmacytoid dendritic cells are crucial for the initiationof inflammation and T cell immunity in vivo: Takagi
H, Fukaya T, Eizumi K, Sato Y, Sato K, Shibazaki A,
Otsuka H, Hijikata A, Watanabe T, Ohara O, Kaisho T,
Malissen B, Sato K Immunity. 2011 35 ( 6) :958-71.

A

B
Figure 1: Analysis of NLRP3 somatic mosaicism by NGS(A) The reference error rate map for the amplicon of NLRP3
exon 1 on the upper and lower strands. The colors ofbars represent the error category (insertions: cyan;deletions: red; mismatches: green; ambiguous base calls:
purple). The orange and blue lines depict the primer
and target regions, respectively. The yellow shaded area
depicts the homonucleotide stretches (n>3) region.
(B) Scatter plot of the observed variation frequency on
both strands. The colors depict mosaic mutations(magenta), heterozygous mutations (orange),
single-nucleotide polymorphisms (SNPs, green)and sequencing errors (gray), respectively.

Frequency

20

BB
from the top 10%
69%
from the bottom 90%

10

N.D.

N.D.

N.D.

Pre

90%

4-5hr
2-3hr
0-1hr
20

40

60

Pre
80

1

0-1 hr 1-2 hr 2-3 hr 4-5 hr

1-2hr

0

100

IL-6 (ng/mL/hr)120

from the top 10%
53%
from the bottom 90%

100

0

0.5

0

150

TNFA (ng/mL/hr)

1

30

140

Frequency

IL-6 (ng/mL/hr)

AA

50
0

58%
Pre

21%

31%

32%

0-1 hr 1-2 hr 2-3 hr 4-5 hr

4-5hr
0.5

2-3hr
1-2hr
0-1hr

0

0

20

40

60

Pre

80

100

TNFA (ng/mL/hr)

120

140

Figure 2: Single-cell measurements reveal a large
variation in cytokine secretion activities
of J774.1 cells after LPS stimulation.
IL-6 (panel A) and TNFα (panel B) secretion by single
cells was measured by a microengraving method before
and after stimulation with LPS. These histograms show
the frequency (vertical axis) of J774.1 cells with eachsecretion activity (ng/mL/hr, horizontal axis).
Inset bar graphs show the secretion activityof the whole cell population. Each coloredpart of bar indicates the contribution fromthe higher secretor cells in the top 10%.

develop a new pipeline to detect even a low-frequencyof responses to external stimuli. Here, we introduced a new

NLRP3 allele with statistical significance. To solve the prob-

method named “microengraving”, which allows simultane-

lem of how to discriminate a low-level allele from sequencing

ous measuring of secretion activity of thousands of single

errors, we first constructed position- and strand-specificcells, to establish an approach to describe a secretion activ-

error rate maps of 14 PCR products covering the entire cod-ity distribution measurement at the single-cell level in a celling NLRP3 exons from 50 control samples without mosaicism on a Roche 454 GS-FLX sequencer ( Fig. 1) . Based on

population. In this experiment, we used a macrophage-likethese results, we formulated a statistical confidence value

cell line, J774.1. After the J774.1 cells were activated withbacterial lipopolysaccharide ( LPS ) , the secretion activity dis-for each sequence variation on each strand to discriminate

tribution was measured for IL-6 and TNFα by the “microen-

sequencing errors from real genetic variation even in a low-

graving” method. The IL-6 response to LPS stimulation was

frequency allele, and thereby realized detection of base sub-observed with a short delay of 2 hrs while TNFα was secreted

stitutions at an allele frequency as low as 1% with a 99.9%

immediately after LPS stimulation. The secretion activity ofor higher significance level. This new method can easily beapplied to other cases of somatic mosaicism, e.g., various

IL-6 and TNFα 4 hrs after stimulation varied widely from cellto cell ( Fig. 2A ) . For IL-6, this heterogeneity meant that thetypes of cancers to detect disease-causing mutations.

high secretor cells in the top 10% were able to account forthe majority of total secreted cytokine. On the other hand,

Monitoring of secretion of cytokines from single
macrophages measured by a “Microengraving”
methodshowed a positively skewed bell-shaped distribution ( Fig.
2B ) . The more limited heterogeneity of TNFα secretion

To maintain a dynamic and integrated immune system,

among the cells was reflected in the lower contribution of theimmune cells closely communicate with each other by means

secreted TNFα from the top 10% cells to the total secretedof humoral factors and cell surface molecules. Traditionally,

TNFα, especially at early time points. Such large cell-to-cellsecreted humoral factors are quantified to estimate the func-

variations in cytokine secretion could never have been

tions of the cells based on their concentrations in culture

detected by a bulk cytokine measurement in culture medium

supernatants from a population of purified immune cells.

and thus the microengraving method provides us with unex-

However, recent single cell analyses are revealing great het-

plored lines of information to build a systems view of a multi-

erogeneity even within an isogenic cell population. There-

cellular ensemble.

secretion activity of TNFα after a short period of stimulation

fore, it is interesting to discover just how heterogeneous
immune cells are, even among a clonal population, in terms

4. High incidence of NLRP3 somatic mosaicism inpatients with chronic infantile neurologic, cutaneous,articular syndrome: results of an International
Multicenter Collaborative Study: Tanaka N, Izawa
K, Saito MK, Sakuma M, Oshima K, Ohara O, et.
al. Arthritis Rheum. 2011 63 (11) :3625-32.

5. Quantification of κ-deleting recombination excision
circles in Guthrie cards for the identification of early
B-cell maturation defects: Nakagawa N, Imai K,
Kanegane H, Sato H, Yamada M, Kondoh K, Okada
S, Kobayashi M, Agematsu K, Takada H, Mitsuiki
N, Oshima K, Ohara O, Suri D, Rawat A, Singh S,
Pan-Hammarström Q, Hammarström L, Reichenbach

J, Seger R, Ariga T, Hara T, Miyawaki T, Nonoyama S. J
Allergy Clin Immunol. 2011 128 ( 1) :223-225.e2.

117Research Unit for

Immunoinformatics

Unit Leader :S. Sujatha Mohan
Research Associate : Suresh Kumar RamadossAn important mission of our research unit is to develop

dards in an easily decipherable and usable format.

and maintain an open access bioinformatics platformand data resource in order to gain insights into primary
immunodeficiency diseases ( PID ) through genomic, tran-

RAPID statistics

scriptomic and proteomic data. In this pursuit, we launched

confirmed to cause over 250 PIDs. There are over 4700

a web-based compendium of molecular alterations in PID,

unique mutations annotated from 1704 citations as of Janu-

Resource of Asian Primary Immunodeficiency diseases( RAPID ) at http://rapid.rcai.riken.jp in 2008. RAPID also

ary 2012. The data growth of RAPID since its launch is

At present, RAPID includes a total of 234 genes that are

depicted in Fig. 1.

hosts other pertinent information such as expression proDNA sequencing protocols for recognized PIDs. All mutation
data have been linked with a graphical user interface ( GUI )PhenomeR: A Genotype – Phenotype integrated
approach for mining Primary Immunodeficiency
Disease causal genes using semantic web technology

enabled tool, named Mutation@A Glance, to visualize and

The main challenge for in silico genotype-phenotype corre-

evaluate reported mutations. Using RAPID data, we haveused Support Vector Machine ( SVM ) based parameter clas-

lation for any genetic disease is to standardize phenotype

files, interaction networks, mouse studies and standardizedsification to predict candidate PID genes by scanning genes

ontology terms and the genotype. We have standardized PID
specific phenotype terms using NCBO’s BioPortal ( http://

in the whole human genome. Furthermore, RAPID has been

bioportal.bioontology.org/ ) for mapping and standardizing

updated with a standardized PID expert page, a terms

terms from other ontology resources. We are following asearch option for mouse and human phenotypes and aunique semi-automated method to assign literature collected

mutation data submission tool.

phenotype terms to specific PIDs. This kind of analysis

Our ultimate goal is to provide relevant, up-to-date andshould bridge a gap between genotype and phenotype cor-validated information on PID as per global community stan-relation, thereby improving phenotype-based genetic analy-

Recent publications
1. Raju, R., V. Nanjappa, et al. “NetSlim:high-confidence curated signaling
maps.”Database ( Oxford ) bar032 ( 2011).

118

2. Suresh Kumar Ramadoss and Sujatha Mohan, In Silico
Identification of Prioritized Interacting Domains inPrimary Immunodeficiency Disease Causing Genes. Intl
J Biosci, Biochem Bioinformatics,
1 ( 2) : 84-88 ( 2011) .

3. Hiroshi Masuya, Yuko Makita, Norio Kobayashi, et al.
The RIKEN integrated database of mammals. Nucleic
Acids Res, 39 ( Database issue ) : D861-70 ( 2011) .

Figure 1: RAPID data growth as of January 2012
RAPID data growth as of January, 2012. No. of PubMeds refers to the numberof annotated reference papers with mutation data, No. of mutations indicates
non-redundant entries of mutation data annotated and No. of PIDs represents
unique primary immunodeficiency disease names included in RAPID.

sis of PID genes. Moreover, it should aid clinicians inFigure 2: RAPID - Integrated PID KnowledgeBase
Overview of RAPID integral features and its
elements in the PID integrated KnowledgeBase

edgeBase is shown in Fig. 2.

confirming early PID diagnosis and also be helpful in implementing appropriate therapeutic interventions.

Inception of an integrated Arthritis KnowledgeBasefor high-throughput data analysis

Road map towards RAPID - integrated PID
KnowledgeBase

Integrated Arthritis database ( IntARTdB ) is a web based

RAPID’s ongoing and future tasks are identified as four major

as an integrated platform for arthritis-specific data collec-

components and their salient features are highlighted as fol-tion, storage, annotation and analysis of genomics, tran-lows:

scriptomics, proteomics, metabolomics and other omics

compendium of high-throughput data on arthritis. It servesstudies. The IntARTdB framework is based on the coni)

PID Pathway resource - Generation of a web-based

structed model-view-controller architecture. We intend tointegrated PID-specific immune signaling and regulatory

provide the data annotation tool to registered users, withpathway framework that would aid biomedical investi-

controlled access privilege, to share and contribute analyzed

gators in understanding the molecular mechanisms ofhigh-throughput data to IntARTdB.

PID pathogenesis and also help in identifying diseaseii )This project has been recently initiated as an Interna-causing candidate PID genes.

tional collaboration with the Gobezie lab at Musculoskeletal

PID phenotype resource - Standardize PID phenotype

Proteomics, The Case Center for Proteomics and Systems

terms and mapping to genes, mutation and disease as

Biology, Case Western Reserve University - School of Medi-

well as the construction of PID phenotype ontology

cine, USA.

resource for easy access and query of any observed
iii )

iv )

symptoms against PID using semantic web technology

Collaboration and funding

PID functional domain analysis - Identify functionallysignificant domains that disrupt the interactions of PID

The PID project has been initiated in collaboration with theInstitute of Bioinformatics ( IOB, Bangalore, India ) and thegenes associated with disease mutation through an

Immunogenomics research group at RIKEN RCAI, Japan.

integrated in silico approachThis laboratory was supported by The Asia S&T Strategic

PID structural elucidation tool - Accomplish sequence-

Cooperation Promotion Program, Special Coordination

structural analysis of PID mutations through modelingFunds for Promoting Science and Technology by the Minis-

and docking studies to assess their functional impact

try of Education, Culture, Sports, Science and Technology( MEXT ) between July, 2007 and March 2010. Now, it is being

on PID pathogenesis

funded by RCAI, RIKEN internal funding source.
A brief overview of the RAPID road map to this Knowl-

4. Atsushi Hijikata, Rajesh Raju, Shivakumar Keerthikumar
et al. Mutation@A Glance: an integrative web
application for analyzing mutations from human
genetic diseases. DNA Res, 17 (3 ) :197-208 ( 2010).

5. Shivakumar Keerthikumar, Sahely Bhadra,
Kumaran Kandasamy, et al. Prediction ofcandidate primary immunodeficiency disease
genes using a support vector machine learning
approach - DNA Res,16 ( 6 ) :345-51 ( 2009 ) .

6. Shivakumar Keerthikumar, Rajesh Raju,
Kumaran Kandaswamy, et al. RAPID: Primary
Immunodeficiency disease database. Nucleic
Acids Res, 37 ( Database issue) :D863-7 ( 2009 ) .

119

Laboratory for

Cellular Systems
Modeling

Team Leader :Mariko Okada (Hatakeyama)
Senior Scientist :Shinji Kondo

Research Scientists : Masaki Nomura						Hisaaki Shinohara

R

Technical Staff :Noriko Yumoto						Kaoru Takahashi						Ki SewonStudent Trainee :Hanna Iribe

ecent sequencing projects have begun to reveal a

networks, which determine cell fate in mammalian cells. By

coordinated effect of genomic mutation, epigenetic

doing so, we are able to identify rules specified in immuno-

modification, micro and noncoding RNAs for determining

logical systems.

cellular outcome. Yet, it is still unclear how all these compo-

For this purpose, we take both bottom-up kinetic analy-

nents are dynamically connected and function to produce

sis and top-down omics approaches. These approaches

the desired biological outputs. Systems Biology thus meetscomplement each other in order to understand objects at a

the strong desire of biologists to understand mechanistic
aspects of the molecular and cellular networks. As a result,

system level. For kinetic analysis, we constructed ordinary
differential equation ( ODE ) -based mathematical models to

one day, we may be able to define simple logic that drives

describe digital induction of an early responsive gene, theproliferation, differentiation or death of the cells. One of the
expected outcomes of Systems Biology research is to iden-c-Fos transcription factor, in a cell differentiation process( Cell, 2010 ) . The model explains that AND-gate and negative

tify network designs and functions in the organism and to

feedback loops might shift the cell into the irreversible dif-engineer them for industrial and medical application. Recent
forefront studies of Systems Biology have begun to give us

ferentiated state and that a sustained ERK signal is a driving
force of the system ( Figure 1) . This model is valid for produc-

an idea about universal cellular logic designs and motifs dis-tion of transcription factors in B-cell development and neu-

tributed in living organisms regardless of the cell type.

rite elongation of PC12 cells, therefore it is most likely that

The Laboratory for Cellular Systems Modeling has been

the model captures general network principles of this signal

carrying out research to identify biological networks andnetwork during a cellular differentiation process. Using thisregulatory principles using computational, theoretical andlogic, we were able to convert a transient signal to a sus-

experimental approaches. Our research interest is to identify

tained signal by membrane receptor mutation and, as pre-and reconstruct biological regulatory circuits from experi-

dicted, this network conversion was accompanied by amentally observable data using computational and theoreti-

differentiated cellular phenotype.

cal approaches to reveal the complexity of signal transduction

Recent publications
1. Morita M, Oike Y, Nagashima T, Kadomatsu T, Tabata M,
Suzuki T, Nakamura T, Yoshida N, Okada M, Yamamoto T.
Obesity resistance and increased hepatic expression of
catabolism-related mRNAs in Cnot3 (+ /-) mice. EMBO J.
30, 4678-4691 ( 2011) .

120

2. Oyama M, Nagashima T, Suzuki T, Kozuka-Hata H,
Yumoto N, Shiraishi Y, Ikeda K, Kuroki Y, Gotoh N,
Ishida T, Inoue S, Kitano H, Okada-Hatakeyama M.
Integrated Quantitative Analysis of the Phosphoproteome
and Transcriptome in Tamoxifen-Resistant Breast
Cancer. J. Biol. Chem., 286, 818-829 ( 2011) .

3. Shiraishi Y, Kimura S, Okada M. Inferring Clusterbased Networks from Differently Stimulated
Multiple Time-course Gene Expression Data.
Bioinformatics 6, 1073-1081 ( 2010 ) .

Figure:Network structure of ERK signal-induced c-Fos induction necessary for the earlystage of cellular differentiation. ERK serves as a driving force to produce c-Fos using
AND-gate loops (noise filtering). Thus, the c-Fos network is very robust to noise.

In addition, inspired by a relationship between the signal

integrative analysis indicated that AP-1 and CREB transcrip-

dynamics and cell fate decisions, we attempted to obtaintion factor activation was dysregulated in the drug-resistantbinding kinetics of ligand-membrane receptor interaction inliving cells ( in collaboration with Dr. Yasushi Sako, RIKEN

cells. We proposed and clinically verified that GSK3beta is a

ASI ) . This analysis indicated that ligand-receptor binding

grated analysis of signaling and transcription allows us tokinetics are very similar in the cells whether they are under-

understand drug-resistant properties at a network level andgoing proliferation or differentiation, therefore, intracellular

to identify a therapeutic target. For this kind of large-scale

kinetics rather than ligand-receptor kinetics seem to play andata analysis, we also developed several computer algo-

important role in cell fate decision.

rithms and methods. Particularly, we developed a method to

As for the top-down approach, we perform several

drug-resistance marker. These studies showed that inte-transcriptome studies to unveil transcription networks. For

identify synergic effects of two transcription factors / or histone modifications responsible for gene expression ( Bioin-

an example, we integrated phospho-proteome data andformatics, 2011) . The method further allows us to evaluate

transcriptome data to understand drug-resistance mechanisms in breast cancer cells ( J. Biol. Chem., 2010 ) . This

combinatorial drug therapy for certain diseases using tran-

4. Nakakuki T, Birtwistle MR, Saeki Y, Yumoto N, Ide
K, Nagashima T, Brusch L, Ogunnaike BA, OkadaHatakeyama M*, Kholodenko BN.* Ligand-Specificc-Fos Expression Emerges from the Spatiotemporal
Control of ErbB Network Dynamics. Cell 141,884-896 ( 2010) . * corresponding authors.

scriptome data.

5. Suenaga A, Hatakeyama M, Kiyatkin AB, Radhakrishnan
R, Taiji M, Kholodenko BN. Molecular Dynamics
Simulations Reveal that Tyr-317 Phosphorylation
Reduces Shc Binding Affinity for Phosphotyrosyl
Residues of Epidermal Growth Factor Receptor.
Biophys. J. 96, 2278-2288 ( 2009 ) .

121

Research Unit for

Thymic Environment

Unit leader :Willem van Ewijk
Research scientist : Kathryn Ischi-Schrade

STAT3
We have set out to analyze the role of signaling cascades
other than NFκB in TEC development. Around 10 years ago,

STAT 3 influences the development of thymic
microenvironmentsT

he development of thymic microenvironments formedby thymic epithelial cells ( TECs ) is a two-step phenom-

it was suggested that STAT3 signaling influenced TEC development ( Sano et al., Immunity 15, 261-273, 2001) . In thisreport, cre-mediated deletion of STAT3 under control of the
Keratin-5 ( K5 ) promotor was found to strongly affect TEC
development mainly in the cortex, however, the influence of
STAT3 deletion in the medulla remained less clear.

enon. The embryonic initiation of TECs occurs as a cell-

To study the STAT3 deletion in more detail, we createdautonomous phenomenon, regulated among others by thetissue specific transcription factor Foxn1 ( Nehls et al., Sci-

mice where STAT3 was deleted under control of the Foxn1
promotor. We employed Foxn1-cre mice ( Hauri-Hohl et al.,ence 272, 886-889, 1996; Balciunaite et al., Nat. Immunol 3,1102-1108, 2002 ) , while further expansion of TECs is con-

moto et al., FEBS Lett. 470, 263–268, 2000 ) to constructtrolled by developing thymocytes, a phenomenon earlier
designated as “thymic crosstalk” ( van Ewijk et al., Immunol.

Foxn1-STAT3CKO mice. The thymus of these mice was normal sized, and T cell development ( as determined by expres-Today 15, 214-217, 1994 ) . Analysis of the molecular signathe lymphoid dependent phase of TEC

sion of CD4 and CD8 ) appeared normal ( Fig 1A, B ) .
Immunohistological analysis of cortical TECs ( cTECs )development is at present a ‘hot’ topic in thymic research.

revealed a normal architecture but mTECs, as identified byThis signature is now becoming increasingly clear in thedevelopment of medullary TECs ( mTECs ) , where the NF-kB

the mTEC specific marker ER-TR5, formed long cytoplasmic

signaling pathways controls the proliferation and differentia-

less frequent and they formed smaller medullary domains( Fig 1C ) .

ture underlying

tion of mTECs, and involves cell surface receptors such as
LTβR, RANK and CD40 on mTECs ( Boehm et al., J.Exp.Med.

Blood, 112, 626-634, 2008 ) and GFP reporter mice ( Kawa-

extensions. Moreover, mTECs in STAT3 deleted mice were

We next investigated whether the functional maturation

198, 757-769, 2003; Hikosaka et al., Immunity 29, 438-450,
2008; White et al., J. Immunol. 185, 4769-4776, 2010 ) .

of mTECs in Foxn1-STAT3CKO mice was impaired. For thisHowever, in cortical TECs ( cTECs ) this signature has

mice with antibodies detecting UEA-1 and AIRE, molecules

yet to be unraveled, although in vitro experiments imply

expressed in mature mTEC subsets involved in negative

Notch signaling as an important regulator in functional TEC
development ( 1) .

selection of autoreactive thymocytes. Fig. 2 shows that

purpose, we stained cryosections of the thymus of these

UEA-1- and AIRE-expressing mTECs are present in medullary domains in Foxn1-STAT3CKO thymi. These data there-

Recent publications
1. Masuda, K, Germeraad WTV , Satoh R, Itoi M,
Katsura Y, van Ewijk W, Kawamoto H. Activation of
Notch signaling in thymic epithelial cells induces
development of thymic microenvironments.
Mol. Immunol. 2009, 46: 1756-1767.

122

2. Vroegindeweij, E, Itoi M, Satoh R, Zuklys S, Crobach S,
Germeraad WTV. Cornelissen JJ, Cupedo T, Holländer
G, Kawamoto H, van Ewijk W. Thymic cysts originate
from Foxn1 positive thymic medullary epithelium.
Mol. Immunol. 2010 47 ( 5 ) :1106 -1113.

Figure 1: A. Thymi of Foxn1-STAT3CKO thymi and control
thymi are equal in size.
B. Similar CD4/CD8 profiles of thymocytes inFoxn1-STAT3CKO mice.
C. Cryostat sections of thymi derived from Foxn1STAT3CKO show a size reduction in the medullary
domains, stained by ER-TR5 (red).

Figure 2 : Cryostat sections of thymi derived from Foxn1-STAT3CKO
show mature mTECs as identified by UEA-1 (red; top
panels) and AIRE (green; bottom panels) staining.

fore indicate that TEC development in itself is not impaired inabsence of STAT3, also explaining the normal phenotypicalT cell development observed in the Foxn1-STAT3CKO mice
( Fig.1B ) . Thus, it seems likely that STAT3 controls the expansion rather than the final differentiation of mTECs.
In contrast to data reported by Sano et al ( Immunity 15,
261-273, 2001) , we did not find a cTEC phenotype in Foxn1STAT3CKO mice. Overall, the three dimensional organizationof cTECs in Foxn1-STAT3CKO thymi was found similar to thatin control mice, and functional molecules like MHC I and II,
and β5T were normally expressed by Foxn1-STAT3CKO
cTECs ( data not shown ) .
To exclude the possibility that genetic variations underlie the difference in TEC phenotype between Sano’s paper

Figure 3 : Cryostat sections of thymi derived from Foxn1-Cre::EGF-Rfl/+ (control) and Foxn1-Cre::EGF-R-fl/fl mice (deletion)
stained with ER-TR5 (mTECs; green) and AIRE (red).

EGFR

and our results, we generated our own K5-STAT3 CKO mice.

EGFR and HGFR are known molecules upstream of

Surprisingly, and in contrast to the earlier report by Sano et

STAT3 ( Guo et al., J Clin Invest.;117:3846-3856, 2007) . We

al., the stromal phenotype of these mice was quite similar tothe phenotype observed in Foxn1-STAT3CKO mice ( data

generated EGFR and HGFR floxed mice and mated thesenot shown ) .

TECs. While HGFR-Foxn1CKO mice had no apparent epithe-mice to FoxN1-Cre mice in order to target the deletion to

We have so far no clear explanation for the phenotypiclial phenotype, EGFR-Foxn1CKO mice had a TEC phenotype

difference in the cTECs in the two studies. Since K5 is also

in the thymic medulla, similar (but slightly milder) to that in
STAT3-Foxn1CKO mice ( Fig.3 ) .

expressed in the skin, which is a reason why these mice arealso used as a model for atopic dermatitis ( Sano et al., Embo
J. 18,4657-4668, 1999 ) , one could speculate that induction

In conclusion, our study indicates that (1) TEC develop-of dermatitis induced some stress that indirectly affected
TEC development. However, stress related glucocorticoid

ment in the medulla, but not in the cortex, is under the control of STAT3, ( 2 ) STAT3 regulates expansion of mTECs rather

levels in the K5 STAT3 were reported to be normal (Sano etthan their differentiation. ( 3 ) The EGF receptor acts upstreamal., Immunity 15, 261-273, 2001).

of STAT-3 signaling in this process.

123Research Unit for

Immunoepigenetics

Unit Leader :Miguel Vidal
Research Scientist : Kaori HisadaP

olycomb group ( PcG ) complexes function as transcrip-

poorly characterized, in part due to the embryonic lethal

tional repressors of genes controlling cell identity states

phenotype of constitutive RYBP mutant alleles.

and transitions between them. In stem cells, PcG complexes
orderly deployment during their differentiation through selec-

RYBP function studies in an embryonic stem ( ES )cell modeltive silencing of their targets. PcG complexes act both atEmbryonic stem ( ES ) cells cannot be derived from earlyhigher order chromatin structure and at cis-acting control

embryonic structures constitutively deficient in RYBP andregion levels. Their function depends, at least in part, on
their activities as histone modifiers ( trimethylation of histoneRYBP is found among interactors with Oct-4, the regulatory

H3 and monoubiquitylation of histone H2A ) . Recruiting ofin ES cells. We have generated ES cells carrying both a

PcG complexes to their targets and dissection of functional

floxed RYBP allele and a gene encoding a ubiquitously

diversity of PcG subunits are subjects of active research.

expressed Cre-ER recombinase inducible by hydroxytamox-

One of these subunits, the RYBP protein that we identified

ifen. Inactivation of RYBP results in self-renewing ES cells
that express markers of pluripotency ( Fig. 1) . ChIP on Chipfunction in maintaining their developmental potential and its

as a direct interactor with the PcG histone H2A monoubiquitin ligases Ring1A and Ring1B, appeared to be a goodcandidate for PcG recruiting. RYBP function, however, isRecent publications
1. Hisada, K., Sánchez, C., Endo, T., Endoh, M., RománTrufero, M., Sharif, J., Koseki, H. and Vidal, M. RYBP
represses endogenous retroviruses, preimplantationand germline-specificgenes in mouse embryonicstem cells. Mol. Cell. Biol. 32:1139 -1149 ( 2012)124

master of the pluripotency network of transcriptional control

studies showed that RYBP colocalizes with class I Polycomb
products ( for example Ring1B ) on chromatin of wild type ES

Figure 2: RYBP represses retrotransposons in ES cells.
Top: schematic representation of MuERVs. Bottom:
specific derepression of MuERVs but not otherretrotransposons in RYBP-deficient ES cells.
Figure 1: Pluripotency markers in RYBP-deficient ES
cells. Oct4, SSEA1 and alkaline phosphatase, incontrol and tamoxifen-treated RYBPf/f ES cells

500

Figure 3: Contrasting sensitivity to
H3K27me3 of RYBP and Ring1B
association to chromatin. RYBP
binding to targets in Eed-KO cells(without H3k27me3) was not altered,
whereas Ring1B was acutely depleted
from chromatin in mutant cells.

500
RYBP

Genes

Genes

Ring1B

wt
0
-2

0
2
4
6
Fold enrichment, log2

8

0
-2

Eed-KO
0
2
4
6
Fold enrichment, log28cells. However, RYBP-deficient ES cells showed little or no
derepression of genes repressed by Ring1B ( core Polycomb

This observation, together with the property of RYBP toassociate with chromatin in ES cells lacking H3K27me3 ( Fig.

targets such as developmental regulators ) . Instead, transcripts originating in class III retrotransposons ( MuERVs ) are,3 ) , the mark that recruits Ring1B complexes, and the expression pattern of RYBP ( starting at the two-cell stage ) suggest

together with preimplantation-specific genes, among thehighest upregulated mRNAs in RYBP-deficient cells ( Fig. 2 ) .

a role for RYBP in early developmental stages. Future work

In some cases, at least, transcripts from preimplantation

conditionally deficient for Yaf2, the RYBP paralog.

will address this hypothesis and also the function of ES cells

genes originate from nearby truncated transposon elements.

125Research Unit for

Immune Crosstalk

Unit Leader:

Hilde Cheroutre
Mucosal T cells March to the Beat of a
Different DrummerOgenic conditions. RA promotes the induction of TGF-βdependent

Foxp3

iTreg

whereasitsuppresses

thedifferentiation of inflammatory TGF-β-driven Th17 cells.
ur research aims at understanding and elucidating the

Our research continues to elucidate mechanisms ofdifferentiation, function and immune regulation of T

mucosal immune regulation and in a recent study we uncov-

cells that reside at the mucosal interface of the intestinal

ered an unexpected degree of plasticity for CD4 T helper( Th ) cells which, upon antigenic stimulation, are able to ter-epithelium.
Unlike lymphoid T cells, intestinal intraepithelial lymphocytes ( IEL ) reside at epithelial surfaces and form the first line

minate the expression of the Th transcription factor, ThPOK,
and differentiate into CD8-CTL-like effector cells.

of defense against invading pathogens. Although indispens-

We recently also showed that an affinity-based selective

able for preventing the initial infection, IEL need to balance

process operates at the mucosal interface of the intestine

their protective function with safeguarding the integrity of theand preserves the optimal effector cells to become longlived effector memory T cells ( TEM ) that establish pre-existingand heightened protective immunity ( Huang et al., Naturebarrier and failure to do so compromises homeostasis of theorganism.
These unique challenges drive specialized differentia-

Immunology 2011) .

tion and regulation of IEL and the main goals of our researchare 1) to elucidate regulatory mechanisms that preserve the

Unlike central memory T cells ( TCM ) that reside in lymphoid tissues, TEM gain the capacity to reside long-term inmucosal barrier in the face of potentially destructive immune
protection and 2 ) characterizing unique processes that drive

respond to antigen with a robust clonal expansion, are effec-

the generation of mucosal effector memory T cells that war-tive at protecting against infections by pathogens that repli-

rant optimal pre-existing protective immunity.

cate systemically, but they are inadequate to prevent

non-lymphoid tissues, such as the intestine. TCM cells, whichshowed that, for example, retinoic acid ( RA ) is a key factor intransmission of viruses, including the human immunodeficiency virus ( HIV ) , or intracellular bacteria, which penetrate

controlling mucosal immune responses and under tolero-across mucosal epithelia. Effective resistance against trans-

We have made milestone discoveries in both areas andRecent publications
1. Huang Y, Park Y, Wang-Zhu Y, Larange A, Arens
R, Bernardo I, Olivares-Villagómez D, HerndlerBrandstetter D, Abraham N, Grubeck-Loebenstein B,
Schoenberger SP, Van Kaer L, Kronenberg M, Teitell
MA, Cheroutre H. Mucosal memory CD8+ T cells are
selected in the periphery by an MHC class I molecule.
Nat Immunol. 2011 Oct 2;12 ( 11) :1086-95.

126

2. Ichikawa S, Mucida D, Tyznik AJ, Kronenberg
M, Cheroutre H. Hepatic stellate cells function
as regulatory bystanders. J Immunol.
2011 May 5;186 ( 10 ) :5549-55.

3. Cheroutre H. HIV vaccination: turning the spotlighton effector memory T cells as mucosal gatekeepers.
F1000 Biol Rep. 2009 Nov 26;1:89.

Figure :Proposed model for the roles of TL and CD8αα in selection of high affinity CD8αβ TEM. CD8αα expression is selectively inducedon high affinity/avidity primary effector CD8αβ+ T cells and further enhanced by RA released by mucosal migratory (CCR7+)
DC, which also promote gut-tropism (α4β7+CCR9+) of the effector T cells. Activated migratory DC express the CD8αα highaffinity ligand, TL, which when interacting with CD8αβ on activated T cells leads to TL-induced cell death ( ,TICD). High affinity
primary effector cells that induce CD8αα use it to escape TICD by sequestering TL away from CD8αβ, leading to affinity-based
selective survival. TL constitutively expressed on the intestinal epithelial cells (IEC) mediates affinity maturation of the mucosal
TEM and eliminates low affinity/avidity primary and secondary effector cells that home to the gut and fail to induce CD8αα.

mission of such pathogens requires the presence of local,
highly efficient antigen-specific TEM prior to re-challenge.
Nevertheless, because most of the current knowledge ofcontrolled by TL expressed on APCs, which leads to theselective survival of high affinity, CD8αα+ CD8αβ memory

immune memory has been gained from model systems that

precursor cells. Furthermore, TL on the intestinal epithelial
cells ( IEC ) continues to impose selection pressure anduse systemic immunization routes for the generationof lymphoid TCM, the generation of TEM was very poorly

drives the affinity maturation of the resident mucosal CD8αβ
TEM and warrants the optimal protective capacity of theseunderstood.

memory cells.

We showed previously that the TCR repressor, CD8αα,

Our findings represent a fundamentally new concept

induced on activated CD8αβ T cells, marks those primary

for immune memory differentiation and indicate that aneffector cells that preferentially differentiate into memory
cells ( Madakamutil et al., Science 2004 ) . The expression

affinity-based selective process operates in vivo thatlevel of CD8αα is controlled by signal strength and it is mostpreserves the optimal effector cells to become long-lived
TEM that establish pre-existing and heightened protective

highly induced on those effector cells with the highest antigen-affinity. We found that mucosal TEM are highly enriched

immunity at mucosal borders.

for CD8αα-expressing memory cells, indicating that a selec-

control the pathogen load and delay or prevent the initialtive mechanism might drive the preferential accumulation of

infection and spreading of the pathogen, as well as reducehigh affinity effector cells at the mucosal interface of thethe potential for secondary transmission. Therefore, our find-intestine.

ing that an endogenous TCR quality-based mechanism

In an earlier study we had shown that the high affinity
ligand for CD8αα, the non-classical MHC class I molecule
thymus leukemia antigen ( TL ) , is constitutively expressed on
mucosal epithelial cells. Using oral infection with the foodborne pathogen, Listeria monocytogenes ( Lm ), we demon-

Although unable to provide sterilizing protection, TEM

selects for the most avid effector cells to form mucosal TEM
has significant implications for the design of new andimproved

strategies

toinduceeffective

pre-existing

protective immunity at the most vulnerable entry sites forpathogens.

strated that an affinity-based selection mechanism exists,

4. Murai M, Turovskaya O, Kim G, Madan R, Karp
CL, Cheroutre H, Kronenberg M. Interleukin
10 acts on regulatory T cells to maintainexpression of the transcription factor Foxp3 andsuppressive function in mice with colitis. Nat
Immunol. 2009 Nov;10 ( 11) :1178-84

5. Mucida D, Pino-Lagos K, Kim G, Nowak E, Benson
MJ, Kronenberg M, Noelle RJ, Cheroutre H. Retinoic
acid can directly promote TGF-beta-mediatedFoxp3 (+) Treg cell conversion of naive T cells.
Immunity. 2009 Apr 17;30 ( 4 ) :471-2

127

Central Facilities

C

entral Facilities in RCAI provide all researchers in the Center with access to the most advanced
equipment and technologies. Central Facilities consist of five sections; the FACS, Confocal, andMonoclonal Antibody Laboratories managed by Dr. Takashi Saito, the Genomics Laboratory managed by
Dr. Osamu Ohara, and the Animal Facility managed by Dr. Haruhiko Koseki.

FACS Lab.
Technical Scientist : Hanae Fujimoto
Technical Staff: Yukiko Hachiman

Visiting Scientist: Ikuo Ishige ( BM Equipment Co. Ltd.)The FACS Lab provides a range of support for flow cytometry and cell sorting, procedures that are essential fornearly all immunological experiments. In 2011, the FACS Lab newly installed a CyTOF ( Fig. 1 ) , which combines
the functions of a flow cytometer and a mass spectrometer by using atomic mass spectrometry to allow immunophenotypic detection of up to 25 metal labeled intracellular/extracellular antibodies. Because the mass spectrometer provides at least 3 orders of magnitude resolution between adjacent detection channels, compensation
is not required. This is the first CyTOF installed in Japan and, even throughout the world, only 4-5 institutions
have installed the machine. The FACS Lab invited Dr. Sean C. Bendall from Stanford University and organized
a seminar to introduce the application of this machine for single-cell analysis of immunological research.
Table 1: Instruments in the FACS Lab
Machine types

Machines

Number of machines

FACS cell analyzer

Calibur5

CantoⅡ

1

Imaging flow cytometer

ImageStreamX

1

Mass Cytometer

CyTOF

1

FACS cell sorter

AriaⅠ

2

AriaⅡ

1

Vantage

2

Diva (digital vantage)

1

Figure 1 : CyTOF

For the users of FACS machines ( cell analyzers and cell sorters ) , Hanae Fujimoto and Yukiko Hachiman provide
various services, mainly in the following three areas.
1. Technical support and training
In 2011, the facility offered 12 technical courses ( 5 for cell sorting, 4 for cell analysis and 3 for CyTOF ) bothin English and Japanese. Courses were held for 4 different machine types, Calibur, Canto II, CyTOF and Aria
I/II. A total of 82 RCAI researchers took the courses in 2011.
2. Cell sorting operation service
The FACS Lab provides a cell sorting operation service, in which researchers can ask an experienced
operator to conduct the sorting experiment. In 2011, there were 283 such requests.
3. Management/maintenance of FACS machines
FACS machines are available for registered users 24 hours a day and reservations are accepted up to one
month in advance through an internal website. All the necessary information including instructions, reservations and user fees can be accessed via the intranet. In addition to the in-house FACS Lab staffs, engineersfrom Becton Dickinson visit once a week to provide maintenance and technical support.

128

Monoclonal Antibody Lab.
Technical Staff : Tomomi Aoyama, Kazuyo Uchida

The Monoclonal Antibody ( mAb ) Lab aims to produce mAbs that meet the needs of RCAI researchers, and also
focuses on more targeted development of mAb with a strategic purpose, such as those for allergy-related
molecules. This activity is now being done in cooperation with the program for Drug Discovery and Medical
Technology Platforms for development of monoclonal antibody as diagnosis and drugs. Two technical staff
members, Tomomi Aoyama and Kazuyo Uchida, are engaged in producing, purifying and analyzing mAbs. In
2011, the Lab produced mAbs against 13 different antigens, which were requested by 8 laboratories. The lab
also prepared human chimeric antibodies, such as anti-Cry j 1 human IgE antibody.

Confocal Lab.
Visiting Scientist : Yasutaka Wakabayashi (Leica Microsystems K.K.)

The Confocal Lab provides imaging equipment and technical support. The Confocal Lab is managed in collaboration with Leica Microsystems and there are 6 confocal microscope systems:
1. Inverted system with a visual and multi-photon MP laser suitable for time-lapse imaging of living cells andorgans.
2. Inverted system with a 405 nm laser suitable for a time-lapse imaging of living cells in a controlled environment ( CO2, temperature, and humidity ) .
3. Leica SP5 confocal system, the successor of the SP2. Brighter and more striking images can be produced
because the optical system has been improved.
4. Upright system with visual and UV lasers suitable for standard fixed specimen observation.
5. Newly installed inverted intravital system with a visual, MP, and OPO laser and a high speed scanner which
can be used for in vivo imaging of various tissues. In particular, the OPO laser can excite red fluorophores
and excite them in a deeper region of the tissue because the tunable wavelength is longer than usual MP.
6. Intravital upright system with a single visual laser, double MP lasers and a high speed scanner that can beused for in vivo imaging and for some applications such as light stimulation.
During 2011, Dr. Wakabayashi from Leica Microsystems K.K. provided training for 14 researchers. The total
running time of the microscopes was over 1980 hours.

129

Genomics Lab.
Research Scientist : Takashi Watanabe, Yoko Kuroki
Research Associate : Mai Yamagishi, Atsushi Hijikata
Technical Staff: Tomoko Hasegawa, Nobutake Suzuki, Akio Kobayashi, Fumie Yokoyama, Emi Abe

The Genomics Lab provides a wide variety of services to the members of the Center: proteomics analysis, invitro translation, multiplex suspension array, DNA microarray, DNA sequencing, cDNA/Genomic clone distribution, and Primer/labeled probe distribution for qRT-PCR analysis of immune cells ( Table 2 ) .
Because genome technology has been progressing very quickly, the Genomics lab is keen to provide theCenter with the most up-to-date technology on demand. We have thus decided to introduce two next generation sequencers, a GS Junior [ Roche ] and a HiSeq 1000 [ Illumina ] ( Fig. 2 ) . Both sequencers enable us to obtainhigh massively parallel DNA sequencing data for genome DNA, transcripts, and so forth. The GS Junior isalready available while the members of the Center will be able to access the HiSeq 1000 in early 2012.

Table 2: Services provided by the Genomics Lab in 2011
Proteimics# of samples# of teams

Two-dimentional electrophoresis

102

Mass Spectrometry Analysis

78

2

In vitro translation ( recombinant protein synthesis )# of samples# of teams

1

1Multiplex suspension array# of samples# of teams

4,012

11

Affymetrix Genechip ( Exon array, Gene array, miRNA array )# of samples# of teams

Human

118

5

Mouse

518

17

636

22

DNA sequencing

Total# of samples# of teams

36cm capillary

13,3841950cm capillary

13,152

17

Total

26,53636# of clones# of teams

cDNA clone delivery
Primer/labeled probe delivery

62

7# of sets# of teams

145

3

Figure 2: GS junior (left) and
HiSeq 1000 (right)

130

Animal Facility
Senior Technical Scientist: Takanori Hasegawa

Technical Scientist: Shinobu Mochizuki, Masashi Matsuda: Tomoyuki Ishikura, Isamu Hisanaga, Yusuke Iizuka, Hiromi Tatsumi,
		 Yurie Kawamoto, Chie Yoshida
Technical Staff

Animal caretakers ( outsourced ) : 21 people

Over 10,000 cages of SPF mice are maintained in the Animal Facility. The facility provides the following services
for the users in the RIKEN Yokohama Institute.
1. Maintenance, generation and cryostocks of genetic resources
The Animal Facility has been maintaining over 47,000 mice and 5 rats in the SPF area, 1,700 mice in an
isolated area and several germfree mice. They have newly introduced 553 mouse lines into the SPF area by
a combination of in vitro fertilization ( IVF ) and embryo transfer and generated cryostocks of genetic resources
for 448 lines. They also maintain relatively large colonies for several commonly used strains such as NOD/
SCID/CγKO mice, Rag1KO and cre deleters and provided them to users on demand. In addition, the animal
facility provided technical assistance to generate chimeras ( 249 lines ) , transgenic and BAC-transgenic mice( 12 lines ) , as well as to establish and maintain ES cells. Furthermore, they generated an internally available
database for genetic resources.
2. Introduction of human BAC clones into NOD/SCID/CγKO miceThis work is undertaken in collaboration with Dr. Osamu Ohara ( RCAI, Immunogenomics Group ) and Dr.
Fumihiko Ishikawa ( RCAI Human Disease Model Group ) .
The Animal Facility has launched a new activity to improve the efficacy of transplantation of humanhematopoietic stem cells into NOD/SCID/CγKO mice by “humanizing” the host strain. For this purpose, they
have introduced large genomic fragments containing human genes encoding MHC, cytokines, adhesion
molecules, virus receptors and others into NOD/SCID/C γ KO mice. Up to now, they have established 19
BAC transgenics and confirmed the expression of human genes on a C57BL6 background and begun backcrossing onto the NOD/SCID/C γKO mice using the speed-congenic method.
3. Creation of germfree mice, maintenance and management
This work is undertaken in collaboration with Dr. Hiroshi Ohno ( RCAI, Epithelial Immunobiology Team ) .
The Animal Facility attempted sanitization of knockout mice, and built a system that creates a germ-freemouse at the pace of 1 strain per month. They have created 4 germ-free mice in this year. They are bred in
the germ-free state even now.

Figure 3: Bio-bubble for the maintenanceof immunodeficient mice131

Administrative
Coorddination Office
Table : Members of the Administrative Coordination OfficeSince the Administrative Coordination Office (AdmCO) was
organized in April 2010, the office has been working to support the researchers of RCAI in a variety of ways. This year
they extended their support to the scientists outside RIKEN.

Position

Name

Office Manager

Ichiro Taniuchi

After the Great East Japan Earthquake, RCAI, as a member

Administrationof RIKEN Yokohama Institute decided to provide support toChief Assistants

the researchers in the Tohoku area. AdmCO took a lead in

Hiroko Yamaguchi

arranging to send them research materials, such as celllines and mice, as well as laboratory equipment. AdmCO

Mari Kurosaki

believes that these donations contributed greatly to therecovery of universities and research institutions there.

Assistants

Sachiko Haraguchi

mer 2011, Mr. Ogata, facilities staff, and others actively
cooperated with the power saving committee in reducing

Akiko Imaithe number of power-consuming machines, especially

Akiko Imaizumi

freezers and refrigerators.

Aiko Iyama

The devastating earthquake greatly affected the center’s

Shihoko Kato

events too. RCAI had to cancel most of the Center's international programs, such as RCAI-JSI International Sympo-

Reiko Kimurasium, RCAI International Summer Program, and the Harvard

Satomi Law

intern program after months of preparation. However, a newprogram called the RCAI 10th anniversary seminar series

Rie Morita

was launched instead. AdmCO invited 35 promising young

Rieko Okoshi

scientists both from Japan and abroad. Each AdmCO staff
member prepared for a seminar or two and the IT teamToshiko Nakamura

assisted the speakers, providing for a better presentation

Kazuyo Nomura

environment.

Yuko Ochi

To support the RCAI researchers AdmCO organized three

Norie Takeuchi

working groups in the office. Each group works on MTAs,
mouse import and export support, and the ordering sys-

Yuuki Yamadatem, respectively. The MTA group processed more than 150

Mio Yoshioka

MTAs and the mouse group supported 17 exports. The
order system staff dealt with many daily inquiries, requests

Motoko Yoshioka

and problems, the numbers reaching almost 600 in a half

Next year AdmCO plans to expand the MTA group further

IT-team
Technical Scientists

scattered in discontinued teams. Also, AdmCO plans toimprove the internal website just revamped by the IT team.
AdmCO is always considering better ways of supporting
RCAI and its members.

132

Yasuaki Murahashi
Miho Izawa

and to build the RCAI intellectual property data by consolidating these agreements, which up until now have been

Sachiko Dohi
Chiaki Fukushima

At the same time, to cope with the power shortages in sum-year. The working groups did their tasks quite efficiently.

Hiroko Tanabe

Technical Staff

Aoi Ozawa

Facilities
Technical Scientist

Toshihiko Ogata

2011Part 7

Data and Statistics

Publications (Apr. 2011 - Mar. 2012)

1

2

134

Journals

IF(2010)FY2011Nature

36.1

1

Cell

32.4

1

Cancer Cell

26.9

1

Nat Immunol

25.7

4

Nat Med25.4

1

Immunity

24.2

6

J Exp Med

14.8

6

Genome Res

13.6

1

PLoS Biol

12.5

1

Gastroenterology

12.0

1

Blood

10.6

5

EMBO Journal

10.1

2

Proc Natl Acad Sci USA

9.8

6

Trends Immunol

9.5

1

Cell Res

9.4

1

J Allergy Clin Immun

9.3

2

Arthritis Rheum

8.4

1

Stem Cells

7.9

1

Adv Immunol

7.2

2

Development

6.9

2

Genome Biol

6.9

1

Sci Signal

6.4

2

Mol Cell Biol

6.2

1

Mol Biol Cell

5.9

1

J Immunol

5.7

8

J Biol Chem

5.3

2

J Virol

5.2

1

Eur J Immunol

4.9

1

Br J Haematol

4.9

1

Bioinformatics

4.9

1

DNA Res

4.8

2

Am J Respir Cell Mol Biol

4.4

1

PLoS One

4.4

2

Intl Immunol

3.3

5

Baumjohann, D., Okada, T. & Ansel, K. Cutting Edge: Distinct
waves of BCL6 expression during T follicular helper cell
development. J Immunol Sep. 2011, 187 (185 ) :2089-2092.

6

Bin, B., Fukada, T., Hosaka, T., Yamasaki, S., Ohashi, W., Hojyo,
S., Miyai, T., Nishida, K., Yokoyama, S. & Hirano, T. Biochemical
characterization of human ZIP13 protein: a homo-dimerized zinctransporter involved in the spondylocheiro dysplastic Ehlers-Danlos
syndrome. J Biol Chem Nov. 2011, 286 (246) :40255-40265.

7

Casanova, M., Preissner, T., Cerase, A., Poot, R., Yamada, D., Li, X.,
Appanah, R., Bezstarosti, K., Demmers, J., Koseki, H. & Brockdorff,
N. Polycomblike 2 facilitates the recruitment of PRC2 Polycomb
group complexes to the inactive X chromosome and to target loci inembryonic stem cells. Development Mar. 2011, 138 (138 ) :1471-1482.

8

Ebisawa, M., Hase, K., Takahashi, D., Kitamura, H., Knoop, K., Williams,
I. & Ohno, H. CCR6hiCD11cint B cells promote M-cell differentiationin Peyer’s patch. Int Immunol Apr. 2011, 23 ( 24 ) :261-269.

9

Fujii, H., Ato, M., Takahashi, Y., Otake, K., Hashimoto, S., Kaji,
T., Tsunetsugu-Yokota, Y., Fujita, M., Adachi, A., Nakayama,T., Taniguchi, M., Koyasu, S. & Takemori, T. HIV-1 Nef impairs
multiple T-cell functions in antigen-specific immune responsein mice. Int Immunol Jul. 2011, 23 ( 27 ) :433-441.

10Fujimura, T., Yonekura, S., Horiguchi, S., Taniguchi, Y., Saito,
A., Yasueda, H., Inamine, A., Nakayama, T., Takemori, T.,
Taniguchi, M., Sakaguchi, M. & Okamoto, Y. Increase ofregulatory T cells and the ratio of specific IgE to total IgE are
candidates for response monitoring or prognostic biomarkers
in 2-year sublingual immunotherapy (SLIT) for Japanese cedar
pollinosis. Clin Immunol Apr. 2011, 139 (131) :165-174.

11

Fukada, T. & Kambe, T. Molecular and genetic featuresof zinc transporters in physiology and pathogenesis.
Metallomics Jul. 2011, 3 ( 7 ) :662-674.

12

Fukada, T., Yamasaki, S., Nishida, K., Murakami, M. & Hirano,T. Zinc homeostasis and signaling in health and diseases: Zincsignaling. J Biol Inorg Chem Oct. 2011, 16 (17 ) :1123-1134.

13

Fukuda, S., Hase, K. & Ohno, H. Application of a mouse ligatedPeyer’s patch intestinal loop assay to evaluate bacterial uptake
by M cells. J Vis Exp Jan. . 2011, 10.3791/3225 (Epub.).

14

Galbas, T., Steimle, V., Lapointe, R., Ishido, S. & Thibodeau,
J. MARCH1 down-regulation in IL-10-activated B cells
increases MHC class II expression. Cytokine Apr.
2012, 10.1016/j.cyto.2012.1003.1015 (Epub.).

15

Goel, R., Raju, R., Maharudraiah, J., Kumar, G., S. S.,
Ghosh, K., Kumar, A., Lashmi, P., T., Sharma, J., Sharma, R.,
Balakrishnan, L., Pan, A., Kandasamy, K., Christopher, R.,
Krishna, V., Mohan, S. S., Harsha, H. C., Mathur, P. P., Pandey,
A. & Prasad, T. S. K. A Signaling Network of Thyroid-Stimulating
Hormone. J Proteomics Bioinform Sep. 2011, 238-241.

16

Guo, Y., Miyazaki, M., Itoi, M., Satoh, R., Iwama, A., Amagai,
T., Kawamoto, H. & Kanno, M. Polycomb group gene
Bmi1 plays a role in the growth of thymic epithelial cells.
Eur J Immunol Apr. 2011, 41 (44 ) :1098-1107.

17

Hashimoto-Tane, A., Yokosuka, T., Sakata-Sogawa, K., Sakuma, M.,
Ishihara, C., Tokunaga, M. & Saito, T. Dynein-driven transport of T
cell receptor microclusters regulates immune synapse formation
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Hassan, H., Sakaguchi, S., Tenno, M., Kopf, A., Boucheron, N.,
Carpenter, A., Egawa, T., Taniuchi, I. & Ellmeier, W. Cd8 enhancer E8I
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Hemmi, H., Zaidi, N., Wang, B., Matos, I., Fiorese, C., Lubkin, A.,
Zbytnuik, L., Suda, K., Zhang, K., Noda, M., Kaisho, T., Steinman, R. &Idoyaga, J. Treml4, an Ig superfamily member, mediates presentationof several antigens to T cells in vivo, including protective immunity
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5

Other Journals

28

Total

103

Adoro, S., McCaughtry, T., Erman, B., Alag, A., Van, L., F., Park,J., Tai, X., Kimura, M., Wang, L., Grinberg, A., Kubo, M., Bosselut,
R., Love, P. & Singer, A. Coreceptor gene imprinting governs
thymocyte lineage fate. EMBO J Oct. 2011, 31 (32) :366-377.
Arima, Y., Harada, M., Kamimura, D., Park, J., Kawano, F., Yull,F., Kawamoto, T., Iwakura, Y., Betz, U., M_rquez, G., Blackwell,T., Ohira, Y., Hirano, T. & Murakami, M. Regional neuralactivation defines a gateway for autoreactive T cells to crossthe blood-brain barrier. Cell Feb. 2012, 148 (143 ):447-157.

3Baba, Y. & Kurosaki, T. Impact of Ca2+ signaling on B cellfunction. Trends Immunol Dec. 2011, 32 (12 ) :589-594.

4

Baravalle, G., Park, H., McSweeney, M., Ohmura-Hoshino, M.,
Matsuki, Y., Ishido, S. & Shin, J. Ubiquitination of CD86 is a key
mechanism in regulating antigen presentation by dendritic
cells. J Immunol Sep. 2011, 187 (186 ) :2966-2973.

20

Hijikata, A., Yura, K., Noguti, T. & Go, M. Revisiting gap
locations in amino acid sequence alignments and a proposal
for a method to improve them by introducing solvent
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36

Kong, K., Yokosuka, T., Canonigo-Balancio, A., Isakov, N., Saito, T. &Altman, A. A motif in the V3 domain of the kinase PKC-É∆ determines
its localization in the immunological synapse and functions in T cells
via association with CD28. Nat Immunol Oct. 2011, 12 (11) :1105-1112.

21

Hisada, K., S_nchez, C., Endo, T., Endoh, M., Rom_n-Trufero, M., Sharif,J., Koseki, H. & Vidal, M. RYBP Represses Endogenous Retroviruses
and Preimplantation- and Germ Line-Specific Genes in Mouse
Embryonic Stem Cells. Mol Cell Biol Mar. 2012, 32 (36 ) :1139-1149.

37

Kubota, Y., Karube, F., Nomura, M., Gulledge, A. T., Mochizuki, A.,
Schertel, A. & Kawaguchi, Y. Conserved properties of dendritic trees infour cortical interneuron subtypes. Sci Rep Feb. . 2011, 1: 89 (Epub.).

22

Honke, N., Shaabani, N., Cadeddu, G., Sorg, U. R., Zhang,
D. E., Trilling, M., Klingel, K., Sauter, M., Kandolf, R., Gailus,N., van Rooijen, N., Burkart, C., Baldus, S. E., Grusdat, M.,
Lohning, M., Hengel, H., Pfeffer, K., Tanaka, M., Haussinger, D.,
Recher, M., Lang, P. A. & Lang, K. S. Enforced viral replication
activates adaptive immunity and is essential for the control ofa cytopathic virus. Nat Immunol Jan. 2012, 13 (11) :51-17.

38

Kurokawa, J., Nagano, H., Ohara, O., Kubota, N., Kadowaki,T., Arai, S. & Miyazaki, T. Apoptosis inhibitor of macrophage(AIM ) is required for obesity-associated recruitment ofinflammatory macrophages into adipose tissue. Proc Natl
Acad Sci U S A Jul. 2011, 108 (129 ) :12072-12077.

39

Kurosaka, H., Islam, M., Kuremoto, K., Hayano, S., Nakamura,M., Kawanabe, N., Yanagita, T., Rice, D., Harada, H., Taniuchi, I. &Yamashiro, T. Core binding factor beta functions in the maintenance ofstem cells and orchestrates continuous proliferation and differentiationin mouse incisors. Stem Cells Nov. 2011, 29 (11) :1792-1803.

40

Kurosaki, T. Regulation of BCR signaling. Mol Immunol
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41

Lee, S., Rigby, R., Zotos, D., Tsai, L., Kawamoto, S., Marshall,J., Ramiscal, R., Chan, T., Gatto, D., Brink, R., Yu, D., Fagarasan,
S., Tarlinton, D., Cunningham, A. & Vinuesa, C. B cell priming
for extrafollicular antibody responses requires Bcl-6 expression
by T cells. J Exp Med Jul. 2011, 208 (207 ):1377-1388.

42

Li, Y., Gao, X. & Wang, J. Comparison of two POLQ mutants
reveals that a polymerase-inactive POLQ retains significant
function in tolerance to etoposide and É¡-irradiation inmouse B cells. Genes Cells Sep. 2011, 16(19):973-983.

43

Limnander, A., Depeille, P., Freedman, T., Liou, J., Leitges, M.,
Kurosaki, T., Roose, J. & Weiss, A. STIM1, PKC-É¬ and RasGRP
set a threshold for proapoptotic Erk signaling during B cell
development. Nat Immunol May. 2011, 12 (15) :425-433.

44

Linterman, M., Pierson, W., Lee, S., Kallies, A., Kawamoto,S., Rayner, T., Srivastava, M., Divekar, D., Beaton, L., Hogan,
J., Fagarasan, S., Liston, A., Smith, K. & Vinuesa, C. Foxp3+
follicular regulatory T cells control the germinal center
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45

Matsumoto, M., Fujii, Y., Baba, A., Hikida, M., Kurosaki,
T. & Baba, Y. The calcium sensors STIM1 and STIM2
control B cell regulatory function through interleukin-10
production. Immunity May. 2011, 34 (35) :703-714.

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Mishima, Y., Miyagi, S., Saraya, A., Negishi, M., Endoh, M., Endo,T., Toyoda, T., Shinga, J., Katsumoto, T., Chiba, T., Yamaguchi, N.,
Kitabayashi, I., Koseki, H. & Iwama, A. The Hbo1-Brd1/Brpf2 complex
is responsible for global acetylation of H3K14 and required for fetalliver erythropoiesis. Blood Sep. 2011, 118 (119 ) :2443-2453.

47

Miyao, T., Floess, S., Setoguchi, R., Luche, H., Fehling,H., Waldmann, H., Huehn, J. & Hori, S. Plasticity of
Foxp3(+) T cells reflects promiscuous Foxp3 expression inconventional T cells but not reprogramming of regulatory
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48

Mizuno, T., Sakai, H., Nishikomori, R., Oshima, K., Ohara, O.,
Hata, I., Shigematsu, Y., Ishige, T., Tamura, K. & Arakawa, H. Novelmutations of MVK gene in Japanese family members affectedwith hyperimmunoglobulinemia D and periodic fever syndrome.
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Mochizuki-Kashio, M., Mishima, Y., Miyagi, S., Negishi, M., Saraya,
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Morita, M., Oike, Y., Nagashima, T., Kadomatsu, T., Tabata, M., Suzuki,T., Nakamura, T., Yoshida, N., Okada, M. & Yamamoto, T. Obesity
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23

Hori, S. Stability of regulatory T-cell lineage. Adv Immunol
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Imai, T., Kato, Y., Kajiwara, C., Mizukami, S., Ishige, I., Ichiyanagi,T., Hikida, M., Wang, J. & Udono, H. Heat shock protein 90(HSP90) contributes to cytosolic translocation of extracellularantigen for cross-presentation by dendritic cells. Proc Natl
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25

Izawa, K., Hijikata, A., Tanaka, N., Kawai, T., Saito, M.,
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26

Kajikawa, M., Li, P., Goto, E., Miyashita, N., Aoki-Kawasumi,M., Mito-Yoshida, M., Ikegaya, M., Sugita, Y. & Ishido, S. The
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27

Kano, C., Hanaoka, F. & Wang, J. Analysis of mice deficient in both
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Kano, C., Ouchida, R., Kokubo, T. & Wang, J. Rapid cell divisioncontributes to efficient induction of A /T mutations during Ig gene
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Kendal, A., Chen, Y., Regateiro, F., Ma, J., Adams, E., Cobbold,S., Hori, S. & Waldmann, H. Sustained suppression by Foxp3+
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Kenmochi, R., Hanai, T., Nakakuki, T., Okada, M. &Ishii, C. Particle simulation of epidermal growth factor
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Kimura, S., Araki, D., Matsumura, K. & Okada-Hatakeyama,
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Kimura, S., Nakakuki, T., Kirita, S. & Okada, M. AGLSDC: a genetic local
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Kitano, M., Moriyama, S., Ando, Y., Hikida, M., Mori, Y.,
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Kitano, M. & Okada, T. Four-dimensional tracking of lymphocyte
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Kometani, K., Yamada, T., Sasaki, Y., Yokosuka, T., Saito, T.,
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Murakami, M. & Hirano, T. A four step model for the IL-6
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Murtagh, V., O’Meally, D., Sankovic, N., Delbridge, M., Kuroki, Y.,
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Naito, T., Tanaka, H., Naoe, Y. & Taniuchi, I. Transcriptional control
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Nakagawa, N., Imai, K., Kanegane, H., Sato, H., Yamada, M.,
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S., Pan-Hammarstr_m, Q., Hammarstr_m, L., Reichenbach,J., Seger, R., Ariga, T., Hara, T., Miyawaki, T. & Nonoyama, S.
Quantification of kappa-deleting recombination excision circles
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Nishida, K., Yamasaki, S., Hasegawa, A., Iwamatsu, A.,
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Otsuka, A., Kubo, M., Honda, T., Egawa, G., Nakajima, S.,
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Renfree, M., Papenfuss, A., Deakin, J., Lindsay, J., Heider, T., Belov,
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Saeki, Y., Nagashima, T., Kimura, S. & Okada-Hatakeyama, M. An
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Tanno, H., Yamaguchi, T., Goto, E., Ishido, S. & Komada, M.
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Shiraishi, Y., Okada-Hatakeyama, M. & Miyano, S. A rank-based
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Song, S., Chew, C., Dale, B., Traum, D., Peacock, J., Yamazaki,T., Clynes, R., Kurosaki, T. & Greenberg, S. A requirement
for the p85 PI3K adapter protein BCAP in the protectionof macrophages from apoptosis induced by endoplasmic
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Troutman, T., Hu, W., Fulenchek, S., Yamazaki, T., Kurosaki, T., Bazan,
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Ushio, H., Ueno, T., Kojima, Y., Komatsu, M., Tanaka, S., Yamamoto, A.,
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137

Invited Presentations
Date

Speaker

Title

Meeting

Place

Apr - 11

Cheroutre, H.

Stretching CD4 Effector T Cells Beyond Plasticity

Keystone Symposium on Immunoregulatory Networks

Breckenridge, USA

Apr - 11Wang, J.-Y.

The lysosomal protein LAPTM5 negatively regulates cellsurface TCR and BCR expression and T and B cell activation

Peking University Special Seminar

Beijing, China

Apr - 11

Fagarasan, S.

PD-1 deficiency skews the gut microbiota by altering the IgA
selection in gut

The 2012 Spring Conference of the Korean Association of
Immunologists

Seoul, Korea

Apr - 11

Kurosaki, T.

Function of Calcium in Lymphocytes

Advances in Immunology and Cancer Biology

Istanbul, Turkey

Apr - 11

Kurosaki, T.

Function of Store-Operated Calcium Entry in B Lymphocytes

Keystone Symposia, B Cells: New Insights into Normal versus
Dysregulated Function

Whistler, Canada

Apr - 11

Fukuda, S.

Elucidation of the mechanisms of host-bacterial crosstalk in
the gut by multiple omics approach

Global COE program, The University of Tokyo

Kashiwa, Japan

Apr - 11

Taniuchi, I.

Transcriptional control of thymocytes development

Seminar at SIgN

Singapore, Singapore

Apr - 11

Ohara, O.

Integration of New Technologies for Accurate and Rapid
Molecular Diagnosis of Primary Immunodeficiencies

The 7th Congress of Asian Society for Pediatric Research

Denver, USA

May - 11

Tokunaga, M.
& SakataSogawa, K.

Strategies adapted by cells examined by single molecule
quantification using fluorescence imaging“The next technical breakthrough” Bio-finance Guild Seminar

Tokyo, Japan

May - 11

Ohara, O.

Integration of Microfluidics Technology with Advanced
Genomics for Prognosis/Diagnosis of Allergic and
Immunological Diseases

150 Jahre Wirtschaftsbeziehungen Deutschland-Japan

Dusseldorf, Germany

May - 11Ohno, H.

Mechanism of protection of mouse O157-infectious death bybificobateria using multi-omics approach

1st JAXA Space Medicine and Biology Research Workshop in2011 "How to prevent immune suppressor - from astronautes
to the elderly -"Tokyo, Japan

May - 11

Kaisho, T.

Dendritic cell sensing and responses for nucleic acidadjuvants

KSBMB 2011 Annual Meeting

Seoul, Korea

May - 11

Fagarasan, S.

Bacteria and retinoic acid impact on follicular dendritic cells
function in Peyer's patches

The 76th Japanese Society of Interferon and Cytokine
Resesarch The 19th International Symposium on Molecular
Cell Biology of Macrophages

Osaka, Japan

May - 11

Saito, T.

Visualizing the dynamics of immune synapse by TIRFXXVI Congress of the International Society for Advancement of
Cytometry ( CYTO2011 )Baltimore, USA

May - 11

Ishikawa, F.

Targeting chemotherapy-resistant human AML stem cells

XXVI Congress of the International Society for Advancement of
Cytometry (CYTO2011 )Baltimore, USA

May - 11

Koseki, H.

Polycomb-dependent regulation for differentiation programsof stem cells and progenitors

5th Annual Meeting of Japanese Society for Epigenetics

Kumamoto, Japan

May - 11

Kawamoto, H.

Lineage restriction process from hermatopoietic stem cells
to T cell progenitors: An essential developmental checkpoint
for the production of T cell lineage

Hematology Lecture at Erasmus University

Rotterdam , The
Netherland

May - 11

Taniuchi, I.

Transcriptional control of thymocytes development

Seminar at Max Planck Institute, Freiburg

Freiburg, Germany

May - 11

Taniuchi, I.

Transcriptional control of thymocytes development

Seminar at University of Tubingen

Tubingen, Germany

Jun - 11

Fagarasan, S.

T cell-independent and T cell-dependent lgA synthesis in
the gut

Master Class
Istituto di Ricerca in Biomedicina.

Bellinzona, Switzerland

Jun - 11

Hirano, T.

IL-6 amplifier and autoimmune

The 2th Symposium on Joint Research Core Network Institutefor Enzyme Research, Institute for Enzyme Research,The
University of Tokushima

Tokushima, JapanJun - 11Ishii, Y.

Uncovering adjuvant potential of natural killer T (NKT) cell
ligand, alpha-galactosylceramide

5th World Vaccine Congress Asia 2011

Singapore

Jun - 11

Saito, T.

Spatiotemporal regulation of T cell co-stimulation

FASEB Summer Conferences-Signal Transduction in the
Immune System

Snowmass Village,USA

Jun - 11

Kurosaki, T.

Calcium signaling in immune cells

FASEB Summer Conferences-Signal Transduction in the
Immune System

Snowmass Village,USA

Jun - 11

Hase, K.

Biological significance of M cells in gut mucosal immunity

The 15th Annual Meeting of Intestinal Microbiology

Tokyo, JapanJun - 11Ohno, H.

Multi-omics analysis for elucidating the host-commensal
bacteria interaction

The 15th Annual Meeting of Intestinal Microbiology

Tokyo, JapanJun - 11Ohno, H.

Multi-omics analysis for elucidating the host-commensal
bacteria interaction - Mechanism of protection of mouse
O157-infectious death by bificobateria

The 16th Tokyo Liver Symposium "Next generation sequenceand genomics"

Tokyo, JapanJun - 11

Fukuda, S.

Carbohydrate transporters confer a probiotic effect tobifidobacteria

The International Scientific Conference on Probiotics andPrebiotics

Kosice, Slovakia

Jun - 11

Taniuchi, I.

Epigenetic regulation of T cell developemnt

Environment and Hormone Meeting

Tokyo, Jaoan

Jun - 11

Ishikawa, F.

Leukemia stem cell research using humanized mouse

21st Japan Soceity of Cytometry Meeting

Kyoto, JapanJun - 11

Kawamoto, H.

Lineage commitment process during hematopoiesis:
Visualization of developmental checkpoints

21st Japan Soceity of Cytometry Meeting

Kyoto, JapanJun - 11

Okada, T.

Imaging of lymphocyte migration and interactions during
immune responses

The 63rd Annual Meeting of The Japan Society for Cell Biology(Satellite Symposium 1 )Sapporo, Japan

Jul - 11

Ishikawa, F.

Investigating human immunity and diseases using
humanized mouse system

15th Japan Society of Cancer Immunology meeting

Osaka, Japan

Jul - 11

Fagarasan, S.

Dynamic interactions between bacteria and immune cells
in the gut

Seminar Series at the Max Planck Institute of Immunobiology
and Epigenetics

Freiburg, Germany

Jul - 11

Fukada, T.

Slc39a14/Zip14: its involvement in regulation of systemic
growth and GPCR signal transduction

The 22nd Annual Meeting of the Japan Society for Biomedical
Research on Trace Elements

Kyoto, Japan

Jul - 11Ishii, Y.

Therapeutic potential of immunoregulatory liposomal alphaGalCer

38th BMS Conference

Hakone, Japan

Jul - 11

Tanaka, M.

Regulation of anti-tumor immunity by clearance of deadtumor cells

Gordon Research Conferences Apoptotic Cell Recognition &Clearance

Lewiston, USA

Jul - 11Ohno, H.

Multi-omics approach reveals that bifidobacteria protectfrom enteropathogenic Escherichia coli infection throughproduction of acetate

The 9th International Workshop on Advanced Genomics

Tokyo, Japan

Jul - 11

Ishikawa, F.

Targeting AML stem cells

9th Japan Soceity of Medicao Oncology meeting

Yokohama, Japan

Jul - 11

Taniuchi, I.

Transcriptional control of thymocytes development

Seminar at RIMD Osak Univ

Osaka, Jaoan

Jul - 11

Koseki, H.

SAM domain-mediated polymerization of mammalianpolyhomeotic homologues mediates recruitment of PRC1 to
Polycomb target genes and their condensation

3rd X-Inactivation Meeting

Oxford, UK

Jul - 11

Hori, S.

Control of autoimmunity by regulatory T cells

The 35th Aso Symposium

Kumamoto, Japan

Aug - 11

Kaisho, T.

Molecular mechanisms for dendritic cell functions

JSI Immunology Summer School

Sendai, Japan138

Aug - 11

Saito, T.

Dynamic regulatory mechanism of T cell activation

JSI Immunology Summer School

Zao, Japan

Aug - 11

Taniuchi, I.

Transcriptional and epigenetic control of helper vs cytotoxic
lineage decision in thymus

Seminar at Stanford University

Stanford, USA

Aug - 11

Taniuchi, I.

Genetic and molecular analyses of Runx-dependentsilencers during T cell development

18th International Runx Workshop

San Diego, USA

Aug - 11Ohno, H.

Analysis of Host-intestinal microbiota interaction by multiomics approach

The 23rd Takato Symposium " The cell revisited"

Ina, Japan

Aug - 11

Fukuda, S.

Understanding the gut ecosystem by multiple omics
approach

The 6th Seminar of the Creative Research Program, Universityof Tsukuba

Tsukuba, Japan

Sep - 11

Saito, T.

Spatiotemporal regulation of T cell activation andcostimulation

1st POSTECH International Symposium of Bio-Imaging

Pohang, Korea

Sep - 11Ohno, H.

Function and differentiation of M cells, a unique subset ofintestinal epithelial cells important for mucosal immunity

ComBio2011, Plenary Lecture

Cairns, Australia

Sep - 11Ohno, H.

Multi-omics approach reveals that bifidobacteria protectfrom enteropathogenic Escherichia coli infection throughproduction of acetate

ComBio2011, symposium

Cairns, Australia

Sep - 11Ohno, H.

The role of M-Sec in tunneling nanotube formation

The EMBO Meeting 2011

Vienna, Austria

Sep - 11

Ishikawa, F.

Phenotype, Distribution, and Heterogeneity of Leukemic
Stem Cells

2011Working Conference on Cancer Stem Cells

Vienna, Austria

Sep - 11

Fukada, T.

How does the zinc homeostasis system control mammalian
growth? Lessons from molecular and genetic studies of zinctransporters Zip13 and Zip14

The 5th International Conference on Metals and Genetics( ICMG 2011)

Kobe, Japan

Sep - 11

Taniuchi, I.

Transcriptional and Epigenetic Regulation of CD4/CD8
Lineage ChoiceCold Spring Harbor Asia Conference

Suzhou, China

Sep - 11

Cheroutre, H.

Protecting the Border with a Fake ID: CD4 T Cells Stretch
Beyond Plasticity

4th International Conference on Crossroads between Innate
and Adaptive Immunity

Mykonos, Greece

Sep - 11Ishii, Y.

Generation of vaccines for polliniosis

59th allergy meeting for kanto otolaryngologist

Tokyo, Japan

Sep - 11

Taniguchi, M.

Early NKT Precursor

6th International Symposium on CD1 and NKT Cells

Chicago, USA

Sep - 11Ohno, H.

Food Allergy, Intestinal Immune System, and Oral Tolerance

The 18th Annual Meeting of The Japanese Society of
Immunotoxicology

Chiba, Japan

Sep - 11

Taniguchi, M.

iPS-derived NKT cells as a clinical application for cellularimmunotherapy

The 39th Annual Meeting of the Japan Society for Clinical
Immunology

Tokyo, JAPANSep - 11

Taniuchi, I.

Transcriptional Control of Helper versus Cytotoxic Lineage
ChoiceAustrian Society for Allergology and Immunology Annual
Meeting 2011

Graz, Austria

Sep - 11Ishido, S.

Novel immune regulation by ubiquitination

The 84th Annual Meeting of the Japanese Biochemical Society

Kyoto, Japan

Sep - 11

Fukada, T.

The zinc transporter Zip14 controls systemic growth and
GPCR signal transduction

The 84th Annual Meeting of the Japanese Biochemical Society

Kyoto, Japan

Sep - 11

Nishida, K.

Role of adaptor molecule Gab2 in mast cell-mediatedanaphylaxis response

The 84th Annual Meeting of the Japanese Biochemical Society

Kyoto, Japan

Sep - 11

Yamasaki, S

The role of intracellular Zn signaling on mast cell activation

The 84th Annual Meeting of the Japanese Biochemical Society

Kyoto, Japan

Sep - 11

Okada, M

Kinetic analysis of transcriptional signal network for
activation of c-Fos transcription factor

The 84th Annual Meeting of the Japanese Biochemical Society

Kyoto, Japan

Sep - 11

Fukuda, S.

Multi-omics analysis elucidates gut environmental
ecosystems

Research Workshop for Lactic Acid Bacteria and Intestinal
Bacteria

Tokyo, Japan

Oct - 11

Okada, M.

Quantitative and predictive analysis of cancer signaling andtranscription network

The 70th Annual Meeting of the Japanese Cancer Association

Nagoya, Japan

Oct - 11

Fujii, S.

New type of immunotherapy using artificial adjuvant vector
cells (aAVCs) linking innate and adaptive immunity.

The 70th Annual Meeting of the Japanese Cancer Association

Nagoya, Japan.

Oct - 11

Watarai, H.

Function of Pathogenic Invariant Natural Killer T Cells
Contribute to the Induction of Airway Hyperreactivity

Joint Congress of Asia Pacific Association of Pediatric Allergy,
Respirology and Immunology (APAPARI 2011) & 48th Annual
Meeting of Japanese Society of Pediatric Allergy and Clinical
Immunology ( 48th JSPACI )Fukuoka, JAPANOct - 11Ohno, H.

Mechanism of protection of mouse from enterohemorrhagic
E. Coli O157-infectious death by bifidobacteria using multiomics approach

The 94th Meeting of the Japanese Society for Bacteriology
Kanto BranchTokyo, Japan

Oct - 11

Fujii, S.

New type of immunotherapy using NKT cell-induced DC
activation in situ

The 73rd Annual Meeting of Japanese Society of Hematology

Nagoya, Japan.

Oct - 11

Ishikawa, F.

Creating therapeutic strategies targeting AML stem cells

The 73rd Annual Meeting of Japanese Society of Hematology

Nagoya, Japan.

Oct - 11

Fukuda, S.

Multi-omics analysis uncovers an ecosystem of gut
environment

The 6th Metabolome Symposium, Japan

Osaka, Japan

Nov - 11

Okada, M.

Identifying logics of signal-transcription network for cellulardifferentiation

IFReC / IPR Joint Seminar

Osaka, Japan

Nov - 11

Okada, M.

Cell fate decisions determined by switch-like responses ofgenetic networks

Theory of Biomathematics and Its Applications VIII

Kyoto, Japan

Nov - 11

Kurosaki, T.

Function of Calcium Signaling in B Lymphocytes

The 1st International Meeting on Ion Channel Signaling
Mechanisms: from Basic Science to Clinical Application

Marrakesh, Morocco

Nov - 11

Hirano, T.

The role of Interleukin 6 amplifier in inflammatory diseases.

Bio-Rheumatology International Congress (BRIC) Tokyo, The
8th GARN Meeting

Tokyo, Japan

Nov - 11Sato, K.

Molecular mechanism for the establishment of oral tolerance

Japan Bioindustry Association Seminar

Tokyo, Japan

Nov - 11

Taniuchi, I.

Transcriptional and epigenetic regulation of thymocytes fatedecision

Seminar at Albany University

Albany, USA

Nov - 11

Taniuchi, I.

Transcriptional and epigenetic control of helper versus
cytotoxic lineage choice

Seminar at Washington University

St. Louis, USA

Nov - 11

Saito, T.

Regulatory mechanism of T cell activation by imaging
analysis

The 61st Annual Meeting of Japanese Society of Allergology

Tokyo, Japan

Nov - 11

Nishida, K.

Role of Zinc in immmune/inflammatory response

The 61st Annual Meeting of Japanese Society of Allergology

Tokyo, Japan

Nov - 11

Okada, T.

Imaging of lymphocyte dynamics during the germinal center
formation

The 61st Annual Meeting of Japanese Society of Allergology

Tokyo, Japan

Nov - 11

Taniguchi, M.

A novel NKT cell subtype which contributes to the inductionof allergic reactions

The 61st Annual Meeting of Japanese Society of Allergology

Tokyo, JAPANNov - 11

Tanaka T.

PDLIM2 inhibits T helper 17 cell development andgranulomatous inflammation through degradation of STAT3

The 40th Annual Meeting of the Japanese Society for
Immunology

Chiba, Japan

Nov - 11

Tanaka, M.

The role of CD169 macrophages in anti-tumor immunity

The 40th Annual Meeting of the Japanese Society for
Immunology

Chiba, Japan139

Nov - 11

Yokosuka, T.

The PD-1 microclusters: negative costimulatory
signalosomes directly targeting TCR downstream

The 40th Annual Meeting of the Japanese Society for
Immunology

Chiba, Japan

Nov - 11

Kurosaki, T.

Contribution of transcription factors to rapid responsivenessof IgG type memory B cells

The 40th Annual Meeting of the Japanese Society for
Immunology

Chiba, Japan

Nov - 11

Cheroutre, H.

Activation-induced silencing of Thpok defines a new type of
CD4 effector T lymphocytes

The 40th Annual Meeting of the Japanese Society for
Immunology

Chiba, Japan

Nov - 11Wang, J.-Y.

The Fc receptor for IgM (FcμR ) positively regulates B cell
survival and activation

The 40th Annual Meeting of the Japanese Society for
Immunology

Chiba, Japan

Nov - 11

Okada, T.

Imaging of lymphocyte dynamics during the germinal center
formation

The 40th Annual Meeting of the Japanese Society for
Immunology

Chiba, Japan

Nov - 11

Fukuda, S.

Acetate-producing bifidobacteria equipped with ‘probiotic
transporters’ protects the host against enteropathogenic
infection

The 40th Annual Meeting of the Japanese Society for
Immunology

Chiba, Japan

Nov - 11

Hori, S.

Analysis of IPEX mutations reveals a critical role of theKLRG1+ subset of regulatory T cells in the regulation of
autoimmunity

The 40th Annual Meeting of the Japanese Society for
Immunology

Chiba, Japan

Nov - 11

Kawamoto, H.

Molecular mechanisms for the production and maintenanceof the T cell lineage

The 40th Annual Meeting of the Japanese Society for
Immunology

Chiba, Japan

Nov - 11

Cheroutre, H.

Mucosal Immunity: At the Borders, It Matters Who You Are

The 6th Chiba University Global COE Symposium

Chiba, Japan

Dec - 11

Yokosuka, T.

Dynamic regulation of T cell activation by TCR microclusters

Japan-China-Korea Immunology Symposium 2011

Osaka, Japan

Dec - 11

Hase, K.

Epithelial immune functions at the interface between self
and non-self

Japan-China-Korea Immunology Symposium 2011

Osaka, Japan

Dec - 11

Cheroutre, H.

Activation-induced silencing of Thpok defines a new type of
CD4 effector T lymphocytes

University of Michigan-RIKEN RCAI Joint Workshop

Yokohama, Japan

Dec - 11

Fujii, S.

DC targeting immunotherapy by antigen mRNA-transfected,
allogeneic cells loaded with NKT cell ligand as artificial
adjuvant vector cells (aAVC)

University of Michigan-RIKEN RCAI Joint Workshop

Yokohama, Japan

Dec - 11Ohno, H.

The epithelia-specific membrane trafficking factor AP-1B
secures gut immune homeostasis in mice

University of Michigan-RIKEN RCAI Joint Workshop

Yokohama, Japan

Dec - 11

Tanaka T.

HSP70 is essential for PDLIM2-mediated termination of
NF-κB signaling

University of Michigan-RIKEN RCAI Joint Workshop

Yokohama, Japan

Dec - 11

Taniuchi, I.

Mechanisms of Helper versus Cytotoxic Lineage Choice

University of Michigan-RIKEN RCAI Joint Workshop

Yokohama, Japan

Dec - 11

Udono, H.

Dendritic cell requires HSP90 as a cytosolic translocator of
extracellular antigen for cross-presentation

University of Michigan-RIKEN RCAI Joint Workshop

Yokohama, Japan

Dec - 11

Yokosuka, T.

Imaging of T cell activation regulation: - Negative regulationof TCR signaling by PD-1 microcluster –University of Michigan-RIKEN RCAI Joint Workshop

Yokohama, Japan

Dec - 11

Nomura, M.

Practical application of the cmgraph method in using realdata

Workshop on Applied Dynamical Systems

Kyoto, Japan

Dec - 11

Hase, K.

Functional roles of GP2 and M-Sec in intestinal M cells

Matrix of Infection Phenomena Syposium on "The future of
Infectious Disease Study"

Tokyo, Japan

Dec - 11Sato, K.

Crucial role of mesenteric lymph node dendritic cells in theestablishment of oral tolerance

The 32nd Special Seminar on Institute of Natural Medicine,
University of Toyama

Toyama, JapanJan - 12

Fagarasan, S.

PD-1 deficiency skews the gut microbiota by altering the IgA
selection in gut

IFReC-SIgN Winter School on Advanced Immunology

Awaji, JapanJan - 12

Fagarasan, S.

Regulation of IgA synthesis in Peyer’s patches: new aspects

The 4th Symposium for the MEXT Priority Research on
Immunological Self

Kyoto JapanJan - 12

Hori, S.

Plasticity of Foxp3 + T cells: its implications in Treg celllineage commitment

The 4th Symposium for the MEXT Priority Research on
Immunological Self

Kyoto, JapanJan - 12

Hirano, T.

Zinc as a signaling moleculeInternational Society for Zinc Biology 2012 Conference

Melbourne, Australia

Feb - 12

Fagarasan, S.

New aspects of T cell-dependent IgA synthesis in gut

New Horizons in the Immune System

Tokushima, Japan

Feb - 12Kaisho, T.

Molecular mechanisms for regulating dendritic cell subset
functions

The 29th Annual Meeting of Japanese Society of Immunology& Allergology in Otolaryngology

Ooita, Japan

Feb - 12

Hori, S.

Resolving the controversy over regulatory T cell plasticity

The 14th International Conference on Lymphocyte Activation
and Immune Regulation: T Cell Differentiation and Plasticity

Newport Beach, USA

Feb - 12

Saito, T.

Negative regulation of TCR signaling by PD-1 microcluster

RCAI-LIAI Joint Workshop

La Jolla, USA

Feb - 12

Hori, S.

Genetic and environmental control of regulatoy T cell fitness
in peripheral tissues

RCAI-LIAI Joint Workshop

La Jolla, USA

Feb - 12Ohno, H.

M Cells and Antigen Delivery to DCs in the Gut

Keystone Symposium on Dendritic Cells and the Initiation of
Adaptive Immunity

Santa Fe, USA

Feb - 12Sato, K.

Control of immune response by dendritic cells

Seminar on Nippon Institute for Biological Science

Tokyo, Japan

Feb - 12Cheroutre, H.

Protecting the Border with a Fake ID: CD4 T Cells Stretch
Beyond Plasticity

Keystone Symposium on Mucosal Biology: A Fine Balance
between Tolerance and Immunity

Vancouver, Canada

Feb - 12Sato, K.

Plasmacytoid dendritic cells in innate and adaptive immunity

Seminar on WPI Immunology Frontier Research Center, Osaka
University

Osaka, Japan

Feb - 12

Koseki, H.

Application of iPS Cell Technology for NKT Cell-targeted
Adjuvant Therapy for Tumors in Mice

14th US-Japan Cellular and Gene Therapy Conference

Bethesda, USA

Feb - 12

Kawamoto, H.

A revised model of hematopoiess

2011 Japan Research Association For Immunotherapeutics

Tokyo, Japan

Feb - 12

Kawamoto, H.

Myeloid-based model of hematopiesis: revison of theclassical myeloid-lymphoid dichotomy concept

The 34th Annual Meeting of the Japan Society for
Hematopoietic Cell Transplantation

Osaka, Japan

Mar - 12

Fagarasan, S.

Dynamic interactions between bacteria and immune cells
in gutESF-JSPS Frontier Science Conference for Young
Researchers Cutting Edge Immunology and its ClinicalApplication

Hulshorst, Holland

Mar - 12

Saito, T.

Negative regulation of TCR signaling by PD-1 microcluster

Keystone Symposium

Keystone, USA

Mar - 12

Tokunaga, M.

Dynamic feature of life examined by single molecule imaging

Public lecture on frontier researches at Tokyo Institute ofTechnology

Tokyo, Japan

Mar - 12Ohno, H.

Mechanism of protection of mouse O157-infectious death bybificobateria using multi-omics approach

Bridge Seminar Series (Biken/Buinkai-Ridge Seminar Series )Osaka, Japan

Mar - 12Cheroutre, H.

Stretching the CD4 T helper (Th)-Lineage Beyond Plasticity

2011 Cancer and Immunology Colloquium Seminar Series atCedars-Sinai Medical Center

Los Angeles, USA

Mar - 12

Nishida, K.

Role of adaptor molecule Gab2 in mast cell-mediated allergy
response

The 132th Annual Meeting of the Pharmaceutical Society ofJapan

Hokkaido, Japan140

RCAI Seminars 2011
Date

Title

Speaker

Affiliation

Country

05 - Jun - 11

Regulation of fate determination andplasticity of germ cells

Yasuhisa
Matsui

Institute of Development, Aging
and Cancer, Tohoku University

Japan

29 - Jun - 11

Coupling of metabolism and epigenome
by nuclear protein complex
SAMIT

Kazuhiko
IgarashiTohoku University School of
MedicineJapan

14 - Jul - 11

The role of the immune system in prion
neuroinvasion from the intestine

Neil A
Mabbott

The Roslin Institute & Royal(Dick)
School of Veterinary Sciences
University of EdinburghUK

28 - Jul - 11

Exploration of Host-Parasite Interface
Possibly Reveals a New Aspect of
Immunology

Masahiro
Yamamoto

Graduate School of Medicine,
Osaka University

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 1

01- Aug - 11

Donor mesenchymal stem cells trigger
chronic graft-versus-host disease
following minor antigen mismatchedbone marrow transplantation

Yumi
Matsuzaki

Center for Integrated Medical
Research, Keio University

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 2

03- Aug - 11

Proteinomic analysis of cell signaling
networks with micro-western arrays

Richard B.
Jones

Institute for Genomics andSystems Biology, The Universityof Chicago

USA

The RCAI 10th
Anniversary Seminar
Series/Seminar 3

08 -Aug - 11

Systems Approach to Hematopoietic
Stem/Progenitor Cell Biology

Jun Seita

Institute for Stem Cell Biology& Regenerative Medicine, andDepartment of Pathology,
Stanford University School of
Medicine

USA

The RCAI 10th
Anniversary Seminar
Series/Seminar 4

29 -Aug - 11

Signaling pathways activated by innate
receptors and their roles in host defense

Taro Kawai

Laboratory of Host Defense,
Immunology Frontier ResearchCenter, Osaka University

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 5

05 - Sep - 11

Role of Crosstalk between Adipocytes
and Endothelium in Sepsis

Kiichiro Yano

Cardiovascular-Metabolic
Laboratories, Daiichi-SankyoCo., Ltd.

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 6

05 - Sep - 11

Current status and Prospect of iPS Cell
Research

Takashi Aoi

Center for iPS Cell Research andApplication , Kyoto University

Japan

12-Sep - 11

Natural helper cells play a critical role in
IL-33-dependent eosinophilia in the lungKazuyo Moro

Department of Microbiology and
Immunology, Keio University
School of Medicine

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 7

28 - Sep - 11

High-speed interstitial T cell migration in
lymph node involves LFA-1-dependentand -independent mechanisms controlled
by stromal cell network

TomoyaKatakaiKansai Medical University

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 8

06 - Oct - 11

MicroRNA Regulation of Energy
Metabolism in Mammalian Immune
SystemTaro Fukao

Germany

The RCAI 10th
Anniversary Seminar
Series/Seminar 9

10 - Oct - 11

Retinoic acid and gut-homing T cells in
intestinal immune homeostasis

J. Rodrigo
Mora

Massachusetts General Hospital& Harvard Medical School

USA

The RCAI 10th
Anniversary Seminar
Series/Seminar 10

12- Oct - 11

LRF Transcription Factor Maintains HSC
Homeostasis by Preventing LymphoidPrimed LT-HSCs from Excessive
Differentiation

Takahiro
Maeda

Hematology Division, Brigham
and Women’s Hospital

USA

The RCAI 10th
Anniversary Seminar
Series/Seminar 11

17- Oct - 11

Forced expression of reprogramming
factors in vivo

Yasuhiro
YamadaCenter for iPS Cell Research andApplication, Kyoto University

Japan

18 - Oct - 11

C-type lectins: their roles in the hostdefense against fungal infection

Shinobu Saijo

Medical Mycology ResearchCenter, Chiba Univerisity

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 12

24 - Oct - 11

Regulatory B cells, lessons from themodel of IBD

AtsushiMizoguchi

Harvard Medical School,
Massachusetts General Hospital

USA

The RCAI 10th
Anniversary Seminar
Series/Seminar 13

01- Nov - 11

Molecular mechanisms required forthymic self-tolerance

TaishinAkiyama

Institute of Medical Science,
University of TokyoJapan

The RCAI 10th
Anniversary Seminar
Series/Seminar 14

15 - Nov - 11

Mediator Lipidomics in Inflammation
Research

Makoto Arita

Graduate School of
Pharmaceutical Sciences, The
University of TokyoJapan

The RCAI 10th
Anniversary Seminar
Series/Seminar 15

20 - Nov - 11

Transcription factors in cytotoxic T cell
development

Takeshi
Egawa

Washington University School of
Medicine

USA

The RCAI 10th
Anniversary Seminar
Series/Seminar 16

20 - Nov - 11

Towards an Experimental and Systems
Biology Framework for Cancer Cell
Therapeutics

Petar M.
Mitrasinovic

Indian Institute of Technology
Roorkee, India & Belgrade
Institute of Science andTechnology, Serbia

Serbia24 - Nov - 11

The nature of B and T cell memory
generated in germinal centers

David
Tarlinton

Walter and Eliza Hall InstituteUSA

07- Dec - 11

Drosophila models to study chronic
inflammation

Hidehiro
Fukuyama

Institute of Molecular and Cellular
Biology

FranceMax-Planck Institute of
Immunobiology and Epigenetics

The RCAI 10th
Anniversary Seminar
Series/Seminar 17

141

07-Dec - 11

Targeted gene correction of a1antitrypsin deficiency in induced
pluripotent stem cells

Kosuke Yusa

Wellcome Trust Sanger Institute

UKThe RCAI 10th
Anniversary Seminar
Series/Seminar 18

11-Dec - 11

Revisiting ‘Thymic Nurse Cell’: Uniquelympho-epithelial complexes in thethymic cortex

Takeshi Nitta

National Center for Global Healthand Medicine

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 19

15 -Dec - 11

Primary immunodeficiency in Japan

Kosuke Imai

Department of Community
Pediatrics, Perinatal andMaternal Medicine
Tokyo Medical and Dental
University

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 20

20 - Dec - 11Bone marrow niches for hematopoietic
stem and progenitor cells

TakeshiNagasawa

Institute for Frontier Medical
Sciences, Kyoto University

Japan

21- Dec - 11

Intestinal microbiota shapes the immune
system

Kenya Honda

Department of
Immunology,Graduate Schoolof Medicine, The University of
Tokyo

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 21

25 -Dec - 11

Central nervous system autoimmunityand gut flora

Takashi
Yamamura

Department of Immunology,
National Institute of
Neuroscience, NCNPJapan

The RCAI 10th
Anniversary Seminar
Series/Seminar 22

18 - Jan - 12

Deciphering pancreas development anddiseases

Matthias
HebrokUCSF Diabetes Center

USA

The RCAI 10th
Anniversary Seminar
Series/Seminar 23

23 - Jan - 12

HLA variation and disease susceptibility

KatsushiTokunaga

Department of Human Genetics,
Graduate School of Medicine,
University of TokyoJapan

RCAI-CGM Joint
Seminar23 - Jan - 12

HLA-DRB1polymorphism and anticitrullinated peptide antibody inrheumatoid arthritis

Yuta Kochi

RIKEN Center for Genomic
MedicineJapan

RCAI-CGM Joint
Seminar24 - Jan - 12

Germinal Center Selection and theDevelopment of Memory B and Plasma
Cells

Mark
ShlomchikLaboratory Medicine and
Immunolobiology, Yale University

USA

The RCAI 10th
Anniversary Seminar
Series/Seminar 24

30 - Jan - 12

The role of the ERM protein moesin in
lymphocyte homeostasis

Takako Hirata

Kyoto University Graduate
School of Medicine

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 25

31- Jan - 12

The role of the thymus in populating the
T cell pool and some thoughts on thecommercialisation of basic research

RolandScollay

Nursing & Health Science,Monash University, Melbourne,
Australia

Australia

06 - Feb - 12

Microbial and cytokine detection controls
antigen degradation by autophagy
and subsequent endogenous MHC IIrestricted presentation in dendritic cells

Philippe
PierreCentre d’Immunologie deMarseille Luminy

France12- Feb - 12

Interpreting the CpG island signal

Rob Klose

Department of Biochemistry,
Oxford University

UK

The RCAI 10th
Anniversary Seminar
Series/Seminar 26

13 - Feb - 12Signaling functions of reactive oxygen
species and electrophiles: regulatory
mechanisms and implications in chronic
inflammation-associated diseases

Tomohiro
Sawa

Department of Microbiology,
Graduate School of Medical
Sciences, Kumamoto University

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 27

19 - Feb - 12

Paradox of Immunodeficiency andInflammation & Autoimmunity in Human
Aging

Sudhir Gupta

University of California

USA

The RCAI 10th
Anniversary Seminar
Series/Seminar 28

19 - Feb - 12

Lipid, vitamin, and nucleotide in theregulation of gut immunity

Jun Kunisawa

Division of Mucosal Immunology,
Institute of Medical Science, The
University of TokyoJapan

The RCAI 10th
Anniversary Seminar
Series/Seminar 29

22- Feb - 12

A systems biology approach to
Parkinson’s disease: Research at the
new Luxembourg Centre of Systems
Biomedicine

Rudi Balling

Luxembourg Centre for Systems
Biomedicine (LCSB)

Luxembourg

The RCAI 10th
Anniversary Seminar
Series/Seminar 30

22- Feb-12

Identification of claudin-expressing
medullary thymic epithelial stem cells
that maintain the functional medulla
during life

Yoko
Hamazaki

Graduate School of Medicine,
Kyoto University

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 31

27-Feb - 12

Unraveling governing principles from
immune cell dynamics

Kumar
SelvarajooInstitute for Advanced
Biosciences, Keio University

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 32

27- Feb - 12

Immune regulatory functions of DOCK
family proteins in health and disease

Yoshinori
Fukui

Medical Institute of Bioregulation,
Kyushu University

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 33

07-Mar - 12

Allele-specific profiling of genomewide epigenetic marks and sequencepolymorphisms

Yoshiko Mito

Massachusetts General Hospital/
Harvard Medical School

USA

The RCAI 10th
Anniversary Seminar
Series/Seminar 34

22- Mar - 12

Regulatory effects of IgG on B cellfunctions

Toshiyuki
Takai

Institute of Development, Aging
and Cancer, Tohoku University

Japan

The RCAI 10th
Anniversary Seminar
Series/Seminar 35

142

Budget, Personnel and Patents

100 million yen

RCAI Budget ( JPY 100 Million)
70

RCAI Budget FY2001-2011(JPY 100 Million)60

Note: Budgets for FY2001-2003 include
construction expenses for RCAI facility
RCAI Budget (JPY 100 Million )

50
40
30
20
10
0
2000

2002

2004

2006

2008

2010

2011

2001

41.74

2002

54.23

2003

60.48

2004

40.10

2005

39.02

2006

35.90

2007

34.56

2008

32.61

2009

31.86

2010

30.83

2011

31.06

Fiscal Year

RCAI Personnel

450

RCAI staff composition(as of Mar. 2012)

400
350

RCAI Personnel

300
250
200
150
100
50
0
2000

2002

2004

2006
Fiscal Year

RCAI Personnel

2008

2010

2011

Category

NumberDirector

1

Senior Advisor

2

Group Director

8Team Leader

12

Unit Leader

11

Coordinator

1

Senior Scientist

12

Research Scientist

49

Special Postdoctoral Researcher

9

Foreign Postdoctoral Researcher

1

Research Associate

6

Junior Research Associate

11International Program Associate

8Student Trainee

63

Research Fellow

1

Research Consultant

3

Visiting Scientist

68

Senior Technical Scientist

1

2001

84

2002

156

2003

191

2004

328

2005

363

Technical Scientist

6

2006

366

Technical Staff I

40

2007

339

Technical Staff II

63

2008

326

Assistant

24

2009

380

2010

400

Temporary Employment

4

2011

404

Total

404

RCAI Patents FY2011 ( as of Mar. 2012 )There were 17 patents filed from
April 2011- March 2012.

143

Cover and Section Heading Photo Legends
Cover: Two-photon live imaging analysis reveals that
Bcl6 is essential for entry of antigen-engaged B cells
into the germinal center
Using two-photon microscopy, The Research Unit for Immunodynamics visualized a 3D-reconstructed slice ( 360 μm x 360 μm x 30 μm ) ofa B cell follicle in a mouse lymph node and viewed from an angle.
Bcl6-sufficient antigen-engaged B cells ( labeled with cyan fluorescentprotein, light blue) are forming a nascent germinal center in the B cellfollicle. Migration of Bcl6-deficient, antigen-engaged B cells (labeledwith green fluorescent protein, green) was analyzed to show that they
were unable to enter the nascent germinal center just like the nonantigen-specific polyclonal, mostly naïve B cells ( labeled with rhodamine, red) . This study highlights the critical role of Bcl6 in the entry ofantigen-engaged B cells into the germinal center. Changes in the colors of the migration tracks of Bcl6-deficient, antigen-engaged B cells
indicate the time after the beginning of the 60-min time-lapse recording ( from dark blue to purple to red to orange to yellow ) . The lymph
node capsule, which was visualized by detecting second harmonic
generation signals, is also shown ( dark purple) .

Image courtesy of the Research Unit for Immunodynamics

Front page of Part 1: CD169-positive macrophages in the lymph
node sinus engulf subcutaneously injected dead tumor cells
The lymph sinus is a highway that links afferent and efferent lymph and is believed
to be a filtering zone for the lymphatic system. The Laboratory for Innate Immunity
discovered that CD169-positive macrophages ( green ) residing within the lymph
sinuses rapidly consume the dead tumor cells ( red ) and play a critical role in crosspresenting tumor antigens to cytotoxic T cells.

Image courtesy of the Laboratory for Innate Immunity

Front page of Part 2: Alteration of responsiveness to gliogenic cytokines
Young Chief Investigator Laboratory for Stem Cell Competency has been investigating the molecular mechanisms governing thedifferentiation of neural stem cells ( NSCs ) . They identified Coup-tfI and II ( Coup-tfs ) as critical molecular switches for the neurogenicto-gliogenic transition of NSCs. Coup-tfs knockdown neurospheres continued to produce neurons even in the late developmental
stage and resisted the induction of gliogenesis by gliogenic cytokines, LIF and BMP2.

Image Courtesy of the Young Chief Investigator Laboratory for Stem Cell Competency

144

Front page of Part 3 and 6:
Crosstalk between prokaryotic
and eukaryotic kingdoms
A Gram stain of a section from the small
intestine showing Gram-positive segmented bacteria ( violet) trapped in the
mucus ( yellow layer) or interacting withimmune cells.

Image courtesy of the Laboratory for Mucosal
Immunity

Front page of Part 4: Bacteria shielding by IgA in
the gut
The production of IgA by plasma cells in the gut is critical for thecontainment of the gut commensal microbiota. The Laboratory for
Mucosal Immunity demonstrated expansion of segmented filamentous bacteria ( SFB ) in the absence of IgA coating, such as in
AID-/- mice. Pictures show IgA plasma cells ( green ) in lamina propria and secretory IgAs coating bacteria in the intestinal lumen.
Blue stains the DNA of eukaryotic and prokaryotic cells. Note theabsence of IgA and the attachment of SFB directly to the epithelium in AID-/- mice.

Image courtesy of the Laboratory for Mucosal Immunity

Front page of Part 5: Introduction of Cy3-labeled calmodulin in
LAT-GFP expressing Jurkat cells
The Research Unit for Single Molecule Imaging, in collaboration with the Research
Unit for Molecular Systems Immunology, established a method to visualize cellsignaling using single molecule fluorescence microscopy. Calmodulin was labeledwith a single molecule of Cy3 in vitro and introduced into Linker of Activated T cells( L AT ) -GFP Jurkat cells by electroporation. The yellow fluorescence represents
successful Cy3-labeling of calmodulin and its colocalization with LAT, a raft-resident
protein. Bar: 10 μm.

Image courtesy of the Research Unit for Single Molecule Imaging, Research Unit for Molecular Systems Immunology and Dr. Nobuhiro Hayashi at the Tokyo Institute of Technology

Front page of Part 7: Enlarged germinal centers andexpanded TFH cells in Peyer’s patches of PD-1-/- mice
The Laboratory for Mucosal Immunity discovered that PD-1 regulates repertoire selection of IgA in the germinal centers of Peyer's patches. PD-1 deficiency leads to an expansion of follicular helper T ( TFH ) cells, resulting in alarge number of IgA B cells receiving TFH help and differentiating into IgAplasma cells.

Image courtesy of the Laboratory for Mucosal Immunity