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

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

International Advisory Council
Max D. Cooper, Chair, M.D.

Tadamitsu Kishimoto, M.D., Ph.D.

Klaus Rajewsky, M.D.

Rudolf Aebersold, Ph.D.

Bernard Malissen, Ph.D.

Kiyoshi Takatsu, Ph.D.

Antonio Coutinho, M.D., Ph.D.

Ruslan Medzhitov, Ph.D.

Dale Umetsu, M.D., Ph.D.

Alain Fischer, M.D., Ph.D.

Hiromitsu Nakauchi, M.D., Ph.D.

Arthur Weiss, M.D., Ph.D.

Ronald N. Germain, M.D., Ph.D.

William E. Paul, M.D.

Paul W. Kincade, Ph.D.

Susan K. Pierce, Ph.D.

Deputy Directors

Research Coordination
OfficeAdministrative
Coordination Office

Shigeo Koyasu, Ph.D.

Peter Burrows, Ph.D.

Ichiro Taniuchi, M.D., Ph.D.

Takashi Saito, Ph.D.

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

Haruhiko Koseki, M.D., Ph.D.

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

Group

Team

Lab. for Immune Cell System Shigeo Koyasu, Ph.D.

Lab. for Mucosal Immunity Sidonia Fagarasan, M.D., Ph.D.

Lab. for Human Disease Model Fumihiko Ishikawa, M.D., Ph.D.

Lab. for Infectious Immunity Satoshi Ishido, M.D.,Ph.D.

Lab. for Developmental Genetics Haruhiko Koseki, M.D., Ph.D.

Lab. for Vaccine Design

Lab. for Lymphocyte Differentiation Tomohiro Kurosaki, M.D., Ph.D.

Lab. for Lymphocyte Development Hiroshi Kawamoto, M.D., Ph.D.

Lab. for Immunogenomics Osamu Ohara, Ph.D.

Lab. for Epithelial Immunobiology Hiroshi Ohno, M.D., Ph.D.

Lab. for Cell Signaling Takashi Saito, Ph.D.

Lab. for Cellular Systems ModelingLab. for Immunological Memory Toshitada Takemori, M.D., Ph.D.

Lab. for Dendritic Cell Immunobiology Katsuaki Sato, Ph.D.

Lab. for Immune Regulation Masaru Taniguchi, M.D., Ph.D.

Lab. for Immune Diversity Ji-Yang Wang, Ph.D.

Lab. for Cytokine Signaling Masaru Taniguchi, M.D., Ph.D.

Lab. for Immunogenetics Hisahiro Yoshida, M.D., Ph.D.

Lab. for Transcriptional Regulation Ichiro Taniuchi, M.D., Ph.D.

Yasuyuki Ishii, Ph.D.

Mariko Okada-Hatakeyama, Ph.D.

RIKEN
Research Center forAllergy and
Immunology (RCAI)
Organization

Unit

International Research Unit

Unit for Cellular Immunotherapy Shin-ichiro Fujii, M.D., Ph.D.

Unit for Immune Crosstalk Hilde Cheroutre, Ph.D.

Unit for Immune Homeostasis Shohei Hori, Ph.D.

Unit for Allergy Toshiaki Kawakami, Ph.D.

Unit for Immunoinformatics S. Sujatha Mohan, Ph.D.

Unit for Thymic Environments

Unit for Single Molecule Imaging Kumiko Sakata-Sogawa, Ph.D.

Unit for Immunoepigenetics Miguel Vidal, Ph.D.

Willem van Ewijk, Ph.D.

Unit for Therapeutic Model Kanako Shimimzu, M.D., Ph.D.
Unit for Inflammatory Regulation Takashi Tanaka, M.D., Ph.D.
Unit for Immunodynamics Takaharu Okada, Ph.D.
Unit for Molecular Systems Immunology Makio Tokunaga, Ph.D.

i

Contents
Organization  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iContents  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ii

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

Development of artificial adjuvant vector cells(aAVCs) as a novel therapy . . . . . . . . . . . . . . . . . . . . . . . . 27
Longitudinal birth cohorts and the humanizedmouse model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Establishing an efficient delivery system for mucosal
vaccines  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Intestinal Microbe Symbiosis Laboratory, Integrated
Collaborative Research Program with Industry . . . . . . . . . 30

Part 1 Creation of New Paradigms
Skewed gut microbial communities induce
hyperactivation of the systemic immune system  . . . . . . .

2

Raising an antitumor army  . . . . . . . . . . . . . . . . . . . . . . . .

3Part 3 Nurturing Young Scientists

Giving antitumor immunity a helping hand  . . . . . . . . . . . .

4Young Chief Investigator Program . . . . . . . . . . . . . . . . . . 32

Enabling rapid recall of familiar foes . . . . . . . . . . . . . . . . .

5

YCI Laboratory for Stem Cell Competency . . . . . . . . . . . . 33

Reassigning cells to fight infection  . . . . . . . . . . . . . . . . . .

6

YCI Laboratory for Mathematical Modeling of
Immune System  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

A blueprint for the gut’s antimicrobial defenses  . . . . . . . .

7

Medical Innovation World Initiative (MIWI) Kick-off
Meeting  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

YCI Laboratory for Immune Regeneration  . . . . . . . . . . . . 39

Tackling the enigmas of influenza infection . . . . . . . . . . . .

9RCAI International Summer Program 2012 . . . . . . . . . . . . 41

From genetic analysis to mechanistic interpretation of
immune diseases  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

7th RCAI-JSI International Symposium on Immunology  . . 43

YCI Laboratory for Integrative Genomics  . . . . . . . . . . . . . 37

RIKEN International Program Associate . . . . . . . . . . . . . 45
RCAI Advisory Council 2012 . . . . . . . . . . . . . . . . . . . . . . . 11
RIKEN Special Postdoctoral Researcher (SPDR ) . . . . . . . 46
Outstanding Contribution of the Year 2012 . . . . . . . . . . . . 13
RIKEN Foreign Postdoctoral Researcher  . . . . . . . . . . . . . 46
Prize winners 2012  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
RIKEN Junior Research Associate (JRA )  . . . . . . . . . . . . . 47
Excellent Paper of the Year 2012  . . . . . . . . . . . . . . . . . . . 22
Adjunct Professorship Programs  . . . . . . . . . . . . . . . . . . . 48

Part 2 Technology and Innovation

Part 4 Collaborative Networks

RCAI and Torii collaborate for development of anallergy vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Open Laboratory at RCAI . . . . . . . . . . . . . . . . . . . . . . . . . 50
Open Laboratory for Signal Network . . . . . . . . . . . . . . . . 50

ii

iPS technology development for immunologicalresearch and therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . 24

Open Laboratory for Bioenvironmental Epigenetics . . . . . 52

RCAI innovation projects in drug discovery andmedicine  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

The 2nd RIKEN RCAI-University of Michigan Medical
Center Joint Workshop  . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

2nd New Zealand-Japan Joint Immunology Workshop . . . 55

Laboratory for Infectious Immunity . . . . . . . . . . . . . . . . . . 90

Harvard Summer School at RCAI  . . . . . . . . . . . . . . . . . . . 57

Research Unit for Inflammatory Regulation  . . . . . . . . . . . 92

RCAI-ZIBI student exchange program  . . . . . . . . . . . . . . . 57

Research Unit for Immunotherapeutic Model . . . . . . . . . . 94

International Research Collaboration Award . . . . . . . . . . . 58

Research Unit for Immune Homeostasis . . . . . . . . . . . . . . 96
Laboratory for Immune Regulation . . . . . . . . . . . . . . . . . . 98
Laboratory for Dendritic Cell Immunobiology . . . . . . . . . . 100

Part 5 Outreach Activities
RCAI-Novo Science Forum . . . . . . . . . . . . . . . . . . . . . . . . 60
Open Seminar “When cells determine their fate” . . . . . . . 60
Science Café “Gut bacterium and probiotics”  . . . . . . . . . 61
Science Café “Do computers cure diseases?” . . . . . . . . . 61
RIKEN Yokohama Open Campus . . . . . . . . . . . . . . . . . . . 62
Science Café “Towards the cure for leukemia” . . . . . . . . . 62

Laboratory for Cytokine Signaling . . . . . . . . . . . . . . . . . . . 102
Laboratory for Immunogenetics . . . . . . . . . . . . . . . . . . . . .  104Research Unit for Allergy . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Laboratory for Vaccine Design  . . . . . . . . . . . . . . . . . . . . . 108
Laboratory for Human Disease Models . . . . . . . . . . . . . . . 110
Research Unit for Cellular Immunotherapy . . . . . . . . . . . . 112
Laboratory for Immunogenomics  . . . . . . . . . . . . . . . . . . . 114
Research Unit for Immunoinformatics . . . . . . . . . . . . . . . . 116

Part 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 Cellular System Modeling . . . . . . . . . . . . . 118
Research Unit for Thymic Environment . . . . . . . . . . . . . . . 120
Research Unit for Immunoepigenetics  . . . . . . . . . . . . . . . 122
Research Unit for Immune Crosstalk  . . . . . . . . . . . . . . . . 124
Central Facilities  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Administrative Coordination Office . . . . . . . . . . . . . . . . . . 130

Laboratory for Lymphocyte Differentiation . . . . . . . . . . . . 74
Research Unit for Immunodynamics . . . . . . . . . . . . . . . . . 76
Research Unit for Molecular Systems Immunology  . . . . . 78
Laboratory for Immune Cell System . . . . . . . . . . . . . . . . . 80
Laboratory for Epithelial Immunobiology  . . . . . . . . . . . . . 82
Laboratory for Mucosal Immunity . . . . . . . . . . . . . . . . . . . 84

Part 7 Data and Statistics
Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Invited presentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
RCAI Seminars 2012 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Budget, Personnel and Patents  . . . . . . . . . . . . . . . . . . . . 141

Laboratory for Immune Diversity . . . . . . . . . . . . . . . . . . . . 86
Laboratory for Immunological Memory . . . . . . . . . . . . . . . 88

iii

T

his is the final annual report as the RIKEN Researchcrocluster. This discovery was brought about throughCenter for Allergy and Immunology (RCAI). In April

the use of single molecule fluorescence microscopy

2013 RCAI will merge with the RIKEN Center for Genom-developed by Dr. M. Tokunaga. Dr. Tokunaga re-

ic Medicine (CGM) to form a new institute, the Center for

ceived the Commendation for Science and Technology

Integrative Medical Sciences (IMS) focusing on humanawarded by the Minister of Education, Culture, Sports,

immunology.

Science and Technology (MEXT) in 2011. Dr. T. Hirano
discovered that zinc functions as a signaling molecule

It has been twelve years since RCAI was estab-in two phases, from the early induction of a zinc wave inlished on the RIKEN Yokohama Campus. On this par-response to a stimulus to the late induction of specificticular occasion, I would like to reflect on the promisestransporters. He received the Crafoord Prize in 2009that I made upon becoming the Founding Director ofand the Japan Prize in 2011 for his IL-6 research. For

RCAI; at that time I decided to define seven objectives inlymphocyte development, Dr. H. Kawamoto establishedestablishing the Center.

a new myeloid-based model of hematopoiesis that isquite different from the classical model with its lym-

The first was to establish a system to allow cutting

phoid-specific progenitors. Dr. I. Taniuchi defined a tran-edge research. The outcomes are now quite clear andscriptional circuit regulated by Runx and ThPOK for lin-

there have been various epoch-making discoveries ineage determination in the development of CD4 and CD8

the field of immunology by RCAI investigators.

T cell subsets. Drs. Kawamoto and Taniuchi received theJapanese Society for Immunology (JSI) Award in 2010iv

For lymphocyte signaling, Dr. T. Saito discovered

and in 2013, respectively. For Th2 cell differentiation, Dr.

the minimal unit for T cell activation, called the TCR mi-

M. Kubo identified a specific region of the IL-4 enhancer

that regulates Th2 responses and a gene, Mina, whichand discovering the principles for diagnosis and therapy

determines Th2 bias. Dr. T. Kurosaki identified the in vivoof human diseases. For this purpose, we established the

localization of long-term memory B cells adjacent to theMedical Immunology World Initiative (MIWI), which is an

germinal center and received a Commendation for Sci-

interdisciplinary research platform and an international

ence and Technology awarded by MEXT in 2012.

consortium of multiple research groups.

Dr.

T. Okada utilized two-photon microscopy technology
to track the process of B cell development in germinal

Third, we have established the Young Chief Investi-

centers. For mucosal immunity, Dr. H. Ohno identified

gator Program with its mentor system to nurture talented

GP2, a pathogen uptake receptor on M cells that pro-young investigators who will lead immunology into thevides a target for oral vaccines. Dr. S. Fagarasan, in

next generation and establish interdisciplinary researchcollaboration with Dr. S. Hori, showed that Foxp3 T

areas. We have recruited talented young researchers,

cells in Peyer’s patches have unique characteristics that

Drs. S. Nakaoka (mathematical modeling), H. Kaneda

promote IgA generation. Drs. Fagarasan and Hori re-(stem cell competency), T. Ikawa (immune regeneration),ceived the Young Scientist Award from MEXT, Japan in

K. Shiroguchi (integrative genomics), K. Hase (bioen-

2005 and 2006, respectively. Regarding innate immu-

vironmental epigenetics) and T. Kitami (cellular bioen-

nity, Dr. T. Kaisho determined that IκBα is the key player

ergetics network). Dr. Ikawa received Young Scientist

in mediating interferon induction by two Toll-like receptor

Award from MEXT, Japan in 2012.

+

family members. He received a JSI Award in 2009. Dr.
T. Tanaka identified a pathway by which PDLIM2 termi-

Fourth, we have built up a platform from basicnates NF-κB activation through intranuclear sequestra-

research to clinical applications. Dr. F. Ishikawa, in col-tion and subsequent degradation. For disease models,laboration with Drs. H. Koseki and O. Ohara, has been

Dr. H. Yoshida identified a genetic mutation causing

creating the third generation of humanized mice with a

atopic dermatitis through the analysis of ENU-mutant

humanized microenvironment. They succeeded in gen-mice and found that atopic dermatitis is not primarily an

erating mice with a humanized environment in thymus

immune disorder. For systems immunology, Dr. M. Oka-

and bone marrow, supporting human T cell develop-

da developed a systems biology model of a signaling

ment and human HSC maintenance, and showed thatcascade to discriminate activation vs. differentiation ofhuman CD8 T cells could develop and differentiate into

cells through ERK activity.

cells with cytotoxic function in these mice.

RCAI’s strong publication track record indicates

Fifth, we succeeded in producing six original seeds

that the research conducted at RCAI is world class. As

toward clinical applications. The first two were anti-al-of 2012, 11.4% of RCAI papers were ranked in the top

lergy drugs. The PEGylated Cryj-1/2 fusion recombinant

1% of papers based on citations. This figure was theprotein has been licensed to the Torii pharmaceuticalhighest amongst RIKEN institutes and it clearly demon-

company to develop as a biochemical drug for cedar

strates the high quality of research conducted at RCAI.

pollinosis. Another version, a chemical compound

As of 2013, the average citation per RCAI paper wasdeveloped by Dr. Y. Ishii and me, selectively induces

46.13, which ranked us third in the world according to

apoptosis of IgE but not IgG B cells and preferential-

Thomson Reuters Essential Science Indicators (Seely suppresses IgE production. Therefore, it potentially

Table 2 in p. 132). Our strong publication record has

could be applied to any type of IgE-mediated allergic

been maintained through FY2008-2012. About 30% of

disorder, such as pollinosis and food allergy, as well as

the papers in these five years have been published inallergic asthma. The third seed was NKT cell-targetedjournals with impact factors above 10. I recognize thetherapy. In collaboration with Professors Nakayama,tireless effort of RCAI researchers and appreciate theirMotohashi and Okamoto at Chiba University, I havescientific contributions during these ten years.

conducted NKT cell-targeted adjuvant therapy as aphase I/II study for cancer patients. Advanced lung

The second purpose was to create a platform forcancer patients had a very significant prolonged medianunderstanding the mechanisms of disease development

survival time (MST) of more than 30 months with only av

primary treatment compared to the best supportive care,research activities at the Center as well as the Center’swhich had an MST of 4.6 months, or the MST of current

overall research strategy, future plans, organizational

advanced therapy, 10 months. This NKT cell-targeted

policies, and research management, is held every threetherapy was authorized by the Japanese government

years. The system has been extremely helpful for RCAIas Advanced Medical Treatment B (Senshin Iryo B) in

researchers and I would like to express my sincere

2011 for advanced non-small lung cancers and in 2013

thanks to all AC members: Drs. M. Cooper (chair), R. Ae-for head & neck tumors. As a medical scientist, it is abersold, A. Coutinho, A. Fischer, R. Germain, P. Kincade,great honor and gives me tremendous satisfaction that I

T. Kishimoto, S. Koyasu, B. Malissen, R. Medzhitov, M.

could discover NKT cells and then achieve their clinicalMiyasaka, D. Mathis, H. Nakauchi, W. Paul, S. Pierce,

application. The fourth seed was the artificial adjuvant

K. Rajewsky, T. Sasazuki, R. Steinman, K. Takatsu, D.

vector cell as an anti-tumor vaccine developed by Drs.

Umetsu, and A. Weiss.

S. Fujii and K. Shimizu. This vaccine can be dosed withmRNA encoding antigens specific to the malignant tu-

Finally, I am extremely proud of RCAI researchers,mor, and activates both innate and acquired protective

including the seventeen researchers who became univer-

immunity and induces long-term memory for more thansity professors during the last 10 years: Dr. K. Hase, YCI,

one year in mice and dogs with a single administration of(The Institute of Medical Science, Univ. of Tokyo), Drs. H.

the vaccine. The fifth seed was the establishment of iPS

Kawamoto, M. Hikida and T. Watanabe (Kyoto Univ.), Dr.

cells from human cytotoxic T cells specific for melanoma

K. Hoshino (Kagawa Univ.), Dr. M. Hoshino (Gifu Univ. ofand of human NKT cells by H. Koseki’s group, as well

Medical Science), Dr. S. Ishido (Showa Pharmaceuticalas murine NKT-iPS cells generated by my group. The

Univ.), Drs. T. Kaisho and Dr. T. Kurosaki (Osaka Univ.),

human iPS project was accepted as a Center for Clinical

Dr. M. Kubo (Tokyo Univ. of Science), Dr. M. Sakaguchi

Application Research (Type B) in the Research Center(Azabu Univ.), Dr. K. Sato (Miyazaki Univ.), Dr. K. Seino

Network for Realization of Regenerative Medicine, Japan(Hokkaido Univ.), Dr. M. Tanaka (Tokyo Univ. of Pharma-

in 2013. The sixth seed was Dr. Ishikawa’s work on iden-cy and Life Sciences), Dr. H. Udono (Okayama Univ.), Dr.

tification of leukemia stem cells and also development of

M. Tokunaga (Tokyo Institute of Technology), and Dr. S.

a drug candidate for effective treatment of leukemia. He

Yamasaki (Kyusyu Univ.).

received a Young Scientist Award from MEXT, Japan in2009 and a Japan Society for the Promotion of Science(JSPS) Prize in 2013.

The new Center aims to create an innovative research field, focusing on the prevention of human disorders using genomic and systems biology approaches in

Sixth, we have developed a strong support system

human immunology. The research endeavors by RCAIfor the Center’s researchers. I am very proud of theresearchers will continue at a high level in the new center,

Central Facilities— the Animal Facility managed by Dr.

and the name of our institute will remain as a Research

Koseki and Dr. T. Hasegawa, Dr. O. Ohara’s Genomics

Core for Advanced Immunology, IMS ( IMS-RCAI ).

Lab, and the FACS Lab managed by Dr. T. Saito and Ms.
H. Fujimoto. Also, the Administrative Coordination Officeheaded by Ms. H. Tanabe, including Mr. Y. Murahashi’s
IT-team and Mr. T. Ogata’s Facility Support, is operating

Mar. 31, 2013at a very high level. I also appreciate Drs. P. Burrows,
Professor of Alabama Univ., as a science advisor, Drs. T.
Takemori and H. Iwano as research coordinators in RCAI.
Seventh, we established a unique Advisory Council
system with a two-tiered approach. The Annual Scientific
Reviews have a mentoring function and are limited to areview of the research undertaken by a given laboratory whereas the Term Review Meeting, which covers allvi

Masaru Taniguchi
Director,
RIKEN Research Center for Allergy
and Immunology (RCAI)2012
Part 1Creation of New Paradigms

Sidonia Fagarasan

Skewed gut microbial communities
induce hyperactivation of the systemic
immune system
Figure:Research HighlightsT

he gut must keep its trillions of microbial inhabitants contained, and Immunoglobulin A (IgA ) playsa central role in the process, maintaining the symbioticbalance between gut bacterial communities and thehost immune system. In a process that is not well understood, B cells that produce IgA specific for the gut
microflora are selected in Peyer’s patches in the gut.
Sidonia Fagarasan’s group (Laboratory for Mucosal
Immunity) used genetically manipulated mice and could
show that the inhibitory co-receptor programmed cell
death-1 (PD-1) regulates microbial communities and IgA
production in the gut.
In PD-1-deficient mice, the total numbers of “healthy”
bacteria, such as Bifidobacterium and Bacteroides,
were markedly reduced. By contrast, bacteria of theEnterobacterobacteiaceae family, which are minor representatives in wild-type mice, were expanded about 400fold in PD-1-deficient mice. Flow cytometric analyses offecal bacteria revealed that the proportion of bacteriacoated with IgA was reduced in PD-1-deficient mice,
while the concentration of free IgA in intestinal secretions
was higher. Thus, the team suspected that the IgAs
produced in PD-1-deficient mice have reduced bacte-

Schematic representation of IgA selection within
and outside the GCs. Discrimination of GC IgA B
cells with different affinities for gut antigens requires
BCR engagement and competition for TFH cell help. A.
In WT mice, the limited number of TFH cells implies
that only the B cells able to present more antigen(high affinity) will receive T FH help, with B cells
capturing less antigen (low affinity) or those loosing
the integrity of the BCR undergoing apoptosis. B. In–/–
PD-1 mice, both the high and low affinity B cells
will receive help from the abnormally numerous
TFH present within the GCs. In both cases, the IgA
B cells generated in the PP GCs migrate out to the
MLN where they further proliferate and differentiate
into plasmablasts, which via the thoracic duct andblood will reach the lamina propria (LP). In addition
to selection in the GCs, IgAs appear to undergo asecond, likely commensal-driven selection within
the LP. In this way, the proliferating plasmablasts
are reselected to fit the geographical distribution ofbacteria along the intestine. (This does not exclude
the possibility that IgAs are also undergoing selection
at their first post-GC station, in MLN). C. The
possible sequential events leading to generation of–/–
auto-reactive antibodies in PD-1 mice.

ria-binding capacity. By sequencing the immunoglobulin
heavy chain variable region genes of IgA-producing
cells, they found that the IgA repertoire in PD-1-deficient
mice is altered, which could cause the observed alterations in the composition of the microflora.
To investigate the cause of the altered IgA repertoire, they examined the Peyer’s patches in mice deficient in the inhibitory receptor PD-1. The absence ofPD-1 affected the differentiation of T follicular helper
cells (TFH ), which provide important differentiation signals
to B cells. PD-1 deficiency resulted in an excess number
of TFH cells with altered phenotypes, and the ratio of TFH
to B cells was increased 2-fold in the germinal centers ofPeyer’s patches (Fig.). This observation is very telling; it
indicates that deregulation of TFH cells leads to inappropriate GC B cell selection when B cells compete for T
cell help in the GC.
Along with T and B cell hyperplasia in PD-1-deficient
mice, the gut microbiota induced hyperactivation of thesystemic immune system. They found that serum from
PD-1-deficient mice contained antibodies specific forcomponents of commensal bacteria, a response that
does not occur in wild type mice.
“The skewed gut microbial communities that result
from the dysregulated selection of IgAs drive the expansion of self-reactive B and T cells,” says Fagarasan. “Ourstudies have implications for how modulation of PD-1
expression promotes tolerance or uncontrolled immune
reactions leading to autoimmunity.”
Original paper(from left to right) Thinh Huy Tran, Shimpei Kawamoto and Mikako Maruya

2

Kawamoto, S., Tran, TH., Maruya, M., Suzuki, K., Doi, Y., Tsutsui, Y., Kato, LM., Fagarasan,
S. The inhibitory receptor PD-1 regulates IgA selection and bacterial composition in the gut.
Science 336, 485-489 (2012 )Raising an antitumor army
Reprogrammed immune cells might give doctors an edge
in rallying the body’s defenses against tumor growth

Hiroshi Kawamoto

Figure:Research Highlights

Haruhiko Koseki

Scientists reprogrammed purified CTLs into embryonic stem cell-like iPSCs by forcing themto express genes known as the ‘Yamanaka factors’. After allowing these reprogrammed cells to
proliferate, they then cultivated them alongside specialized cells (OP9/DLL1) that promote
their maturation into transplantation-ready, MART-1-specific CTLs.© 2013 ElsevierS

tem cells represent an ideal resource for the production of patient-specific antitumor cytotoxic T
lymphocytes (CTLs), but the production process poses
special challenges. To deal with the vast array of potential threats that might endanger our health, the immune
system uses gene recombination to generate various
armies of cells that each expresses receptors capable ofrecognizing a different target. This means that CTLs produced from either embryonic or adult stem cells would
rarely retain the target-recognition capabilities needed tokill a tumor.
Hiroshi Kawamoto’s team (Laboratory for Lymphocyte Development), in collaboration with Haruhiko
Koseki’s group in RCAI, therefore pursued an alternative
approach involving the use of a genetic reprogramming
technique to transform mature human CTLs into induced
pluripotent stem cells (iPSCs). From this undifferentiated state, iPSCs can be cultivated under conditions thatfavor their development into mature CTLs (Fig.). “All ofthese lymphocytes will come to express the same antigen receptor as the original T cells,” says Kawamoto.
“The technique allows us to regenerate antigen-specific
T cells with very high efficiency.”

After proving the soundness of this approach using
an isolated pool of mixed human CTLs, the researchers applied their method to a specific line of CTLs that
selectively recognize MART-1, a protein commonly overexpressed by the skin cancer melanoma. After reprogramming, they obtained two iPSC lines that contained
the same recombined MART-1-specific receptor genefound in the parental CTL cell line. Using one of these as
starting material, the researchers were able to produce a
95%-pure pool of new anti-MART-1 CTLs.
Initial experiments using purified MART-1 confirmed that these iPSC-derived CTLs were producing
the specialized receptor required to bind this melanoma
antigen, and Kawamoto and his colleagues confirmedthat their CTLs produced appropriate cytokines when
cultured alongside human cells that express MART-1 on
their surface. This result is encouraging, as the experiment roughly replicates the context in which CTLs might
encounter this antigen in a melanoma patient.
The question of whether this apparent tumor-specific immune response will translate into improved patient
outcomes remains to be determined. “We wish to test
whether the regenerated T cells can kill tumor cells in invivo situations using mouse models,” Kawamoto says.
“We are also planning to try the same experiments using
different tumor antigens to confirm that this method canbe generally used,” says Kawamoto. “If this technology
can be applied to cancer therapy, an extremely large
number of patients would benefit.”

Original paper(from left to right) Raul Vizcardo Sakoda, Hiroshi Kawamoto,
Kyoko Masuda and Daisuke Yamada

Vizcardo, R., Masuda, K., Yamada, D., Ikawa, T., Shimizu, K., Fujii, S., Koseki, H. & Kawamoto,
H. Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature
CD8 + T cells. Cell Stem Cell 12, 31-36 (2013 )This article is summarized from RIKEN Research with permission
http://www.rikenresearch.riken.jp/eng/hom/7228.html

3Giving antitumor immunity a helping
handShin-ichiro Fujii

A two-pronged approach to immune activation could lead tovaccines that effectively shut down tumor expansion
Research Highlights

Kanako Shimizu

Figure:Artificial adjuvant vector cells (aAVC) were produced by introducing mRNA encoding tumor-specific antigens into humanembryonic kidney cells dosed with the synthetic compound α-galactosylceramide, which activates NKT cells.
© 2013 Shin-ichiro Fujii, RIKEN Research Center for Allergy and ImmunologyTumor cells often express proteins that set themapart from their healthy neighbors. These verysame proteins can also help the immune system torecognize and destroy the cancer. Several researchgroups and companies have already demonstrated
proof-of-concept for antitumor therapeutic vaccines
based on this principle, typically employing ‘retrained’
dendritic cells ( DCs ) harvested from a patient’s own
immune system. To date, however, such vaccines havedemonstrated only limited effectiveness in beating backtumor progression. The research of Shin-ichiro Fujii(Research Unit for Cellular Immunotherapy), Kanako
Shimizu (Research Unit for Therapeutic Model) andcolleagues led to development of a technique that could
supercharge the potency of future cancer vaccines.
DC-based vaccines directly stimulate ‘adaptive’
immunity, which is directed against specific moleculartargets. “DCs play a pivotal role in determining the character and magnitude of an immune response,” explains
Fujii, “and the loading of patients’ DCs in vivo with tumor-specific antigens is one of the most promising current immunotherapeutic strategies.”
Using a mouse model, Fujii’s team demonstrated
that the natural antitumor DC response can be considerably ramped up by stimulating invariant natural killer
T ( iNKT ) cells from the ‘innate’ immune system, which
triggers a more generalized response against disease.
Key to their i NK T cell-based strategy is theuse of artificial adjuvant vector cells, derived from humankidney cells (Fig.). The artificial adjuvant vector cells are
induced to take up mRNA encoding a tumor-specific antigen, which is processed by the host DC and expressedon the cell surface, where it can be recognized by T cells
as coming from an ‘enemy’. The artificial adjuvant vector
cells are also treated with α-galactosylceramide, a synthetic glycolipid that is a potent activator of iNKT cells.
The artificial adjuvant vector cells prepared this way arethen injected into patients, where the iNKT cells accelerate maturation of host DCs, which in turn promote
adaptive immune response against the selected tumor
antigen.
Initial experiments demonstrated that artificial
adjuvant vector cells producing the model protein ovalbumin activated DCs in situ in mice and effectively
stalled growth of grafted tumors expressing this protein.
Importantly, this treatment proved more effective than aconventional vaccine approach using ex vivo DCs transfected with ovalbumin mRNA. Subsequent experiments
confirmed that artificial adjuvant vector cells could alsoelicit immunity against the melanoma antigen MART-1.
“Even the injection of one cell could evoke both the innate and adaptive immune response,” says Fujii.
Further tests using dog models revealed a clear
immune response with no notable adverse effects, evenin animals receiving multiple doses of artificial adjuvant
vector cells. These promising preclinical results could
potentially pave the way for human clinical trials in thenear future. “We have some candidate tumor antigens,”
says Fujii, “and now we need to examine the possibilityof using this technique for these antigens in terms of efficacy and safety.”

Original paper(from left to right) Jun Shinga, Miki Asakura, Yusuke Satoand Satoru Yamasaki

4

Shimizu, K., Mizuno, T., Shinga, J., Asakura, M., Kakimi, K., Ishii, Y., Masuda, K., Maeda, T.,
Sugahara, H., Sato, Y. et al. Vaccination with antigen-transfected, NKT cell ligand-loaded,human cells elicits robust in situ immune responses by dendritic cells. Cancer Res. 173, 6273 ( 2013 )This article is reproduced from RIKEN Research with permission
http://www.rikenresearch.riken.jp/eng/research/7263

Enabling rapid recall of familiar foes
A ‘rapid response’ pathway for immune cell development
may improve the body’s ability to recognize and fightback against recurring infectious threats

Research Highlights

Figure:Toshitada Takemori

Fluorescent labeling reveals that both conditionally Bcl6-deficient (top) and wild-type(bottom) mice are able to produce functional memory cells (left column, arrowheads).
However, these genetically modified animals lack germinal center (GC) B cells (rightcolumn, yellow), revealing a GC-independent pathway for memory cell development.
© 2012 Toshitada Takemori, RIKEN Research Center for Allergy and Immunology

Efficient immune protection requires the ability to
rapidly recognize intruders that the body has encountered in the past. This is achieved via ‘memory’ B
cells, which develop following immune system activation
by a virus, bacterium or other threat.
“Scientists have known about immunological memory for centuries,” explains Toshitada Takemori (Laboratory for Immunological Memory), “but certain criticalaspects of this process remain incompletely defined.” Asa case in point, Takemori’s team and Klaus Rajewsky atGermany’s Max-Delbrück-Center for Molecular Medicine
recently uncovered striking proof of a novel memory B
cell production pathway with a potentially distinct role inimmune defense.
The initial appearance of an immunity-triggering
antigen fuels interaction between B and T cells, which inturn yields activated B cells. These can either differentiate into cells that produce antibodies against the target
antigen or migrate to structures called ‘germinal centers’( GCs ) where their antibody-encoding genes undergo
extensive mutation. This somatic hypermutation (SHM )process generates antibodies with optimized target affinity and specificity, and the resulting cells mature into
antibody-secreting plasma cells or memory B cells.
However, Takemori has observed evidence thatsome memory B cells never undergo SHM, apparently
developing via a GC-independent pathway. This has
proven difficult to verify experimentally: mice lacking the
Bcl6 gene fail to develop GCs but also suffer other de-fects, making them a poor research model. To overcome
this, the researchers engineered rodents where Bcl6
inactivation is limited to a subset of relevant cells.
These animals lacked GCs, but nevertheless generated memory B cells after an immune challenge in numbers roughly equivalent to normal mice (Fig.). Closer examination confirmed that the memory B cells producedby conditionally Bcl6-deficient animals did not undergo
SHM. The researchers also isolated non-mutated memory B cells from wild-type animals, although these were
eventually outnumbered by mutated memory B cells,
indicating that these non-mutated cells represent a distinct subset of memory B cells that develop in advanceof the GC maturation process. “Our analysis indicatesthat immunological memory is established as soon as
possible after the onset of an immune response,” says
Takemori.
As non-GC memory B cells produce relatively
low-specificity antibodies, the researchers hypothesizethat these cells may complement optimized, post-SHM
memory B cells by broadly responding to related but
distinct threats: for example, influenza viruses in generalrather than one specific strain. “We are now determining
whether the GC-independent memory pathway assists
the GC-dependent pathway to protect hosts against viral infections,” says Takemori.
Conditional Bcl6-deficient mice, generated by
Takemori and his colleagues, were selected the “Mouseof the Month” in June, 2013, by RIKEN BioResource
Centerht tp://w w w.brc.riken.jp /lab /animal /mailnews /nm
201306_01.html

Original paper
Kaji, T., Ishige, A., Hikida, M., Taka, J., Hijikata, A., Kubo, M., Nagashima, T., Takahashi, Y.,
Kurosaki, T., Okada, M. et al. Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory. J Exp Med. 209, 2079-2097 (2012 ).

Related publication

Tomohiro Kaji ( left ) and Akiko Ishige ( right )Takahashi, Y., Ohta, H. & Takemori, T. Fas is required for clonal selection in germinal centers
and the subsequent establishment of the memory B cell repertoire. Immunity 14, 181-192( 2001)This article is reproduced from RIKEN Research with permission
http://www.rikenresearch.riken.jp/eng/research/7186.html

5Reassigning cells to fight infection
Unexpected flexibility in immune cell development could
help the body rapidly marshal its defenses at sites
vulnerable to infection

Ichiro Taniuchi

Research Highlights

Hile Cheroutre

Figure:J

ust as a uniform helps distinguish a soldier from a
police officer, scientists use proteins that immune
cells wear on their surfaces to determine their job in the
body. T cells, for example, that display the CD8 protein
are classified as ‘cytotoxic lymphocytes’ (Fig.), which killoff cancerous or infected cells, whereas those displaying
the CD4 protein are identified as ‘helper’ T cells that coordinate the immune response.
Immunologists have previously viewed these roles
as fixed endpoints in development, but research by Ichiro Taniuchi (Laboratory for Transcriptional Regulation) incollaboration with Hilde Cheroutre (Research Unit for Immune Crosstalk, RCAI, and La Jolla Institute for Allergy
and Immunology in California) and colleagues now reveals that helper T cells retain opportunities for a ‘careerchange’.
Cheroutre had noticed that some helper T cells
transplanted into immunodeficient recipients unexpectedly began expressing CD8. “This suggested reprogramming of cells from a helper fate into a cytotoxic
lineage,” says Taniuchi, whose research group studies
ThPOK, a ‘master’ protein that coordinates helper T cell
development and suppresses CD8 production.
+
Teaming up to examine these enigmatic CD4

Electron microscope image of T
lymphocytes from the intestinal epithelium.
© 2013 I. Taniuchi, RIKEN ResearchCenter for Allergy and Immunology

cytotoxic lymphocytes ( CTLs ) , the groups of Taniuchi
and Cheroutre developed genetically modified micehaving fluorescently labeled ThPOK-expressing cells,corresponding primarily to helper T cells. They also
isolated unlabeled CD4-expressing cells from theintestine, and closer examination showed that theseCD4-positive, ThPOK-negative cells now expressed CD8
and behaved like CTLs.
The researchers followed up with a strategy called‘fate mapping’, using genetically modified mice whosecells become fluorescently labeled if they express
ThPOK at any point—even transiently—in their lifetime.
+
These experiments showed that intestinal CD4 CTLs
begin as ThPOK-expressing helper cells, but switch toa CD8-expressing CTL fate when external factors cause
these cells to inactivate ThPOK production via a genetic‘switch’ called the ‘ThPOK silencer’. “This demonstrates+
that CD4 T cells retain unappreciated plasticity for CTL
development,” says Taniuchi.
This flexibility appears to arise in response to infectious threats, as mice cultivated to be ‘germ-free’ do+not develop CD4 CTLs. The researchers subsequently
determined that helper T cells must be properly exposed
both to foreign antigens as well as interleukin-15 tochange roles. Although the intestinal wall is a particularly
critical point of entry for pathogens, this mechanism mayapply more generally as a means of rallying local defenses by retraining helper T cells in a crisis. “I believe other‘barrier sites’ in the body may have these cells as well,”says Taniuchi.

Original paper

Sawako Muroi

6

Mucida, D., Husain, M.M., Muroi, S., van Wijk, F., Shinnakasu, R., Naoe, Y., Reis, B.S., Huang,+Y., Lambolez, F., Docherty, M. et al. Transcriptional reprogramming of mature CD4 helper
T cells generates distinct MHC class II–restricted cytotoxic T lymphocytes. Nat Immunol. 14,
281-289 ( 2013 )This article is reproduced from RIKEN Research with permission
http://www.rikenresearch.riken.jp/eng/research/7272

A blueprint for the gut’s
antimicrobial defenses

Figure:Every bite of food or drink of water is an invitation
for potentially harmful bacteria and viruses to set
up shop in the body. In order to protect against such
invaders, the mucous membrane that lines the intestine
contains clusters of specialized microfold cells ( M cells ),which can absorb foreign proteins and particles from thedigestive tract and deliver them to the immune system.
New work from Hiroshi Ohno’s group (Laboratory for Epithelial Immunobiology), in collaboration
with Ifor Williams and colleagues at Emory University in Atlanta, Georgia, has revealed valuable insights
into how these M cells develop. Previous researchfrom Williams’ group showed that a signaling protein
called RANKL switches on M cell development but
virtually nothing was known about the subsequent
steps in this process. To find out, Ohno and Williams
looked for genes that are switched on when intestinal
cells undergo differentiation in response to RANKL
exposure.
They discovered that treatment with RANKL causes
immature intestinal epithelial cells to sharply increase theproduction of Spi-B, a protein that regulates the expression of other developmental genes. To test the specificcontribution of this protein to M cell maturation, the researchers collaborated with Tsuneyasu Kaisho’s groupat Osaka University, which had engineered a genetically

Hiroshi Ohno

Research Highlights

The identification of a developmental ‘master switch’helps scientists explore the function of intestinal cells
that help prevent infection

Fluorescent labeling of GP2, a protein
expressed by M cells in wild-type mice(left) reveals that this cell population
is virtually absent in mice lacking
the gene encoding Spi-B (right).
© 2012 Nature Publishing Group

modified mouse strain lacking the gene encoding Spi-B.
The resulting animals were devoid of mature M cells( Fig.) . On the other hand, intestinal development as awhole was unaffected by the absence of Spi-B, demonstrating that this protein’s impact is limited to this specific class of cells within the gut.
M cells normally localize to immune structures
known as Peyer’s patches (PPs ). Bacteria such as Salmonella enterica Typhimurium (S. Typhimurium ) normally
accumulate within these PPs shortly after inoculation.
This uptake was considerably reduced in Spi-B-deficient mice, indicating the absence of a functional M cell
population. The mice showed a considerably weakened
immune response following oral administration of S. Typhimurium bacteria relative to wild-type animals, demonstrating the importance of M cell-mediated microbial
uptake.
The identification of this critical ‘master switch’ for
M cell development opens exciting new avenues ofresearch into these mysterious cells. Ohno is eager toinvestigate the details of how the cells perform their critical immunity-training function. “These questions could
not be answered previously because of the lack of M
cell-deficient mice,” he says. “But now, ‘knockout’ micethat specifically lack Spi-B in their mucosal epithelium
will provide the ideal tool for such studies.”

Original paper
Kanaya, T., Hase, K., Takahashi, D., Fukuda, S., Hoshino, K., Sasaki, I., Hemmi, H., Knoop, K.A., Kumar, N., Sato M. et al. The Ets
transcription factor Spi-B is essential for the differentiation of intestinal microfold cells. Nat Immunol. 13, 729-736 ( 2012 )Related publication
Knoop, K.A., Kumar, N., Butler, B.R., Sakthivel, S.K., Taylor, R.T., Nochi, T., Akiba, H., Yagita, H., Kiyono, H. & Williams, I.R. RANKL isnecessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium. J Immunol. 183, 5738-5747(2009)
Takashi Kanaya

This article is reproduced from RIKEN Research with permission
http://www.rikenresearch.riken.jp/eng/research/7094.html

7Medical Innovation World Initiative (MIWI) Kick-off Meeting Immune System and Primary Immunodeficiency ( PID)

MIWI

is an international humanized mouse users’consortium and, as well, an integrative network for human immunology research in the next generation.
As a part of the MIWI initiative, RCAI launched a collaborative
project with INSERM/Hospital Necker in France, Tokyo Medical and Dental University ( TMDU ) and the Center for Genomic
Medicine ( CGM ) in RIKEN Yokohama Institute for the elucidation of the human immune system and diseases. To celebratend
the opening of MIWI, a Mini-symposium was held on May 22 ,2012 at RCAI.
Dr. Shigeo Koyasu opened the meeting. He described
the recently identified innate lymphocytes, including the natural helper (NH ) cell. These cells have various effector functions
such as limiting the expansion of microorganisms including
viruses, bacteria, and parasites. However, unlike T and B cells,
innate lymphocytes act without antigen-specific receptors, but
cooperation between innate lymphocytes and antigen-specific
T and B cells is likely important in protective immunity against
specific types of microbes.
The next speaker, Dr. Tomohiro Morio ( TMDU ) talked
about X-linked agammaglobulinemia ( XLA ), a human diseasecaused by Bruton’s tyrosine kinase (Btk ) deficiency. 11-30%
of XLA patients have neutropenia, but it is distinct from thatseen in common variable immunodeficiency (CVID) in that theneutropenia seen in XLA is induced by infection and is usuallyameliorated after supplementation with immunoglobulin andis not mediated by an autoimmune response. Using neutrophils from patients with XLA, they found that the productionof reactive oxygen species (ROS ) was augmented. Excessive
production of ROS was associated with neutrophil apoptosis,
which was reversed by the transduction of wild-type Btk protein, suggesting that Btk is a negative regulator of signal transduction and prevents excessive neutrophil responses.
The third speaker, Dr. Yoshiyuki Minegishi ( TMDU ) discussed hyper IgE syndrome (HIES ). HIES is classified as two
diseases, type 1 and type 2. Type 1 HIES is the most common form, in which pneumonia is frequently followed by theformation of pneumatocele and is complicated by fungal infections. A dominant-negative mutation in the signal transducer
and activator of transcription 3 ( STAT3 ) has been identified
as a major molecular cause of the Type 1 HIES. Type 2 HIES

8

shows no skeletal abnormalities but the patients suffer fromrecurrent viral infections. A null mutation in tyrosine kinase 2( TYK2 ) was identified as a molecular defect in type 2 HIES.
The last speaker, Dr. Anne Durandy ( INSERM/Hospital
Necker ) described immunoglobulin class switch recombination (CSR ) deficiencies in humans. CSR deficiencies are rare
primary immunodeficiencies whose frequency is estimated as
1 in 100,000 births. They are characterized by normal or increased serum IgM levels and a contrasting, marked decreaseor absence of IgG, IgA and IgE. Mutations in the gene encoding the CD40 ligand, a molecule highly expressed by activatedfollicular helper T cells, result in reduced CSR. Another CSR
deficiency, caused by mutations in the AICDA gene encoding
AID, is characterized by the impairment of both CSR and somatic hypermutation ( SHM ).
During the meeting, researchers rediscovered the important insights gained from the study of primary immunodeficiencies for the understanding of the normal human immune
system. They agreed to collaborate for the analysis of primary
immunodeficiencies, especially targeting common variable
immunodeficiency (CVID ) and CSR deficiency. Using samples
collected from TMDU and Necker Hospital, RCAI, in collaboration with CGM, will conduct multiomics analyses, including whole exon sequencing, to identify the causative genes.
In addition, once candidate genes are identified, RCAI will
generate equivalent
humanized miceand gene targeted
mice to analyze themechanisms of thedisease onset in vivo.
To achieve a seamless collaboration,
there will be an exchange of scientists,
on a short-term basis, between RCAI,TMDU and INSERM/
Hospital Necker.

Tackling the enigmas of influenza infection

I

nfluenza virus is one of the most hazardous
human pathogens. Despite intensive study
in this field, the viral infection pathways andmechanisms of viral escape from the immune
system remain obscure. One of the major reasons for this knowledge deficit is the lack of agood animal model. Although some human influenza virus isolates can infect mice, and ferrets are sensitive to most of human viruses, wecannot examine the human immune response
to influenza in these animals.
T he 3rd generation ( 3G ) humanizedmouse is a newly established human hematopoietic cell transfer model based on improvements in the original NOD/SCID IL2Rg KO. The 3G mice
are also transgenic for a human MHCII, and this successful
genetic engineering will now allow us to analyze human helper T cell-based immune responses. Taking advantage of thishumanized mouse, RCAI launched a collaborative project with
Dr. Yoshihiro Kawaoka ( The Institute of Medical Science, The
University of Tokyo ). Under the project, the collaborative teamwill carry out the following experiments to tackle current enigmas of influenza virus infection.

1) Analysis of influenza virus HA antigenic drift in the3G humanized mice
Influenza virus pandemics are a major hazard to humankind. On a population scale at present, global surveillance andanti-influenza virus drugs are effective defenses. On the individual scale, innate and acquired immune systems are both
important. The viral protein Hemagglutinin (HA) is a dominantantigen in influenza virus infection, and anti-HA neutralizing
antibodies play a role in protection against the virus. However, the high frequency of viral genome mutations generates
mutated HA, so-called antigenic drift, which allows the virus
to escape from the humoral immune response. Therefore, influenza virus expressing a newly mutated HA could be a great
public safety risk, since it would not be covered by existing
vaccines.
Global surveillance is useful to follow antigenic drift during
an infection cycle, but the experimental study of influenza virus
antigenic drift is limited, since it is carried out using in vitro
systems, which do not recapitulate the complexity of virus-antibody competition in vivo.
The team will take advantage of the 3G humanized micethat can reconstitute the human acquired immune system todetermine which HA mutations result in escape form antibody
protection in vivo. Specifically, humanized mice will be infectedwith influenza A virus to induce neutralizing antibodies, thenthe mice will be infected with viruses that carry random mutations in the HA gene. Only viruses that can escape from theexisting neutralizing antibodies will be able to replicate, so we
can identify the escape HA mutants by simple sequencing ofisolated viruses.
These experiments will provide us information about HAmutations in influenza virus that potentially may cause pandemics.

2) Analysis of pathological mechanisms of highly
pathogenic Influenza A (HPIA) infection using the 3G
humanized mice
Seasonal influenza virus infection and pathological changes are usually limited to the respiratory system. However, in
HPIA H5N1 virus infection, the virus can enter into various
types of cells, not just respiratory epithelial cells, where it
causes intense and destructive immune responses. These
aberrant immune reactions (e.g. a cytokine storm) can cause
acute respiratory distress syndrome (ARDS) and also damage
non-respiratory organs. However, the mechanism by which
HPIA infection causes such severe pathology is largely unknown.
The team will dissect HPIA infection in the 3G humanized
mice to elucidate which human cells are infected, and what
immune factors determine the damage in each organ, ultimately forcing the system to collapse. Specifically, the teamwill utilize an infected cell visualization system, which consistsof HPIA H5N1 encoding a fluorescent protein in the viral genome, which will allow us to monitor the systemic spread of
HPIA all throughout the humanized mice. The team will thenisolate mRNA from infected cells sorted based on HPIA fluorescence at various time points after infection. The mRNA will
be analyzed by RNA-seq and the team will establish a map of
the cytokine and chemokine network during HPIA spreading,including chronological information.
These experiments will provide new insights into HPIA
infection and will provide a framework for further studies toestablish new biomarkers for more effective therapeutic strategies and more efficient vaccine development.

9

From Genetic Analysis to Mechanistic Interpretation of
Immune Diseases

Important features of the immune system have been elucidated during the long history of immunology. However, there
still remains a large black-box in understanding of immune
homeostasis and its dysregulation. This lack of knowledge,
as a matter of course, is a large hurdle in the development ofbetter therapies for many immune diseases. To address this,
we must bridge numerous gaps between 1) the information
about how the immune system is regulated in mouse and human, 2 ) human genetic information obtained by genome-wide
association studies (GWAS) and mechanistic interpretation of
disease etiology, and 3 ) global cell populations and particularsubsets of immune cells.
Because human genetic data based on advanced genome technologies have already proven to be very powerful indetecting the association between genotype and phenotype,close collaborations with human genetics groups are very
likely to bear fruit in tackling unsolved mechanisms of immune
diseases. Our efforts to understand how the immune system
is regulated have been confined largely to studies in mice, but,after serious deliberation, we think that it is now time to boldly
take a step towards conquering human immune diseases. In
this regard, it is fortunate that RIKEN has a strong and prominent research institute in this field, the Center for Genomic
Medicine ( CGM ) , in the same Yokohama campus. Because
the mission of CGM is to understand individual genetic variations and to apply this knowledge to healthcare, we certainly
have several overlaps in our research interests.
Besides intramural collaboration with CGM in RIKEN, wehave begun to discuss the possibility of formal collaboration
with extramural genetics research groups carrying out theSardiNIA project ( http://sardinia.nia.nih.gov/Project_Team/

10

project_team.html ) because they are particularly interested in
the relationship between genotype and immune-phenotype.
The SardiNIA project is a joint effort of investigators at the
National Institute of Aging in the National Institutes of Health( USA ) , at the Italian National Research Council Institute of
Neurogenetics and Neuropharmacology in Cagliari, and statistical geneticists at the University of Michigan. Most countries
have a heterogeneous population, and the unique advantageof the SardiNIA project is that the lineage of most Sardinians
can be traced directly back approximately 8,000 years to theisland’s original settlers. Their relative genetic homogeneity
greatly simplifies population genetic analyses, so that study ofthis highly interrelated population cohort is expected to have ahigh probability of identifying genetic factors affecting specified phenotypic traits.
It is quite likely that collaboration with human genetics
groups will give us many insights into the etiology of immune
diseases. However, there still remains the issue of how toapproach mechanistic interpretations. In particular, because
the immune system is a highly sophisticated and complex biological system, approaches using human samples have an
obvious limitation since only non-invasive analyses are possible in most cases due to ethical reasons. The use of model
animals is thus an indispensable approach towards this end,
and the question becomes, how to fill the gap between knowledge obtained from human and model animals. One possible
approach is to use and analyze knock-in animal models witha disease-related genetic variation identified in human genetic
analysis by integrative genomic approaches from molecularto whole body levels. This course of endeavor has just beenlaunched in a systematic manner at the center.

RCAI Advisory Council 2012T

he RCAI Advisory Council met on October 1-2, 2012 and what follows is a summary of comments
and recommendations from that meeting. This was a particularly important meeting since RCAI will
undergo significant changes in the next fiscal year beginning April 2013: Dr. Taniguchi will step down
as director after a remarkably successful ten year tenure. There is a significant new push at RIKEN toprioritize translational medicine coupled with systems biology. RCAI will merge with the RIKEN Center for
Genomic Medicine ( CGM ) to form a new institute called “Center for Integrative Medical Sciences (IMS)”.
Taken together, all of these factors pose an exciting but potentially hazardous challenge to the hard won
successes of RCAI.

W

ithin less than a decade of its inception, RCAI has become a leading world center in immunology research.
Its success is indicated by the high quality, quantity andimpact of (1) publications in the best scientific journals, ( 2 )development of a steady stream of successful independent
investigators and ( 3 ) regional and international educational
contributions. RCAI scientists have achieved leading positionsin understanding the mucosal immune system and immune
cell signaling mechanisms through innovative biochemical, imaging and gene targeting studies in mouse models. Although
successful translation of research findings into health benefits
typically requires 12-15 years, RCAI investigators have alreadydeveloped promising leads for a cedar pollen allergy vaccine
and NKT cell-based therapy for refractory lung cancer. RCAI
has truly become a crown jewel of RIKEN, for which RCAI,
Director Taniguchi and all of the RCAI researchers and staff
are to be heartily congratulated. Future plans for changes in
research direction or administrative organization should havethe preservation of this excellence as a major priority.

Merger of RCAI and CGM
The merger of RCAI and CGM will have major, unpredictable
and likely unforeseen consequences on the productivity, direction and future of both institutes as they combine into the new
hybrid institute. It was unclear to the AC whether or not this fusion is a fait accompli, and thus whether any of our comments
and recommendations will have any impact. Despite some
general descriptions about the new institute, it remains unclear
at this point how the new institute will be structured, precisely
what each side will bring to the merger, and how the two willinteract in a synergistic way. These topics are apparently under current intensive discussion; however, since the merger isscheduled to take place by April 1, 2013, there is concern that
there is not yet a clearer vision as of October 1, 2012.
The proposed merger seems only weakly justified from ascientific perspective. RCAI is a world-class immunology pow-

11

erhouse that could likely draw from the scientific expertise of
CGM as needed on an ad hoc basis by natural collaborations
arising between RCAI and CGM investigators. The merger
seems to be based more on administrative considerations
than on a firm scientific rationale.
As a leading immunology institute, RCAI has brought RIKEN into the international limelight of this important discipline.
The RCAI “brand” has become very well known in a remarkable eight short years. To have it disappear and be subsumed
into an institute titled “Center for Integrative Medical Sciences(IMS)” without the word “Immunology” in the name is, in theopinion of the AC, an incomprehensible move. In the absenceof this label, the new institute will, at least initially, no longer be

12

so attractive and visible to outside scientists and potential recruits.
The administrative and budgetary framework of the new
institute has not been worked out; in particular the incipient
administrative leadership vacuum in the new hybrid at this latedate is a concern. Dr. Taniguchi is stepping down as RCAI
director and, based on their English website, Dr. Kubo is presently acting and not permanent director of CGM. During the
AC meeting it was revealed that Dr. Koyasu has recently been
appointed to a position where he will oversee the transitionand establishment of the new institute and help identify potential director candidates. Such essential movement should
proceed as quickly as is judiciously possible.

RCAI Outstanding Contributionof the Year 2012
RIKEN RCAI Outstanding Contribution of the Year 2012 wasawarded to Dr. Shin-ichiro Fujii, and Ms. Hiroko Tanabe.
Dr. Shin-ichiro Fujii ( Photo 1), Leader of Research Unit for Cellular Immunotherapy, received the award for the establishment of human artificial
adjuvant vector cells, a new cell-based therapy to coordinate the activityof innate and adaptive immunity against tumors.
Dendritic cells (DC ) are extremely efficient at activating T cells, andthe use of DCs in anti-tumor therapy has been a goal of many investigators since the 1990’s, when it was found that DCs could be generated
ex vivo by culture with GM-CSF and that inclusion of tumor-derived
peptides or irradiated tumor cells could help eradiated tumor cells. Development of effective DC anti-tumor therapy has been a lifelong dreamof Dr. Fujii, who spent several years in the laboratory of Professor Ralph
Steinman, the Novel Laureate who discovered these cells. Dr. Fujii published several important studies on the roles of DCs against cancer, but
was looking for a new breakthrough to make their application more effiPhoto 1 : Dr. Shin-ichiro Fujii
cient and powerful.
Around that time, Dr. Masaru Taniguchi discovered a unique anti-cancer immune system. He showed that α -GalCer activates NKT cells and induces very strong
direct anticancer effects. Dr. Fujii was very surprised to hear the story, and wanted to know how NKT
cells work on DCs. He was able to show that activated NKT cells induce DC maturation in vivo (Fujii
et al., J Exp Med., 2003), but this result contradicted those from in vitro experiments, where α-GalCer
had no such effect on DC maturation.
Dr. Fujii devoted his efforts to understand the difference between in vivo and in vitro, andfound a specific adjuvant for DC, CD40 ligand, that could link innate and adaptive immunity(Fig. 1).

Figure 1: Bridge 1. From bench
to bedside=research
to the clinic

13

Outstanding Contribution of the Year 2012

This result gave Dr. Fujii a new idea; co-administration of NKT ligand (α-GalCer ) and irradiateddead tumor cells might trigger both adaptive and innate immunity. However, in testing this idea inmice he discovered a serious problem; very large numbers of tumor cells were required for an effective
treatment, and many animals died because of pulmonary embolisms due to entrapment of the cells in+
the lung. Pressing forward, they were able to overcome this problem by injecting CD1d cells expressing tumor antigen and α-GalCer, which required many fewer cells (Shimizu et al., J Immunol, 2007,
Shimizu et al., J Exp Med, 2007).
This year, Dr. Fujii published another important breakthrough in the application of this anti-tumor+
therapy. They can now use allogenic CD1d cells carrying α-GalCer and mRNA encoding tumor antigen, artificial adjuvant vector cell, to efficiently generate both innate and adaptive anti-tumor immunity(Fig. 2) ( Shimizu et al., Cancer Res. 2013 ). Because artificial adjuvant vector cell utilize RNA to generate protein, this therapy is different from gene therapy. Dr. Fujii rather thinks that this therapy should be
used as a prodrug. Prodrugs undergo chemical conversion by metabolic processes before becoming
an active pharmacological agent. Indeed, artificial adjuvant vector cells activate NKT cells and release
IFN-γ which activates NK cells and innate immunity. Also, neighboring DCs capture the artificial adjuvant vector cell, undergo maturation, and induce adaptive immunity, along with immunological memo-

Figure 2: Artificial adjuvant vector
cells (aAVC) as CD1d+ cells
carrying NKT-L and TAA

Figure 3: Bridge 2: From bedside tobench=clinic to the research14

ry that persists for longer than a year (Fig. 3 ).
When artificial adjuvant vector cells were administered to dogs, there were no adverse events.
+
NKT cells initially increased but one month later had almost disappeared; however CD8 T cell responses were still present ( Fig. 4 ). Currently, artificial adjuvant vector cells therapy is being tested using human samples.
Dr. Fujii thinks that the artificial adjuvant vector cell makes three bridges. First, it bridges innate and adaptive immunity. The American Cancer Research Association gave this comment to
Dr. Fujii, “preclinical proof-of-principle studies establish a new cell-based therapy to coordinate
the activation of innate and adaptive immunity against tumors, as an effective strategy for cancer immunotherapy.” The second bridge is between the clinical and basic sciences. Artificial
adjuvant vector cells are expected to be a new type of cell therapy that can be used as a prodrug. Thirdly, artificial adjuvant vector cell has come out of two immunology fields; the NKT field,
pioneered by Masaru Taniguchi, and dendritic cells, pioneered by Ralph Steinman. Dr. Fujii thinks ofartificial adjuvant vector cell as an artificial cell that bridges NKT and DC ( Fig. 5 ). “We are honored toconcentrate on using these systems in humans. I would like to thank all the laboratory members, Drs.

Figure 4: Preclinical study ofartificial adjuvant vector
cell using large animals

Figure 5: Bridge 3: Artificial
adjuvant vector cells=NKT
field + DC field15

Outstanding Contribution of the Year 2012

Shimizu, Shinga, Sato, Yamasaki, and Ms. Asakura. We’d like to thank Dr.
Ishii, Dr. Taniguchi, and external collaborators, especially Dr. Schuler for
generation of GMP grade mRNA,” Dr. Fujii said in his speech at the award
ceremony.
Ms. Hiroko Tanabe ( Photo 2 ) , Chief Assistant of the Administrative
Coordination Office, received the award for the establishment and management of the Administrative Coordination Office ( AdmCO ) . The AdmCO was established within the RCAI in April, 2010 to manage a wide
range of administrative tasks related to RCAI laboratories and the Center.
Until then, each leader had an assistant and the assistant’s tasks
differed greatly depending on the laboratory. In addition, tasks related to
the Center were managed by the Director’s assistant or by the organizers’ Photo 2 : Ms. Hiroko Tanabe
assistants, and they had to ask for volunteers to support the Center’s
events.
To make these dispersed administrative duties more efficient, Ms. Tanabe centralized all of theirwork, as well as IT related work and facility support. Thanks to the tremendous efforts by Ms. Tanabe and other chief assistants, Mses Hiroko Yamaguchi and Mari Kurosaki, the reorganization madegreat progress in a short time and enhanced the Center’s activities. A new data sharing system has
been introduced, which enables assistants to share information, creating an environment where theyeasily interact and teach each other. Ms. Tanabe has also launched an RCAI ordering system, which
completely separates three parties’ activities; order placement by laboratories, delivery verification by
the Yokohama Research Promotion Division ( YRPD), and payment processing by the AdmCO. Thishas proven a useful system to manage various budgets simultaneously and to prevent any potential
fraudulent activity. Ms. Tanabe centralized the MTA related tasks, and AdmCO currently processes
200 MTAs per year. Using this centralized approach, the AdmCO is now able to provide the same highstandards to all RCAI laboratories.
It is not an exaggeration to say that the RCAI could not have been reorganized smoothly to become the IMS without the support of the AdmCO and Ms. Tanabe. The AdmCO arranged construction of new laboratories and provided people arriving and leaving with constant administrative support. Although many people longed for her continued efforts in the new Center, she decided to leave,handing over her work to Ms. Yamaguchi. “I have no words to express our appreciation. Tanabe-sanalways showed us how to work with pride, and she always stood by us when we felt so small,” said
Ms. Yamaguchi. Ms. Tanabe in her speech thanked all the members of AdmCO; all assistants, IT
members and Mr. Ogata. “Without them, I could not have done anything,” she said.

Dr. Taniguchi retires from the Director.
The ceremony was the last chance for all RCAI members to gather
together. In the end of the ceremony, Dr. Cooper (Photo 3 ), the chairmanof the RCAI Advisory Council, sent a video message to Dr. Taniguchi(Photo 4).
In the message, he said, “I want to tell you that anyone who
was ever associated with you was very impressed with what you Photo 3 : Dr. Max D. Cooper
and your colleagues accomplished with RIKEN. In a decade, you’ve
become an international leader in the field of immunology. I realize you owe a lot to your colleagues and to RIKEN as well, but I
would like to pay a special tribute to your leadership. You’ve done
the job in exceptional and incredible ways to draw cooperationfrom everyone around you. You’ve had a lot of difficult situations, Photo 3 : Dr. Max Cooper
both personal and professional and through all of that, you maintained a calm leadership, listened to people around you, and responded to advice from Scientific16Advisory Board members. You’ve treated all this with an even-handedness that was absolutely
remarkable and admirable. I think you had learned a lot from being a dean of a medical school, andyou brought that experience and knowledge with you when you came to RCAI. I think though, thatprobably you’ve been able to maintain your balance and even serenity by the fact that you continued
to run your own laboratory and do research, and thus had a way to test whether your opinions orideas were right or wrong. It is easy to get off track, but having laboratory-based research activity cankeep you on track to a much better extent. I don’t know what you will be doing next, but I hope thatyou maintain the research activities. That will continue to give you pleasure. You will have many goodmemories of your times as the Director of RCAI, and all of us who had any chance to experience yourleadership or to be with you will treasure those memories, and we hope you will, too.”In the end of the message, he performed a song by Louis Armstrong, “Summer Day.”I hear laughter, from the swimming hole
Kids out fishin’, with the willow pole
Boats come float, round the bend
Why must summer, ever end...
Love, to me, is like a summer day
If it ends, the memories will stay
Warm and sweet, and tenderI can hear my summer song

Dr. Taniguchi closed the ceremony by citing a phrase from Samuel Ullman’s poem Youth and one of Gen. Douglas MacArthur’s
speeches.

Nobody grows only by merely living a number of years;
people grow old only by deserting their ideals.
You are young as your faith, as old as doubt;
as young as your self-confidence, as old as your fear;
as young as your hope, as old as your despair.
Old soldiers never die, they just fade away.

Photo 4 : Dr. Masaru Taniguchi

References:
Fujii, S., Shimizu, K., Smith, C., Bonifaz, L., and Steinman, R.M. Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein. J Exp Med. 198,267-279 (2003)
Shimizu, K., Goto, A., Fukui, M., Taniguchi, M., and Fujii, S. Tumor cells loaded with alpha-galactosylceramide induce innate NKT and NK cell-dependent
resistance to tumor implantation in mice. J Immunol. 178, 2853-2861 (2007)
Shimizu, K., Kurosawa, Y., Taniguchi, M., Steinman, R.M., and Fujii, S. Cross-presentation of glycolipid from tumor cells loaded with alpha-galactosylceramide leads to potent and long-lived T cell mediated immunity via dendritic cells. J Exp Med. 204, 2641-2653 (2007)
Shimizu, K., Mizuno, T., Shinga, J., Asakura, M., Kakimi, K., Ishii, Y., Masuda, K., Maeda, T., Sugahara, H., Sato, Y., et al. Vaccination with antigen-transfected, NKT cell ligand-loaded, human cells elicits robust in situ immune responses by dendritic cells. Cancer Res. 73, 62-73 (2013)17

Prize Winners 2012
Tomohiro Kurosaki (Photo 1), Group Director of the Laboratory for Lymphocyte Differentiation, received The Commendation for Science and Technology
from the Minister of Education, Culture, Sports, Science and Technology (MEXT ),2012. He won the Research Category Prize for his studies on identification ofregulatory factors in allergic reactions. The prize is awarded to investigators
whose research is highly original and contributes to the science and technologyof Japan.
Photo 1 : Tomohiro Kurosaki

Sidonia Fagarasan (Photo 2), Team Leader of the Laboratory for Mucosal

Photo 2 : Sidonia Fagarasana

Immunity, received the 15th Japanese Society for Immunology Award for her
research on IgA synthesis: a form of functional immune adaptation extending
beyond gut. Through this research, she discovered that IgA production is essential for the symbiosis of microorganisms and immunity in the gut, and it evenhas strong effects on systemic immunity. She is currently expanding her studies
on the relationship between gut bacteria and autoimmune diseases, and further
important research achievements in this new area are very likely.

Tomokatsu Ikawa (Photo 3 ), Young Chief Investigator of the Laboratory for
Immune Regeneration, received the MEXT Prize for Young Investigators. Thisprize is awarded to young scientists ( under 40 years of age ) in recognition ofcreative and original research and an outstanding ability to develop scientificresearch projects. He received the award for his research on molecular mechanisms of T cell lineage developmental decisions.
Photo 3 : Tomokatsu Ikawa

Tadashi Yokosuka (Photo 4), Senior Researcher of the Laboratory for Cell
Signaling, received The Chiba Medical Society Award 2012. He won the basic
medical science category prize for his research on immune signal units that
achieve T cell antigen recognition and immune responses. This award is given
to scientists whose research is greatly expected to contribute to advanced medical science, and the winners receive ¥500,000.
Photo 4 : Tadashi Yokosuka

Hayato Kaneda (Photo 5), Young Chief Investigator of the Laboratory for Stem
Cell Competency, received The Keio University School of Medicine Sanshikai
Encouragement Award 2012. He won the basic medical science category prize
for his research on requirement for COUP-TFI and II in the temporal specificationof neural stem cells in centeral nervous system development. This award is given to young scientists under 40 years of age who has greatly contributed to thefields of basic or clinical medical sciences.
Photo 5 : Hayato Kaneda

18

Masanaka Sugiyama (Photo 6 ), Junior Research Associate of the Research
Unit for Inflammatory Regulation, received the Young Investigator Award at
the 20th International Symposium on Molecular Cell Biology of Macrophages(MMCB ) 2012. He received the award for his poster presentation: “In vivo ablation of XC chemokine receptor 1-expressing dendritic subset.”

Photo 6 : Masanaka Sugiyama

Osamu Masui (Photo 7), Researcher of the Laboratory for Developmental Genetics, received a Genetic Society of America Poster Award at the 2012 Mouse
Molecular Genetics Conference held at Pacific Grove, CA, USA. He received theaward for his presentation: “Live-cell imaging of X-chromosome inactivation indifferentiating ES cells: visualization of X-inactivation centre locus and Xist RNA”.

Photo 7 : Osamu Masui

Shimpei Kawamoto (Photo 8 ), Special Postdoctoral Researcher of the Laboratory for Mucosal Immunity, and Takashi Kanaya ( Photo 9 ) , Researcherof the Laboratory for Epithelial Immunobiology, received Poster Awards at The
34th Naito Conference “Infection, Immunity and their Control for Health: Mucosal
Barrier” held on October 16-19, 2012, in Sapporo. Kanaya’s presentation “Etstranscription factor Spi-B is essential for the differentiation of intestinal microfold
cells” and Kawamoto’s presentation “The inhibitory receptor PD-1 regulates IgA
selection and bacterial composition in the gut” were selected as excellent poster presentations, and The Naito Foundation Subsidiy for Promotion of SpecificResearch Projects (¥500,000 each ) were granted to them.

Photo 8 : Shimpei Kawamoto

Photo 9 : Takashi kanaya

The RIKEN Research and Technology Incentive Awards 2012 were presented to Raul Eduardo Vizcardo Sakoda (Photo 10 ) and Shimpei Kawamoto (Photo 8 ). Sakoda, Researcher of the Laboratory for
Developmental Genetics, received the award for his study on regenerationof human tumor antigen-specific T cells from iPSCs derived from mature
CD8 + T cells. Kawamoto, Special Postdoctoral Researcher of the Laboratory for Mucosal Immunity, received the award for his study of how theinhibitory receptor PD-1 regulates IgA selection and bacterial compositionin the gut. The RIKEN Research Incentive Award is given to researchers under age 40 who have contributed to furthering RIKEN’s ideals byachieving exemplary results in their research.

Photo 10: President Ryoji Noyori (left) and
Raul Eduardo Vizcardo Sakoda (right)

19

Excellent Paper of the Year 2012T

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 torecognize excellent publications by RCAI scientists. Although the funds were depleted
by 2008, RCAI’s strategic committee decided that there was great value in awarding
excellent achievements by young researchers and encouraging their efforts, so they
provided the funding to continue this prize.
In 2012, 6 excellent papers were selected from 11 candidates for this award.

Takashi Kanaya, Tsuneyasu Kaisho and Hiroshi Ohno
Laboratory for Epithelial Immunobiology“The Ets transcription factor Spi-B is essential for the differentiation of intestinal M cells”
Nature Immunology, Vol. 13, pp. 729-736, 2012

Tomohiro Kaji and Toshitada Takemori
Laboratory for Immunological Memory“Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory”
The Journal of Experimental Medicine, Vol. 209, pp. 2079 -2097, 2012

Sawako Muroi, Chizuko Miyamoto, Ichiro Taniuchi and Hilde Cheroutre
Laboratory for Transcriptional Regulation and Research Unit for Immune Crosstalk“Transcriptional reprogramming of mature CD4 T helper cells generates distinct MHC class II restricted cytotoxic
T lymphocytes”
Nature Immunology, Vol. 14, pp. 281-289, 2013

Kanako Shimizu and Shin-ichiro Fujii
Research Unit for Therapeutic Model and Research Unit for Cellular Immunotherapy“Vaccination with antigen-transfected, NKT cell ligand – loaded, human cells elicits robust in situ immune responses by dendritic cells”
Cancer Research, Vol. 73, pp. 62-73, 2013

Shimpei Kawamoto, Mikako Maruya and Sidonia Fagarasan
Laboratory for Mucosal Immunity“The inhibitory receptor PD-1 regulates IgA selection and bacterial composition in the gut”
Science, Vol. 336, pp. 485-489, 2012

Raul Vizcardo, Kyoko Masuda, Daisuke Yamada, Haruhiko Koseki and Hiroshi Kawamoto
Laboratory for Developmental Genetics and Laboratory for Lymphocyte Development+“Regeneration of human tumor antigen-specific T cells from iPS cells derived from mature CD8 T cells”
Cell Stem Cell, Vol. 12, pp. 31-36, 2013

20

2012
Part 2

Technology and Innovation

RCAI and Torii collaborate fordevelopment of an allergy vaccine

Figure:J

Subcutaneous vaccination significantly prevents the increase of specific IgE after Cry j 1 sensitization.
The recombinant vaccine was administered subcutaneously into Cry j 1-sensitized mice four times; the mice were sensitized again with Cry j 1 again after the vaccination. The increase of Cry j 1-specific IgE after the last sensitization withCry j 1 was significantly attenuated in the vaccinated mice compared the control saline injected mice. The figure shows
the average Cry j 1-specific IgE titer in each group. *p<0.01 by Mann-Whitney U-test.

apanese cedar (Cryptomeria japonica ) pollinosis is
a common allergy in Japan, with a prevalence estimated to be 26.5% in a nationwide survey conducted
in 2008. Antigen-specific immunotherapy ( SIT ) is
considered to be the only curative treatment for allergy. Only a crude extract from Japanese cedar pollen
has been approved for clinical use by the Ministry of
Health, Labor and Welfare in Japan. However, it will
be very difficult to gather enough cedar pollen fromthe male flower of Japanese cedar to prepare enoughvaccine for pollinosis patients, because of the limited
source of the pollen and the limited pollen scattering season, usually early February to middle March.
Therefore, development of a recombinant vaccine independent of the natural source is expected to be very
valuable for clinical use as a SIT vaccine. Vaccines
using allergoids and modified Cry j 1, a major allergenof Japanese cedar pollen, have been developed andused in pre-clinical trials; however, none of them has
become commercially available for medical use due
to poor clinical outcomes at later stage clinical trials
or to the failure to find a cooperative pharmaceutical

22

company to introduce them into market. To fill thecritical gap between basic research and later stage ofdrug development, RCAI offered a program named the‘exchange zone’, where RCAI, universities, hospitals,
and pharmaceutical companies work together for drug
development, including allergy vaccine development,
especially for Japanese cedar pollinosis. Based on
this program, RIKEN and TORII pharmaceutical Co.,
Ltd. set up joint research laboratory in RCAI in May
2010 and started research and development of the recombinant SIT vaccine. The aims of the joint researchlaboratory are to develop a recombinant SIT vaccinewith high safety and tolerability, and to introduce thevaccine into the Japanese market.

Development of recombinant SIT vaccine
For preparation of the vaccine, recombinant technology is used to conjugate two major allergens fromJapanese cedar pollen, namely Cry j 1 and Cry j 2,
and then it is further modified with polyethylene glycol( PEG ) to prevent binding with immunoglobulin E ( IgE )and for improvement of its solubility. The research

team conjugates PEG with recombinant Cry j 1 andCry j 2 fusion protein via a cysteine residue on the fusion protein. To accomplish this, all cysteine residues
except one in the cDNA encoding the fusion protein
had to be substituted with serine residues to control
the number and location for conjugation with PEG.
The vaccine is expected to be unable to bind human
IgE, which could induce adverse events after vaccine
administration in vivo.
The research team compared the yield of the recombinant fusion protein using several host/vector combinations in eukaryotic cells. Based on this analysis,
they selected the best combination and, using a jar
fermenter and, based on knowledge of safety and usage to produce vaccine or medicine already approvedby the Ministry of Health, Labor and Welfare in Japan,
are optimizing the culture conditions to produce themaximum amount of fusion protein. They are also developing simple methods for purification of the recombinant SIT vaccine from the host cells, taking into consideration cost-performance and purity. They isolated
hybridomas that produce vaccine-specific IgG and set
up a procedure to specifically determine the quantityof the vaccine by sandwich ELISA. They also set upa system to evaluate uniformity and purity of the vaccine using a high performance liquid chromatographysystem.

Therapeutic potential of the vaccine in amouse model of Japanese cedar pollinosis
Systemic injections of Cry j 1-sensitized mice with thevaccine prevented the increase of serum Cry j 1-specific IgE following sensitization with native Cry j 1. ( seeFig.) The research team evaluated therapeutic potential of the vaccine by comparing the reduction of local
inflammation or IgE production among various doses
and frequencies of vaccination using sensitized micein the mouse model of pollinosis. They confirmed theability of the vaccine to prevent the increase of specific IgE following systematic or local sensitization in adose-dependent manner. To improve the therapeutic
efficacy of the vaccine, elucidation of the therapeuticmechanisms and identification of biomarkers are important issues. It is also important to find biomarkers
to distinguish responders and non-responders to thevaccine. Therefore, they are looking for biomarkers tomonitor therapeutic responses during and after vaccination. The research team plans to evaluate therapeutic effects of the vaccine in humans and the availabilityof biomarkers in translational research, after which
they will produce the vaccine at GMP grade.

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

Senior Research Scientist :Takashi Fujimura

Visiting Scientist :Hiroyuki Miyazaki, Yasushi Okumura, Koji Fujinami, Ryosuke Ishikawa

Technical Staff :Hisako Motokawa

Administrative Staff :Akiko Imai

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 ofregulatory 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: apreliminary 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 )23

iPS technology development for
immunological research and therapeutics

Figure:Efficient generation of antigen-specific mature T cells in vitro from T-iPS cells originally derived from antigen-specific T cells(A) Flow cytometric profile of JKF6 cells.
(B) Photomicrograph of iPS cells derived from JKF6 cells.
(C) Efficient generation of antigen-specific mature T cells in vitro from T-iPS cells. Left panel shows the CD4 vs. CD8 profile ofcells generated in the coculture system using OP-9/DLL1 cells. Right panel shows the CD3 vs. MART-1 tetramer staining of
CD8+ T cells gated in the rectangle in the left panel.
(D) Production of IFNg by CD8+ T cells upon antigen -specific stimulation. IFNg secretion was measured after co-culturing CD8
SP cells for 24 hours with EBV-lymphoblastoid cells (CIRA0201) pulsed or not with MART-1 peptide.

I

nduced pluripotent stem cells (iPSCs) possess tremendous therapeutic potential, not only in the fieldof regenerative medicine but also for immune therapy.
RCAI has started an activity to apply iPS technology
for mouse and human immunology research and fortherapeutic development. The iPS Group is engaged
in developing efficient protocols to reprogram various
lymphocytes and induce differentiation of iPS cells into
the lymphoid lineage. These studies are being doneon a collaborative basis with individual researchersin RCAI. This activity is partly supported by a CREST
award from JST.

sKO by an iPSCs establishment assay. These results
indicate that polycomb impact during reprogramminghas at least two phases ( Early and Late ) . Polycomb
represses the reprogramming in the early phase while
promoting it in the late phase. Thus, aberrant reprogramming by polycomb may be occurring in the latephase. Polycomb impact during reprogramming in theearly phase may be addressed using a reprogrammable mouse strain ( kindly provided from CiRA, Kyoto
University ) .

Aberrant reprogramming of polycomb protein
Ring1B in B-iPSCs

The Group used an improved version of a temperaturesensitive ( TS ) SeV vector to generate iPSCs in which
the SeV could be efficiently depleted by a simple temperature shift. Pluripotency of the resulting iPSCs wasvalidated by generating germ line chimeric mice. They
then showed that human T cells were reprogrammed
to iPSCs ( T-iPSCs ) , which are also virus-free in thehost genome, using the TS SeV vector. Furthermore,they showed more efficient induction of human T-iPSC
using SV40 combined with defined Yamanaka factors( SV40+OSKM ) , compared to using defined Yamanaka
factors alone ( OSKM ) . Finally, they induced T-iPSCs
from several types of human T cells such as CD4 +or CD8 +. These results suggested that the TS SeV isqualified as a future standard vehicle to reprogram human cells for clinical purposes.

The iPS Group successfully established B cell (B)-iPSCs in mouse and human by using overexpression ofdefined factors. Furthermore, by transcriptome analysis, the Group identified polycomb ( Ring1B ) target
genes as candidates for an aberrant reprogramming
region. To address this issue, they generated B-iPSCs
from Ring1A/B conditional knockout mice. They foundthat Ring1 deleted B-iPSCs showed more apoptotic cells than ESCs. Moreover, a Ring1B target gene,Ink4a/Arf, was upregulated in B-iPSCs after Ring1
protein deletion. These results suggest that B-iPSCs
are reprogrammed when cells are in a pluripotent
state, but have aberrant phenotypes when cells are ina differentiated state. Next, they addressed the impact
of polycomb during somatic cell reprogramming topluripotency using Ring1B and Cdkn2a double knockout (dKO ) or Cdkn2a single knockout ( sKO ) mice. They
found that there were two fold more ESC-like colonies
in dKO than in the sKO by an alkaline phosphatase( ALP ) assay. Although they were statistically low, moreclones were established as B-iPSCs in dKO than in the24

Efficient induction of iPSCs from human
circulating T cells

Generation and analysis of NKT-iPSC-derived
cloned mice
The Group has previously succeeded in generating
iPSCs harboring NKT cell-specific rearranged TCRloci by a conventional protocol with retroviral vectors
starting with highly purified B6 splenic NKT cells. The

iPSC clones can efficiently develop into NKT cells producing large amounts of IFN-g in vitro by co-culturingwith the OP9/Dll-1 stromal cell line in the presence of
IL-7 and Flt3L. These NKT cell-derived iPSCs also generated coat color chimeras when injected into BALB/
c blastocysts. The male chimeras tested were found tobe germline chimeras that transmitted the rearranged
Va14-Ja18 and Vb7-Db2-Jb2.3. The NKT-iPSC-derived
cloned mice have reduced numbers (1/5 to 1/20) of total MNCs in the thymus but not in the periphery. They
also had a 5- to 100-fold increase in absolute numberof NKT cells and a severe reduction of abT cells ( 1/201/100 ) and loss of gdT cells. The function and phenotype of NKT cells in cloned mice is comparable tothose in B6 mice, indicating that the cloned mice area feasible model for the analysis of NKT cells and areadapted for NKT cell-targeted therapy.

eage specification. iPS-derived CD34 + CD38 - cells at
day 7 and at day 14 expressed GATA3, GFI1, GATA2
at levels similar to those in normal BM/CB HSCs. Consistent with the transcription profile of human iPS-derived CD34 + CD38 - cells, a colony forming capacity( CFC ) assay demonstrated that these iPS-derived
CD34+ CD38- cells generated granulocyte-, monocyte-,
and erythroid colonies in methylcellulose agar. Furthermore, 750-3000 CD34 + CD38-CD43 + cells exhibited potent capacity to differentiate into T cells and NK
cells in the OP9-DLL1 culture system. Together, human
iPSCs generate human CD34 + CD38 -CD43 + cells withmulti-lineage differentiation capacity, and the putativeprogenitor could be a key cellular component in developing hematopoiesis therapies in humans.

Induction of hematopoietic lineages fromhuman iPSCs

In this study, the Group established iPSCs from mature
cytotoxic T cells specific for the melanoma epitope
MART-1 (Fig. A). JKF6 cells are long-term cultured
tumor infiltrating lymphocytes that were originallyderived from a melanoma patient, and have been
maintained at the Surgery Branch of the National Cancer Institute ( Yang et al., PLOS One, e22560, 2011) .
JKF6 cells are specific for the complex of MART-1peptide and HLA-A*02:01, which can be visualized
as MART-1-tetramer+ cells by flow cytometry ( Fig. A ) .
MART-1-specific T cells were transduced with Yamanaka factors, and they established a clone that forms
colonies with human ESC-like morphology ( MART-1iPSCs, Fig. B ) . When co-cultured with OP9/DLL1 cells,
these MART-1-iPSCs efficiently generated CD8 + T
cells, and more than 90% of these cells were specificfor the original MART-1 epitope ( Fig. C ) . Stimulation ofthese CD8 + T cells with HLA-A*02:01-expressing cells
pulsed with MART-1-peptide resulted in the secretionof IFNg (Fig. D ). The present study thus provides a novel method for cloning and expanding functional CD8 + T
cells specific for a given antigen, which can potentiallybe applied for cell therapy against cancer.

The Group aims to induce hematopoietic stem/progenitor cells from human iPS cells to understand humanhematopoietic development and to elucidate whether human iPS cells can recapitulate hematopoiesis
through progenitor cells with myeloid, lymphoid, anderythroid potential.
To this end, they set up in vitro culture of human
iPSCs using stromal cells. Compared with primary
human osteoblasts or mesenchymal stem cells, OP9
induces human CD34 + CD38 - cells from iPSCs more
efficiently, regardless of Notch signaling. The Groupthen examined the extent to which human iPS-derived
CD34 + CD38- cells recapitulate the transcriptional signature of normal HSCs. Expression levels of iPS-associated genes including NANOG, SOX2 and OCT3/4 inhuman iPS-derived CD34+ CD38- cells were downregulated to the levels seen in CB/BM HSCs. Of the genesets associated with the HSC-specific transcriptionalsignature, HOXB4, HOPX, STAT5 and CBFA2TE wereexpressed by iPS-CD34 + cells at comparable levels
to those in HSCs. Furthermore, they analyzed theexpression of transcription factors associated with lin-

Regeneration of antigen-specific T cells from iPS
cells derived from human CD8 + mature T cells

Members of the iPS Group
Leader :		
Hiroshi Kawamoto, Fumihiko Ishikawa, Osamu Ohara
Senior Research Scientist :Hiroshi Watarai
Research Scientist : Daisuke Yamada, Kyoko Masuda, Yoriko Saito, Raul Eduardo Vizcardo Sakoda
Technical Staff :Momoko Okoshi-Sato, Genta Kitahara, Masako Fujita, Chieko Tezuka, Sakura Sakata, Yuko Nagata,			Mariko Tomizawa
Recent Publications
1. 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 T, Toyoda T, Ohara O, Harigaya KI, 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 ).
2. Pereira CF, Piccolo FM, Tsubouchi T, Sauer S, Ryan NK, Bruno L, Landeira D, Santos J, Banito A, Gil J, Koseki H, Merkenschlager M, Fisher AG. ESCs require
PRC2 to direct the successful reprogramming of differentiated cells toward pluripotency. Cell Stem Cell 6, 547-556 ( 2010 )3. Watarai H, Yamada D, Fujii S, Taniguchi M, Koseki H. Induced pluripotency as a potential path towards iNKT cell-mediated cancer immunotherapy. Int J Hematol. 95, 624-631 (2012 ).

25

RCAI innovation projects in
drug discovery and medicineIn order to conduct research and development of novel drugs and medical technologies, RIKEN establishedthe Program for Drug Discovery and Medical Technology
Platforms (DMP) in April, 2010. Since then, based on its
guidelines, DMP has selected Project Leaders, who promote projects for drug discovery and medical technology,
focusing on the areas of diseases with low therapeutic
satisfaction, orphan drugs and new concepts. The Project
Leaders are researchers at RIKEN, but DMP supports the
projects by providing the assistance of a portfolio manager
with experience in drug discovery and by providing techni-

cal platforms established in DMP, linked to RIKEN ResearchCenters.
Several RCAI researchers collaborate with DMP.
Toshitada Takemori is the Unit Leader of Drug Discovery
Antibody Platform Unit of DMP, in order to develop monoclonal antibodies that can be used as therapeutic drugs fortreatment of cancer and other diseases.
Besides the Antibody Platform Unit, six other R&D projects were conducted under the collaboration between RCAIand DMP ( Table 1).

Table 1
Project

Project Leader

NKT cell therapy for cancer

Masaru Taniguchi

Vaccine for cedar pollinosis

Masaru Taniguchi

Artificial adjuvant vector

Shin-ichiro Fujii

Stem cell-targeting therapy for leukemia

Fumihiko Ishikawa

Mucosal vaccine

Hiroshi Ohno

Antibody therapy for CD8+ T cell-related diseases

Tsuneyasu Kaisho

NKT cell-targeted adjuvant celltherapy for cancer patientsR

IKEN and the National Hospital Organization( NHO ) reached an integrative collaborationagreement for NKT cell-targeted therapy and medical innovations in 2012. NHO manages a Japanese
network of 144 hospitals including 53,000 beds and astaff of 54,000. Under this agreement, Nagoya Center
and Kyusyu Cancer Center of the NHO will begin NKT
cell-targeted therapy for lung cancer.
A phase I/IIa clinical study of the application of
the NKT cell-targeted therapy for advanced lung cancer had been conducted in collaboration with Chiba
University ( Drs. Toshinori Nakayama and Shin-ichiro
Motohashi ) and RIKEN RCAI. In this study, the median survival time ( MST ) of the responder group was
significantly longer than the poor-responder group( 29.3 months versus 9.7 months ) . Following the outcome of this clinical trial, NKT cell-targeted therapy for26lung cancer was approved by the Advanced Medical
Care Assessment System by the Japanese Ministry of
Health, Labor and Welfare (MHLW ) in 2011. This system allows partial health insurance coverage to easethe financial burden on patients who need state-of-theart medical treatment.
After the agreement between RIKEN and NHO, a
collaboration between RIKEN RCAI, Chiba University
and NHO was started. NHO created Cell Processing
Centers in Nagoya Center and Kyusyu Cancer Center.
RCAI coordinated the agreement, provides α-GalCer
to stimulate the NKT cells, and conducts SNP analysis

of responder and poor-responder groups in collaboration with the RIKEN Center for Genomic Medicine and
NHO. Chiba University provides advice on the clinicalaspects of the therapy and technical training to the
NHO.
In 2012, NKT cell-targeted therapy for head andneck squamous cell carcinoma, in collaboration withProfessor Yoshitaka Okamoto, Chiba University Hospital, was newly approved by the Advanced Medical Care
Assessment System. Although translational researchon NKT cell-targeted therapy has been progressing,
there are still problems in its clinical applications.
There is no clear regulation of cell therapy in Japan;
some cell therapies are conducted for beauty treatment

or cancer without enough safety studies. Moreover,different regulations are applied depending on where
the cell therapy is conducted, in hospitals or otherplaces. In addition, to apply for the Pharmaceuticals
and Medical Devices Agency’s review, it is not clear
how cell therapy should be categorized. The Japanese Pharmaceutical Affairs Law ( PAL ) has only
two categories, “pharmaceuticals” and “medical
devices”. Regenerative medicine, such as transplantation of skin or cartilage cells is currently categorized as “medical devices”, but NK T cell-targeted
therapy may be rather closer to “pharmaceuticals”.
A draft amendment of PAL was submitted to the parliament and it will be discussed in the Diet in 2013.

Development of artificial adjuvant
vector cells as a novel therapyIn collaboration with Drs. Kakimi ( Univ. of Tokyo ) ,Maeda (Iwate Medical Univ.) and Mizuno ( Yamaguchi
Univ. ) , RCAI’s Shin-ichiro Fujii, Kanako Shimizu andYasuyuki Ishii have been developing unique adjuvant
vector cells that stimulate NKT cell activation. They
generated glycolipid-loaded, mRNA-transfected allogenic fibroblasts that act as artificial adjuvant vector
cells to promote NKT cell activation, leading to dendritic cell maturation and antigen-specific T cell immunity.

In preclinical studies, they observed the safety profile
and immune response. Administration of these artificial adjuvant vector cells to dogs activated NKT cells
and also elicited antigen-specific T cell responses withno adverse events. This unique tool could prove clinically beneficial in the development of immunotherapies
for malignant and infectious diseases. ( See ResearchHighlights p. 4 and p. 13 )A drug lead for leukemia

Acute myeloid leukemia ( AML) is a difficult cancer
to cure. Although most people with the diseaseachieve a period of remission following a standard
dose of chemotherapy, the leukemia will typically return. Now, in a breakthrough that could lead to thedevelopment of a drug that promises to prevent such
relapses, Dr. Fumihiko Ishikawa and his colleagues
discovered a small-molecule drug that wipes out AML
cells in a mouse model ( Saito et al. Sci Trans Res.
2013 ).
Ishikawa and his team previously found that theexpression of hematopoietic cell kinase ( HCK ) — anenzyme involved in blood cell differentiation and prolif-

eration — was greater in leukemia stem cells taken fromindividuals with AML than in blood stem cells obtained
from people without cancer.
In their latest research, Ishikawa’s team assessed
the potential of targeting this enzyme as a possible
therapeutic agent for human use. They found that byknocking down HCK expression in cell culture withcounteracting RNA molecules, they could achieve asignificant reduction in both the growth and survival of
the AML cells.
The team then enlisted the help of Dr. Toshio Gotoand colleagues at the RIKEN Program for Drug Discovery and Medical Technology Platforms to search for a27

chemical agent that possessed the same beneficial activity against AML. A high-throughput screen of 50,000
compounds for HCK inhibitors, in which the most
likely compounds were compared with the results of aparallel computer model-based screen, identified one
molecule as the most promising drug candidate. Thischemical, a pyrrolo-pyrimidine derivative, was structurally analyzed by x-ray crystallography and subsequently optimized to yield the small molecule RK-20449.
Experiments with human AML cells, both in vitro
cultures and implanted into mice, showed that the drug
reduced the number of leukemia cells and shrank thetumor burden of the disease ( Fig.) (Saito, Y. et al. Sci
Trans Med. Vol. 5, 181ra52, 2013). If these results are

Figure:translatable to the clinic, the drug could help preventand overcome cancer relapse in AML patients.
“If RK-20449 or its derivatives can eliminate AML
cells, including leukemia stem cells, in patients, thatwill advance AML treatment significantly,” Ishikawa
says. “After thorough and careful examination of the
toxicity of RK-20449, we hope that our findings can betranslated into a safe and effective treatment for AML.”This article is reproduced from RIKEN Research withpermission.
http://www.rikenresearch.riken.jp/eng/research/7345.
html

Spleens from mice engrafted with human AML cells after 52 days of treatment with the small-molecule
drug RK-20449 (left) and prior to treatment (right).

Longitudinal birth cohorts andthe humanized mouse modelT

he incidence of atopy, allergic disease and asthma
is increasing in industrialized nations. Allergies
are considered to result from the combined effectof genes and environmental factors and their interactions. To clarify the incidence of allergic disease,
RCAI, in a close collaboration with Chiba University
and the National Center for Child Health and Development ( NCCHD ) , has been working on longitudinalbirth cohorts. They recruited babies born of mothers
who, along with their family and relatives, have histories of allergy. In this process, Chiba University
and NCCHD are studying the environmental factors
associated with the development of allergic disease
from birth to 3 years of age ( the original cohort isnow at 2 year of age ) . For its part, RCAI has alreadystored white blood cells ( now 300 samples ) that were

Figure:28

The incidence of allergic diseases

purified from umbilical cord blood of all participants.
After completing the cohort study, RCAI will purify
hematopoietic stem cells from stored samples anduse them to establish humanized mice that will be reconstituted with the immunological state of the allergic
infants before the onset of disease. This approach will
enable us to evaluate the environmental and genetic
risk factors in allergy development.
As of December 2012, 191 babies at 1 year of age
and 55 babies at 2 years of age were examined. Until
now, the presence or absence of atopic dermatitis andsensitization to major food and airborne allergens has
been evaluated in 180 babies at 1 year of age. The rateof atopic dermatitis at 1 year of age was 12.7%. Sensitization to any of the allergens tested was 39.3%, withegg white showing the highest ( 32%) . Colonization of
the cheeks by Staphylococcus aureus at 1 month and6 months of age was 46.1% and 46.7%, respectively.
The clinicians found several early life exposures that
could be risk factors for allergy development in infants
at one year of the study and will continue the analysis
up to two years of age. When candidate factors are
identified for each of the participants, RCAI will be able
to evaluate their roles by using humanized mice reconstituted with the participants’ immune system at birth.

Establishing an efficient delivery
system for mucosal vaccines

Mucosal ( oral ) vaccination can be a better vaccination protocol in that it can induce both mucosal secretory IgA as well as systemic serum IgG,
whereas systemic (e.g., subcutaneous) vaccination can
only induce serum IgG. However, an oral vaccine has
to be resistant to gastrointestinal digestion to reachgut-associated lymphoid tissue ( GALT ) , the inductive
site for mucosal immune responses, in a sufficientquantity to induce an efficient immune response. Because of this requirement, successful oral vaccines todate are often attenuated live viruses, such as poliovirus and rotavirus, which can propagate in the body togenerate enough immunogenicity, even when the initialamount delivered to GALT is very small.

Figure :RCAI’s Hiroshi Ohno and his team identified a bacterial uptake receptor GP2 on M cells, a subset of intestinal epithelial cells that overlay the GALT, and haveshown that GP2 is important for efficient delivery of theinternalized bacteria to GALT for induction of mucosal
immune responses. Therefore, GP2-targeting should
be an ideal vaccination strategy, since this could enable efficient delivery of vaccine antigens to GALT for
mucosal immune responses, even if the vaccines are
killed or attenuated viruses unable to propagate in
the body. With support from the RIKEN Program for
Drug Discovery and Medical Technology Platforms,
they are now trying to develop a novel GP2-targetedmucosal vaccine delivery system.

Schematic overview of the GP2-targeted vaccine delivery system

29Host gastrointestinal immune response
upon exposure to probiotic bacteria
Intestinal Microbe Symbiosis Laboratory,Integrated Collaborative Research Program with Industry,RIKEN Innovation CenterM

any commercial probiotic bacterial strains havebeen developed to help manage a number ofhealth issues such as gastrointestinal ( GI ) disorders,
inflammatory bowel diseases, allergies, and pathogenic infections. Among these probiotic strains, Lactobacillus acidophilus L-92, with anti-allergic effects forpollen allergy, perennial allergy, atopic dermatitis, andthe ability to control GI disorders has been developed
by CALPIS Co．Ltd. To understand the mechanismsof the beneficial effects of L-92, the Intestinal Microbe
Symbiosis Laboratory has been established under the
Integrated Collaborative Research Program with Industry of RIKEN in collaboration with CALPIS and theLaboratory for Epithelial Immunobiology.
The health benefits from L-92, especially immunomodulatory effects, are thought to result from interactions between L-92 and the host mucosal immune
system in the GI tract ( Fig.) . They hypothesized that

Figure:30

these health benefits are most likely initiated with theassociation between the L-92 and host intestinal epithelium, and are focusing on the immune response of
the epithelium.
To understand host GI epithelial immune responses upon exposure to L-92, microarray analysis
was performed by using the well-established humanadenocarcinoma epithelial Caco-2 cell line. Genes
classified as cell adhesion, MAP kinase pathway,transmembrane proteins, and immune related pathway
were up-regulated after 20 h of treatment, whereas
no significantly down-regulated genes were observed.
Information obtained in the present study could be an
important step for consideration of the impact of L-92
on the mucosal immune system in the GI tract and on
host health ( Yanagihara, S. et al., J. Med. Food 15(6):511-519, 2012 ) .

The mucosal immune system in the GI tract forms the largest part of the entire immune system,
where the health benefits from L-92, especially immunomodulatory effects, are induced.

2012Part 3

Nurturing Young Scientists

Young Chief Investigator Program
RIKEN

R

CAI launched the Young Chief Investigator
Other Center
RCAI
Program in 2010 to provide a career path foryoung investigators who conduct multidisciplinary
Host Lab.
1 mentor
research that will bridge immunology with other
Space &
equipment
research fields. In this program, the selected
Other Center
Young Chief
Young Chief Investigator ( age below 40 ) will head
2 mentor
Investigater (YCI)Lab
an independent research program but will havean access to mentoring by multiple senior speFacilities &4 mentor
experimental
cialists in related research fields, i.e. the leaders insupportvarious RIKEN Institutes. Mentors provide guidCentral
Facility
ance for experimental design, preparation of paUniversity/Institutepers and presentations, promotion of international
3 mentor
visibility, and obtaining research funding. The YCIlaboratory will also share space, equipment andfacilities with a host laboratory in RCAI ( Groups,
Figure:Scheme of the Young Chief Investigator Program
Teams or Units ) ( Fig. ) To consider necessarychanges in the level of RCAI support for YCI, the YCIProgram Committee meets twice a year. The committee
In 2012, four researchers conducted their studies aswill also discuss the relevance and value of the research
Young Chief Investigators, Dr. Hayato Naka-Kaneda ( Stemproject as part of the core research projects at RCAI.
Cell Competency) , Dr. Shinji Nakaoka ( Mathematical ModThere will be an initial 5- year appointment, with theeling of Immune System ) , Dr. Tomokatsu Ikawa (Immune
Regeneration) and Dr. Katsuyuki Shiroguchi ( Integrative
possibility of extending for an additional 2 years after evalGenomics ) . Their mentors and host laboratories are listed
uation by the Director and an internal committee. At thatpoint, a Young Chief Investigator can leave RCAI to take aposition at another institution or be promoted to anothertype of position within RCAI.

in Table.

Table: Young Chief Investigators and their mentors 2012
YCI lab
Shinji Nakaoka
Laboratory for Mathematical
Modeling of Immune System

Mentors
Masahiro Ueda
Kenji Kabashima

Host Lab:
Osamu Ohara, RIKEN RCAI

Osamu Ohara
Masato Kubo

Hayato Kaneda
Laboratory for Stem Cell
Competency
Host Lab:
Ichiro Taniuchi, RIKEN RCAI
Tomokatsu Ikawa
Laboratory for Immune
Regeneration
Host Lab:
Haruhiko Koseki, RIKEN RCAI
Katsuyuki Shiroguchi
Laboratory for Integrative
Genomics
Host Lab:
Osamu Ohara, RIKEN RCAI

32

Haruhiko Koseki
Takashi Nagasawa
Piero Carninci
Toshio Kitamura
Haruhiko Koseki

Affiliation
Group Director, Laboratory for Cell Signaling Dynamics, RIKEN QBiC
Associate Professor, Department of Dermatology, Graduate School of Medicine, Kyoto
University
Group Director, Laboratory for Immunogenomics, RIKEN RCAI
Professor, Research Institute for Biological Science, Tokyo University of Science / Senior
Visiting Scientist, Open Lab. for Signal Network, RIKEN RCAI
National Institutes of Health, USA
Team Leader, Laboratory for Pluripotent Stem Cell Studies, RIKEN CDBTeam Leader, Functional Genomics Technology Team, RIKEN OSCTeam Leader, Laboratory for Lymphocyte Development, RIKEN RCAI / Professor, Institutefor Frontier Medical Sciences, Kyoto University
Group Director, Laboratory for Developmental Genetics, RIKEN RCAI
Professor, Institute for Frontier Medical Sciences, Kyoto University
Team Leader, Functional Genomics Technology Team, RIKEN OSC
Professor, The Institute for Medical Science, The University of Tokyo
Group Director, Laboratory for Developmental Genetics, RIKEN RCAI

Yoshie Harada
Yutaka Suzuki
Yoshio Kodera
Osamu Ohara

Professor, Institute for Integrated Cell Material Sciences, Kyoto University
Associate Professor, Graduate School of Frontier Sciences, The University of Tokyo
Director, Center for Disease Proteomics, Kitasato University
Group Director, Laboratory for Immunogenomics, RIKEN RCAI

Ronald Germain
Hitoshi Niwa
Piero Carninci
Hiroshi Kawamoto

YCI Laboratory for

Stem Cell Competency
Young Chief Investigator :Hayato Kaneda
Research associate :Michiko Ohno-Oishi
Technical staff :Shiho NakamuraAge-related disruption of tissue homeostasis reduces regenerative ability, resulting in age-associatedpathologies and a lower quality of life ( QOL) . Japan has
already become an ageing society and the demographic
trends predict a doubling of the world population over 65
years of age within the next 30 years. However, we know
little about the mechanisms of ageing and even less about
how to counteract it. A tight balance between cellular proliferation and cell death maintains tissue homeostasis. In
the former case, somatic stem cells ( SSCs ) play importantroles by supplying tissue-specific cells over the lifespanof the animal. SSCs are generally defined by multipotency
and the ability to self-renew. However, recent studies havedemonstrated that SSCs themselves undergo ageing,changing their functions with age. SSC dysfunction anddecreased regenerative capacity can cause physiologicaldeficiencies, e.g., inefficient muscle repair, reduced bonemass, neurodegenerative diseases, and dysregulation ofhematopoiesis. Therefore, we predict that the restorationof SSC functions towards those in young healthy individ-

uals would contribute to recovery of tissue homeostasis
and improvements in our health.

Competence regulation of aged stem cells
Although neural stem cells (NSCs ) have the capacity to differentiate into neurons and glial cells, neurogenesis largely
precedes gliogenesis during central nervous system ( CNS )development in vertebrates. The neurogenesis-to-gliogenesis switch requires the temporal identity transition of
NSCs. In a previous study, we found that the “competencechange” is a key molecular mechanism for the temporal
identity transition of NSCs. Inhibition of the competencechange causes loss of gliogenesis and sustained neurogenesis. Remarkably, the competence change does notpromote or repress the differentiation processes directly.
Instead, it controls changes in responsiveness to extrinsic
signals such that NSCs cannot respond to the gliogenic
cytokines and express their gliogenic potential before theyacquire the gliogenic competence.
Recently, we found that the competence regula-

33

Figure:Functional restoration of aged stem cells
Forced competence regulation of aged HSCs and MSCs restored diminished lymphopoiesis andosteogenesis ability, respectively.

tion also has important roles in stem cell ageing. Forced
competence regulation of aged hematopoietic stem cells( HSCs ) or mesenchymal stem cells ( MSCs ) restored diminished lymphopoiesis and osteogenesis ability, respectively (Fig.). We will try to reveal the molecular mechanisms
underlying stem cell ageing in terms of competence regulation.

Screening of systemic anti-ageing factors
derived from competence-regulated MSCs

pacity of muscle stem cells by heterochronic parabiosis
of young and old mice suggests that there is a systemic
ageing-control factor. Several studies have describedpro-ageing factors such as CCL11 and C1q, however, no
critical systemic anti-ageing factors have been identified.
Recently, we found that transplantation of competence-regulated MSCs has anti-ageing effects on the reduction of bone mass and the decline of lymphopoiesis.
These effects seem to be due to systemic factors because
transplanted MSCs engrafted in the lungs.

A recent study about restoration of the regenerative ca-

Recent Publications
1. Sato T., Shimazaki T., Naka H., Fukami S., Satoh Y.,
Okano H., Lax I., Schlessinger J., Gotoh N. FRS2alpharegulates Erk levels to control a self-renewal target Hes1
and proliferation of FGF-responsive neural stem/progenitor cells. Stem Cells 28, 1661-1673 (2010 )34

2. Naka-Kaneda H., Shimazaki T., Okano H.
Neurogenesis to gliogenesis switching.
Experimental Medicine 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 mouse embryonicstem cells. J Neurosci Res. 88, 234-247 (2010)

YCI Laboratory forMathematical Modeling of
Immune System
Young Chief Investigator :Shinji NakaokaT

he skin tissue is a primary line of host defense thatacts as barrier and sensor for invading pathogens.
Atopic dermatitis is a common skin disease which may
occur as a consequence of loss of skin barrier function,
followed by extensive allergic/inflammatory immune responses. Despite extensive basic and clinical research,definitive identification of components and mechanismsthat mediate chronic allergic immune responses in the skintissue remains unsolved. Dysregulated immune responses
in the skin tissue of atopic dermatitis patients are typicallyobserved in heterogeneous spatio-temporal scales. Spatio-temporal multiscaleness and participation of multiple
types of immune cells are key issues to be elucidated for
obtaining a comprehensive view of the progression of
atopic dermatitis.
The main research focus of my laboratory is to construct multi-scale mathematical models for atopic dermatitis, psoriasis vulgaris and other inflammatory diseases.
The entire research plan consists of the following four
essential building blocks: ( i ) Construction of a resource/
information-base for atopic dermatitis, ( ii ) Exploration ofgene expression dynamics of skin and immune cells, ( iii )Quantitative study on immune cell dynamics in the skin tissue, and ( iv) Development of a supportive theory for multiscale mathematical modeling -- in silico representation of
immunological events and rules that govern them.

(i) Integrative knowledgebase for atopic dermatitis
The onset of atopic dermatitis (AD) is diverse because
there are several routes to induce skin barrier dysfunction,for example, impaired protease activity and excessiveoxidative stress. To treat static diversity in the progression of AD, bioinformatics offers useful tools. Recently,
I constructed an integrative knowledgebase for atopic
dermatitis that provides gene-centered information on AD
found in the AD literature and on public databases. The
knowledgebase also provides automatic keyword-basedclassification for the entire AD literature with which we cangenerate hypotheses via associative inferences (Fig. 1).
(ii) Stochastic dynamics of skin inflammation
In order to treat dynamic diversity in the progression ofAD, it is necessary to decompose spatio-temporal multiscale dynamics of atopic dermatitis into characteristic

35

Figure 1: Automatic keyword-based classification for
the entire literature on atopic dermatitis.
Each sub-research field in atopic dermatitis
study is represented and summarized in ahierarchical tree.

Figure 2: Left: A scheme of inflammation spread in theepidermis. Right: stochastic simulation result
for the propagation of inflammation among
keratinocytes via paracrine interactions: thehorizontal axis represents successful probability of a cell to become an active secretor of
inflammatory mediators (0-100%), while thevertical axis represents the fraction of active
cells in a local epidermal tissue (0-100%).
For each fixed successful probability, we
carried out stochastic simulations100 times.
The results are shown as a box-plot. A wideranged box-plot for relatively small successfulprobabilities suggests that stochastic variability might largely affect outcomes (success or
failure of inflammation spread).

spatio-temporal time scales. At the microscopic scale,signal transduction pathways associated with the inflammatory response play an important role in determining the
behavior of cells in the onset of atopic dermatitis. We will
investigate the role of the JAK/STAT signaling pathway toreveal how differences in signal transduction can result indiverse outcomes. At the mesoscopic scale, we need todescribe the process of recruitment of immune cells into
the epidermis by sensing chemokines released from cellsof the epidermis. At the macroscopic scale, it is necessary to describe the destruction process of the epidermis
and the invasion of various substances that would initiate
inflammatory responses. We will construct a tissue-level mathematical model to describe macroscopic barrier
function of the epidermis against air-liquid substance or al-

lergen invasion. Epidermal permeability will be also investigated with the tissue-level model to link clinically available
measurements, such as trans-epidermal water loss, withthe degree of barrier integrity. I have recently constructeda mesoscopic-level mathematical model and a performed
simulation study to link intracellular molecular dynamics
with intercellular communication among keratinocytes via
release and receipt of pro-inflammatory cytokines. Preliminary stochastic simulation results suggest that local propagation of the inflammatory response can be determinedby stochastic variability, which might endow an on/off
switch-type information processing machinery within theepidermis to flexibly control the initiation of inflammation(Fig. 2).

Recent Publications
1. Katsuyama C., Nakaoka S., Takeuchi Y., Tago K., Hayatsu M., Kato K., Complementary cooperation between
two syntrophic bacteria in pesticide degradation, J Theor Biol. 256 644-654 (2009 )2. Iwami S., Nakaoka S., Takeuchi Y., Miura Y., MiuraT., Immune impairment thresholds in HIV infection,
Immunol Lett. 123, 149-154 (2009 )36

3. Diekmann O., Gyllenberg M., Metz J.A.J., Nakaoka S.
and de Roos A., Daphnia revisited: local stability and bifurcation theory for physiologically structured population
models explained by way of an example, J Math Biol.
61, 277-318 (2010 )4. Nakaoka S., Aihara K., Mathematical study on kineticsof hematopoietic stem cells -- theoretical conditions for

successful transplantation, J Biol Dyn. 6, 836-854(2011)
5. Nakaoka S., Aihara K., Stochastic simulation of structured skin cell population dynamics, J Math Biol. 66,
807-835 (2013)

YCI Laboratory for

Integrative Genomics
Young Chief Investigator :Katsuyuki ShirogouchiIcame to Yokohama from Boston last summer and joined
this center, RCAI, as a young chief investigator ( YCI ) . I
think my mission here is to expand the research area of
RCAI by creating my own research direction. Since I settled in, I have been thinking of what and how I can generatea new research field, and also have been discussing withmany new colleagues in order to understand what RCAIis, so that I can start unique projects related to this center.
One possible way is to study immunology and related issues, e.g. diseases, based on my own experience and myoriginal techniques. Below, I will introduce my researchbackground and current ideas toward my new research
direction here.
I had been working on a motor protein, myosin V.
Myosin V is a linear molecular motor which moves unidirectionally along actin filaments using energy obtained in
the ATP hydrolysis reaction. Myosin V apparently has two“feet” (each has an actin binding site and an ATP bindingsite ), and is supposed to “walk” like a human. One of theoutstanding questions was how the lifted ( rear) foot canbind a forward site on the actin filament. To answer thisquestion, first, I developed a rod-shape probe and a microscopic observation system. Then, I attached the probe
to a leg of myosin V and observed under an optical microscope the change in the angle of the probe when a single
myosin V molecule was walking along the actin filament.
Using this biophysical approach, I elucidated walking
mechanism of myosin V ( Shiroguchi and Kinosita, Science
2007 ) .
When I joined a research group at Harvard University
in Boston, I dramatically changed my research filed, frombiophysics to genomics. I have developed “Digital RNA
Sequencing” using a next generation sequencer for gene
expression profiling. This technique allows one to countnucleic acid molecules accurately with single molecule
resolution genome-wide, which opens a window of oppor-

37

New techniqueforSingle cells
System-wide analysis

Distribution

Figure:Biological system
Medical Science

tunity to define a cell state with single cell transcriptome
analysis.
Thus, I have been developing techniques to “see”
something unknown. Here at RCAI, based on my experience and skills, I am going to keep developing newtechniques in order to understand biological systems andto contribute to medical science and our understanding of
diseases. Especially, I am interested in visualizing the distribution of cell states by accurate system-wide measure-

My approachA new technique for single cell and/or system wide
analysis may provide a new angle to visualize a new
distribution of cell states that is related to biological systems and diseases.

ments with single molecule and/or single cell resolution(Fig.) I believe that this approach may provide significant
insights in these research fields since heterogeneity ofcell populations may be one of the key factors in medical
science and diseases. For example, at the beginning of adisease, homeostasis starts to be destroyed at the single
cell level. I have just started some projects based on thisapproach. I hope I can show some interesting results nexttime.

Recent Publications
1. Shiroguchi K., Jia T. Z., Sims P. A., Xie X. S. Digital RNA
Sequencing Minimizes Sequence-Dependent Bias andAmplification Noise with Optimized Single Molecule Barcodes Proc Natl Acad Sci USA. 109, 1347-1352 (2012 )38

2. Shiroguchi K.*, Chin H. F., Hannemann D. E., MuneyukiE., De La Cruz E. M.*, Kinosita K. Jr. Direct Observationof the Myosin Va Recovery Stroke That Contributes
to Unidirectional Stepping along Actin” PLoS Biol. 9:e1001031 (2011) (* corresponding author)

3. Kohori A., Chiwata R., Hossain M.D., Furuike S., Shiroguchi K., Adachi K., Yoshida M., Kinosita K. Jr. Torque
Generation in F 1-ATPase Devoid of the Entire Amino-Terminal Helix of the Rotor That Fills Half of the Stator Orifice Biophysical J. 101, 188-195 (2011)

YCI Laboratory for

Immune Regeneration
Young Chief Investigator :Tomokatsu Ikawa
Technical Staff :Asako Shibano-SatohH

ematopoietic stem cells ( HSCs ) give rise to various
cell types in hematopoietic and immune systems,
including erythrocytes, megakaryocytes, myeloid cells(macrophages and granulocytes ) and lymphocytes. HSCs
and their progenitors exhibit multiple patterns of gene
expression during differentiation, and the developmentof hematopoietic cells is regulated by a network of transcription factors. Although many essential transcription
factors, such as PU.1, Ikaros, C/EBPs, GATA1-3, E2A,
EBF1 and Pax5 have been implicated in regulating the cellfate choice of each hematopoietic cell lineage, molecularmechanisms underlying the generation of these patterns
during cell fate determination remain unexplored becauseof an absence of suitable experimental systems.
We have recently established an ideal system thatcan examine gene regulatory networks during lymphoidlineage specification from HSCs. This novel system enabled the analysis of a large set of regulatory moleculesthat control the generation of T and B lymphocytes.
It can also be applied for ex vivo expansion of humanhematopoietic stem/progenitors, which will be requiredfor immune cell therapy or transplantation of HSCs.
Thus, the aims of our study are 1) from a basic science
perspective, to elucidate the mechanisms that orchestrate cell fate specification, commitment and differentiation during lymphocyte development and 2 ) from aclinical medicine perspective, to establish a novel method

to expand human hematopoietic stem/progenitors for thedevelopment of HSC transplantation as a clinical strategy.

Generation of induced Leukocyte Stem ( iLS ) cells
E2A is a basic helix-loop-helix transcription factor essential for B cell development. We have previously shown that
B cell development in the bone marrow of E2A-deficient
mice is blocked at the myelo-lymphoid progenitor stage,
and that such progenitor cells have a multi-differentiation
potential both in vitro and in vivo ( Ikawa et al. Immunity, 2004 ) . These results indicate that (1) the progenitors
whose differentiation potential is blocked at the myelo-lymphoid progenitor stage display self-renewal activity,( 2 ) these progenitors are able to proliferate indefinitely inpro B cell culture conditions. Therefore, we hypothesizedthat myelo-lymphoid progenitors can be expanded bysuppressing E2A activity in multipotent progenitors thatare in the process of differentiating into B lineage cells.
We overexpressed Id3, one of the Id proteins thatare natural inhibitors of E2A, in normal murine HSCs,
and cultured the transduced cells on TSt-4 stromalcells in the presence of added cytokines ( SCF, IL-7 andFlt3-L ) . As a result, these cells expanded extensively
without losing their multilineage differentiation potential.
They were able to generate T, B, and myeloid lineage
cells for several months, upon transfer into irradiatedimmunodeficient ( Rag1-/- ) mice. We named such induced

39

Figure 1: Induced Leukocyte Stem (iLS) cells
We have established multipotent progenitors that haveself-renewal activity and multipotency. This was done byoverexpressing Id3 in hematopoietic stem/progenitor cells
and culturing the cells under B cell differentiating conditions. Id3 suppresses the E2A activity and the multipotent
progenitors acquire self-renewal activity. The cells extensively
proliferate for at least several months, still maintaining theirmultipotency.

Figure 2: Inducible culture system to examine transcriptional networks in
B cell lineage specification
Hematopoietic stem/progenitor cells were transduced with the
Id3-Estrogen receptor (ERT2) retrovirus. The cells were cultured in
the presence or absence of 4-Hydroxytamoxifen (4-OHT). 4-OHT
blocks B cell differentiation, whereas following its removal, B cell
generation ensues. Large numbers of CD19+ B cells were generated
within 7 days.

multipotent cells as iLS ( induced leukocyte stem ) cells(patent applied for, manuscript in preparation; Fig. 1) .
These unique cells provide an ideal system that can be induced to proliferate or differentiate at will.
The advantages of the iLS cells are ( i ) iLS cells arehomogenous, ( ii ) iLS cells are normal and not neoplastic,( iii ) time course analysis of T, B and myeloid lineage celldifferentiation can be performed under appropriate culture conditions, ( iv ) a large number of cells ( >1x10 6 ) areavailable, allowing us to do genome-wide assays suchas transciptome or epigenetic (DNA methylation and histone modification) analyses. Therefore, this system hastremendous potential to answer basic developmental biology questions, especially about the mechanisms of cellfate determination. We are currently translating this novel
method for the expansion of human leukocyte progenitorsthat later can also be applied in clinical application such as
immune cell therapy and reconstitution of hematopoietic
cells in immunocompromised patients.

Establishment of an inducible system that canclarify the transcriptional network in
B-lymphoid lineage specification
We have recenlty succeeded in modifying the iLS cells byusing an Id3-ERT2 ( Estrogen receptor) retroviral constructwhose expression can be induced by 4-Hydroxytamoxifen(4-OHT ). Mouse HSCs were transduced with the Id3-ERT2
retrovirus and cultured on TSt-4 stromal cells in the presence of 4-OHT. One month later, a homogenous population of iLS cells was obtained. The iLS cells kept proliferating and could be stably maintained by continuous additionof tamoxifen. However, once tamoxifen was removed, they
began differentiating into B cells under B cell generating
culture conditions ( Fig. 2 ) . They easily give rise to CD19 +cells, and the B lineage commitment can be induced within 7 days. Using this inducible system, we will attempt toelucidate the transcriptional network regulating B cell fatespecification.

Recent Publications

40

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

3. Seo W., Ikawa T., Kawamoto H., Taniuchi I. Runx1-CBPβ facilitates early B lymphocyte development byregulating expression of Ebf1 gene. J Exp Med. 209,
1255-1262 (2012)

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

4. Isoda T., Takagi M., Piao J., Nakagama S., Sato M.,
Masuda K., Ikawa T., Azuma M., Morio T., Kawamoto
H., Mizutani S. Process for Immune defect and chromosomal translocation during early thymocyte development
lacking ATM. Blood 120, 789-799 (2012)

5. Sakamoto S., Wakae K., Anzai Y., Murai K., TamakiN., Miyazaki M., Miyazaki K., Romanow WJ., Ikawa T.,
Kitamura D., Yanagihara I., Minato N., Murre C., Agata Y.
E2A and CBP/p300 act in synergy to promote chromatin
accessibility of the immunoglobulin κ locus. J Immunol.
188, 5574-5560 (2012)

RCAI International
Summer Program 2012T

he 2012 RCAI International Summer Program ( RISP )was held on June 22-29. This was the seventh annual RISP and was co-sponsored by the Global Centerof Excellence at Chiba University. It was a special event,since RISP 2011 had to be canceled because of theGreat East Japan Earthquake and tsunami on March 11,2011 and subsequent nuclear accident at Fukushima
Nuclear Power Plant. Although repercussions of this disaster are still being felt, the situation in Yokohama wasstable and the RCAI leadership was pleased to resume
the program, which is one of the major annual eventsat the Center. Forty one young scientists from nineteen
countries participated in RISP 2012; approximately 80%
were graduate students and the remainder postdoctoral
fellows. The first part of the program ( June 22-27 ) was
held in Yokohama at RCAI. There were morning oralpresentations by the participants and these were supplemented by two early evening poster sessions, where
there was time for more detailed discussions of the data.
The afternoon sessions featured lectures by prominentscientists invited from RCAI, Japanese universities, andabroad. On June 28, the RISP participants joined theannual RCAI-JSI International Symposium on Immunol-ogy at the Pacifico Yokohama Conference Center. This
year’s theme was “New Horizon in Immune Regulation –Bridging Innate and Acquired Immunity.” Some of the RISP
lecturers also spoke at this venue, but on new topics andthere were also many new speakers, making for an exciting two day meeting. Most of the participants returned
home after the close of the meeting, but six participantsremained at RCAI for a month-long research internship.
The RISP lectures at RCAI were quite diverse. Topics
included lymphocyte development and differentiation, thefunction of microRNAs in these processes, transcriptional
regulatory networks, immunoreceptor signaling, mucosal immunity, and the inflamasome.
B e c a u s e o f t h e i r b r e a d t h,these lectures provided an
expansive overview of the immune system, with experimental approaches ranging fromsophisticated imaging in cancer and in the germinal center
reaction, to whole animal and“humanized” mouse analyses.

41

The invited speakers incorporated introductory material aswell as recent highlights from their own research into theirtalks. The research interests of the participants were similarly varied, making for a unique opportunity for cross-fertilization among immunology subdisciplines during the oral
and poster presentations and in more informal settings.
The question periods following talks by the invited lecturers as well as the participants were spirited and stimulating. Awards for best RISP posters were presented at afarewell party held at the close of the RCAI-JSI meeting.
All of the participants expressed their appreciation to theorganizers for inviting them to RISP, and some of themestablished collaborations as a result of the RISP that arelikely to be long-lasting.
The RISP 2012 participants had a full schedule, but

had some opportunities to explore the Yokohama / Tokyo
area and many of them also visited nearby Kamakura.
RISP provided an exceptional experience from both scientific and cultural perspectives. In a survey completed
after the school, the participants unanimously agreed thatthey would recommend the program to colleagues. Halfindicated that they would consider a postdoctoral position
at RIKEN and nearly all would consider returning to RCAI
for a short-term period of collaborative research. The success of this unique program was due to the efforts of theOrganizing Committee, chaired by Dr. Kurosaki, and the
RISP Secretariat, Mr. Fukushima, Mss. Haraguchi, Iyama,
Nomura and Yoshioka, who kept the entire operation running smoothly, as well as to the efforts of the outstanding
participants. Planning is already underway for RISP 2013.

Table: :Lectures
Toshinori Nakayama, Chiba University, Japan

Protective and pathogenic function of memory Th2 cells

Ichiro Taniuchi, RIKEN RCAI, Japan

Transcriptional regulation of thymocyte development

Hilde Cheroutre, La Jolla Institute for Allergy & Immunology, USA and
RIKEN RCAI, Japan

Mucosal T cells: Shaped by the challenge

Rudolf Grosschedl, Max Planck Institute of Immunobiology andTranscription networks regulating B lymphopoiesis

Thomas Boehm, Max Planck Institute of Immunobiology and

Design principles of adaptive immune systems

Gary A. Koretzky, University of Pennsylvania, School of Medicine, USA

Integration of immunoreceptor signaling pathways by adaptor proteins

Hisataka Kobayashi, National Cancer Institute, NIH, USA

Molecular cancer imaging; New optical diagnostic technologies andbeyondTakaharu Okada, RIKEN RCAI, Japan

Imaging of lymphocyte dynamics during the germinal center formation

Axel Kallies, Walter and Eliza Hall Institute of Medical Research,

Australia

Transcriptional control of conventional and regulatory effector T celldifferentiation

K. Mark Ansel, University of California San Francisco, USA

MicroRNA regulation in helper T cell differentiation

Matthias von Herrath, La Jolla Institute for Allergy and Immunology,

USA

Peripheral insulin recognition determines pathogenic versus Foxp3+
regulatory CD4 T cell formation and susceptibility to type 1 diabetes

Shigeo Koyasu, Keio University School of Medicine and RIKEN RCAI,

Role of natural helper cells in helminth infection

Kiyoshi Takeda, Osaka University, Graduate School of Medicine, Japan

Regulation of gut homeostasis by innate immunity

Jenny Ting, University of North Carolina at Chapel Hill, USA

NLRs: Basic science and disease relevance

Fumihiko Ishikawa, RIKEN RCAI, Japan

Humanized mouse: a research tool to investigate normal & diseased
human immunity

Epigenetics, Germany

Epigenetics, Germany

Japan

42

7th RCAI-JSI International
Symposium on Immunology
New Horizon in Immune Regulation–Bridging innate and acquired immunity–T

he seventh RCAI-JSI International Symposium on
Immunology was held on Jun 28-29, 2012, at Pacifico
Yokohama Conference Center. Three hundred and ninety
people, including 57 from abroad, gathered to learn about
the most advanced topics in immunology and to discuss
their results.
This year’s symposium focused on immune regulation
bridging innate and acquired immunity. Twenty one invitedresearchers introduced their cutting edge research. The
first session ‘Novel innate cell subsets’ started with a hot
discussion on the recently discovered ‘natural helper ( NH )cells’ and ‘nuocytes’, which play critical roles in Th2 immune responses in innate immunity. Shigeo Koyasu (Photo1) introduced GATA-3 as a master regulator for differentiation of NH cells, and then Andrew McKenzie (Photo 2) explained that transcription factor RORα plays an importantrole in nuocyte development. In the ‘Innate cell function’session, David Mosser (Photo 3) explained the characteristics of so-called ‘regulatory macrophages’ that produce
the anti-inflammatory cytokine IL-10. In the ‘Recognition
by innate sensor’ session, Tadatsugu Taniguchi (Photo 4)
beautifully summarized ‘classical’ secreted cytokines and‘new’ cytokines that are present in distinct cell compartments. Alexander Rudensky (Photo 5) discussed the biological functions of thymus Tregs ( tTregs ) and peripheral
Tregs ( pTregs ) in the final session ‘Lymphocyte subsets

and effecter function’.
The RCAI-JSI International
Symposium on Immunology isa series of annual conferences
star ted in 2005 and hosted
by RIKEN RCAI in conjunction
with the Japanese Society for
Immunology ( JSI ) . The symposium provides a forum foractive discussions on the cutting edge of immunological research. The steering committee is composed of representatives of RCAI and JSI, andorganizes the program with advice from an international
advisory committee. The RCAI-JSI international symposium is complementary to JSI International Symposia thatare held during its annual meeting. These meetings promote the progress of immunology and encourage young
researchers not only in Japan but also in Asia and all over
the world.
Direct interaction among people is one of the most effective ways to spread knowledge and inspire researchers
to explore new directions. In this rapidly advancing era ofscience, the symposium connected researchers and provided an opportunity to consider both newly solved andyet unsolved problems in immunological science.

43

Table: Program

Session I: Novel innate cell subsets
Shigeo Koyasu, Keio University / RIKEN RCAI, Japan

Critical role of GATA3 in natural helper cell differentiation and function

Andrew N.J. McKenzie, MRC Laboratory of Molecular Biology, UK

RORα and Notch play important roles in type-2 ILC development

Dmitry I. Gabrilovich, H. Lee Moffitt Cancer Center & Research Institute,

Myeloid-derived suppressor cells in regulation of immune responses

USA

Session II: Innate cell function
Foo Y. Liew, University of Glasgow, UK

The role of Interleukin-33 in infection and inflammation

David M. Mosser, University of Maryland, USA

Macrophage heterogeneity and inflammatory diseases

Joseph C. Sun, Memorial Sloan-Kettering Cancer Center, USA

The NK cell response against viral infection: Crossing the boundaries ofinnate immunity

James P. Di Santo, Institut Pasteur, France

IL-22-producing innate lymphoid cells (ILC22): Regulators of immune
defense and tissue homeostasis

Mitchell Kronenberg, La Jolla Institute for Allergy & Immunology, USA

Recognition and responses to microbial antigens by NKT cells

Session III: Recognition by innate sensor
Jenny P.Y. Ting, The University of North Carolina, USA

NLRs in infection, inflammation and cancer

Eicke Latz, University of Bonn, Germany / University of Massachusetts, USA

Inflammasome activation in chronic inflammatory diseases

Sho Yamasaki, Kyushu University, Japan

Immune responses through C-type lectin receptors

Tadatsugu Taniguchi, The University of Tokyo, Japan

Regulation of innate and adaptive immune responses by old and new
cytokines

Session IV: Interface between innate and acquired immunity
William R. Heath, The University of Melbourne, Australia

Immunity to viral infection of the skinYong-Jun Liu, Baylor Research Institute, USA

Biochemical characterization of nucleic acid sensors in dendritic cells

Hiroshi Ohno, RIKEN RCAI, Japan

Function and differentiation of M cells, a unique subset of intestinal
epithelial cells specialized for mucosal antigen-uptake

Shin-ichiro Fujii, RIKEN RCAI, JapanNKT cell-mediated licensing of dendritic cells (DCs) in vivo

Ken J. Ishii, National Institute of Biomedical Innovation, Japan

Making immune sense of nucleic acids in inflammation and vaccination

Session V: Lymphocyte subsets and effecter function
Alexander Y. Rudensky, Memorial Sloan-Kettering Cancer Center, USA

Biological roles of extrathymic differentiation of regulatory T cells

Claudia Mauri, University College London, UK

Regulatory B cells maintain regulatory T cells whilst inhibiting Th17 and Th1
differentiation.

Masato Kubo, Tokyo University of Science / RIKEN RCAI, Japan

Regulation of cytokine expression in follicular helper T cell and humoral
immunityDavid Artis, University of Pennsylvania, USA

Mechanisms of immune regulation at barrier surfaces

01

44

02

03

04

05

RIKEN Joint Graduate School Program
International Program Associate

R

CAI accepted seven international students as RIKEN International Program Associates (IPA). Under the 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.
The IPA students who studied at RCAI in 2012 were :

01

02

05

03

06

04

07

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

45

RIKEN Special Postdoctoral Researcher (SPDR ) Program

R

IKEN’s program for Special Postdoctoral Researchers was instituted to provide young and creative scientists theopportunity 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, 5 postdocs conducted their research at RCAI through the SPDR program.

01

02

04

03

05

Hirokazu Tanaka ( Laboratory for Transcriptional Regulation ) Photo 1
Yuki Horisawa-Takada (Laboratory for Developmental Genetics ) Photo 2
Shimpei Kawamoto ( Laboratory for Mucosal Immunity) Photo 3
Saya Moriyama ( Research Unit for Immunodynamics ) Photo 4
Shinsuke Ito ( Laboratory for Developmental Genetics ) Photo 5

RIKEN Foreign Postdoctoral Researcher

T

he RIKEN Foreign Postdoctoral Researcher ( FPR ) program offers
aspiring young foreign researchers with creative ideas and whoshow promise of becoming internationally active in the future the opportunity to pursue innovative research at RIKEN under the directionof a RIKEN laboratory head. The FPR program is one of RIKEN’s initiatives to open up its facilities and resources to the forefront of global
science and technology.
In 2012, Jafar Sharif ( Photo ) studied in the Laboratory for Developmental Genetics as a RIKEN FPR.

46

RIKEN Junior Research Associate (JRA) ProgramT

he Junior Research Associate program was launched in 1996 to encourage young scientists with fresh ideas andyouthful enthusiasm to collaborate with, and learn from, senior scientists with years of experience. This programprovides part-time positions at RIKEN for young researchers enrolled in university Ph.D. programs. The JRA program
serves the dual purpose of fostering the development of these young scientists while also energizing RIKEN with theirinnovative thinking.
This year, 16 JRA students studied in RCAI.

01

02

03

04

05

06

07

08

09

10

11

12

13

14

15

16

Norihiko Inoue ( Lab. for Cellular Systems Modeling ) Photo 1

Shinsuke Takagi ( Lab. for Human Disease Model ) Photo 9

Chie Kano ( Lab. for Immune Diversity ) Photo 2

Masanaka Sugiyama ( Research Unit for Inflammatory Regulation) Photo 10

Nanako Shimura ( Lab. for Immunogenomics ) Photo 3

Rikiya Ishikawa ( Lab. for Infectious Immunity ) Photo 11

Misao Hanazato ( Lab. for Epithelial Immunobiology ) Photo 4

Yusuke Sato ( Research Unit for Cellular Immunotherapy ) Photo 12

Yuki Obata ( Lab. for Epithelial Immunobiology ) Photo 5

Hisashi Wada ( Lab. for Transcriptional Regulation ) Photo 13

Akemi Fujiwara ( Lab. for Epithelial Immunobiology ) Photo 6

Takashi Ikeno ( Research Unit for Immunodynamics ) Photo 14

Yuuhou Najima ( Lab. for Human Disease Model ) Photo 7

Satoshi Koga ( Lab. for Immune Cell System ) Photo 15

Yuki Aoki ( Lab. for Human Disease Model ) Photo 8

Ryuichi Murakami ( Research Unit for Immune Homeostasis ) Photo 16

47

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 23 adjunct professors/associate professors in RCAI. Fifty-three students studied at RCAI in 2012.

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 )Toshiyuki Fukada ( Visiting Associate Professor )Graduate School of Medicine,
Chiba University

Takashi Saito ( Visiting 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

Osamu Ohara ( Visiting Professor )School of Biomedical Science,
Tokyo Medical and Dental University

Takashi Saito ( Visiting Professor )International Graduate School of Arts and Sciences,
Yokohama City University

Hiroshi Ohno ( Visiting Professor )Haruhiko Koseki ( 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 )48

Graduate School of Frontier Sciences,
The University of Tokyo

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 )2012Part 4

Collaborative Networks

Open Laboratory at RCAIR

CAI’s Open Laboratory was established in 2009 to provide a framework for researchers
from external institutes, universities or hospitals that would allow them access to theCenter’s resources and the opportunity for collaborative research projects.
In 2011, two researchers, Drs. Masato Kubo (Open Laboratory for Signal Network ) and Koji Hase
( Open Laboratory for Bioenvironmental Epigenetics ), had their own space and staff in RCAI andhave joined various internal research meetings and discussions to promote extensive researchcommunication with RCAI investigators.

Open Laboratory for

Signal Network
Leader:Masato Kubo

( Research Institute for Biomedical Science, Tokyo University of Science )Research Scientist: Kosuke Miyauchi
Visiting Scientist:

Yasutaka Motomura

Technical staff:

Yoshie SuzukiTcells play a central role in the effector and regulatory
functions of immunological surveillance and aberrations in these functions can lead to various immunologicaldisorders. The different subsets of helper T cell secrete
distinct cytokines that define their role in controlling theoutcome of immunological surveillance. Type 1 helper
1 ( TH1) cells secrete IFN-γ and TNF-α during the cellularimmune response to intracellular pathogens. TH2 cells produce interleukin-4 ( IL-4 ) , IL-5, IL-6, IL-10 and IL-13, which
account for protective against extracellular pathogens andallergic immune responses. TH17 cells produce IL-17, whichis associated with autoimmune responses. All the helper
T cell subsets differentiate from a common precursor cell,the naive T cell, and these differentiation programs occur
in milieus of certain types of cytokines. IL-4 was originallyidentified as a T H2 cytokine responsible for class switch
recombination to IgG1 and IgE, and recent studies suggest that follicular helper T cells ( TFH ) cells may be an alternative IL-4 source to regulate humoral immune responses.
However, the regulation of cytokine gene expression in TFH
cells remains a mystery because of the limited availabilityof in vivo tracking systems to follow cell specific cytokine
expression. The overriding goal of our laboratory is to understand the molecular mechanisms underlying cytokine
gene expression in helper T cell subsets and innate type
immune cells.

50

The 3’ enhancer CNS2 is a crucial regulator infollicular helper T cells of interleukin-4 mediatedhumoral immunity
B cells are the antibody factories that selectively target
foreign threats as a component of the humoral immune
response. This process requires T cells to secrete IL-4,
which promotes a mechanism called ‘class switching’ thatenables production of functionally specialized antibody
subtypes. However, it remains unclear exactly which T
cells generate the signal required for the class switching.
Nevertheless, most textbooks say that TH 2 cells control
the antibody response. We provided strong evidence thata recently-discovered class of TFH cells generates the IL-4
signal that facilitates B cell help in the antibody responses.
TFH are a specialized T cell subset localizing in germinal
centers ( GCs ) and there secreting cytokines, IL-4 and IL21. We demonstrated that conserved noncoding sequence
2 ( CNS2 ) is an essential enhancer element for IL-4 expression in TFH cells but not in TH2 cells. Mice with a CNS2
deletion had a reduction in IgG1 and IgE production andin IL-4 expression by TFH cells. Tracking of CNS2 activity
using a GFP reporter mouse demonstrated that CNS2-active cells expressed several markers of TFH cells: CXCR5,
PD-1, and ICOS; the transcriptional master regulator Bcl6;
the cytokines IL-21 and IL-4, and were mainly localized in
B cell follicles and germinal centers. The GFP+ TFH cells
were derived from GFP - naïve T cells after in vivo system-

ic immunization. These results indicate that CNS2 is an
essential enhancer element required for IL-4 expression
in TFH cells that control humoral immunity. Moreover, they
have proven that IL-4 is a critical cytokine for controlling
IgG1 and IgE antibody responses, although its expressionin TH2 and TFH cells is independently regulated by distinct
regulatory elements. Furthermore, TFH and not TH2 cells are
the T cell subset responsible for TH2-type humoral immune
responses.

Dysregulation of suppressor of cytokine signaling 3( SOCS3 ) in keratinocytes caused skin inflammationinduced by pathogenic IL-20 receptor-related
cytokines
Homeostatic regulation of epidermal keratinocytes iscontrolled by the local cytokine milieu. However, a role ofa negative feedback regulator to control the cytokine networks, suppressor of cytokine signaling ( SOCS ) , remains

unclear in skin homeostasis. Keratinocyte specific deletionof Socs3 ( Socs3 cKO ) resulted in severe skin inflammation
with IgE hyper-production, epidermal hyperplasia, and
S100A8/9 expression, although Socs1 deletion caused nosuch inflammation. The inflamed skin showed constitutive
STAT3 activation and up-regulation of IL-6 and IL-20 receptor I ( IL-20RI ) related cytokines, IL-19 and IL-24. Diseasedevelopment was rescued by deletion of the Il6 gene,but not by deletion of the Il23, Il4r, or Rag1 genes. The
expression of IL-6 in Socs3 cKO keratinocytes increased
IL-19 and IL-24 expression, which further facilitated STAT3
hyperactivation, epidermal hyperplasia and neutrophilia.
These results demonstrate that skin homeostasis is strictly
regulated by the IL-6-STAT3-SOCS3 axis. Moreover, theSOCS3 mediated negative feedback loop in keratinocyte
has a critical mechanistic role in the prevention of skin inflammation caused by hyperactivation of STAT3.

Recent Publications
1. Harada, Y., Tanaka, S., Motomura, Y., Harada, Y., Ohno,
S., Ohno, S., Yanagi, Y., Inoue, H., and Kubo, M. The 3 Enhancer CNS2 is a Critical Regulator of Interleukin-4-Mediated Humoral Immunity in Follicular Helper T Cells. Immunity
36, 88-200 (2012)
2. Sawaguchi, M., Tanaka, S., Nakatani, Y., Harada, Y., Mukai,K., Matsunaga, Y., Ishiwata, K., Oboki, K., Kambayashi, T.,
Watanabe, N., Karasuyama, H., Nakae, S., Inoue, H., andKubo, M. Role of mast cells and basophils in IgE responses
and in allergic airway hyperresponsiveness. J Immunol.
188, 1809-1818 (2012)
3. Hill, D.A., Siracusa, M.C., Abt, M.C., Kim, B.S., Kobuley,D., Kubo, M., Kambayashi, T., Larosa, D.F., Renner, E.D.,
Orange, J.S., Bushman, F.D., and Artis D. : Commensal
bacterial–derived signals regulate basophil hematopoiesis
and allergic inflammation. Nat Med. 18, 538-546 (2012)
4. Kurashima, Y., Amiya, T., Nochi, T., Fujisawa, K., Haraguchi, T., Iba, H., Tsutsui, H., Sato, S., Nakajima, S., Iijima,
H., Kubo, M., Kunisawa, J., and Kiyono, H. : Extracellular
ATP mediates mast cell–dependent intestinal inflammation
through P2X7 purinoceptors. Nat Comm. 3, 1034 (2012)

Figure:Localization of IL-4 expressing TFH cells in the Peyer’s
patch. Green indicates GFP, a marker for IL-4 expression,
blue indicates CD4 T cells, and red indicates GL-7 expressing
GC-B cells.

5. Uto-Konomi, A., Miyauchi, K., Ozaki, N., Motomura, Y.,
Suzuki, Y., Yoshimura, A., Suzuki, S., Cua, D., and Kubo,
M. Dysregulation of suppressor of cytokine signaling 3 in
keratinocytes causes skin inflammation mediated by interleukin-20 receptor-related cytokines. PLoS One 7, e40343(2012)

51

Open Laboratory at RCAIOpen Laboratory for

Bioenvironmental
Epigenetics
Leader:Koji Hase
Visiting Scientist: Yukihiro Furusawa
Technical Staff:

Yumiko Fujimura

Student Trainee: Yuuki ObataE

pigenetics is a mechanism that imposes a specificand heritable pattern of gene expression on the progeny of differentiating cells without affecting the nucleotide
sequence of the DNA. The major epigenetic mechanisms
include DNA methylation, chemical modifications of histone tails and changes in higher order chromatin organization. Emerging evidence indicates the importance of
epigenetic events in the development and proper functionsof the immune system. Furthermore, it is assumed thattranscriptional regulation via the epigenetic machinery may
be influenced by environmental factors such as microbial
products and cytokines. However, identification of suchenvironmental factors and their impact on immunologicalhomeostasis remain elusive goals.
Humans harbor over 100 trillion bacteria in the distal
intestine. These commensal bacteria have long been appreciated for the benefits they provide to the host, including their capacity to metabolize otherwise indigestible foodingredients to small metabolites that can be utilized as nutrients by host cells. Moreover, it seems that the presenceof commensal bacteria eventually contributes to shape thegut immune system though promoting the developmentof gut-associated lymphoid tissues, the largest of the secondary lymphoid organs, which are necessary for induction of mucosal IgA responses. For instance, colonizationof germ-free mice with gut-indigenous Clostridia restored
colonic regulatory T cells ( Treg ), which play a pivotal role inhost tolerance to commensal bacteria, to normal numbers.
In further support of this concept, the development of Treg
cells as well as IgA production are defective in germ-freemice. These previous observations led to the notion that52

host-microbe interactions establish immunological homeostasis in the gut, which further raises the important question of how commensal bacteria affect the host immune
system.

Microbial factor (s) responsible for development of
the gut immune system
The enteric microflora serve as a potent bioreactor that
controls several metabolic functions. The main functions
include the fermentation of indigestible food substances such as dietary fibers and resistant starch into simple sugars, absorbable nutrients, and short-chain fatty
acids ( SCFAs ) . Notably, some of the SCFAs have beenreported to exert histone deacetylase ( HDAC ) inhibitory
activity, at least in vitro. We recently found that intestinal
microbiota-derived SCFAs may secure mucosal immune
homeostasis via epigenetic mechanisms ( see Fig ) . A highlevel ( approximately 100 mM ) of SCFAs is constitutively
produced by microbial fermentation in the distal intestineof mammals. Given that down-regulation of a SCFA transporter in the colonic mucosa has been implicated in thepathogenesis of inflammatory bowel disease ( IBD ) , SCFAs
may play a non-redundant role in the maintenance of gut
immune homeostasis.

Host factors responsible for maintenance of gut
immune homeostasis
To determine the host molecule ( s ) responsive to colonization by commensal bacteria, we performed microarray-based transcriptome analysis before and after colonization of germ-free mice with commensal bacteria. We

observed that this colonization upregulated the expression
levels of an adaptor protein for DNA methyl transferase( Dnap ) in colonic CD4 + T cells. Dnap forms a molecularcomplex with Dnmt and HDAC for epigenetic regulation oftranscription, suggesting that colonization by the microbiota may influence the epigenetic status of intestinal CD4 + T
cells. To examine this possibility, we developed mice witha T-cell specific deletion of Dnap using a CD4-driven Cre/
loxP system. The Dnap-deficient mice displayed a defectin colonic Treg expansion in response to bacterial colonization, and spontaneously developed severe colitis characterized by activation of TH1 and TH17 cells. Genome-wide
gene expression profiling followed by molecular network
analysis revealed that cell cycle-dependent kinase inhibitors ( Cdkns ) were derepressed in Dnap-deficient T
cells. This change was associated with hypomethylationof the promoter region of these genes as evidenced by
MeDP-Sequencing analysis. Thus, Dnap-dependent silencing of Cdkns is required for vigorous expansion of colonic Treg cells. This mechanism seems to be essential forthe maintenance of gut immune homeostasis.

Recent Publications
1. Kanaya, T., Hase, K., Takahashi, D., Fukuda, S., Hoshino, K., Sasaki, I.,
Hemmi, H., Knoop, KA., Kumar, N., Sato, M., Katsuno, T., Yokosuka, O.,
Toyooka, K., Nakai, K., Sakamoto, A., Kitahara, Y., Jinnohara, T., McSorley,
SJ., Kaisho, T., Williams, IR. and Ohno, H., The Ets Transcription factor
Spi-B is essential for the differentiation of intestinal microfold (M ) Cells. Nat
Immunol. 13, 729-736 (2012)
2. 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, 543-547(2011)
3. 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, YI., Dohi, T., Katsuno, T., Yokosuka, O., Waguri, S. and Ohno, H. The
Epithelia-specific membrane trafficking factor AP-1B controls gut immune
homeostasis in mice. Gastroenterology 141, 621-632 (2011)
4. Hase, K., Kawano, K., Nochi, T., Pontes, GS., 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)
5. 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, 1427-1432 (2009)

Figure :Schematic diagram of epigenetic regulation of intestinal immunity by commensal bacteria
Intestinal commensal bacteria actively produce small molecule metabolites. These metabolites
may directly or indirectly influence cell differentiation and function of immunocompetent cells,such as T and B lymphocytes, via epigenetic mechanisms.

53

The 2nd RIKEN RCAI-University ofMichigan Medical Center
Joint WorkshopThe second RIKEN RCAI-University of Michigan Medical Center Joint Workshop was held on January 16
and 17, 2013 at the medical school of the University ofMichigan in Ann Arbor. This joint workshop began in 2011
to launch collaborative relationships between RCAI andthe Univ. of Michigan. This year, five researchers fromRCAI visited Ann Arbor to attend the joint workshop.
In the first session, “Inflammation and Pulmonary Diseases”, Steven Kunkel talked about the epigenetic regulation of gene expression in CD4+ T cells and Nicholas W.
Lukacs, demonstrated that an epigenetic demethylase enzyme plays an important role in Respiratory Syncytial Virus
infection of dendritic cells in chronic pulmonary disease.
In the second session, “Stem Cell Development andBone Marrow Transplantation”, Bethany Moore talked
about the mechanisms by which alveolar macrophage (AM )function is inhibited after bone marrow transplantation andJames Ferrara talked about the search for biomarkers of
acute graft versus host disease (GVHD ) using proteomics
approaches. Tomokatsu Ikawa then described how to
expand hematopoietic progenitors by simply overexpressing Id3. He also discussed their model of transcriptional
networks in T cell lineage specification using their novel
culture system combined with Cap Analysis of Gene Expression ( CAGE ).
In the third session, “Antigen-dependent B Cell Differentiation”, two speakers introduced their results using
two-photon imaging. Takaharu Okada showed how follicular helper T cells move into lymph nodes and get activatedduring the adaptive immune response and Irina Grigorova
established the method for iterative application of intravital
imaging and quantitative modeling approaches. Tomohiro
Kurosaki showed that class-switched memory B cells andmemory follicular helper T cells are the major cells responsible for rapid antibody responses and proposed the underlying mechanisms. Marilia Cascalho described the detailed characterization of TACI-deficient mice. In the fourth
session, “Autoimmunity”, Mariana Kaplan explained theprocess of neutrophil extracellular trap ( NET ) formation.
The programmed release of chromatin fibers is thought tostimulate plasmacytoid DC to produce INFα in SLE. She
described other roles of NETs in autoimmune responses,
such as their direct role in vascular damage. David Markovitz discussed the oncoprotein DEK, one of the few known
autoantigens associated with juvenile idiopathic arthritis(JIA ), and he described the connection between DEK and
NET formation. Massimo Pietropaolo described islet cellautoantigen 69 (ICA69 ), a protein that is predominantly expressed by pancreatic islets and neuroendocrine organs.

54

He showed that deletion of ICA69 specifically in Aire-expressing thymus stromal cells results in spontaneous
multi-organ autoimmune disorders.
In the fifth session, “Inflammation and Innate Immunity”, Weiping Zou explained that human tumor infiltrating
Th17 cells are polyfunctional and express multiple cytokines including IFNγ, TNFα, and GM-CSF, and that Th17
cells and Th1 cells in human autoimmune lesions coexist
and work together for disease progression. Marc Peters-Golden presented a series of studies showing that
two eicosanoids, LTB4 and PGE2, have activating andsuppressive functions for macrophages, respectively.
Yuumi Nakamura showed that a mutation in the NLRP3
inflamasome, which was known to cause a skin disease in
humans, caused dysregulated expression of IL-1b by mast
cells. In the sixth session, “Immune Cell Signaling and Activation”, Takashi Saito described how T cell activation is
dynamically and spatially differentially regulated at TCR-microclusters ( TCR-MCs ) and the central-supramolecular
activation cluster ( c-SMAC ) . Joel Swanson described thatin murine macrophages stimulated with M-CSF, actin-richsurface ruffles transform into 1-5 mm diameter circular
ruffles, or macropinocytic cups, which close at their distal
margins, thereby creating endocytic vesicles called macropinosomes.
In the last session, “Genes In and Out of the Immune
System”, Haruhiko Koseki talked about how genes silenced by Polycomb group ( PcG ) proteins are reactivatedduring the developmental process. He identified a tissue-specific enhancer in the promoter region of the Meis2
gene locus and showed how the enhancer, the promoter
and the Polycomb binding site dynamically interact witheach other to reactivate the Meis2 gene during midbrain
development. The last speaker, James Douglas Engel talked about the function of GATA3 in the T cell development.
He generated GFP reporter mice whose expression iscontrolled by a presumptive GATA3 enhancer and showedthat the enhancer specifically functions in T cells.
After the presentations, RCAI researchers had oneon-one discussions with several researchers from the Univ.
of Michigan with common interests. During the workshop,
researchers agreed to continue the collaborative relationships and stimulate some new personal collaborations.

2nd New Zealand - Japan
Joint Immunology WorkshopOn 26 th and 27 th February 2013, the New ZealandJapan Joint Immunology Workshop was held at the
University of Auckland campus. Eight Japanese researchers, Drs. Shin-ichiro Fujii, Shohei Hori, Takaharu Okada,Yasuyuki Ishii and Takashi Saito from RCAI and Drs.
Toshinori Nakayama, Koichi Hirose and Takeshi Tokuhisa
from Chiba University and thirteen researchers from New
Zealand presented talks. In addition to the New Zealanddelegation members who participated in the first meeting
held at RCAI in 2010, sixty researchers and graduate students from the University of Auckland, University of Wellington and the University of Otago participated in this 2 nd
meeting.
The meeting was held at “Fale Pasifika”, one of themore iconic buildings on campus. For most Pacific communities, Fale is more than just a physical structure, but
it is their point of reference for activities that are central totheir cultural expression and it gives them a sense of placeand community. Equally, the University’s Fale Pasifika is asymbol of identity for Pacific Island students and staff, andis the second largest such structure in the world. It was asign of the warmest hospitality by the organizing committee to hold the meeting there.
In his opening remarks, Prof. Rod Dunbar, the University of Auckland said, “We were extremely pleased
with the way in which the presentations at the workshop
highlighted both the collaborative projects that have grownsince 2010, when the New Zealand delegation visited
Japan, and areas where more collaborations could beinitiated. We look forward to supporting RCAI’s continued
collaborations. In case you are interested in contacting anyof the other New Zealand workshop speakers to discussa potential collaborative project, please don’t hesitate tocontact us if you would like some assistance with this.”
Indeed, two collaborations had already started after
the 1st workshop. One is to combine two-photon imaging data of Dr. Takaharu Okada from RIKEN RCAI with anovel computational immunology technology developedby Dr. Gib Bogle, the University of Auckland. It is quite achallenging and innovative program, and may open a newresearch paradigm. The other collaboration is a practicalprogram of Dr. Yasuyuki Ishii from RIKEN RCAI with Prof.
Sarah Hook, the University of Otago to establish new oralvaccines for allergic diseases. Ishii develops a liposomal
allergy vaccine incorporating α-galactosylceramide, a representative ligand for invariant natural killer T ( iNKT ) cells,
and Prof. Hook has a long research career and worldclass technologies in oral administration of drugs. Drs. Ishii
and Hook are collaborating to develop new technologies
for oral drug administration, which will be applicable toasthma, atopic dermatitis, pollinosis, and food allergy.
These collaborations are still ongoing.
During the workshop, there was the impression that
immunologists in New Zealand communicate well witheach other and were very interested in the progress of science and technologies in Japan, and there would be more
possibilities for research collaborations between Japan
and New Zealand. The workshop was successfully completed and all speakers were invited to the Japanese Consulate in Auckland. In the closing remarks, Prof. Tokuhisa
said, “Hopefully, relationships between New Zealand andJapan will be spread much more than now. This joint
Immunologist workshop is one of successful friendshipand will work as a trigger for bridging.”

55

Program
Day 1 – Tuesday 26 th February 2013
Session 1:

Allergy, Autoimmunity & Tolerance

Rod Dunbar

School of Biological Sciences, Faculty of Science,
The University of Auckland

Opening Remarks

Toshinori Nakayama

Department of Immunology, Graduate School of
Medicine, Chiba University

CS4+ T cell memory and airway inflammation

Graham Le Gros

Malaghan Institute for Medical Research

Th2 and innate cell differentiation in allergic and parasitic disease

Koichi Hirose

Department of Allergy and Clinical Immunology,
Graduate School of Medicine, Chiba University

Beta-glucan curdlan induces IL-10 producing CD4+ T cells and inhibits
allergic airway inflammation

Session 2: Allergy, Autoimmunity & Tolerance

Takeshi Tokuhisa

Department of Developmental Genetics, Graduate
School of Medicine, Chiba University

Development of high affinity IgE B cells

Anne La Flamme

School of Biological Sciences, Faculty of Science,
Victoria University of Wellington

Targeting innate immunity during multiple sclerosis

Yasuyuki Ishii

Laboratory for Vaccine Design, RCAI

Research for oral allergy vaccines inducing immune tolerance

Elizabeth Forbes-BlomMalaghan Institute for Medical Research

Understanding the cellular mechanisms underlying food allergensensitisation

Shinichiro Fujii

Research Unit for Cellular Immunotherapy, RCAIiNKT cell-triggered in vivo DC targeting immunotherapy

Ian Hermans

Malaghan Institute of Medical Research

iNKT cells in DC-based immunotherapy

Shinichiro Motohashi

Department of Immunology, Graduate School of
Medicine, Chiba University

Invariant NKT cell targeted immunotherapy for lung cancer

Gavin PainterCallaghan Innovation

Synthetic Vaccines for Cancer Immunotherapy with NKT cells

Rod Dunbar

School of Biological Sciences, Faculty of Science,
The University of Auckland

Targeting human APCs with synthetic peptides

Sarah Hook

School of Pharmacy, University of Otago

Development of immunogenic vaccine formulations

Sarah Young

Department of Pathology, Dunedin School of
Medicine, University of Otago

The use of Virus-like particles as an immune therapy for cancer

Session 3: Immunotherapy

Session 4: Immunotherapy

Day 2 – Wednesday 27 th February 2013
Session 5: Immune regulation & immunodynamics

Takashi Saito

Laboratory for Cell Signaling, RCAI

Dynamic imaging analysis of T cell activation regulation

Franca RoncheseMalaghan Institute of Medical Research

Regulating dendritic cell function

Shohei Hori

Research Unit for Immune Homeostasis, RCAI

Genetic and environmental control of regulatory T cell fitness inperipheral tissues

Session 6: Immune regulation & immunodynamics

Takaharu Okada

Research Unit for Immunodynamics, RCAI

2-photon imaging of adaptive immune responses

Gib Bogle

Auckland Bioengineering Institute, The Universityof Auckland

Computational immunology: helping to elucidate lymph node processes
and structure

Roslyn Kemp

Department of Microbiology and Immunology, Otago Immune Diversity in the Gut Mucosa
School of Medical Sciences, University of Otago

Session 7: Infectious immunology

56

John Fraser

Faculty of Medical and Health Sciences, The
University of Auckland

Joanna Kirman

Department of Microbiology and Immunology, Otago The generation and maintenance of CD4+ Th1 memory
School of Medical Sciences, University of Otago

Christopher Hall

Department of Molecular Medicine and Pathology,
The University of Auckland

Microbial pathogenicity

Immunometabolism: irg1 and mitochondrial ROS production

Harvard Summer School at RCAI
Jun 11 - Aug 17, 2012

R

CAI offers an undergraduate student internship program in collaboration with Harvard University’s Summer School. The participants earn university credits in the
Harvard Summer School Program while at RCAI. In 2011,
RCAI had to cancel the program because of the aftermathof the Great East Japan Earthquake, but RCAI began accepting students again this summer. Three students, Mss.
Malisa Smith, Ning Hou and Natasha M Furtado Dalomba
conducted research projects in the Lab. for Immunog-

enomics, the Lab. for Transcriptional Regulation and theLab. for Epithelial Immunobiology, respectively. They also
participated in a series of basic immunology lectures at
RCAI, the RCAI International Summer Program, and the
RCAI-JSI International Symposium on Immunology, andhad basic Japanese classes. Besides these studies, they
enjoyed a cultural exchange with students of Yokohama
Science Frontier High School (Photo), and an excursion to
Kamakura.

Photo: Japanese culture visit to Yokohama Science Frontier High School (left) and Basic Immunology Lecture at RCAI (right)

RCAI-ZIBI student exchange
programR

CAI International Summer School and the ZIBI Summer School in Berlin launched a student exchange
program in 2011. In 2012, two Ph.D. course students from
RCAI, Hisashi Wada in the Laboratory for Transcriptional
Regulation and Misao Hanazato in the Laboratory for Epithelial Immunobiology participated in ZIBI summer school
held on Jun 9-24, 2012, and two students from ZIBI,
Kerstin Westendorf from German Rheumatism ResearchCenter and Stefan Riebel, from Max Planck Institute for
Infection Biology attended the RCAI International Summer
Program.
ZIBI ( Interdisciplinar y Center of Infection Biology
and Immunity ) merges the activities of the Humboldt
University and non-university research institutes in thefield of infection biology and immunity in Berlin, including
Robert Koch Institute, Charite (a joint institution of the Freie
Universität Berlin and the Humboldt-Universität zu Berlin ) ,The German Rheumatism Research Center Berlin ( DRFZ ) ,Leibniz Institute for Zoo and Wildlife Research, and Max
Planck Institute for Infection Biology. ZIBI summer pro-gram 2012 provided a one-week lecture course, two days
scientific symposium “Global Challenge of Chronic Tropical Infections” and three days of practical courses andsight-seeing in Berlin. Twenty-two master/Ph.D. students
participated from Germany, Turky, Brazil, Ethiopia, South
Africa, India, Indonesia, Bangladesh, Greece, Slovakia, Israel and Japan. “It was my first experience to visit abroad
and I was a little worried about English. However, I could
have so many discussions with various people and it was
my honor to receive the poster award during the symposium,” said Hanazato.

57

International Research
Collaboration AwardT

he RCAI International Research Collaboration Awardis a unique program supporting researchers outsideof Japan in setting up semi-independent research units
within the laboratory of their collaboration partner at theCenter. The program provides up to 10 million JPY/year toeach collaborative research project for up to three years.
( Until 2007, the program awarded 15 million JPY/year but
the amount was reduced in 2008 )Since the program began in 2004, 18 projects havebeen funded ( Table ) and the collaborations have resulted
in several important papers. Drs. Vidal and Koseki’s collaborative project on Ring1 genes resulted in 5 papers,
Dev Cell. (2004), Development (2006 ), Nat Cell Biol. (2007),

Mol Cell Biol. ( 2008 ) and Development ( 2008 ) . Drs. Dustin
and Saito published their work on T cell microclusters in
Nat Immunol. ( 2005 ) and Immunity ( 2006 ) . Drs. Ewijk and
Kawamoto’s work on thymic progenitor cells resulted inthree papers in Development ( 2006 ) and Mol Immunol.
( 2009 and 2010 ) . Drs. Bix and Kubo published their workon the IL-4 repressor in Nat Immunol. ( 2009 ) , and Drs. Ellemeier and Taniuchi published their study on transcription
factor network for CD4/CD8 lineage choice in Nat Immunol. ( 2010 ) , and Dr. Cheroutre published transcriptional
reprogramming of CD4/CD8 lineage in Nat Immunol. (2013 )with Drs. Ellemeier and Taniuchi.

Table: Awardees of RCAI International Collaboration Award Program
Host Lab.

Title of ResearchAwardee20042006

Year

Takashi Saito

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

New York University School of Medicine

20042006

Hiroshi
Kawamoto

Regulatory role of lymphoid progenitors during developmentof thymic microenvironments

Leiden University Medical Center

20042006

Haruhiko
Koseki

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

20042005

MasaruTaniguchi

Role of NKT cells in TSLP-mediated allergic inflammation

20042006

Ji-Yang Wang

Expression and function of FcRY-a novel Fc receptor-related Dr. Peter D. BURROWS
gene expressed in B cells
University of Alabama at Birmingham

20052007

Ichiro Taniuchi

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

20052007

Masato Kubo

Understanding genetic regulation of interleukin 4 production Dr. Mark BIX
by a CD4 (+) T cell-intrinsic mechanism.
University of Washington, Seattle, Washington

20052006

Yasuyuki Ishii

Gene-array analysis and proteomics of Th2 tolerance

20052007

Osami
Kanagawa

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

20052007Tomohiro
KurosakiRole of signaling molecules in B cell synapse formation andits maintenance20062008

Masato Tanaka Hepatocytes and Examination of Its Effect on the20072008

TakeshiWatanabe

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

20072009

Ichiro Taniuchi

Understanding of tumor suppressive mechanism of Runx
complexes against leukemia and gastrointestinal cancer

20072009

Sidonia
Fagarasan

Nuclear reprogramming of terminally differentiated plasma
cells to study the specific role of IgA in mucosal andsystemic immunity and B cell development

20102012

Mariko Okada- Proteomics based-quantitative analysis of signalHatakeyamatranscriptional network

20112013

Understanding and Engineering of Dendritic Epidermal T
Ichiro Taniuchi
Cells ( DETC ) development

20122013

Yasuyuki Ishii

Oral vaccine design for asthma, atopic dermatitis andpollinosis

Dr. Sarah HOOK

Hiroshi Ohno

Identification and analysis of transcription factors directing
intestinal M cell differentiation

Dr. Ifor WILLIAMS

20122013

58

Identification of Novel Necrotic Molecules from Necrotic
Inflammatory Response

Dr. Michael DUSTIN

Dr. Willem van EWIJK
Dr. Miguel VIDAL

Centro de Investigaciones Biologicas, CSIC

Dr. Steven F. ZIEGLER

Benaroya Research Institute at Virginia Mason Medical Center

Dr. Wilfried ELLEMEIER Institute of Immunology, Medical University

ViennaDr. Hilde CHEROUTRE La Jolla Institute for Allergy and Immunology

Dr. Yun-Cai LIU

La Jolla Institute for Allergy and Immunology
Dr. Kenneth M. MURPHY

Howard Hughes Medical InstituteWashington University School of Medicine

Dr. Facundo Damian BATISTA

Cancer Research UK London

Dr. Sunhwa KIM and Dr. Michael KARIN Department ofPharmacology, Univ. of California, San Diego, USA
Dr. Andrea BRENDOLAN

Cornell University Medical Center, Department of Cell andDevelopmental Biology
Dr. Motomi OSATO and Dr. Yoshiaki ITO

Institute of Molecular and Cell Biology, National University ofSingapore
Dr. Stefano CASOLAIFOM-The FIRC Institute of Molecular Oncology Foundation, Milano,
Italy
Dr. Boris KHOLODENKO

University College Dublin, Ireland
Dr. Richard JONES

University of Chicago, USA
Dr. Florent GINHOUXSingapore Immunology Network ( SIgN ) , Singapore
University of Otago, New Zealand
Emory University, USA

2012Part 5

Outreach Activities

RCAI-Novo
Science Forum

Open Seminar“When cells
determine their fate”

April 19, 2012

April 21, 2012

IOn April 19, 2012, RCAI and Novo Nordisk, a global
healthcare company headquartered in Denmark,held a joint scientific forum at RCAI. Fifteen Novo Nordisk
researchers and senior managers in the fields of biopharmaceuticals, immunology, pharmacology, target discovery, biotechnology, development, and disease models,visited RCAI. After the introduction of sourcing innovation
at Novo Nordisk by Jørn Roland Müller, Corporate Vice
President of Biopharmaceutical Sourcing in Novo Nordisk,
Takashi Saito, Deputy Director of RCAI, described thestructure of the Center. After these introductions, nine
RCAI researchers, Toshitada Takemori, Mariko Okada, Tomohiro Kurosaki, Ji-Yang Wang, Hiroshi Ohno, Tsuneyasu
Kaisho, Takashi Tanaka, Shohei Hori and Sidonia Fagarasan described their recent research discoveries. At theend of the forum, Anan Gautum, Director of Biopharmaceutical Sourcing in Novo, commented that the research in
RCAI was very impressive and that Novo would continue
to look for collaborative possibilities. This workshop was
one of the rare opportunities for RCAI researchers to discuss science with a pharmaceutical company. Takashi
Saito told that it is difficult to consider pharmaceutical or
clinical applications of the research results from the Center
without collaboration and support from pharmaceuticalcompanies.

60

chiro Taniuchi, Group Director of Laboratory for Transcriptional Regulation, spoke at the Open Seminar heldin the RIKEN Wako Open Campus. The seminar “Whencells determine their fate” attracted an audience of 230,
which exceeded the capacity of the lecture hall, so that
more than 50 people watched on a live remote screen in a
separate room.
His talk started with a simple question, “What is immunity?”. He effortlessly explained that immunity is a strategy that allows us to survive environmental dangers. To
answer the second question, “What is the environment?”,
he asked the audience to close their eyes. Our eyes sense
the light, and similarly, ears perceive the sound, nose perceives the smells, and tongue perceives the taste. Immune
cells recognize the surrounding environment, too, but indifferent ways.
After he explained the basics of the immune system,he talked about helper and killer T cell fate during their development. He had discovered a mechanism that controls
expression of the transcription factor ThPOK, the master
regulator to choose a helper or killer fate in a cell fate decision. Interestingly, his recent study found that helper
T cells could switch their function to become killer-like T
cells. He compared the cell fate decision to the college
preparation courses in Japanese high schools. In Japan,
high school students choose natural science or humanities courses to prepare for the college entrance exams.
Students who took natural science courses would major inscience in college, but some students could change theirmajor to humanities. Helper T cells can change their fate,too, he explained.
Various individuals participated in the seminar, including university students, company workers, teachers,
housewives, retired folks, etc. “It was interesting that thestory started from the surface and connected to a deep
part. I wanted to know more about transcriptional regulation,” said one man, but another said, “It was easy in the
beginning but difficult at the end.” Taniuchi said, “It was
not easy to make the story easy-to-understand but I was
glad that so many people were interested in my research.”

Science Café
“Gut bacterium
and probiotics”

Science Café
“Do computers
cure diseases?”

April 22, 2012

September 1, 2012

On April 22, Hiroshi Ohno, Team Leader of the Laboratory for Epithelial Immunobiology, spoke at the RIKEN Yokohama Science Café held at Kanagawa Prefectural
Kawasaki Library. Among 170 applicants, 50 participants
were selected by a random drawing.
Ohno first described how many bacteria live in a human body. Our body consists of 60 trillion human cells
and has 30 thousand human genes. Meanwhile, one hundred trillion bacteria live in the gut, consisting of more than
1,000 species, and 3 million gut bacteria genes. Consequently, in the gut, humans have more than 100 fold morebacterial genes than human genes. Our body is actually
maintained by many organisms, and this concept wastermed “superorganism” by Joshua Lederberg, a Nobel
laureate.
Recently, metagenomics, which means genomics ona large scale, has emerged as a powerful tool to analyze
bacterial communities. Metagenomics is based on the
genome sequencing of samples collected directly from theenvironment. It enables a survey of the different bacterial
species in the environment, and metagenome analysis
of human gut microbiota has been conducted mainly inEurope ( MetaHIT; Metagenomics of the Human Intestinal
Tract ) and the US ( HMP; Human Microbiome Project ) as
the national/international projects funded by their government .
In the latter half of the talk, Ohno explained his discovery that Bifidobacteria can protect from enteropathogenic
E. coli O157 infection through production of acetate.
There were many questions from the participants
during and after his talk, and so the café ran out of time
for any more questions and answers. Most of the questions were related to health. “What is the relation between
gut bacteria and colon cancer?” “Are the supplements
beneficial to colon health?” “Do the gut microbiota recoverafter an endoscope examination?” Ohno tried to answer
as much as possible, and the participants had favorable
impressions of his friendly attitude.

On September 1, 2012, Mariko Okada, Team Leaderof Laboratory for Cellular Systems Modeling, spokeat the Science Café held at Yokohama City Central Library.
Forty people, including three junior high and senior highstudents attended the event.
The talk started by defining the notion of systems
biology. Okada easily introduced various pathways andcomponents in biology. She showed multiple examples ofmetabolic pathways, signaling pathways, central dogma,
and explained that adequate regulation of the pathways isnecessary for the normal function of biological systems.
She compared systems biology to a car control system.
To make a car, all parts must be assembled properly andinput-output pathways must be adequately controlled.
Then she described the challenges of systems biology: To understand the whole body as a system, it isnecessary to transcend the different layers of body components and pathways and create an integrated network.
In a human body, there are 12 kinds of organs, 4 kinds oftissues, 300 kinds of cells, 100,000 different proteins and23,000 genes. The size and time scales of these systems
are also different. Molecular or ionic changes occur in
10 -6 seconds, signaling events occur in 10 -3-1 second, cell
movement and cell division occur in 1-104 seconds, protein
turnover occurs in 10 6 seconds and the human lifespan isabout 10 9 seconds.
Lastly, she explained how computer models could
contribute to the cure of diseases. Using an American
venture company, Entelos, as an example, she explainedthat computer model platforms are actually used for theinterpretation of physiological state, disease state andtherapeutic decisions. In addition, computer models could
calculate uncertainties between individuals, life style differences and genetic differences.
“It was new for me to know that animals were systems, and it is composed of various networks,” a participant told. “It was new, too, to know that researcher’scommunications and networks are important in systems
biology.”

61

RIKEN Yokohama
Open Campus
September 29, 2012

Science Café
“Towards the curefor leukemia”
March 2, 2013OT

he RIKEN Yokohama Institute Open Campus was held
on Sep. 29, 2012. Under clear autumn skies, 1,749
people visited the campus. Haruhiko Koseki, Group
Director of the Laboratory for Developmental Genetics,
gave a seminar “Is the calico cat’s pattern inheritable?”
There were 104 people at the seminar, ranging in age fromelementary school students to retired folks. He explained
the genetic combinations that encode the fur color of cats
and their genetic linkage to the X or Y chromosome. Then
he introduced the calico cat cloning experiment. Because
one of the X chromosomes was already inactivated in thesomatic cell used for cloning, the cloned cat turned out tohave a different fur pattern, a brown tabby. “It sounded
simple in the beginning, but the epigenetic control was actually a little complicated,” a participant said.
Twenty RCAI teams showed posters or movies. Highlights were the hands-on experiences provided by four
teams. Ohno and his team members provided hands-on
practice of Gram staining of mouse gut bacteria. Twenty-five participants each stained two mouse fecal samples,
before and after the administration of antibiotics, andcompared the change in the gut microbiota using a microscope. Yoshida and his team prepared stained samplesof fetal mice and newborn mice at various developmental
stages. Mariko Okada and her team members held “Fillinga pipette tip box competitions.” Participants competed forthe best time for filling 100 micropipette tips into a box.
The top results of the day were 1 min 6 sec for 1000ml pipette tips and 1 min 21 sec for 200ml tips. Social networksites, Facebook, Twitter and YouTube, were set up for the
open campus this year. A photo of “Filling a pipette tip
box competitions” gathered re-tweets and comments on
Twitter. “What’s this?” “Wow, I didn’t have this idea.” “Iwant to take my time!” The RIKEN Yokohama Open Campus seems to evolve year by year.

62

n March 2nd, Fumihiko Ishikawa, Group Director of
the Laboratory for Human Disease Model, talked
at a Science Café held at Yokohama City Central Library.
Among 82 applications, forty participants were selectedby a random drawing.
The first half of the café was a lecture by Dr. Ishikawa.
He talked about his experience as a hematologist and how
he decided to become a researcher to study the recurrence of acute myeloid leukemia. Then he introduced his
current research on the development of novel therapies
targeting leukemic stem cells.
“I had not heard of the humanized mouse before, but
I could understand how it could contribute to the development of new therapies.” “I really look forward to theanti-leukemia drug RK-20449. I felt Dr. Ishikawa’s passion.
His dream will come true,” were some of the comments by
the participants.
The second half of the café was a Q & A session.
Participants wrote down questions on Post-it notes and
Dr. Ishikawa responded to them. The 24 questions varied from the difference between hematopoietic stem cells
and leukemic stem cells, characteristics of the humanizedmouse, future medicine, and even the future of RIKEN.
One question, “There are many questions ( gene therapy,
iPS therapy etc. ) which cannot be explained by current
basic sciences ( chemistry, physics, quantum scienceetc.) . Isn’t it necessary to conduct more fundamental research to solve the questions?” induced a lively discussion
from the floor. “What is the research that RIKEN can dobut other institutions or universities cannot do?” “RIKEN
should pursue the research irrelevant to profits.” “RIKEN can do the advanced research and integrate various
fields.” “Dr. Ishikawa’s research would be an example ofwhat RIKEN can do.”After the café, a participant said to a RIKEN staff
member, “It was like Michael Sandel’s ‘Justice’ TV program from Harvard. You can rename this RIKEN’s programto ‘Passionate Science’ café.”

2012Part 6

Laboratory Activities

Laboratory for

Developmental
Genetics
Group Director :Haruhiko Koseki

Senior Research Scientist :Kyôichi Isono

Research Scientist : Yûichi Fujimura, Mitsuhiro Endô, Osamu Masui,			 Takashi Kondô, Yixin Dong, Kit Wan Ma,
			 Nayuta Yakushiji, Yang Li,
			 Raul Eduardo Vizcardo Sakoda,
			 Yuki Takada (SPDR ), Shinsuke Ito (SPDR ),			 Jafar Sharif (FPR )Technical Staff :								

Tamie Endo, Naoko Ônaga, Rie Suzuki,
Kayoko Katsuyama, Yôko Koseki, Kaori Kondô,
Mami Kumon, Fuyuko Kezuka, Miho Urabe

Student Trainee:

Misaki Ôno, Marika Shibata

T

he Developmental Genetics Research Group fulfills adual role within RCAI. A large portion of the manpower and financial resources of the group is devoted to themaintenance of a high-standard mouse facility in RCAI.
Through the Animal Core Facility, the group is also responsible for the generation of knock-out and transgenic
animals for the various research laboratories at the center.
At the same time, the laboratory is pursuing a research
program to elucidate the molecular mechanisms underlying organ development and stem cell functions. Particularemphasis has been put on epigenetic regulation mediatedby combinatorial actions of Polycomb group ( PcG ) geneproducts and DNA methylation mechanisms.

The role of epigenetic regulators during
development and differentiation
Two distinct Polycomb complexes, PRC1 and PRC2,
collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells ( ESCs ) . PRC1
and PRC2 have histone modifying activities, catalyzing
mono-ubiquitination of histone H2A ( H2AK119u1 ) andtrimethylation of H3 lysine 27 ( H3K27me3 ) respectively.
The interplay of these modifications and their contribution to Polycomb repression, however, remains not fully
understood. We now show that high levels of H2AK119u1
deposition occur at a subset of target loci that are critical for ESC maintenance. We could further demonstratethat H2AK119u1 deposition occurs at these targets in

PRC2-deficient ESCs albeit at lower levels, suggestinga presence of H3K27me3-independent compensatory
mechanisms for recruitment of PRC1 function. Finally, we
have found that the H2A ubiquitination activity of PRC1 isessential for target gene repression and ESC maintenance,
and that distinct PRC1 functions mediate chromatin compaction and contribute to silencing activity, most notablyat Hox loci. Based on these findings, we now propose that
PRC1 utilizes these diverse sensing and effector mechanisms, which provides a means to maintain a repressive
state that is robust yet highly responsive to developmental
cues during ES cell self-renewal and differentiation.

Regulation of large scale chromatin structures by
epigenetic regulators
Accumulating evidence documents a role for PcG proteinsin regulating higher order chromatin structures, but themechanisms and impact of such structures on transcriptional regulation remain obscure. We have identified PcG
bodies in mouse primary fibroblasts as distinct foci, at
which PRC1 and H3K27me3 are colocalized and canonical PcG target genes are condensed. We found that PcG
body formation requires Phc2-SAM polymerization, which
critically contributes to condensation and repression of
PcG target genes. We further showed that Phc2-SAM
polymerization limits the dynamic nature of PRC1, andthereby promotes stable association of PRC1 with PcGtarget genes. These findings suggest a novel model bywhich SAM polymerization of Phc2 modulates the struc-

Recent Publications
1. 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 )64

2. 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 differentiallymodulate Polycomb activity at both the Hox gene cluster
and at Cdkn2a genes. Mol Cell Biol. 31, 351-364 ( 2011)

3. Takada Y, Naruse C, Costa Y, Shirakawa T, Tachibana
M, Sharif J, Kezka-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 in mice. Development 138, 4207-4217 ( 2011)

Figure :DNA topology of promoter (PRT)/enhancer (ENH)/silencer (SIL) during midbrain development.
DNA FISH analysis indicates that PcG/PRT/SIL associate together when gene (Meis2) transcription is suppressed. The ENH sequence transiently associates with this complex in the 8 dpc midbrain primordium justbefore the onset of Meis2 expression. SIL dissociates from the complex at the stage when Meis2 expression
begins (9 dcp midbrain). Although the tri-DNA complex formation required PcG activity, PcG is no longer
necessary for maintenance of the PRT/ENH association.

tural organization of PcG complexes to enable robust yet
reversible PcG-mediated repression during development.
Since Hox gene clusters retain a special genomic
configuration, we went on to test whether PRC1 uses
this mechanism also at solitary target genes. Intriguingly,
by re-examining ChIP-seq data for Ring1B distributions
in ESCs, we found that PRC1 is distributed to not only
promoter regions but also genomic regions surrounding
stop codons of repressed target genes. We hypothesizedthat promoter regions are associated with 3’ regions ofrespected targets. We focused on the Meis2 gene, which
occupies a large genomic segment spanning around
200Kb in mammals. Meis2 has complex expression
patterns during development and mutations in the locus are lethal with various defects. We identified severaltranscriptional regulatory sequences for the Meis2 gene,which are located at large distances from the promoter inthe linear DNA sequence, and analyzed their topologicalrelationships at transcription activity transitions during development using knockout mice, 3C, transgenic analysis
and histological FISH. In the transcription repressive state,such as in early embryonic stages, the promoter/silencer( Polycomb binding sequence ) associate with each otherin a Polycomb-dependent manner, while the promoter/enhancer are in close proximity without silencer association in the active state ( Figure ) . Detailed observationof midbrain expression during midbrain organogenesis
revealed that all three sequences-promoter/enhancer/
silencer transiently associate into close proximity in the

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

preparative state just prior to Meis2 expression in midbrain
primordium neural plate, then the silencer dissociates fromthe complex to activate gene expression. This three-region-association is also dependent on Polycomb activity,
while maintenance of the active state of expression does
not require Polycomb activity.

A novel role for tubulins to organize transcriptionally
repressive domains at the nuclear periphery
In metazoans, the inner surface of nuclear envelope islined by nuclear lamina ( NL) , a filamentous protein network
primarily consist of lamins. Physical interactions of NL
and intranuclear molecules are engaged in regulation ofchromosome status and of transcriptional activity. Mostof the genes associating with the NL are transcriptionally
repressed. Consistent with this, the NL also associateswith a variety of chromatin modifying factors that correlatewith gene silencing such as histone deacetylases (HDACs ).
However, the molecular mechanisms which link the NL
and its binding factors in living cells are poorly understood.
We have shown that tubulin proteins persistently associate with the NL and are indispensable for association of
NL-chromatin, and of NL-HDACs. Loss of tubulin from the
NL results in the disengagement of these factors, and upregulation of histone acetylation at the nuclear periphery.
These findings suggest novel functions of tubulins as a
scaffold for molecular assembly of genomic DNA and determinants for chromatin status at the nuclear periphery.

5. Endoh M, Endo TA, Endoh T, Isono K, Sharif J, Ohara O,
Toyoda T, Ito T, Eskeland R, Bickmore WA, Vidal M, Bernstein BE, Koseki H. Histone H2A Mono-Ubiquitination Is
a Crucial Step to Mediate PRC1-Dependent Repressionof Developmental Genes to Maintain ES Cell Identity.
PLoS Genet. 8:e1002774 (2012 )65

Laboratory for

Lymphocyte Development
Team Leader:

Hiroshi Kawamoto
Research Scientist: Kiyokazu Kakugawa
Kyoko Masuda
Research Associate: Rumi SatohTechnical Staff:

Midori Kawauchi

Visiting Scientist:

Nagahiro Minato
Yoshimoto Katsura
Toshio Kitamura
Takeshi WatanabeThe major aim of our team is to elucidate the molecularmechanisms that regulate cell fate decisions in theprocess of lineage restriction from multipotent hematopoietic stem cells to unipotent progenitors. A series of studies
from our laboratory on early hematopoiesis has led to afundamental redefinition of lymphoid progenitors as well as
the ontogeny and phylogeny of T- and B-cell development.
We thus have proposed our original model of hematopoiesis, the myeloid-based model, in which myeloid potential
is retained along with the specification pathways towards
erythroid, T, and B cell lineages.

Individual hematopoietic stem cells in human bonemarrow stably give rise to limited cell lineages
To sustain hematopoiesis, hematopoietic stem cells (HSCs )must, on the one hand, replenish themselves by self-renewal and, on the other hand, produce differentiatingprogenitor cells. It is also known that most HSCs remain
dormant and are only rarely and randomly activated. It has
been estimated that each human possesses a total of 104
HSCs, and that ~400 HSCs actively contribute to hematopoiesis, replicating once per year 1, 2. However, the actualdynamics of hematopoiesis by HSCs remains uncertain.
For example, it is unclear how long an individual HSC
maintains hematopoiesis and whether all major lineages ofcells are produced from a single HSC. These issues havebeen difficult to address due to the lack of appropriate experimental systems, regardless of animal species.

GPI-APs- cells were seen in limited lineages of blood
cells from most BM failure patients
Mutation of the PIG-A gene in hematopoietic stem cells( HSCs ) results in the loss of glycosylphosphatidylinositol-anchored proteins ( GPI-APs ) on HSCs, but minimallyaffects their development and thus can be used as a clonal marker of HSCs. We analyzed GPI-AP expression on
six major lineage cells in a total of 574 patients with bonemarrow ( BM ) failure in which the microenvironment itself isthought to be unaffected, including aplastic anemia ( AA )and myelodysplastic syndrome ( MDS ) . We determined the
proportion of GPI-AP-deficient ( GPI-APs- ) cells in six major
lineages, namely granulocytes ( G ) , monocytes, erythrocytes, T cells, NK cells and B cells, in peripheral blood (PB )cells from BM failure patients using a highly sensitivity flow
cytometric analysis.
GPI-APs- cells were detected in 250 patients. Whereas the GPI-APs - cells were seen in all six lineages in a
majority of patients who had a higher proportion (>3%) of
GPI-APs- cells, they were detected in only limited lineages
in 92.9% of cases in the lower proportion ( < 3%) group. In

Recent Publications
1. Katagiri T*, Kawamoto H*, Nakakuki T*, Ishiyama K, ,Okada-Hatakeyama Ohtake S, Seiki S, Hosokawa K and
Shinji Nakao. (*equal contribution ) Individual hematopoietic stem cells in human bone marrow of patients withaplastic anemia or myelodysplastic syndrome stably giverise to limited cell lineages. Stem Cells 31, 536-546,
2013 (*equal contribution )66

2. Vizcardo R, Masuda K, Yamada D, Ikawa T, Shimizu K,
Fujii S-I, Koseki H, Kawamoto H. Regeneration of humantumor antigen-specific T cells from iPS cells derived
from mature CD8 + T cells. Cell Stem Cell 12, 31-36,( 2013 ).

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

Figure :all 250 cases, the same lineages of GPI-APs- cells weredetected even after 6-18 month intervals, indicating that
the GPI-APs- cells reflect hematopoiesis maintained by aself-renewing HSC in most cases. The frequency of clones
with limited lineages seen in mild cases of AA was similar
to that in severe cases, and clones with limited lineages
were seen even in two healthy volunteer cases. These results strongly suggest that most individual HSCs produce
only a restricted set of lineages even in the normal steadystate.

A model for the hematopoietic microenvironment to
explain the production of limited cell lineages froma single HSC
While the observed restriction could reflect heterogeneity
in the developmental potential of HSCs, we propose analternative model in which the BM microenvironment ismosaic in its capacity to support commitment of progenitors towards distinct lineages. For simplicity, we reclas-

Simulation of HSC differentiation recapitulates the clinicalobservations.
A. The percentage of GPI-APs- granulocytes vs. the number of
GPI-APs- cell lineages in individual patients. For simplicity,
granulocytes and monocytes are placed into the myeloid lineage
while T and NK cells are placed into the T lineage, consequently
all lineages can be classified as M, E, T, or B lineages. The redline at 0.25% indicates the expected value for an average size ofa single HSC clone.
B. Frequency of clone types among cases where the percentage of
GPI-APs- granulocytes is between 0.3 to 3 %.
C. A model assuming that the BM microenv ironment isheterogeneous in its capacity to support the commitmentof progenitors. A certain range where the commitment ofprogenitors mainly occurs (with a probability of >97%)
is termed the “commitment sphere”. Areas colored beige,
pink, light green, or blue represent environments supporting
commitment only towards M, E, T or B cells, respectively.
If a HSC happens to reside in a location in which thecommitment sphere contains only M and E regions, thenthis HSC can eventually produce only M and E cells, even
if committed progenitors expand enormously and become
widely scattered. The depicted simulation goes as follows: avirtual HSC undergoes several cell divisions at fixed intervals
while randomly migrating. The uncommitted progenitors thenundergo a fate decision based on their ultimate location in thecommitment sphere. Committed progenitors can also randomly
move around, but can proliferate only in the lineage-specificsupporting environment. After several additional fixed time cell
divisions as committed progenitors, cells become mature andlineage restricted.
D. One example illustrating the frequency of clone types simulated
as shown in (C). A total of 100 virtual HSCs were simulated toform hematopoietic clones, and the numbers of the resulting
clone types are illustrated.
E. Simulation of individual HCSs for size and number of progeny
lineages. The simulation well recapitulated the clinicalobservations shown in (A).

sified the six lineages into four lineages ( Fig. A, B ) . In themodel, we assume that a self-renewing HSC is located ina particular site in BM and that uncommitted progenitors
derived from this HSC can reach to a certain defined area
( Fig. C ) , which we term here the “commitment sphere”. If
the BM microenvironment is mosaic in terms of function in
inducing the commitment of progenitors towards a particular lineage, and the size of such a mosaic is as large as
the commitment sphere, then production of progenitorsof limited lineages can occur. We have named our model“mosaic commitment-inducing microenvironment model”.
To test whether our model can explain the in vivo
findings, we applied a computer simulation approach,in collaboration with Mariko Okada ( RCAI ) and Takashi
Nakakuki ( Kougakuin University ) . Simulations basedon this model nicely recapitulated the observed in vivo
findings ( Fig. D, E ) .

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

67

Laboratory forTranscriptional Regulation
Group Director:

Ichiro Taniuchi
Research Scientist : Taku Naito
Mari Tenno
Hirokazu Tanaka (SPR)
Wooseok Seo (JSPS)
Technical Staff :Sawako Muroi
Chizuko Miyamoto
Risa Chihara
Mayu Okoshi
Mika Ikegaya

Student Trainee :Sebastian Nieke (IPA)
Hisashi Wada (JRA)O

ne of fundamental questions in developmental biology is how the fate of progenitor/precursor cells
differentiating into opposing lineages is determined upon
exposure to environmental cues. Even as we learn more
about the link between cellular signaling and cell fate determination in the nucleus, other questions arise about
how genetic programming leads to establishment of cell
identity, which is maintained in differentiated cells in manycases. Research in my laboratory is directed toward understanding ( a ) how precursor cells sense external differentiation stimuli and then integrate them into a genetic
program for regulating fate decisions and ( b ) how the lineage-specific gene expression pattern established during
the commitment process is stably inherited in differentiated cells. We have been addressing these questions bystudying the mechanisms that govern lineage choice by
CD4+ CD8 + double-positive ( DP) thymocytes that differentiate into either CD4 + helper- or CD8 + cytotoxic-lineage T
cells after positive selection. The specificity of the TCR to
the MHC has been documented to correlate well with theoutcome of the CD4/CD8 lineage decision. In addition, recent genetic studies in mice demonstrated that expressionof the ThPOK transcription factor with accurate kinetics
and lineage-specificity is essential to link TCR/MHC specificity with CD4/CD8 lineage choice. Thus Thpok gene

regulation can serve as an ideal model to study how theanalog differences in TCR signals are converted into digitaloutcomes of cell fate, i.e. either CD4+ helper or CD8 + cytotoxic lineage cells.
Another research focus in my laboratory is on thefunction of Runx/Cbfb transcription factor complexes inthe development and homeostasis of the immune system.
By analyzing several mouse strains harboring mutations inRunx family genes, we have reported an essential requirement for Runx complexes in the development of a varietyof hematopoietic cells such as B-lymphocytes, lymphoidtissue inducer ( LTi ) cells and basophils. To gain a better
understanding of Runx/Cbfb complex functions, we are
exploring how these complexes are involved in regulationof immune cells and immune responses.

Timing of lineage choice after positive selection in
the thymus
Our previous studies have shown that a transcriptionalsilencer in the Thpok locus is essential to repress Thpok
expression specifically in cells developing into the cytotoxic-lineage, while a transcriptional enhancer in proximity to
the Thpok gene is essential to upregulate ThPOK expression with correct kinetics in cells developing into the helper-lineage. Given that the silencer-mediated Thpok repres-Recent Publications
1. Tanaka H, Naito T, Muroi S, Seo W, Chihara R, Miyamoto
C, Kominami R and Taniuchi I. Epigenetic Thpok silencing
limits the time window to choose CD4 + helper-lineage
fate in the thymus. EMBO J. 32, 1183-94 (2013)

68

2. Mucida D, Husain M.M, Muroi S, van Wijk F, Shinnakasu
R, Naoe Y, Reis B, Huang Y, Lambolez F, Docherty M,
Attinger A, Shui J.W, Kim G, Lena C, Sakaguchi S, Miyamoto C, Wang P, Atarashi K, Park Y, Nakayama T, Honda
K, Ellmeier W, Kronenberg M, Taniuchi I* and Cheroutre
H*. Transcriptional Reprogramming of Mature CD4 T

helper Cells generates distinct MHC class II restricted
Cytotoxic T Lymphocytes. Nat Immunol. 14, 281-9(2013) *corresponding author

Figure:sion already operates in precursors prior to exposure todifferentiation cues, i.e. positive selection signals by TCR
engagement, reversal of the Thpok silencer is required for
efficient induction of the Thpok gene. This also suggeststhat the Thpok silencer would function as a nuclear sensor and converter of TCR signals into regulatory cues tocontrol the expression status of the Thpok gene, a major
factor that discriminates these two lineages. These uniquecharacteristics of the Thpok gene have prompted us to extensively study mechanisms regulating its expression.
In the last few years, we have examined whether
epigenetic mechanisms are involved in Thpok gene regulation. By using a genetic strategy that allows conditionalremoval of the Thpok silencer, we obtained evidence
showing that an epigenetic silent state, which is established at the Thpok locus during commitment to the cytotoxic-lineage in thymus, can be then maintained without
the silencer in differentiated CD8 + T cells. Interestingly, two
promoters in the Thpok gene are repressed through distinct repressive epigenetic mechanisms. Strikingly, when
the silencer activity is enhanced by increasing its copy
number, epigenetic sealing of the Thpok locus can evenoccur in helper-lineage cells. Thus, the epigenetic machinery that confers crucial epigenetic marks onto the Thpok
locus can work independently of commitment to the cytotoxic-lineage. Collectively, our results provide a novel
epigenetic insight into the timing of the helper/cytotoxic
lineage decision. Even once signals through an MHC class
II-specific TCR open a gate to the developmental path
toward the helper-lineage, the gate is closed again if such
TCR signals are perturbed. Thus the decision to become

3. Muroi S, Tanaka H, Miyamoto C and Taniuchi I. Fine-tuning of Thpok gene activation by an enhancer in close
proximity to its own silencer. J Immunol. 190, 1397401 (2013)

Essential requirement for Runx3/Cbfb2 in development of
DETC and Langerhans cells
Our body is continuously exposed to a variety of pathogenic
microorganisms at the interface to the outer environment.
Primary immune soldiers are placed at such a frontier of theimmune battlefield during embryogenesis. In mice, Langerhans
cells (LC) and dendritic epidermal T cells (DETC) are present
for primary defense and provide a link between the frontiersand central systemic immunity. We found that Runx3/Cbfb2
complexes are essential for development of both cell types. In
mice deficient for Cbfb2, one of the RNA splicing variants of
Cbfb, both LC and DETC are undetectable in skin epidermis
by immune histochemical analyses. Immuno-histochemistry(A) and flow cytometer analyses (B) demonstrating theabsence of Langerin expressing LC and CD3 expressing
DETC cells in the epidermis of Cbfb2-deficient mice.

a helper-lineage cell must be made within a particular time
window, during which TCR signals must be sustained tokeep the gate open. Our on-going studies aim to identify
molecule ( s ) that work as the gate-keepers that switch thegate from close to open upon receiving TCR signals to
initiate ThPOK expression. We are also working to provide
evolutionary insights to understand how such a regulatory
mechanism that finely converts environmental cues into a
correct fate decision process is acquired during evolution.

Role of Runx complexes for placing immune cells at
the interface to the environment
Our body is continuously exposed to microorganisms at
the interfaces between body and environment. The skin is
one of such interface tissue, and inhibits invasion of microorganism mainly by forming a mechanistic and primitive
biological barrier of tightly connected keratinocytes layers. However, immune cells are present beneath this skinbarrier to sense invaders and initiate systemic immune
responses, if necessary, for eliminating them. In mice,
two types of immune cells are placed at the front of theimmune battle before the birth: Langerhans cells (LC ) anddendritic epidermal T cells ( DETC ) . Interestingly, we foundthat both of these cells are missing in a mutant mousestrain that lacks Cbfb2 protein ( Fig.) , encoded by one of
the RNA splicing variants of the obligatory Runx partner
protein Cbfb. Our ongoing analyses of these Cbfb2-deficient mice will provide valuable insights to understand thedevelopmental pathway of these primary immune soldiersand their roles in defense against pathogenic microorganisms at one of the immunologic frontiers.

4. Seo W., Ikawa T., Kawamoto H., Taniuchi I. Runx1/Cbfb
is essential for early B lymphocyte development throughregulation of Ebf1 expression. J Exp Med. 209, 125562 (2012 )5. Setoguchi R., Tachibana M., Naoe Y., Muroi S., AkiyamaK., Tezuka C., Okuda T., Taniuchi I. Repression of theTranscription Factor Th-POK by Runx Complexes in
Cytotoxic T Cell Development. Science 319, 816 -19( 2008 )69

Laboratory for

Cell Signaling

Group Director:

Takashi Saito

Senior Research Scientist: Tadashi Yokosuka
Research Scientist: Takayuki Imanishi, Reiko Onishi, Yasuo Shikamoto, Arata Takeuchi, Akiko Hashimoto-Tane, Shin-ichi Tsukumo
Visiting Scientist: Hiroshi Ike, Masato Tanaka, Yasutaka Wakabayashi, Sho Yamasaki
Technical Staff: Wakana Kobayashi, Machie Sakuma, Chitose Suzuki, Midori Unno
Student Trainee: Mohamed El Sherif Gadelhaq Gadlehaq Badr (IPA ), Yuya Anai
RCAI Central Facility FACS Laboratory: Hanae Fujimoto, Yukiko Hachiman
RCAI Central Facility Confocal Laboratory: Yasutaka Wakabayashi (Leica Microsystems), Ikuo Ishige (BM Equipment)
RCAI Central Facility mAb Laboratory: Tomomi Aoyama, Kazuyo Uchida (Program for Drug discovery and Medical technology platform)Tcells play central roles in immune regulation and are
responsible for immune defense against pathogens
and cancer. Because of their critical functions, impairment of the T cell activation process and function resultsin immune diseases. The group aims to determine themolecular mechanisms of activation, differentiation andhomeostasis of T cells in order to modulate T cell activation/function from the signal transduction perspective in
immune disorders. Particularly in studies of T cell activation, the group has been using real-time imaging analysis
to elucidate the spatiotemporal dynamic regulation of the
TCR signaling complex and signal transduction of related
downstream pathways. These studies also include theroles of positive/negative co-stimulation signals, innate-related signals and cytoskeletal regulation as well as tran-

scriptional regulation in T cell activation and functional differentiation. We also analyze regulation at later phases ofT cell activation for cell migration, functional differentiation,and establishment of peripheral effector functions andtolerance.

Dynamic regulation of T cell activation byco-stimulation
We have studied the dynamic movement of signaling molecules in the process of formation of the immunologicalsynapse ( IS ) and T cell activation upon antigen recognitionat the single-cell level by using a combination of imaging
techniques with model planar bilayers and total internalreflection fluorescence microscopy ( TIRF ) . We have identified TCR-microclusters ( MCs ) as the signalsome to as-

Recent Publications
1. Yokosuka T., Takamatsu M., Kobayashi-Imanishi W.,
Hashimoto-Tane A., Azuma M., Saito T.: Programmed
cell death 1 forms negative costimulatory microclusters
that directly inhibit T cell receptor signaling by recruiting
phosphatase SHP2. J Exp Med. 209, 1201-1217 ( 2012 )70

2. Hashimoto-Tane A., Yokosuka T., Sakata-Sogawa K.,
S akuma M., Ishihara C., Tokunaga M., S aito T.
Dynein-driven transport of T cell receptor microclusters
regulates immune synapse formation and T cell activation. Immunity 34, 919-931 (2011)

3. Kong K-F., Yokosuka T., Canonigo-Balancio A J., IsakovN., Saito T., Altman, A. A motif in the V3 domain of thekinase PKC-q determines its localization in the immunological synapse and functions in T cells via association
with CD28. Nat Immunol. 12, 1105 -1112 ( 2011)

Figure:semble signaling molecules and induce activation signals,
which then translocate into the center of the IS and form
the central supramolecular activation cluster (cSMAC).
We then analyzed the dynamic features of co-stimulation signals by the positive co-stimulatory receptor CD28
and its negative counterpart CTLA-4 and their relationshipwith TCR-MCs. CD28 is initially co-localized with TCRMCs and then accumulates into the cSMAC together with
PKCq for sustained co-stimulation. Later on, CTLA-4 also
accumulates in the same area of the cSMAC as CD28,
where it inhibits activation by competing with CD28 forligand binding. We next analyzed the dynamic regulationof another inhibitory co-stimulation receptor PD-1. Unlike
CTLA-4 accumulation in the cSMAC, PD-1 accumulates
in the TCR-MCs and there mediates inhibition of TCR
proximal signals by dephosphorylation through recruiting
the phosphatase SHP-2. PD-1-mediated inhibition of T
cell activation was observed in normal T cells expressing
PD-1 induced by repeated antigen immunization. Thus,
we have clarified the spatio-temporal dynamic regulationof positive/negative co-stimulation and how these signals
exhibit quantitatively fine-tuned regulation.

Regulation of T cell activation and differentiation
We have analyzed genes up-regulated during T cell development. To this end, we found that the putatively B
cell-specific transcription factor Bach2 is well-expressed

4. Yokosuka T., Kobayashi W., Takamatsu M., Sakata-Sogawa K., Zeng H., Hashimoto-Tane A., Yagita H.,
Tokunaga M., Saito T. Spatiotemporal basis of CTLA-4
costimulatory molecule-mediated negative regulation of
T cell activation. Immunity 33, 326-339 ( 2010 )Dynamic regulation of PD-1-mediatedinhibition of T cell activation throughPD-1-microclusters(Top) AND-TCR transgenic T cells
expressing PD-1-GFP and pre-stained
DyLight-anti-TCRb Fab were stimulated
on a planar bilayer containing PD-L1. Real
time imaging analysis revealed that PD-1
(green) was initially (2 min) co-localizedwith TCR-microclusters (red), and moves
to the cSMAC at 20 min where PD-1
accumulates in the TCR lo area, where
CD28 and CTLA4 also accumulate. For
PD-1-mediated suppression, it is criticalto be within the same TCR-microclusters.
(Bottom) T cells expressing both PD-1YPet and SHP2-CFP were stimulated on
the planar membrane with specific peptideMHC and analyzed for specific FRET
between PD-1 and SHP2. SHP2 wastransiently recruited to TCR-microclusters
upon stimulation in a PD-L1-dependent
manner and dephosphorylated upstream
signaling molecules.

even in T cells and up-regulated during T cell differentiation. Bach2 deficiency resulted in the reduction of naïve T
cells and up-regulation of genes related to effector-memory T cells, particularly to Th2-type cells in naïve T cells.
Bach2-deficient T cells induce rapid secretion of Th2
cytokines and memory-effector related molecules. We
identified the direct Bach2 target genes, which are also
regulated in effector-memory cells. Collectively, Bach2 isa critical repressor that maintains the naïve state of T cells
by suppressing effector-memory related genes.
We have also analyzed the regulation of T cell activation by signals important in innate immunity. After we
identified the critical roles of IRAK4 in T cell activation, we
analyzed the function of innate signal molecules, particularly TLRs and RLRs expressed in T cells. Initial analysis
revealed that TLR2 directly activates Th1 cells to induce
cytokine production independently of TCR stimulation.
Whereas T cell co-stimulation through TLR2 is TLR/
MyD88-dependent, surprisingly, the co-stimulation of T
cells by nucleic acids was found to be independent of
MyD88/TRIF. We found that for the optimal T cell co-stimulator y activity, nucleic acids have to be aggregatedby DNA-binding histones or other proteins. The direct
co-stimulation of T cells by nucleic acids independently of
the recognition by dendritic cells/macrophages provides
new insights into in vivo induction of inflammation and related immunological diseases.

5. 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 raftclustering. Mol Cell Biol. 30, 3421-3429 ( 2010 )71Research Unit forSingle Molecule Imaging

Unit Leader:

Kumiko Sakata-Sogawa

S

ingle molecule approaches enable us to capture transient intermediates and heterogeneous behavior, thus
avoiding ensemble averaging. This ability is powerful inelucidating mechanisms of cellular functions: which molecule interacts with what, when, where, and how it works
in living cells. Thus fluorescence imaging and quantitative
analysis of single molecules are valuable methods to study
the individual behavior of biological systems.
The main target of the Research Unit for Single Molecule Imaging is to elucidate immunological responses andsignaling processes with the technique of single molecule
imaging and quantification. To this end we installed amicroscope system and we have focused on the development of novel types of fluorescence microscopy ( HILO )for use in single cell/single molecule studies. HILO has anadvantage when it comes to observations inside cells withthe high signal to noise ratio of 7.6. In addition, we are developing analysis software tools for imaging and quantitative analysis. We are also applying a quantitative approach
to simulation of cell signaling. Our unit has a tight collaboration with the Research Unit for Molecular Systems Im-

munology for development of new microscopy techniques
and analysis software.

Spatial-temporal regulation of NF-κB inactivation by
PDLIM2
NF-κB is an important transcription factor that activates
expression of inflammatory genes in response to various
stimuli. Although the inflammatory response is an essential
reaction against invaders, overreaction could damage thehost. Accordingly, both activation and suppression are inevitable.
In resting cells, NF-kB is sequestered in the cytoplasmby binding to IκBα. Stimulation leads to phosphorylationof Ser residues of IκBα and the subsequent ubiquitination
results in degradation by the proteasome. As a result ofIκBα degradation, liberated NF-κB translocates to the nucleus and activates expression of target genes, including
the IκBa gene. The nascent IkBa binds to nuclear NF-kB
and exports it to the cytoplasm, resulting in negative feedback loop. In addition to this canonical IκBα regulatory
pathway, PDLIM2 was found to work as an E3 ubiquitin

Recent Publications
1. Hotta K, Nashimoto A, Yasumura E, Suzuki M, Azuma M,
Iizumi Y, Shima D, Nabeshima R, Hiramoto M, Okada A,
Sakata-Sogawa K, Tokunaga M, Ito T, Ando H, Sakamoto
S, Kabe Y, Aizawa S, Imai T, Yamaguchi Y, Watanabe
H, Handa H.: Vesnarinone Suppresses TNFa mRNA
Expression by Inhibiting Valosin-containing Protein.
Mol Pharmacol. 83, 930-938 (2013)72

2. 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)

3. Yokosuka T., Kobayashi W., Takamatsu M., Sakata-Sogawa K., Zeng H., Hashimoto-Tane A., Yagita H., Tokunaga
M., Saito T.: Spatiotemporal basis of CTLA-4-mediatednegative regulation of T-cell activation. Immunity 33,
326-339 (2010 )Figure :PDLIM2 translocates into the nucleus upon stimulation with PMA and ionomycin.
CHO cells expressing GFP-PDLIM2 and p65-RFP are imaged at the indicated time points after
stimulation. The upper images are focused on the cell membrane. The lower images are focusedon the nucleus. Bar: 10 μm

ligase responsible for the regulation of NF-κB activation(Tanaka, T. et al. Nat Immunol., 2007) . Despite the important role of PDLIM2 as a regulatory protein of inflammation,
little is known about its regulation mechanism.
Aiming to investigate why PDLIM2 works only after
stimulation, we performed a series of imaging analyses.
When a GFP-PDLIM2 fusion protein was expressed in cultured cells, the protein was localized, not in the nucleus,
but at the stress fibers near the plasma membrane. In fact,
PDLIM2 has been reported to bind α-Actinin1, an actin
binding protein, and to play an important role in cell adhesion and cell migration. Recent studies of monocytes indicates that PDLIM2, located in the nucleus in the immaturestate, is accumulated in the cytoplasm after PMA-induced
differentiation and nuclear activity of NF-κB is increased.
When we stimulated the GFP-PDLIM2 expressing cellswith PMA and ionomycin, PDLIM2 was translocated into
the nucleus within 10 min ( Fig ) . These findings suggestthat the function of PDLIM2 is in tight relation to its localization in the cell. This finding clearly explains why PDLIM2
degrades NF-κB only after stimulation. If PDLIM2 was freein the cytoplasm, it might ubiquitinate NF-κB there.
The PDLIM2 molecule consists of PDZ and LIM domains. We expressed the GFP fusion protein of the mutant PDLIM2 protein ( ΔPDZ ) in HeLa cells and observed ahomogenous distribution including the nucleus. This indicates that the PDZ domain is responsible to the sequestration at the stress fibers. Next we tested the effect of mutation of serine phosphorylation sites in the PDZ domain.
We mutated 5 serine residues into alanine or aspartic acid.
Although the alanine substitutions showed no difference
from WT, mutation into aspartic acid resulted in a homogenous distribution of PDLIM2. This result suggests thatPDLIM2 localization is regulated by the phosphorylation ofserine residue ( s ) in the PDZ domain.

73

Laboratory for

Lymphocyte
Differentiation
Group Director:

Tomohiro Kurosaki
Research Scientist : Yuich Aiba, Kohei Kometani			

Ryo Shinnakasu

Visiting Scientist :						Yoshihiro Baba, Wataru Ise
Takeshi Inoue, Masanori Matsumoto
Rinako Nakagawa

Technical Staff :Yoko Fujii, Shiori Maeda, Miwako TochigiBcells play an essential role in the regulation of immune
responses. Upon first encountering their cognate
antigen, B cells exert multiple functions including antibody
production, antigen-presentation, and induction of T celldifferentiation. When B cells recognize the same antigen the second time, memory antibody responses canbe induced. These are typically seen in the response to
T-cell-dependent antigens and are characterized by the
rapid production of high-titers of high-affinity antigen-specific antibody. Classical studies have focused on isotype-switched memory B cells ( mainly of the IgG isotype ) .
However, recent advances in monitoring antigen-experienced B cells have revealed the existence of un-switched
IgM type memory B cells, suggesting the functional heterogeneity of memory B cells. Our laboratory has now focused on clarifying the functional differences between IgM
and IgG type memory B cells and on revealing the mechanisms underlying the robustness of memory antibody
responses.

Unique function of IgM memory B cells
Fate mapping-methods have allowed us to monitor bothun-switched ( IgM-type ) and switched ( IgG-type ) memory
B cells after injection of NP-CGG into mice. When B cells
encounter antigens in vivo, almost all the cells induce AID
at the transcriptional level. Thus, we can genetically label
the antigen-experienced B cells by using AID expression.
Employing this system, we have isolated two subsets of

IgM type memory B cells ( IgM + IgD + and IgM + IgD - ) andclarified that IgM + IgD + memory B cells undergo somatic
mutation, whereas the IgM + IgD - cells do not. These observations suggest that IgM type memory B cells are generated through both GC-dependent ( IgM + IgD + ) and independent ( IgM + IgD - ) pathways.
When IgM type memor y B cells were adoptivelytransferred together with activated T cells, these B cells
re-initiate the GC reaction. In contrast to IgM type memory
B cells, IgG type memory B cells showed less expansion
activity and have a high propensity to differentiate into
plasma cells ( Figs. 1 and 2 ) . Thus, IgM type memory
B cells can be considered as a more central type ofmemory cells.
Furthermore, activation of IgM type, but not IgG type,
memory B cells was severely inhibited by addition of anti-NP specific IgG antibodies, suggesting that only IgMtype memory B cells are negatively regulated by generated
Abs. Thus, the following scenario can be envisaged; once
titers of antigen-specific IgG Abs that are produced in the
primary response are sufficiently reduced, then IgM type
memory B cells could get activated. Together, the above
results suggest that IgM and IgG type memory B cells
have distinct features.

Contribution of the transcription factor Bach2 togeneration and function of IgG type memory B cells
Two-non-mutually exclusive models ( BCR-intrinsic andRecent Publications
1. Yasuda T., Kometani, K., Takahashi, N., Imai, Y., Aiba,
Y. and Kurosaki, T. Erk kinases control plasma celldifferentiation by regulating expression of Blimp-1.
Sci Signal. 4, (169 ) ra25 ( 2011)74

2. 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, 14471457 ( 2011)

3. Onodera, T., Takahashi, Y., Yokoi, Y., Ato, M., Kodama,Y., Hachiman, S., Kurosaki, T. and Kobayashi, K. Memory
B cells in the lung participate in protective humoral immune responses against recurrent pulmonary influenza
virus infection. Proc Natl Acad Sci USA. 109, 24852490 ( 2012)

Figure 1: Differentiation of IgM and IgGmemory B cells after antigen
re-exposure
IgM type memory B cells highly
re-enter the germinal center(GC) reaction, but are less able
to differentiate into plasma cells
after re-exposure to the antigen.
IgG type memory B cells showless expansion and preferentiallydifferentiate into plasma cells.

Figure 2: IgM and IgG memory B cells differentiate
via distinct pathways.
IgM type memory B cells mainly differentiate
into germinal center B cells and generate
descendent memory B cells. On the contrary,
IgG type memory B cells preferentiallydifferentiate into plasma cells and protect our
body from pathogens.

BCR-extrinsic ) to explain the robust antibody responsesof IgG type memory B cells have been long debated. By
establishing a mouse model in which antigen-non-experienced B cells express an IgG1 BCR, we previously
addressed this question, and showed that a BCR-intrinsic
mechanism alone is not sufficient to explain the heightened differentiation activity of IgG type memory B cells.
As an extrinsic model, we proposed that reorganizationof transcription factors such as Bach2 takes place during
generation of IgG1 type memory B cells after primary antigen exposure, and is critical for rapid responsiveness of

4. Dong, Z., Davidson, D., Perez-Quintero, L.A., Kurosaki,T., Swat, W., and Veillette, A. The Adaptor SAP Controls
NK Cell Activation by Regulating the Enzymes Vav-1 andSHIP-1 and by Enhancing Conjugates with Target Cells.
Immunity 36, 974-985 ( 2012 )IgG1 memory B cells ( BCR-extrinsic model ).
To directly test our model, we first demonstrated thatthe level of Bach2 was repressed after antigen encounter.
Then we have established mice in which Bach2 could beoverexpressed or knocked out in an inducible manner.
When Bach2 was overexpressed after IgG1 class-switching, more IgG1 type memory B cells were generated, but
the secondary Ab responses were decreased. These
results clearly suggest that the expression level of Bach2
is involved in generation of memory B cells as well as intheir activation.

5. Takahashi, Y., Onodera, T., Kobayashi, K., and Kurosaki,
T. Primary and Secondary B-Cell Responses to Pulmonary Virus Infection. Infect Disord Drug Targets. 12,
232-240 ( 2012 )75

Research Unit for

Immunodynamics
Unit Leader:

Takaharu Okada
Research Scientist:
		 Saya Moriyama (SPDR)
Technical Staff:
		 Noriko Takahashi
		Kyounga Seo
		Akiko Takumi
Student Trainee:
		 Takashi Ikeno (JRA)T

he goal of our research is to understand the mechanisms regulating cell migration and interactions in thetissues that shape immune responses. For this purpose
we use real time imaging, in particular two-photon microscopy, to analyze in vivo cellular migration and interactions.
This microscopy method, which was introduced recently
to the field of immunology, has been revealing striking
dynamics of immune cells in the lymphoid organs, underlining the importance of this approach to resolve the complexity of the immune system. Last year, we started new
projects to understand mechanisms controlling dynamics
of two different T cell types, follicular helper T ( Tfh ) cells
and cytotoxic T cells, which play pivotal roles in humoral
and cellular immunity, respectively. This year, we havecontinued to work on these two topics.

Dynamics of GC Tfh cells
Tfh cells are essential for initiation and maintenance ofT-dependent B cell responses, particularly germinal center( GC ) reactions, which ultimately lead to long-term, highaffinity antibody production. A subpopulation of Tfh cells
that has physical access to the GC is believed to be responsible for controlling GC reactions. However, little has
been known about mechanisms that regulate dynamics ofGC Tfh cells. Last year, we performed two-photon imaging

analysis of the migration of Tfh cells within the GC area
and the surrounding follicular mantle ( FM ) . The analysis
clearly showed that Tfh cells in the GC and those in the FM
tended to be retained in the respective regions, suggesting
differential expression of molecules responsible for theirlocalization. Then, we performed gene expression analysis
of Tfh cells harboring hypomorphic mutations in the geneencoding Bcl6, which is the essential transcription factor
for Tfh cells, because the Bcl6 hypomorphism led to a selective reduction of GC Tfh cells while leaving FM Tfh cells
relatively intact. As a result of this gene expression analysis, we found that expression of a G coupled-receptor
gene might be enriched in GC Tfh cells. Then, we generated a fluorescent reporter animal for this gene. By using thereporter mouse, we could show by histology that a Tfh cellsubset highly expressing this G protein-coupled receptor
was localized in the GC but not in the FM. Furthermore,
deficiency of the receptor led to a reduction of the Tfh cellnumber within GCs. Two-photon imaging analysis revealed
that the reduction of GC Tfh cells was due to impaired
retention of Tfh cells in the GC. We are currently investigating the impact of this impaired Tfh cell retention in the GC
on long-term antibody responses.
Overall, our work during the last several years contributed to understanding of the relationship between Bcl6

Recent Publications
1. Okada T., Moriyama S., Kitano M. Differentiation ofgerminal center B cells and follicular helper T cells
as viewed by tracking Bcl6 expression dynamics.
Immunol Rev. 247, 120-132 (2012 )76

2. Hirata E., Yukinaga H., Kamioka Y., Arakawa Y., Miyamoto S., Okada T., Sahai E., Matsuda M. In vivo
fluorescence resonance energy transfer imaging
reveals dif ferential activation of Rho-family GTPases in glioblastoma cell invasion. J Cell Sci. 125,858-868 (2012 )3. Kitano M., Okada T. Four-dimensional tracking of lymphocyte migration and interactions in lymph nodes by
two-photon microscopy. Method Enzymol. 506, 437454 ( 2012 )Medulla / Red Pulp

T cell zone

B-Tboundary

Bcl6lo/int T cell

Figure :IFR

Bcl6hi T cell

OFR

GC

Blimp1int-hi Eﬀector
T cell (precursor)

Bcl6lo/int pre-GC cell

Bcl6hi pre-GC / GC B Cell

Blimp1lo Plasmablast

Blimp1int-hi
Plasmablast/Plasma cell

Bcl6-/loCentralmemoryT cellChange label: Bcl6 lo/int pre-GC B cell
A model for the relationship between antigen-specific interactions and differentiation of CD4+ T cells andB cells in various microenvironments. Solid arrows indicate the connections supported by relatively direct
evidence. Dashed arrows indicate possible connections suggested by circumstantial evidence. IFR: interfollicular region, OFR: outer follicular region.

expression dynamics and Tfh cell differentiation. It is now
becoming clear that Tfh cells contain diverse subsets withdifferent localization and different roles in B cell responses.
Furthermore modulation of Bcl6 expression is proposed to
be an important event for development of memory helper
T cells from Tfh cells (Fig.) .

Cellular interaction dynamics during cytotoxic T celldifferentiation
Cytotoxic T cells are known to be generated through interactions of antigen-specific CD8 + T cells with dendritic
cells ( DCs ) that cross-present antigens. In skin-draining
lymph nodes, there are two subsets of cross-presenting
DCs, namely CD103 + migratory DCs immigrating from the

4. Baumjohann D, Okada T, Ansel KM. Cutting Edge:
Distinct waves of BCL6 expression during T follicularhelper cell development. J Immunol. 187, 2089-2092(2011)

skin and CD8 + lymph node-resident DCs. However, thespatio-temporal regulation of their interactions with CD8 +T cells is poorly understood. In collaboration with Dr. Kaisho’s group ( Lab for Host Defense, RIKEN RCAI; and Immunology Frontier Research Center, Osaka University) , wedeveloped novel methods that allow visualization of these
two subsets of cross-presenting DCs in the lymph node.
Two-photon microscopy analysis suggested differential
contribution of the cross-presenting DC subsets to activation of antigen-specific CD8 + T cells. Furthermore, ourresults have begun to reveal a previously unknown compartmentalization of the DC subsets within the T cell zoneof the lymph node. The physiological significance of thiscompartmentalization is currently under investigation.

5. 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)77

Research Unit for

Molecular Systems Immunology
Unit Leader :Makio Tokunaga
Student Trainee: Yuma Ito
		
		
		
		
		
		
		

Jun Takimoto
Katsuo Ichinomiya
Masahiro Shimozawa
Ryuta Okada
Naomichi Inaba
Satoshi Ikeda
Hiroshi OyamaOur laboratory has developed technologies that allow
immune responses and signaling processes to bevisualized at the single-molecule level. Single molecule
imaging coupled with the ability to simultaneously visualize
several different proteins in cells has enabled the quantification of molecular dynamics, interactions, and kinetics.
Based on these three-dimensional and temporal parameters, we examine numerical modeling and computer simulations of cell functions. Using the combination of single
molecule quantification and “in silico” modeling, we aim toopen up new frontiers for understanding immune cells as
molecular systems.

Single Molecule Imaging and Molecular
Quantification in Cells
We have demonstrated that clear visualization of single
molecules in cells enables their molecular quantification.
Clear single-molecule visualization was achieved using
TIRF and HILO microscopy. The main technical challengeof single-molecule fluorescence imaging is increasing thesignal/background ratio. We have been involved in thedevelopment of total internal reflection fluorescence ( TIRF )microscopy, a light-microscopic technique. TIRF is now
widely used for single-molecule imaging at cell surfaces,
but cannot be used for molecular imaging inside cells.
To overcome this limitation, we have devised a new ap-

proach, called highly inclined and laminated optical sheet( HILO ) microscopy for single molecule imaging inside cells( Fig. 1a ) . We have achieved notable success in increasing
the signal/background ratio by inclining the illumination
beam and by minimizing the illumination area. The incident
laser beam is highly inclined by a large refraction, and islaminated as a thin optical sheet at the specimen side.
In HILO microscopy, this thin optical sheet is used forillumination.
To evaluate the HILO microscopy technique, we visualized single molecules of the Sp1 transcriptional factor
Sp1 in a living cell ( Fig. 1b ) . Clear point-like images of single Sp1 molecules were obtained without the need for deconvolution to remove out-of-focus haze. The fluorescenceintensity of the Sp1 point images corresponded well to thetheory of image formation. Further, there was much lessphotobleaching than in conventional confocal microscopy
because of the lower intensity and non-focused nature of
the illumination.
Reduction of the background intensity of images
yields clear images. The background intensity of images
depends on the volume of the illuminated region in specimens. To examine the thickness of the illuminated region,
we obtained intensity profiles of illumination along the z-direction, that is, the depth direction in specimens. We devised a method to decrease the illumination thickness by

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 )

abaFigure 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 incells. Illumination by a highly-inclined and thin beam increases image
intensity and decreases background intensity, yielding a signal/
background ratio up to about eightfold greater than that of epiillumination. A high ratio yielded clear single-molecule images andthree-dimensional images. (b) To evaluate the HILO microscopy
technique, we visualized single molecules of the Sp1 transcription
factor in a living cell. Molecular dynamics and interactions can bequantified by image analysis using these single molecule images. Bar,
5 µm.

narrowing the illumination area using a field stop. Notably,
we have achieved the reduction of the illumination thickness to less than 10 µm ( for example, 7 µm of thickness
with a field stop for 20 µm-diameter illumination ) .
Further, we evaluated the signal/background ratio ofimages in HILO. Inclination of the illumination beam increases intensities of the fluorescence images up to 2.8fold compared with epi-illumination. The 2.8-fold increaseis in excellent agreement with the theory. By contrast,
the background intensity is substantially decreased byillumination inclination. As the background is composedof out-of-focus images, the decrease is explained by the
reduction of the illuminated range. As a result, illumination
inclination increased the ratio of image to background( signal/background ) up to 3.1-3.5-fold. Reduction of theillumination diameter further decreased the background intensity. Consequently, reduction of the diameter increased
the ratio of signal to background up to 2.2-2.9-fold. Overall, the HILO illumination microscopy approach notably
increased the signal/background ratio up to 7.6-fold.
To explore potential new uses of this technology,
we performed quantitative analysis of nuclear import todemonstrate its application to kinetic studies. We could
visualize single molecules of GFP-importin β mediating theimport of cargo through nuclear pores in cells as brightspots on the nuclear envelope. Molecular interactions
with the assembled Nuclear Pore Complexes (NPC) were

4. Fukagawa, A., Hiroshima, M., Sakane, I., Tokunaga M.:
Stochastic emergence of multiple intermediates detectedby single-molecule quasi-static mechanical unfolding ofprotein. Biophysics 5, 25-35 (2009 )b

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 can be quantified
by image analysis using these single molecule images. Bar, 5µm. (b) Aiming at understanding immune cells as molecularsystems, we plan to construct “in silico” cell models basedon single-molecule quantification. Bidirectional researchis essential to reconstruct cell functions in silico; researchfrom molecules to systems by single molecule analysis, andfeedback research from systems to molecules.

quantified by single molecule analysis. Retention times,
the number of associated molecules, the dissociation constant, and stoichiometry of import were all determined.

“In silico” Modeling and Simulation
As shown above, molecular interactions with the assembled NPC were quantified by single molecule analysis. In
order to understand the molecular mechanism of nuclear
import, a numerical model of import was constructed using these kinetic parameters. Computer simulation was
carried out based on the model with two types of bindingsites. The simulation fit very well with both the results ofsingle-molecule experiments and the molecular kinetic
features in cells.
We are now expanding the simulation studies into
whole-cell simulation of single lymphocytes based on
single molecule imaging and quantification ( Fig. 2 ) . Directcomparison with molecular imaging is indispensable forthe simulation, since the values of the parameters have ahuge number of degrees of freedom.
We demonstrated that clear visualization of single
molecules in cells enabled accurate quantification. The
combination of single molecule quantification and “in silico” reconstructions of cell functions opens new approaches for developing molecular system biology in immunology
and other fields.

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

Immune Cell
SystemGroup Director:

Shigeo Koyasu

Senior Research Scientist: Kazuyo Moro
Technical Staff:

Miho Mochizuki
Natsuki Takeno
Sachika Wada

Student Trainee:

Jun-ichi Furusawa
Takaharu Sasaki
Takeshi Tanoue
Satoshi Koga (JRA)Yuji Nagano

Visiting Scientist:

Kenya Honda
Koji AtarashiThe immune system plays a critical role in host defense
against invading microbes. Immune reactions are also
involved in the onset and pathophysiology of many diseases including allergic and autoimmune diseases. Different
types of both innate and adaptive immune cells interact
with each other in order to induce complex immune reactions. Recent studies have identified multiple subsets
among innate lymphocyte corresponding to Th subsetsof the adaptive immune system. NK cells, LTi cells andNH cells play distinct roles in innate immune responses
by producing Th1, Th17 and Th2 cytokines, respectively.
Cooperation between these innate lymphocytes and antigen-specific T and B cells are likely important in various
inflammatory responses including protective immunity
against various microbes and allergic diseases such asasthma. We are elucidating the immune cell network inorder to understand how immune cells induce complex
immune reactions in our body.

and IgA production. Upon helminth infection or allergic
response, NH cells respond to a combination of IL-2 andIL-25 or IL-33 to produce large amounts of Th2 cytokines,
most notably IL-5 and IL-13, which induce eosinophilia andgoblet cell hyperplasia and play an important role in anti-helminth immunity and pathogenesis of allergic diseases in the lung such as asthma ( Fig. 1) . After we reported
NH cells, other groups also reported novel Th2 cytokine
producing innate cells, such as MPP type2 cells, nuocytes,
and innate helper type 2 ( Ih2 ) cells. There are similarities
and differences among these newly identified cell populations and NH cells, e.g., MPP type2 cells can differentiate
into other myeloid cells, making this cell type distinct fromthe others. MPP type2 cells, nuocytes, and Ih2 cells werereported to respond to IL-25 alone, but NH cells do notrespond to IL-25 without IL-2 but respond strongly to IL-33.
Localization of NH cells in FALC is also one of intriguing
differences from the other cell types, which are found inlymph node and/or spleen.

Studies on natural helper (NH ) cells
We currently focus on an innate lymphocyte populationthat we first identified in a new lymphoid tissue in mouseand human mesentery that we termed fat-associated lymphoid cluster ( FALC ) . We discovered in the FALC a new
innate lymphocyte subset producing Th2 cytokines andnamed this new lymphocyte “Natural Helper ( NH ) Cell”.
NH cells constitutively produce IL-5, IL-6 and IL-13 andsupport the proliferation of B1 cells in the peritoneal cavity

Studies on Th17 cells
In the adaptive immune system, we have been interested
in the role and differentiation of IL-17-producing helper T( Th ) , Th17. We have shown that the phosphoinositide 3-kinase ( PI3K )- Akt-mammalian target of rapamycin complex
1 ( mTORC1) axis plays a critical role in the differentiation of
Th17 cells. We demonstrated that the suppression of thePI3K-Akt-mTORC1 axis by deletion of p85α or with PI3K/

Recent Publications
1. Kurebayashi Y., Nagai S., Ikejiri A., Ohtani M., IchiyamaK., Baba Y., Yamada T., Egami S., Hoshii T., Hirao A.,
Matsuda S., Koyasu, S. PI3K-Akt-mTORC1-S6K1/2 axis
controls Th17 differentiation by regulating Gfi-1 expression and nuclear translocation of RORγ. Cell Rep. 1,
360-373 (2012 )80

2. Ouchi T., Kubo A., Yokouchi M., Adachi T., Kobayashi
T., Kitamura D. Y., Fujii H., Clausen B. E., Koyasu S.,
Amagai M., Nagao, K. Langerhans cell antigen capture
through tight junctions confers pre-emptive immunity
in experimental staphylococcal scalded skin syndrome.
J Exp Med. 208, 2607-2613 (2011)

3. Moro K., Yamada T., Tanabe M., Takeuchi T., Ikawa
T., Kawamoto H., Furusawa J.-I., Ohtani M., Fujii H.,
Koyasu S. Innate production of Th2 cytokines by adipose
tissue-associated c-Kit + Sca-1+ lymphoid cells. Nature
463, 540-544 ( 2010 )Figure 1: Functions of NH cells(a) NH cells constitutively produce IL-5 andIL-6 and support IgA production by B cells.
In addition, IL-5 supports the growth of B1
cells in the peritoneal cavity. (b) Upon helminth infection, IL-33 produced by epithelial cells, endothelial cells and/or adipocytes
induces high levels of IL-5, eotaxin and IL13 production by NH cells, resulting in eosinophilia and goblet cell hyperplasia, whichplay important roles in the early phase ofanti-helminth immune responses. Similarly, acombination of IL-2 and IL-25 also induces
large amounts of IL-5 and IL-13 from NH
cells.

Figure 2 : The PI3K-Akt-mTORC1 axis and HIF-1
play critical roles in Th17 induction.
Downstream of the PI3K-Akt-mTORC1
axis, S6K1 downregulates Gfi1, a negative
regulator of Th17 differentiation, and S6K2,
a nuclear counterpart of S6K1, binds RORγ,
a critical transcription factor for Th17 differentiation, and carries it to the nucleus. In
addition, a positive feedback loop between
HIF-1 and mTORC1 accelerates Th17 celldifferentiation.

mTORC1 inhibitors, as well as T cell-specific deletion of
raptor, an essential component of mTORC1, impairs Th17
differentiation in vitro and in vivo in a S6K1/2-dependent
fashion. Inhibition of the PI3K-Akt-mTORC1-S6K1 axis
impairs the downregulation of Gfi1, a negative regulator of
Th17 differentiation. Furthermore, S6K2, a nuclear counterpart of S6K1, is induced by the PI3K-Akt-mTORC1 axis,
binds RORγ, and carries RORγ to the nucleus. These results point toward a pivotal role of the PI3K-Akt-mTORC1S6K1/2 axis in Th17 differentiation. During this study,
we have noticed that, in addition to mTORC1, hypoxia
inducible factor-1 (HIF-1, consisting of HIF-1a and HIF-1b)
regulates Th17 differentiation positively as well. Oxygen(O2 ) tension is generally low in these secondary lymphoidtissues compared to the bloodstream or atmosphere.
However, the effect of changes in O 2 concentration on the

4. Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara
T, Momose Y, Cheng G, Yamasaki S, Saito T, Ohba Y,
Taniguchi T, Takeda K, Hori S, Ivanov II, Umesaki Y, Itoh
K, Honda K. Induction of colonic regulatory T cells byindigenous Clostridium species. Science 331, 337-41(2010 )

differentiation of Th cells remains unclear. We primed naïve
CD4 + T cells under 5% O 2, which has been observed in
the lymph node and spleen, and then reoxygenated thecultures under normoxia that mimicked the O 2 concentration in blood. In this model, the differentiation of Th17
cells, but not Th1 or iTreg cells, was enhanced. At 5% O 2,mTORC1 was activated and this led to the stabilization of
HIF-1a in Th17 cells. The activation of mTORC1 and the acceleration of Th17 cell differentiation, which occurred whencells were primed under 5% O2, were not observed in theabsence of HIF-1a but were accelerated in the absenceof von Hippel-Lindau tumor suppressor protein ( vHL ) ,a factor critical for HIF-1a degradation. Thus, a positive
feedback loop between HIF-1a and mTORC1 induced byhypoxia followed by reoxygenation accelerates Th17 celldifferentiation ( Fig. 2 ) .

5. Savitsky DA, Yanai H, Tamura T, Taniguchi T, Honda K.
Contribution of IRF5 in B cells to the development ofmurine SLE-like disease through its transcriptional control of the IgG2a locus. Proc Natl Acad Sci USA. 107,10154-9 (2010 )5. Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T,
Karaoz U, Wei D, Goldfarb KC, Santee CA, Lynch SV,
Tanoue T, Imaoka A, Itoh K, Takeda K, Umesaki Y, Honda
K, Littman DR. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485-98 (2009)81

Laboratory for

Epithelial Immunobiology
Team Leader:

Hiroshi Ohno
Research Scientist:
Shinji Fukuda (~May, 2012) ,Zijin Guo, Takashi Kanaya,
Gaku Nakato, Eiji Miyauchi
Research Associate: Tamotsu Kato
Technical Staff:
Yumi Chiba, Kumiko Nakai,
Masumi Ohmae (=June, 2012) ,Sayuri Sakakibara, Chikako Uetake,Ayako Yamashita
Student Trainee:
Toshi Jinnohara (JRA), Kazunori Kadokura,
Hideaki Shima (JSPS), Misato Hanazato (JRA),Akemi Fujiwara (JRA), Masami Hachisuka,
Kairi Nakamura, Masaki TakahataThe mucosal epithelium that lines the inner surfaces ofthe body, especially within the intestine, is exposed
to a wide range of antigens, including food-derived macromolecules and microorganisms as well as numerous
commensal bacteria, collectively called the intestinal microbiota. Appropriate recognition of these antigens is vitalfor maintaining immune homeostasis. Epithelial cells thatoverlay the gut-associated lymphoid tissue ( GALT ) , such
as Peyer’s patches ( PPs ) and isolated lymphoid follicles,
are distinct from absorptive epithelial cells of the villi andare termed follicle-associated epithelium ( FAE ) . The FAE
contains a specialized subset of epithelial cells, the M
cells, which play a pivotal role in mucosal immune surveillance by delivering luminal microorganisms to the underlying lymphoid cells via transcytosis. One of the primaryaims of our laboratory is to understand the mechanismsthat underlie the differentiation and function of FAE and M
cells. Our research team is also investigating the interaction of commensal microbiota with the intestinal epithelium
and its systemic influence on the physiology and pathologyof the host. These studies may lead to the developmentof novel and more efficient mucosal vaccination protocols/
drug delivery systems as well as functional foods/preven-tive medicine based on host-microbiota interactions.

Understanding of M-cell differentiation steps
M cells are atypical epithelial cells specialized for uptakeof luminal antigens, including the phagocytosis of viruses
and bacteria, to deliver them to dendritic cells that accumulate beneath the FAE. Recently, Dr. Ifor I. Williams’
group discovered that RANKL expressed by the stromalcells underneath the FAE is essential for the induction of
M cells. They have further shown that systemically administered exogenous recombinant RANKL induces ectopic
M-cell differentiation in the small intestinal villi. In collaboration with Dr. Williams, we employed RANKL-induced invivo M-cell differentiation and identified Spi-B as an M-cell
specific transcription factor highly induced within a few
hours upon RANKL treatment. In collaboration with Dr.
Tsuneyasu Kaisho, who has established Spi-B KO mice,
we have found that there are no mature M cells in FAE of
the KO mice. Accordingly, translocation of Salmonella enterica serovar Typhimurium and Yersinia enterocolitica into
PP is markedly diminished in Spi-B KO mice compared towild-type mice. As a consequence, S. enterica Typhimurium-specific T-cell activation in PP is also strongly sup-Recent Publications
1. Kanaya T, Hase K, Takahashi D, Fukuda S, Hoshino
K, Sasaki I, Hemmi H, Knoop KA, Kumar N, Sato M,
Katsuno T, Yokosuka,O, Toyooka K, Naka, K, Sakamoto
A, Kitahara Y, Jinnohara T, McSorley SJ, Kaisho T,
Williams IR, Ohno H. The Ets transcription factor Spi-B
is essential for the differentiation of intestinal M cells.
Nat Immunol. 13, 729-736 ( 2012 )82

2. Nakato G, Hase K, Suzuki M, Kimura M, Ato M, Hanazato M, Tobiume M, Horiuchi M, Atarashi R, Nishida N,
Watarai,M, Imaoka K, Ohno H Brucella abortus exploits a cellular prion protein on intestinal M cells as aninvasive receptor. J Immunol. 189, 1540-1544 ( 2012 )3. Fukuda S., Toh H., Hase K., Oshima K., Nakanishi Y.,
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 protect from enteropathogenic infection through production of acetate. Nature
469, 543-547 (2011)

Figure:Lack of M cells in Spi-B KO mice
Whole mount GP2 staining of PPs from wild-type and Spi-B KO mice was performed. The FAEof Spi-B KO mice is devoid of GP2+ mature M cells. GP2 is shown in green, while F-actin isstained in blue. Scale bar: 80 μm.

pressed. These observations clearly indicate that Spi-B
serves as a key transcription factor intrinsic to intestinal
epithelial cells for M-cell differentiation, and that Spi-B KO
mice provide a useful animal model for elucidating in vivo
roles of M cells.

Elucidation of host-intestinal microbiota
interactions important for host immunityand defense mechanisms
Intestinal microbiota impacts on human physiology andpathology. Certain of the commensal microbiota, such as
those belonging to the bacterial genus Bifidobacterium,
have beneficial effects on our health. Among the mostdistinctive benefits of these bacteria are the modulation ofhost defense responses and protection against infectious
diseases. Nevertheless, the molecular mechanisms underlying these beneficial effects have barely been elucidated. To address this important but complex question, we
have developed a comprehensive ‘multi-omics’ approach,where exhaustive analyses such as (meta )genomics, (meta )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 )transcriptomics and metabolomics are combined. We employed a simplified model of lethal infection of germ-freemice with enterohaemorrhagic Escherichia coli O157:H7( O157 ) associated with certain bifidobacterial strains toprove that our multi-omics approach is useful for analyzing host-microbial interactions. We showed that genes
encoding ATP-binding-cassette ( ABC ) -type carbohydratetransporters present in certain bifidobacteria contribute toprotecting mice against death induced by O157. The bacteria with these transporters were able to produce acetatefrom fructose. Our data strongly suggest that the acetateproduced in large amounts by these bifidobacteria exerts
its action on the colonic epithelium by inducing anti-inflammatory and/or anti-apoptotic effects, which prevent translocation of the O157 Shiga toxin from the gut lumen into
the blood. We are now extending our study to elucidate
the effects of commensal microbiota, as well as probiotics
and prebiotics, on the host immune systems and disease states in mice and humans with normal commensal
microbiota.

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 )83

Laboratory for

Mucosal Immunity
Team Leader:Sidonia Fagarasan
Research Scientist:
Shimpei Kawamoto
Michio Miyajima
Lucia Megumi Kato
Duncan Sutherland (JSPS)
Visiting Research Scientist:
Giuliana Magri
Research Assistant:
Mikako Maruya
Technical Staff:
Yasuko Doi
Yumi TsutsuiThe gut is colonized by trillions of bacteria that perform
vital metabolic and defensive functions for the host.
These bacteria, generally called indigenous bacteria or the
microbiota, coevolved with mammals and for this reason
are integral in digestive and immune system function andhomeostasis. Besides serving as a natural defense against
invading pathogens, indigenous bacteria make important
contributions to nutrient processing, generation of immunomodulatory products ( vitamins, short-chain fatty acidsetc.) and to the development of the immune system. In
spite of having available a wealth of immune sensing andeffector mechanisms capable of triggering inflammationin response to microbial intrusion, we can live together
with our body’s bacteria without any adverse effects. Thisis made possible by a continuous “dialog” between bacteria and host cells that generates finely tuned signaling
programs ensuring a state of non-responsiveness against
dietary antigens and controlled responses against indigenous bacteria. In turn, the immune system maintains
diverse and healthy bacterial communities in the gut, facilitated by innate and adaptive immunity, by mechanismsthat remained to be clarified.
Our long-term goal is to understand the host-microbial relationship in the gut and to apply such knowledge forhealth maintenance and disease prevention/treatment.

Biology of gut germinal centers: roleof PD-1 in selection of IgAs in germinal
centers of Peyer’s patches
Immunoglobulin ( Ig ) A, the most abundantly produced antibody isotype in mammals, is secreted into the gut lumen
as dimeric molecules linked by a ‘joining’ chain [ also called
secretory ( S ) IgA ] . The protective role of SIgA was elucidated in the context of mucosal infections. However, it isincreasingly clear that IgA was selected and maintained
throughout evolution, not just to stop pathogen entry
through the epithelium, but rather to maintain the complex
interplay between microbiota, epithelium and the immune
system.
Diversification of the IgA repertoire by somatic hypermutation ( SHM ) appears critical to maintain intestinal
homeostasis. SHM takes place mostly in the specialized
microenvironment of the germinal center ( GC ) , in the presence of activation-induced cytidine deaminase (AID) and asubset of CD4 T cells named T follicular helper ( TFH ) cells.
TFH cells are defined as T cells expressing high levels of thechemokine receptor CXCR5 and the inhibitory co-receptor
programmed cell death-1 ( PD-1) . We sought to elucidate
the role of PD-1 on TFH cells and GC biology.
We discovered that, in gut, PD-1 is an essential component of the program that regulates IgA selection re-

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 compositionin the gut. Science 336, 485-489 (2012 )84

2. Wei M., Shinkura R., Doi Y., Maruya M., Fagarasan S.,
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-dependenttranscriptional activation of the nuclear factor of activated T cells c1 (NFATc1) is critical for the development andsurvival of B1 cells. Proc Natl Acad Sci U S A. 108,722-727 (2011)

Figure :quired for the maintenance of a fit microbiota. We foundthat PD-1 affects the dynamics of GC B cells by controllingthe number and the nature of T helper cells in the PPs.
The deregulation of the T cell compartment impacts on
the selection of IgA plasma cells leading to gut dysbiosis. Defects in IgA selection, by perturbing the microbial
composition and gut microenvironment, have the potential
to increase autoimmune susceptibility. In several mousemodels of autoimmune diseases, the development of thedisease required intestinal bacteria and expansion of specific subsets of T helper cells (e.g., SFB and Th17) . Indeed,PD-1-deficient mice develop autoimmune diseases only inthe presence of microbiota and AID, suggesting that the
autoreactive antibodies in PD-1-deficient mice may arise
after AID-induced genetic alterations in GCs, which are
driven by stimulation from the gut microbiota.
These findings suggest that the maintenance ofappropriate composition and density of microbiota by
dynamic IgA production in the gut confers resistance toautoimmunity in peripheral compartments (See Fig. on p. 2,
Creation of New Paradigms). Our analysis of PD-1-/- mice
opens new perspectives for studies aimed at understanding the contribution of gut microbiota to the developmentof autoimmune diseases.

Linking innate and adaptive responses: The role ofinnate lymphoid cells (ILCs) in conditioning marginalzone B cell antibody responses through granulocyte
recruitment
Collaborative project with the laboratory of Andrea Cerutti, Institut Municipal d’Investigació Mèdica-Hospital del Mar, Barcelona, Spain

ILCs are innate effector cells whose development depends
on the transcriptional repressor Id2 and the common
g-chain of the IL-2 receptor. However ILCs are rather heterogeneous and different ILC subsets show distinct tran-

4. Suzuki K., Maruya M., Kawamoto S., Sitnik K., Kitamura
H., Agace W.W., Fagarasan S. The sensing of environmental stimuli by follicular dendritic cells promotes
immunoglobulin A generation in the gut. Immunity 33,
71-83 (2010 )Splenic ILCs are bridging innate withadaptive responses via neutrophils andmarginal zone B cells by facilitating
T-independent antibody production.
Representative sections of spleen stained
as indicated, revealing reduced numbers ofneutrophils associated with impaired IgG3producing plasmablasts or plasma cells in
RORγgt-/- mice.

scription factor requirements and discrete cytokine secretion patterns, which mirror those of conventional T helper
cells. ILC1 release interferon-γ and require the transcription factor T-bet, ILC2 secrete IL-5 and IL-13 and require
the transcription factor GATA3. ILC3 represent cells thatinduce development of lymphoid structures (LTi ) or secrete
IL-22 and require the transcription factors RORγt and arylhydrocarbon receptor ( AhR ) .
We studied the spleen in RORγt-deficient mice lacking
ILC3. RORγt-deficient mice have an overrepresented B
cell compartment, yet they produce reduced homeostatic
IgG3 antibodies and post-immune IgG3 responses to a TI
antigen mimicking bacterial polysaccharides. This humoral deficiency depended on the lack of maturation and/or
survival signals provided by splenic ILCs to IgG3-secreting
plasmablasts and plasma cells ( Fig.) , because IgG3 classswitched B cells were conserved in RORγt-deficient mice.
Indeed, splenic ILCs normally expressed APRIL, a CD40L( and BAFF ) -related molecule that supports plasma cell
survival.
Interestingly, in RORγt-deficient mice the impairmentof IgG3 responses to TI antigens was associated with adeficiency not only of ILCs but also of neutrophils ( Fig.) .
RORγt-deficient mice showed fewer neutrophils than either
WT or T cell-deficient mice, suggesting that ILC3 contribute to recruitment, activation or survival of neutrophils in
spleen. Certainly, ILCs produce the neutrophil-attracting
chemokine IL-8 and the neutrophil-stimulating cytokine
GM-CSF. In turn, the stimulated neutrophils release various
cytokines including the proliferation/survival factor BAFF.
Thus our study revealed that the deficiency of RORγt
and ILCs impairs marginal zone (MZ) B cell responses in
spleen, indicating that ILCs orchestrate an innate antibody-inducing network at the MZ-blood interface.

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 )

85

Laboratory for

ImmuneDiversity
Team Leader:

Ji-Yang Wang, Ph.D.
Research Scientist: Rika Ouchida		Yohei Kawai
Student Trainee:				

Chie Kano
Shuyin Li (IPA)
Yanfei Zhang (IPA)

Technical Staff:

Hiromi MoriA

ntibodies are crucial effectors for host defense mediated by neutralization and opsonization of pathogens
and by activation of the complement system. Antibodies
are also important regulators of immune responses, acting
through binding to the Fc receptors expressed by various
immune cells. Insufficient or excess production of antibodies can lead to immunodeficiencies or autoimmune
diseases, respectively. The goal of our current research isto understand how B cell activation and differentiation are
positively and negatively regulated to allow appropriate
production of diversified antibodies of different classes
and to achieve effective humoral immunity.

Uncover new pathways that regulate B cell activationand differentiation through analysis of mice withinactivating mutations in B cell-specific genes
With the tools provided by completion of human andmouse genome projects, the availability of microarray
data, and the technical advancements in generationof gene-manipulated mice, we have taken a molecular
genetic approach to study B cell activation and differentiation. We performed comprehensive searches of the
RCAI RefDIC and public microarray databases, and haveidentified several uncharacterized genes that are exclusively expressed in B cells, including the germinal center( GC ) subset of B cells and other subsets. We have established knockout mice for these genes and obtainedinteresting preliminary results. For example, deficiency ofa B-cell-specific transcription elongation factor caused adecrease of B cell survival after antigen stimulation and resulted in immune deficiency. Further analysis of these andadditional mice with defective B cell-specific genes should
allow us to uncover new pathways that regulate various
aspects of B cell survival, activation, differentiation andantibody production.

Explore the mechanism of A /T mutations in
GC B cells
High-affinity antibodies are central to humoral immunity.
Somatic hypermutation ( SHM ) of Ig genes in GC B cells
is an essential process for generating high-affinity B cells.
SHM is initiated by the activation-induced cytidine deaminase ( AID ) , which is thought to convert cytosine ( C ) touracil ( U ) and generate U:G lesions on DNA. Consistent
with its substrate specificity, ectopic expression of AID in
fibroblasts induces mutations predominantly at C/G bases. However, remarkably, half of the mutations in the Ig
genes of GC B cells are induced at non-targeted A/T bases and thus their origin is difficult to explain. We found thatinduction of A/T mutations is dependent on the GC B cell
environment but independent of the target gene. We also
found that high levels of AID caused the strand-bias of A/
T mutations in Burkitt’s lymphoma cells and established amodel system to study this unsolved mystery in the hypermutation field.

Recent Publications
1. Kano, C. and Wang, J.-Y. High levels of AID cause strand
bias of mutations at A versus T in Burkitt’s lymphoma
cells. Mol Immunol. 54, 397-402 (2013 )86

2. Li, S., Zhao, Y. and Wang, J.-Y. Analysis of Ig gene
hypermutation in Ung -/-Polh -/- mice suggests that UNG
and A:T mutagenesis pathway target different U :G
lesions. Mol Immunol. 53, 214-217 (2013 )3. Ouchida, R., Mori, H., Hase, K., Takatsu, H., Kurosaki, T.,
Tokuhisa, T., Ohno, H. and Wang, J.-Y. Critical role of the
IgM Fc receptor in IgM homeostasis, B cell survival andhumoral immune responses. Proc Natl Acad Sci U S A.
109, E2699-2706 (2012 )Figure 1: Impaired GC formation in FcμR-/- mice.
WT and FcμR-/- mice were immunized with 10 μgof NP-chicken gamma globulin in alum and theirspleen sections were stained with anti-AID todetect GC B cells and anti-CD3 to detect T cells.
Representative staining results of 4 WT and 5
FcμR-/- mice are shown.

Figure 2: FcμR-/- mice produce autoantibodies.
Hep2 cells were stained with sera (100-folddilution) from WT and FcμR-/- mice, followed
by FITC-anti-mouse IgG. Results of 8 WT (1male and 7 females) and 8 FcμR-/- (2 males and 6
females) mice are shown.

Elucidate the function of the IgM Fc receptor in the
humoral immune response
The existence of a receptor for IgM ( FcμR ) was suggested since 40 years ago, but the gene encoding FcμR was
identified only recently. FcμR is expressed exclusively in
B cells in mice and in B and T cells in humans. In collaboration with the Laboratory for Epithelial Immunobiology

4. Li, Y., Li, S., Hoshino, M., Ishikawa, R., Kajiwara, C.,
Gao, X., Zhao, Y., Ishido, S., Udono, H. and Wang, J.-Y.
HSP90α deficiency does not affect Ig gene hypermutation and class switch but causes enhanced MHC class II
antigen presentation. Int Immunol. 24, 751-758 (2012 )of RCAI and the University of Alabama at Birmingham,
we have shown that FcμR positively regulates B cell activation and germinal center formation, and is required for
efficient humoral immune responses ( Fig. 1) . Intriguingly,
Fcμ R -/- mice produced autoantibodies as they age (Fig. 2 ) ,suggesting that FcμR is also required for maintaining tolerance to self-antigens.

5. 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 generation of C
to G and G to C transversions during Ig gene hypermutation. Int Immunol. 24, 169-174 (2012 )87

Laboratory for

Immunological Memory
Group Director:

Toshitada Takemori
Research Scientist: Tomohiro Kaji
Technical Staff:

Akiko Ishige
Natsumi Yoneda

I

mmunological memory is an important element of the
mammalian immune system. We have known about immunological memory for centuries, but critical aspects of
its development and maintenance remain poorly defined.
A minimal definition of memory cells would be the population of lymphocytes that persists long term in a restingstate after antigen clearance. The critical feature of memory cells is their ability to undergo rapid differentiation into
secondary effectors with potent functional activity.
We have studied the development of antigen-specificmemory B and T cells in response to soluble protein antigens or influenza virus and characterized their properties,
genetic signature, and functional activity.

Two pathways of memory B cell development
The germinal center reaction is a critical event in T celldependent responses, during which antigen-activated
B cells accumulate somatic hypermutation and undergo
isotype class switching of their immunoglobulin genes.
Classically, memory B cells have been considered to behigh-affinity isotype-switched variants of precursors that
had been generated from the germinal center reaction.
However, this simplistic view is not consistent with manyobservations that have recently been made in mice andhumans, for example some memor y B cells express
unmutated VH genes. This finding raised the idea thatmemory B cells may also develop in the absence of GC.
We have recently presented experimental findingsthat clearly encapsulate the previous predictions withvery convincing data using novel mouse models of TD
immune responses, and which reveal that immunologicalmemory is established early during an immune response,
prior to GC formation. The GC-dependent and -independent memory B cells differ from one another with respect
to turnover and gene expression and also in their dependence on different subsets of T helper cells, whereas theyshare the important function of protecting the host against
pathogens. In this context, we have observed that the
GC-independent memory pathway functions in mice in
response to influenza viruses and assists the GC-dependent memory pathway to protect the host against viral
infections.
We observed that both GC-independent and -dependent memory cells accumulate mutations optimally adapted to the recall challenge. Therefore, it seems likely thatboth unmutated and mutated memory B cells can enter
the GC upon secondary challenge and undergo accumulation of mutations by SHM in the GC reaction and create
a diverse antibody repertoire in response to infections withpathogens, thus providing effective and efficient protection
against mutated pathogens.

Recent Publications
1. Kishi Y, Aiba Y, Higuchi T, Furukawa K, Tokuhisa
T, Takemori T, Tsubata T. Augmented antibody response with premature germinal center regression in
CD40L-transgenic mice. J Immunol. 185, 211-219(2010 )

88

2. 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. 23, 433-441 (2011)

3. Kuwahara, K., Nakaya, T., Phimsen, S., Kitabatake, M.,
Kaji, T., Takemori, T., Watanabe, T., and Sakaguchi,
N. Lyn signaling to upregulate GANP is critical for thesurvival of high-affinity B-cells in germinal centers oflymphoid organs. J Immunol. 189, 3472-3479 (2012 )Figure:Bcl6 is required for CD4+ memory T cell development

Bcl6 is required for CD4 + T cell development tomemory T cells that function to help memory
B cell terminal differentiation.
The transcriptional repressors Bcl-6 and Blimp-1 counteract each other in many types of cells and play roles
in T cell polarization to different lineages. Expression ofhigh levels of Bcl-6 represses the expression or functionof transcription factors necessary for effector CD4+ T celldifferentiation pathways and leads to T follicular helper ( TFH )cell development (Fig.) . We observed that conditional Bcl6
deletion in CD4 T cells abrogated TFH development andthe secondary memory response with its high affinity antibodies. Therefore, we decided to determine whether Bcl6
plays a role in memory T cell development.
We chose to evaluate memory CD4 + T cell function
by measuring help for the secondary antibody response,
which better reflects those functions of memory T cells
that protect the host. We observed that conditional Bcl6deletion in OVA-specific CD4 + T cells abrogated theirdevelopment to memory T cells with the ability to initiate
an anti-NP memory B cell response upon stimulation
with NP-OVA.
It has been predicted that, after the GC reaction subsides, some of the TFH cells within the GC may become
quiescent memory cells while retaining low expression ofsome TFH cell markers. However, our analysis suggeststhat the GC is required for development of TFH cells but not
for CD4+ memory T cells, suggesting that memory T cells
are not derived from TFH at their terminal differentiation in
the GC.

4. Kaji T, Ishige A, Hikida M, Taka J, Hijikata A, Kubo A,
Nagashima T, Takahashi Y, Okada M, Kurosaki, T., Ohara
O, Rajewsky K and Takemori T. Distinct cellular pathways
select germ-line encoded and somatically mutated antibodies into immunological memory. J Exp Med. 209,
2079-2097 ( 2012 )We observed that CD4 memory T cells consist ofcentral ( cm ) and effector ( em ) memory subsets, which are
distinguishable by surface markers and genetic signature,
whereas both displayed the activity to help in a memory B
cell response. Tcm attains functional activity by interaction
with antigen-specific B cells (cognate interaction ), whereas
Tem does not require cognate interaction for their functional development. These results support the idea that T
cells upregulate Bcl6 expression during dendritic cell (DC )priming and then polarize to the Tem pathway or to Tcm or
TFH pathway upon cognate B-cell interaction. Whether Tcm
precursors are derived from cells that undergo differentiation along the pathways to TFH remains unknown.
Thus the immune system utilizes a different set ofpathways for immunological memory in both B cell and T
cell compartments. Memory B cells localize in the B cellarea for a long period of time, whereas memory T cells
localize in the T cell follicle. Memory B cells require memory T cells for their terminal differentiation, thus they arequiescent during the primary response. Once reactivated,memory T cells enter the B cell follicle, initiating memory B
cell terminal differentiation.

Acknowledgement
We are grateful to Klaus Rajewsky for discussion, help andencouragement and Masaru Taniguchi for reviewing ouractivity and support. We thank Peter Burrows, Yoshimasa
Takahashi, Masaki Hikida, Atsushi Hijikata, Osamu Ohara,
Tomo Kurosaki and Masato Kubo for help.

5. Ato, M., Takahashi, Y., Fujii H., Hashimoto, S.-I., Kaji,T., Yamamoto, K., Itamura, S., Horiuchi, Y., Arakawa, Y.,
Tashiro, M., Takemori, T. Influenza A whole virion vaccine
induces a rapid reduction of peripheral blood leukocytes via interferon-α-dependent apoptosis. Vaccine
31, 2184-2190 (2013 )89

Laboratory for

Infectious
Immunity
Team Leader:

Satoshi Ishido
Research Scientist: Mari Ohmura-Hoshino
Mizuho Kajikawa
Student Trainee:

Rikiya Ishikawa

Technical Staff:

Masami Kawasumi-Aoki
Mari Yoshida-Mito
Naoko Tachibana

Antigen-presentation is a critical process for the initiation of immunity and tolerance. Invading pathogens
are phagocytosed by antigen-presenting cells ( APCs ) andtheir protein constituents are processed and presented
through MHC class II ( MHC II). The MHC II-peptide complex stimulates CD4 T cells through its interaction withpeptide-MHC-specific T cell receptors. With the maturation signals for APCs, peptide-bound MHC II ( pMHC II )can initiate immunity against the pathogens. Thus, it isimportant to reveal how the MHC II-mediated antigen presentation is regulated in vivo. In this regard, we and othergroups found that the expression level of pMHC II is regulated on the surface of APCs by ubiquitination. In addition,
we used genetically modified mice to identify MARCH-I asa physiological E3 ubiquitin ligase for MHC II.
To examine the role of MARCH-I-mediated pMHC
II ubiquitination, several groups including ours examined
how MARCH-I and pMHC II ubiquitination are regulated
in the context of immune responses. At present, it has
been demonstrated that several signals inducing theactivation/maturation of dendritic cells ( e.g. TLR stimuli )stabilize the pMHC II though inhibition of its ubiquitination
by down-regulation of MARCH-I. Thus, loss of pMHC II
ubiquitination is one of the mechanisms for activation ofdendritic cells. Based on these findings, most researches
in this field believe that loss of pMHC II promotes pathogen immunity through enhancement of antigen-presentation. However, at present, there are no findings supporting

such an attractive hypothesis. On the contrary, we havereported that splenic DCs in mice whose pMHC II is not
ubiquitinated have impaired functions, suggesting that lossof pMHC II ubiquitinaiton provides negative feedback toimmune responses. Therefore, we are examining how lossof pMHC II ubiquitination influences the function of cDCs
in a cell-intrinsic manner by using in vitro generated bonemarrow-derived DCs.
Since our results suggest that stabilization of pMHC
II by loss of MARCH-I inhibits immune responses, we propose that loss of pMHC II ubiquitination induces a negative
feedback signal to dendritic cells. At present, we are investigating the mechanism for MARCH-I regulation. Once
we discover this regulatory mechanism, we will test our
hypothesis by perturbing regulation of MARCH-I.

Inhibition of CD4 T cell activation by loss of pMHC II
ubiquitination
In order to examine the role of loss of MHC II ubiquitination
on CD4 T cell activation, we generated cDCs from bonemarrow cells ( BM cells ) of MARCH-I KO mice, in which
pMHC II is not ubiquitinated. During the differentiation of
BM cells into DCs with Flt3-L, DCs were loaded with OVA
as a model antigen. OVA-presenting BM-derived DCs werepositively selected from Flt3-L-treated BM cells on thebasis of CD11c expression and cDCs were purified fromselected BM-derived DCs by depletion of pDC with B220.
Naïve CD4 T cells isolated from OT-II transgenic mice were

Recent Publications
1. Furuta, K., Ishido, S. and Roche, PA. Encounter with antigen-specific promed CD4 cells promotes MHC class IIdegradation in dendritic cells. Proc Natl Acad Sci USA.
109, 19380-19385 (2012)90

2. Hunt, D., Wilson, JE., Weih, KA., Ishido, S., Harton, JA.,
Roche, PA. and Drake, JR. Francisella tularensis elicits
IL-10 via a PGE 2-inducible factor, to drive macrophage
MARCH1 expression and class II down-regulation. PLoS
One 7( 5 ):e37330 (2012)

3. Galbas, T., Steimle, V., Lapointe, R., Ishido, S. andThibodeau J. MARCH1 down-regulation in IL-10-activated B cells increases MHC class II expression. Cytokine
59, 27-30 (2012)

Figure:Inhibition of DC function by stabilization of pMHC class II
In immature DCs, MHC class II-peptide complexes (pMHC II) are continuously degradedby MARCH-I-mediated ubiquitination (left). During the process of maturation, the functionof MARCH-I is inhibited and pMHC II is stabilized through loss of ubiquitination. Stabilized
pMHC II suppresses one signaling molecule (shown as “X”), and this regulation may be criticalfor negative feedback.

co-cultured with OVA-presenting MARCH-I-deficient cDCs
or OVA-presenting wild type cDCs. This experiment revealed that MARCH-I-deficient cDCs had a lower capacity
to stimulate naïve CD4 T cells than wild type cDCs, sinceIL-2 secretion was lower in cultures with MARCH-I-deficient cDCs than with wild type cDCs. This was due to thelow production of IL-2, because the amount of IL-2 mRNA
was low in the MARCH-I-deficient cDCs. Finally, we confirmed that activation of ERK was low in the mixture of OTII CD4 T cells and MARCH-I-deficient cDCs. These results
clearly demonstrate that MARCH-I-deficient cDCs havethe poor ability to activate CD4 T cells, and suggest thatloss of pMHC II ubiquitination suppresses the function of
cDCs in a cell intrinsic manner.

4. Tanno, H., Yamaguchi, T., Goto, E., Ishido, S. and Komada, M. The Ankrd 13 family of UIM-bearing proteinsregulates EGF receptor endocytosis from the plasma
membrane. Mol Biol Cell. 23, 1343-1353 (2012)

Inhibition of stimulatory molecule expression byloss of pMHC II ubiquitination
Previously, we showed that loss of pMHC II ubiquitination
enhances antigen-presentation. Therefore, we expectedthat loss of pMHC II ubiquitination would enhance the activation of CD4 T cells. However, as mentioned above, we
found the unexpected result that loss of pMHC II ubiquitination inhibits CD4 T cell activation. This result suggeststhat loss of pMHC II ubiquitination might induce a negative
signal to CD4 T cells, or suppress an activating signal. At
present, several cDC molecules have been reported as
activating or inhibitory molecules; therefore the candidate
molecules were examined. Among them, we found that
one important signaling molecule was significantly inhibited in MARCH-I-deficient cDCs or MHC II-KI-derived cDCs.
At present, we are examining how this molecule contributes to inhibition of DC functions.

5. Kajikawa, M., Li PC., Goto, E., Miyashita, N, Aoki-K awasumi, M, Mito-Yoshida, M, Ikegaya, M.,
Sugita,, Y. and Ishido, S*. The inter-transmembraneregion of KSHV MIR2 contributes to B7-2 downregulation. J Virol. 86, 5288-5296 (2012)91

Research Unit for

Inflammatory Regulation
Unit Leader:Takashi Tanaka		Technical Staff: Emiri Haga
Azusa Sibazaki
Student Trainee: Masanaka Sugiyama (JRA)Rumiko OnoThe inflammatory response is an important host defense mechanism to sense and eliminate invading
microbial pathogens. Dendritic cells first detect pathogens using their pathogen sensors (e.g. Toll-like receptors[ TLR ] ) . TLR signaling then leads to the activation of thetranscription factor NF-kB, which enters the nucleus andinduces the expression of a series of inflammation-related
genes, including those encoding proinflammatory cytokines such as interleukin-6 ( IL-6 ) and IL-12. These inflammatory responses then direct T-helper ( Th ) lymphocyte
differentiation into distinct effector T cell subsets, such as
Th1, Th2 and Th17, through the activation of STAT4, STAT6
and STAT3 transcription factors, respectively, to combat
various pathogens. These initially helpful inflammatory
responses must be terminated at the appropriate time
point, however, otherwise, excessive responses can damage normal tissue and may cause autoimmune or allergic
diseases. Our research goal is to identify key regulatorsof inflammation-related signal transduction pathways andto clarify the molecular mechanisms for regulating inflammatory responses. These studies should contribute to thedevelopment of new therapeutic tools to control the exaggerated inflammation seen in certain human diseases. Our
research now focuses on the role of PDLIM2 ( PDZ and
LIM-domain protein-2 ) and related LIM proteins in the neg-

ative regulation of inflammatory responses.

The role of LIM proteins in the regulation of T-helper
cell differentiation
The LIM protein family is classified into several groups.
LIM proteins containing a PDZ domain at the N-terminus
and LIM domains at the C-terminus, including PDLIM2, aredesignated as the PDZ-LIM protein subfamily. To date, seven proteins in this subfamily have been identified. It is wellknown that both PDZ and LIM domains are involved in protein-protein interactions, but the functions of PDZ-LIM proteins in the immune system remain unclear. PDLIM2, also
known as SLIM (STAT-interacting protein ), has been shown
to be a nuclear ubiquitin E3 ligase for the STAT4 andSTAT3 transcription factors in CD4 + T cells, suppressing
Th1 and Th17 cell differentiation ( Tanaka et al., Immunity,
2005, Tanaka et al., Sci. Signal., 2011) . We recently foundthat PDLIM4, another PDZ-LIM protein family member, isalso a negative regulator of STAT-mediated signaling. In
contrast to PDLIM2, however, PDLIM4 is located in thecytoplasm. PDLIM4 bound to STAT3, STAT4 and STAT6,
and suppressed their STAT-mediated gene activation. Interestingly, PDLIM4 did not promote polyubiquitination anddegradation of these STAT molecules, but instead inhibited the phosphorylation of a tyrosine residue essential for

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 and granulomatous
inflammation through degradation of STAT3. Sci Signal.
4 ( 202 ), ra85 ( 2011)92

2. Yan, P., Fu, J., Qu, Z., Li, S., Tanaka, T., Grusby, M.J.,
Xiao, G. PDLIM2 suppresses HTLV-I Tax-mediatedtumorigenesis by targeting Tax into the nuclear matrix
for proteasomal degradation. Blood 113, 4370-4380( 2009 )3. Kaisho, T. and Tanaka, T. Turning NF-kB and IRFs on
and off in DCs. Trends Immunol. 29, 329-336 ( 2008 )Figure 1: PDLIM2 and PDLIM4 regulate Th17 cell-mediatedinflammatory responses through different mechanisms.
PDLIM2 interacts with STAT3 in the nucleus and promotes
its ubiquitin/proteasome-dependent degradation, whereas
PDLIM4 binds to STAT3, recruits PTP and enhances its
dephosphorylation and inactivation in the cytoplasm.

Figure 2: PDLIM2 and PDLIM1 negatively regulate NF-kB
signaling through different mechanisms.
PDLIM2 binds to and promotes polyubiquitination of the
p65 subunit of NF-kB in the nucleus, and then targets p65
into discrete intranuclear compartments, called PML nuclear
bodies, for degradation. By contrast, PDLIM1 sequesters
the p65 subunit of NF-kB in the cytoplasm and inhibits its
nuclear translocation.

cytokine-induced STAT activation. We further demonstrated that PDLIM4 bound to and recruited PTP-BL, a protein
tyrosine phosphatase, and facilitated dephosphorylationof STAT proteins. In CD4 + T cells, PDLIM4-deficiency resulted in augmented tyrosine phosphorylation of these
STAT proteins and consequently enhanced Th1, Th2 andTh17 cell differentiation. Our findings delineate an essential role of PDLIM4 in negatively regulating STAT-mediatedeffector T cell differentiation through a mechanism quitedistinct from PDLIM2. Moreover, we demonstrated that asingle nucleotide polymorphism ( SNP ) in the LIM domain
of PDLIM4 is associated with susceptibility to rheumatoid
arthritis, a Th17-related autoimmune disease, suggestingthat PDLIM4 may prevent the onset of human autoimmune
diseases by negatively regulating Th17 responses.

NF-kB. ( Tanaka et al., Nat. Immunol., 2007 ) . PDLIM2 binds
to p65 and promotes its polyubiquitination. In addition,
PDLIM2 targeted p65 to discrete intranuclear compartments, called PML nuclear bodies, where polyubiquitinated p65 was degraded by the proteasome. We recently
found that PDLIM1, another LIM protein family member,is also a negative regulator of NF-kB-mediated signaling.
PDLIM1 is located in the cytoplasm and inhibits NF-kB
p65-mediated gene activation. PDLIM1 did not promote
polyubiquitination of NF-kB, but instead sequestrated
p65 in the cytoplasm, possibly by interaction with actin
stress fibers through binding to a-actinin, an actin binding
protein, and suppressed nuclear translocation of p65 protein. Consistently, PDLIM1 deficiency lead to decreased
cytoplasmic and increased nuclear p65 protein levels, andthus enhanced proinflammatory cytokine expression in
response to TLR stimuli. These data delineate an essential
role of PDLIM1 in suppressing NF-kB activation throughmechanisms different from PDLIM2. Collectively, PDZ-LIM
proteins appear to be a new family of adaptors that cannegatively regulate signal transduction pathways in the immune system.

The role of LIM proteins in the regulation of
inflammatory responses
We previously demonstrated that PDLIM2 negatively
regulates TLR-mediated NF-kB activity in dendritic cells
and subsequent inflammatory responses, acting as anuclear ubiquitin E3 ligase targeting the p65 subunit of

4. Tanaka, T. Grusby, M.J. and Kaisho, T. PDLIM2-mediated termination of transcription factor NF-kB activation
by intranuclear sequestration and degradation of the
p65 subunit. Nat Immunol. 8, 584-591 ( 2007 )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 )93

Research Unit for

Immunotherapeutic Model
Unit Leader:

Kanako Shimizu
Technical staff: Miki AsakuraT

he goal of our laboratory is to develop immunotherapeutic models for cancer. We have been focusing on
the biological role of dendritic cells (DCs ) in vivo as a link
between innate and adaptive immunity. NKT cells haveunique immunoregulatory features that include the ability
to rapidly produce large quantities of cytokines. We haveattempted to generate an approach for inducing adaptive
immunity based on 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. Previously, we have established a unique strategy using a-galactosylceramide (a-GalCer) loaded on tumor cells ( Tumor/
Gal ) for enhancing innate and adaptive immunity. This
strategy induces antigen-specific T cell immunity via invivo DC maturation and we have been optimizing systems
for tumor antigen delivery to DCs. One of the remarkable
points is that this strategy also induces memory T cells.
Thus, we have been evaluating the memory T cells in the
immunized animals in detail in order to develop a more effective immunotherapeutic strategy.
We have recently established an immunotherapeuticstrategy using an artificial adjuvant vector cells ( aAVCs ) ,composed of glycolipid-loaded, antigen-encoding mR-

NA-transfected allogeneic fibroblasts for enhancing bothinnate ( NKT and NK cells ) as well as adaptive immunity( T cells ) . We focused on the mechanism of DCs in detail.
Based on these findings, we have attempted to launchpreclinical studies with human aAVCs, in collaboration with
Dr. Fujii ( Cellular Immunotherapy Research Unit) .

The strategy of using in vivo DC maturation by
NKT Cells to generate adaptive immunity andmemory T cells
Although CD8 + memory T cells have been intensely studied in a range of acute infectious pathogen models, thegeneration of memory T cells reactive with cell-associatedantigens, such as those found in cancer, by vaccination isless well understood and has not been established. Withthe “Tumor/Gal” vaccine, we found that T cells responding
to the specific tumor antigen can be maintained as memory T cells, resulting in long-term antigen-specific antitumor
protection even 12 months after vaccination ( Fig.) . These
memory T cells had a central memory phenotype and amultifunctional cytokine response to antigen. We havebeen elucidating the mechanism of how to most effectively
generate this memory T cell response.

Recent Publications

94

1. Shimizu, K., Mizuno, T., Shinga, J., Asakura, M., Kakimi,
K., Ishii, Y., Masuda, K., Maeda, T., Sugahara, H., Sato,Y., Matsushita, H., Nishida, K., Hanada, K., Dorrie, J.,
Schaft, N., Bickham, K., Koike, H., Ando, T., Nagai, R.,
and Fujii, S. Vaccination with antigen-transfected, NKT
cell ligand-loaded, human cells elicits robust in situ
immune responses by dendritic cells. Cancer Res. 73,
62-73. 2013

2. Fujii, S., Shimizu, K. DC-based immunotherapy targeting
NKT cells. In Terabe, M. and Berzofsky, JA. (eds ), Natural killer T cells: Balancing the regulation of tumor
immunity. Springer New York Dordrecht Heidelberg
London, 95-110. 2011

3. 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, 5927-5937. 2011

Figure:Efficacy of adjuvant vector cells for induction of long-term antitumor responses
C57BL/6 mice were injected i.v. with the CD1dhi-B16 cell line loaded with a-GalCer (tumor/Gal). The mice werechallenged s.c. with parental B16 tumor cells 4 wk later. Moreover, when the mice were re-challenged with B16
one year later, they rejected the B16 tumor cells, but not EL4 tumor cells, demonstrating long-term immunity.

Vaccination with antigen-transfected, NKT cell
ligand-loaded, human cells elicits a robust in situ
immune responses by dendritic cells
We previously demonstrated in mice that a-GalCer-loaded, tumor antigen-expressing allogeneic cells can act as
cellular adjuvants, linking the innate and adaptive immune
systems. We have now established human “artificial adjuvant vector cells ( aAVC ) ” consisting of human HEK293
embryonic kidney cells stably transfected with CD1d, load-

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 natural killer T cells. J Clin
Invest. 120, 2610-2618. 2010

ed with a-GalCer and then transfected with antigen-encoding mRNA. When administered to mice or dogs, theseaAVCs activated invariant NKT ( iNKT ) cells and elicitedantigen-specific T cell responses with no adverse events.
By harnessing the innate immune system and generating
an adaptive immune response to a variety of antigens, thisunique tool could prove clinically beneficial in the development of immunotherapies for malignant and infectious
diseases.

5. Shimizu, K., Hidaka, M., Bickham, K., Moriwaki, M.,
Fujimoto, K., Fujii, S. Human leukemic cells loaded witha-GalCer activate murine NKT cells in situ. Int J Hematol. 92, 152-160. 2010

6. Fujii, S., Goto, A., Shimizu, K. Antigen mRNA-transfected, allogeneic fibroblasts loaded with NKT-cell ligand
confer antitumor immunity. Blood 113, 4262-4272.
2009

95

Research Unit for

Immune Homeostasis
Unit leader:

Shohei Hori
Research Scientist:
		 Norihito Hayatsu		Maria Encarnita Mariotti-Ferrandiz		 (until November 2012)
Visiting Scientist:
		 Ruka Setoguchi
Technical Staff:
		 Takahisa Miyao		
Kuniko Kamiya
Student Trainee:		
Ryuichi MurakamiT

he ultimate goal of our laboratory is to understand thebasic principles governing immunological tolerance
and homeostasis. While the clonal selection theory has
predicted the importance of cell intrinsic mechanisms
such as clonal deletion and anergy, their significance innaturally acquired self-tolerance still remains elusive. Overthe last two decades, evidence has accumulated that natural self-tolerance may be rather dominant and based oncell-extrinsic regulation of autoaggressive lymphocytes byother functional classes of lymphocytes, in particular by
regulatory T ( Treg ) cells exhibiting suppressive functions.
Our studies have demonstrated that Treg cells characterized by expression of the transcription factor Foxp3 plays
an essential role in the establishment and maintenance of
natural self-tolerance and immune homeostasis, particularly at the environmental interfaces.
In order to understand the principles of immunologicaltolerance and homeostasis, it is crucial to understand how
the immune system robustly maintains tolerance to “self”
yet allows for aggressive responses toward “non-self” in
the face of diverse and unpredictable perturbations fromthe internal as well as external environments. To address
this question, we are currently investigating how Foxp3 +Treg cells respond to extrinsic signals and deal withenvironmental changes to ensure the robustness of selftolerance and homeostasis.

Plasticity of Foxp3+ T cells: the controversy resolved
The emerging notion of environment-induced Treg cell
reprogramming remained controversial. We and others have previously shown that at least some of Foxp3 +T cells can differentiate into Foxp3 - helper T ( Th ) cells in
response to environmental changes such as lymphopenia or inflammation, whereas other recent reports argue
against the plasticity phenomena. We have recently proposed that this controversy can be resolved by considering the “heterogeneity model” of plasticity, which hypothesizes that the observed plasticity of Foxp3 + T cells does
not reflect lineage reprogramming of Treg cells but stems
from a minor population of uncommitted Foxp3 + T cells.
By genetic fate mapping and adoptive transfers, we haveaddressed the nature and origin of such uncommitted
Foxp3 + T cells and identified them as a minor population of
non-regulatory cells exhibiting promiscuous and transient
Foxp3 expression (Fig.). Such cells give rise to Foxp3- Th
cells and selectively accumulate in inflammatory cytokine
milieus or in lymphopenic environments including those inearly ontogeny. By contrast, Treg cells did not undergo reprogramming under those conditions irrespective of theirthymic or peripheral origins. Moreover, although a few Treg
cells transiently lose Foxp3 expression, such “latent” Treg
cells retain phenotypic memory and robustly re-express
Foxp3 and suppressive function upon activation. The sta-

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 expression in conventional T cells but not reprogramming of regulatory T cells.
Immunity 36, 262-275 ( 2012 )96

2. Hori S: Stability of regulatory T-cell lineage. Adv
Immunol. 112, 1-24 ( 2011)

3. Kendal AR, Chen Y, Regateiro FS, Ma J, Adams E, Cobbold SP, Hori S, Waldmann H: Sustained suppression by
Foxp3 + regulatory T cells is vital for infectious transplantation tolerance. J Exp Med. 208, 2043-2053 ( 2011)

Figure:A model of Treg cell lineage commitment
During thymic or peripheral Treg cell differentiation, TCR and other signals lead to Foxp3 up-regulation.
(The RFP reporter visualizes Foxp3+ cells and their progeny, independently of continuous 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 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. (The extent of
TSDR demethylation is color-coded in the schematic of the Foxp3 gene in the nucleus.) Nevertheless, Treg cells
may transiently lose Foxp3 expression in environments where TCR and/or IL-2 signals are limited 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 initialsignals that lead to the “imprinting” of a stable Treg cell phenotype remain to be uncovered however.

ble differentiated state of Treg cells was associated withcomplete DNA demethylation of a conserved non-coding
region termed Treg-specific demethylation region ( TSDR )within the Foxp3 locus. This study established that Treg
cells constitute a stable cell lineage, whose committed
state in a changing environment is ensured by an epigenetic mechanism, irrespective of ongoing Foxp3 expression.
Our next focus is to discover the signals that lead to irreversible commitment to the Treg cell fate.

Genetic and environmental control of Treg cell
fitness in peripheral tissues
In an attempt to understand the molecular mechanisms
by which Foxp3 controls Treg cell differentiation and function, we have addressed whether and how Foxp3 genemutations that had been identified in human IPEX impingeon Treg cell development and function, using retroviralgene transduction strategies and knock-in mutagenesis inmice. Serendipitously, we have identified one hypomorphic
mutation that had no effect on the in vitro suppressive

4. Hori S: Regulatory T cell plasticity: beyond the controversies. Trends Immunol. 32, 295-300 ( 2011)activity, thymic differentiation, or global gene expression
profile of Foxp3 + T cells. This mutation, however, led to
impaired fitness of CCR7lo “tissue-seeking” Foxp3 + T cells.
Accordingly, we have found that the mutant Foxp3 + T cells
are much less competitive than wild-type Foxp3 + T cells
in peripheral tissues, although the extent of the reductionof mutant Foxp3 + T cells differed from tissue to tissue,
suggesting an environmental control of Treg cell fitness.
Importantly, the impaired fitness of mutant Foxp3 + T cells
was accounted for, at least in part, by reduced expressionof one transcription factor. We suggest that fitness of Treg
cells in tissue environments is controlled by both genetic
and environmental factors and represents an essential
determinant of self-tolerance and immune homeostasis.
We are currently investigating i ) the molecular nature ofsuch environmental factors; ii ) how such extrinsic cues
are translated into Foxp3-dependent intracellular control
mechanisms, and iii ) how “fit” Treg cells in turn ensure thefitness of peripheral tissues.

5. Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara
T, Momose Y, Cheng G, Yamasaki S, Saito T, Ohba Y,
Taniguchi T, Takeda K, Hori S, Ivanov II, Umesaki Y, Itoh
K, Honda K: Induction of colonic regulatory T cells byindigenous Clostridium species. Science 331, 337-341( 2011)

97

Laboratory for

ImmuneRegulation
Group Director :Masaru Taniguchi
Senior Research Scientist: Hiroshi Watarai
Research Scientist: Takuya Tashiro
		 Michishige Harada
		 Diana Eissens (until Apr 2012 )		 Nyambayar Dashtsoodol
Technical Staff: 		
		
		
		
		
		

Sakura Sakata
Yuko Nagata
Tomokuni Shigeura
Ritsuko Ozawa
Minako Aihara
Kayo Nagata (until Aug 2012 )Student Trainee:		

Yue Ren ( IPA )Sabrina Hamouche ( Intern until Jun 2012 )Tcells are a diverse group of immune cells involved incell-mediated acquired immunity. One unique type ofT cell is the innate-like invariant natural killer T ( iNKT ) cell,which recognizes glycolipid ligands instead of peptides ontarget cells.
NKT cells act as innate immune cells but also bridge
the innate and adaptive immune systems. The nature of
the NKT cell response is dictated by the initial cytokine environment: interaction with IL-10-producing cells induces
negative regulatory TH2/regulatory T cell-type NKT cells,
while that with IL-12-producing cells results in pro-inflammatory TH1-type responses. A prime example of one such
NKT cell bridging function is adjuvant activity: NKT cells
augment anti-tumor responses by their production of
IFN-γ, which acts on NK cells to eliminate MHC negative
target tumor cells and also on CD8 cytotoxic T cells to kill
MHC positive tumor cells. Thus, upon administration of
α-GalCer-pulsed DCs, both MHC negative and positive
tumor cells can be effectively eliminated. Based on thesefindings, we have developed human NKT cell-targetedadjuvant cell therapies with strong antitumor activity.

A phase I-II clinical study of -galactosylceramide( - GalCer)-pulsed IL-2/GM-CSF-cultured peripheral
blood mononuclear cells in patients with advanced
and recurrent non-small cell lung cancer
A phase I/IIa clinical trial has been carried out on 17 pa-tients with advanced lung cancer. 60% of patients withhigh IFN-γ production had a median survival time ( MST ) of
31.9 mo ( p=0.0015 ) , and remained as stable disease with
only the primary treatment. This outcome is significantly
more effective than molecular target drug treatment, with
an MST of 10 mo. These results are encouraging andwarrant further evaluation of the survival benefit of thisnew immunotherapy.
For the next step, we have applied in 2011 for theadvanced medical care assessment system establishedin 2008 by the Japanese Ministry of Health, Labor andWelfare ( MHLW ) , so that the patients’ immunotherapy
treatment will be covered in part by health insurance.
The system was established because advanced medical
technologies using medical devices or pharmaceuticals,such as immune cell therapy, that are not yet approved
under the Pharmaceutical Affairs Law ( PAL ) , are not covered by health insurance in Japan. However in responseto recent rapid progress in medical technology and thepatients’ need to have safe and lower cost treatment withsuch technologies, this system was introduced to allow
health insurance payments. It is also the case that thecollection of appropriate clinical research data is facilitated by this system, and thus can then lead to approval of
the advanced medical technologies under the PAL. The
NKT cell-targeted therapy was recently ( October, 2011)

Recent Publications
1. Watarai H, Yamada D, Fujii S, Taniguchi M, Koseki H.
Induced pluripotency as a potential path towards iNKT
cell-mediated cancer immunotherapy. Int J Hematol.
95, 624-631 ( 2012 )98

2. Nagato K, Motohashi S, Ishibashi F, Okita K, Yamasaki
K, Moriya Y, Hoshino H, Yoshida S, Hanaoka H, Fujii
SI, Taniguchi M, Yoshino I, Nakayama T. Accumulationof activated invariant natural killer T cells in the tumor
microenvironment after α-Galactosylceramide-pulsed
antigen presenting cells. J Clin Immunol. 32, 10711081 (2012 )3. 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 Function of Invariant
Natural Killer T cells Producing TH 2- and TH17-cytokines.
PLoS Biol. 10, e1001255 ( 2012 )Figure :approved by the government to qualify for the advanced
medical care assessment system and was started beginning in Jan 2012 at Chiba University.

Identification of NKT cell precursors in a subset of
CD4/CD8 double-negative (DN ) thymocytes
It is still under intense debate whether the developmental
pathway of the NKT cell lineage is as unique as their nature and behavior suggests, or whether it is similar to thatof conventional β T cells. Currently, the majority opinion
holds that NKT cells are derived from the same precursor
cells as conventional T cells after CD1d selection at theCD4/8 double-positive ( DP ) thymocyte stage. However,
we have previously shown the presence of NKT cell precursors in DN4 stage thymocytes, that is before reaching
the DP stage, in both wild-type and CD1d-deficient mice.
These results provided new insights into the early development of NKT cells prior to surface expression of theirinvariant Va14 antigen receptor and suggested a uniquedifferentiation pathway of NKT cells, different from thatof conventional T cells. Our current goal is to characterize further these NKT cell precursors with the aim ofelucidating the factors and the mechanisms underlying
this unique developmental pathway of NKT cells from DN
precursors. A better understanding of NKT cell biology
should provide us with theoretical foundations for future
clinical interventions to treat various immune-compromised situations.

4. 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 mammalian basic
leucine zipper transcriptinal factor, is a critical regulator of IL-10 and IL-13 production by CD4 T cells. Nat
Immunol. 12, 450-459 ( 2011)

The NKT cell subsets
NKT cells can be divided into four subsets
based on the expression of IL-17RB andCD4. The newly identified IL-17RB+ CD4and IL-17RB+ CD4+ NKT cells respond to
IL-23 and IL-25, respectively, triggering
i n f l am mat i o n by t h e p rod u c t i o n o f
TH2/9/17 cytokines, especially in the lung.

Development and function of NKT cell subtypes
We know that functionally distinct subtypes of NKT cells
are involved in specific pathologies, yet the development,
phenotypes, and functions of these cells are not well understood. We have determined the relationship between
various mouse NKT cell subsets, identified reliable molecular markers for these subsets, and shown that thesecontribute to their functional differences. We identified four
NKT cell subsets that arise via different developmental
pathways and exhibit different cytokine profiles. Importantly, we these subsets can be isolated from the thymus, aswell as from peripheral tissues such as spleen, liver, lung,and lymph nodes. Contrary to the general understandingthat NKT cells mature after their exit from the thymus andtheir migration into peripheral tissues, we could show that
distinct phenotypic and functional NKT cell subsets canbe distinguished in the thymus by virtue of the presence orabsence of the cytokine receptor IL-17RB and another cellsurface molecule CD4, and that these subsets then migrate to peripheral tissues where they retain their phenotypic and functional characteristics. Regarding functionalsignificance, we have shown that the NKT cell subsets thatlead to airway hyper-responsiveness to respiratory viruses
are different to those that lead to allergen-induced airway
hyperreactivity. These findings should enable researchers
to focus on specific subsets as potential targets for therapeutic intervention.

5. 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 )99

Laboratory for

Dendritic Cell
Immunobiology
Team Leader :Katsuaki Sato
Technical Staff: Kaori Sato
Yumiko Sato
Haruna Otsuka

D

endritic cells ( DCs ) are essential antigen-presenting
cells ( APCs ) that initiate primary immune responses. DCs are heterogeneous, and are mainly classified asconventional DCs ( cDCs ) and plasmacytoid DCs ( pDCs ) ,distinguishable by surface and intracellular phenotypicmarkers, immunologic function, and anatomic distribution.
Immature DCs ( iDCs ) serve as sentinels, recognizing thepresence of invading pathogens or virus-infected cells
through various pattern-recognition receptors. Subsequently, under inflammatory conditions they become mature DCs ( mDCs ) , with up-regulated expression of MHC
and costimulatory molecules. The mDCs then move via
the afferent lymphatics into the T-cell areas of secondary
lymphoid tissues where they prime rare antigen-specificnaive T cells for differentiation into effector T ( Teff ) cells, including T helper type (TH ) 1 cells, TH2 cells, and TH17 cells,
depending on environmental cues. DCs thereby play a
crucial role in the link between innate and adaptive immunity. Conversely, iDCs are also crucial for the induction of
immunological tolerance under steady-state conditions.
Their tolerogenic mechanisms include recessive tolerance mediated by clonal deletion and anergy as well as
dominant tolerance involving active immune suppression
by CD4 + Foxp3 + regulatory T ( Treg ) cells in the periphery,a function of likely importance in self-tolerance as well as
in immune disorders and transplant rejection. However,the precise functional role of each DC subset in immune
responses remains unclear. Our goal is to clarify the roleof DC subsets in the immune system in vivo and to identify
the molecular basis for the regulation of their function.

Plasmacytoid dendritic cells are crucial for the initiation of inflammation and T cell immunity in vivo
Plasmacytoid dendritic cells ( pDCs ) are specialized immune cells that are capable of producing large amountsof type I interferon ( IFN ) by sensing single-stranded RNA
or unmethylated CpG DNA through endosomal toll-likereceptors ( TLRs ) . Sialic acid binding Ig-like lectin ( Siglec ) -H is exclusively expressed on murine pDCs and is
unique among Siglec proteins in that it associates with
the ITAM-bearing adaptor molecule, DAP12. However, therole of pDCs and their Siglec-H-mediated regulation in thecontrol of immune responses in vivo remains unclear. Here
we report a critical role for pDCs in the regulation of inflammation and T cell immunity in vivo using gene-targeted
mice with a deficiency of Siglec-H and conditional ablationof pDCs. Siglec-H controlled the threshold of responsiveness to TLR ligands for the production of type I IFN andproinflammatory cytokines and this was mediated throughthe regulation of a MyD88 downstream signaling pathway.
pDCs were required for inflammation triggered by a TLR
ligand as well as by bacterial and viral infections. pDCs
also controlled homeostasis of CD4 + Teff cells and CD4 +Foxp3 + Treg cells. Upon antigenic stimulation and microbial
infection, pDCs suppressed the induction of CD4 + T-cell
responses and participated in the initiation of CD8 + T-cell

Recent Publications
1. Fukaya T, Murakami R, Takagi H, Sato K, Sato Y, Otsuka
H, Ohno M, Hijikata A, Ohara O, Hikida M, Malissen B,
Sato K. Conditional ablation of CD205 + conventional
dendritic cells impacts the regulation of T cell immunityand homeostasis in vivo. Proc Natl Acad Sci USA.
109, 11288-11293 (2012)

100

2. 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. Plasmacytoid dendritic
cells are crucial for the initiation of inflammation and T
cell immunity in vivo. Immunity 35, 1-14 (2011)

4. 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 expressedon mesenteric lymph node dendritic cells in the generation of antigen-specific CD4 + Foxp3 + regulatory T cells in
the establishment of oral tolerance. Blood 116, 22662276 (2010)

Figure 1: Splenic localization of pDCs is disrupted in
pDC-ablated mice.
Immunofluorescent microscopic analysis was
performed on frozen horizontal sections of
spleen in WT mice and pDC-ablated mice.
Sections were stained for CD19 (green), CD3e(blue) and BST2 (red). Original magnification;
10x.

Figure 2: The Multiple roles of pDCs in immune responses in vivo
pDCs secrete massive amounts of type I IFNs
after activation with viral or bacterial nucleic
acids via TLR7 and/or TLR9. Type I IFNs
are key cytokines that link innate and adaptive
immune responses during viral or bacterial infection. The pDC-ablated mouse model demonstrates the following in vivo roles of pDCs: (1)
TLR9-mediated inflammation: pDCs wereshown to initiate TLR-mediated inflammation
triggered by CpG DNA; (2) regulation of adaptive immunity: pDCs were shown to control
antigen-specific CD4 + T cells responses bygenerating CD4+Foxp3+ Treg cells and to induce
differentiation of CD8+ T cells after exposure toexogenous antigens through the cross-presentation pathway ; (3) mucosal tolerance: pDCs
maintained mucosal tolerance by generating
CD4+Foxp3+ Treg cells; (4) bacterial immunity:
pDCs induced bacterial septic shock and further exacerbated bacteria infection by impairing
bacterial clearance; and (5) viral immunity:
pDCs controlled viral infection by producing
type I IFNs and by supporting antiviral CD8+ T
cell differentiation through the cross-presentation pathway. Dysregulation of these pDC functions could be involved in the pathogenesis ofinfectious disease, inflammatory autoimmune
disease, graft rejection, allergy and cancer.

responses. Furthermore, Siglec-H appeared to modulate
the function of pDCs in vivo. Thus, our findings highlightpreviously unidentified roles of pDCs and the regulationof their function for the control of innate and adaptive
immunity.

Conditional ablation of CD205 + conventional
dendritic cells impacts the regulation of T cell
immunity and homeostasis in vivo.
Dendritic cells ( DCs ) are composed of multiple subsetsthat play a dual role in inducing immunity and tolerance.
However it is unclear how CD205 + conventional DCs( cDCs ) control immune responses in vivo. Here we generated novel knock-in mice that allowed the selective conditional ablation of CD205 + cDCs. CD205 + cDCs contributed
to antigen-specific priming of CD4 + T cells under steadystate conditions, whereas they were dispensable for an-

tigen-specific CD4 + T cell-responses under inflammatory
conditions. By contrast, CD205 + cDCs were required for
antigen-specific priming of CD8 + T cells to generate cytotoxic T lymphocytes ( CTLs ) mediated through cross-presentation. While CD205 + cDCs were involved in the thymic
generation of CD4 + Foxp3 + Treg cells, they maintained thehomeostasis of CD4 + Foxp3 + Treg cells and CD4 + Teff cells
in peripheral and mucosal tissues. On the other hand,
CD205 + cDCs were involved in the inflammation triggered
by TLR ligands as well as by bacterial and viral infections.
Upon microbial infections, CD205 + cDCs contributed tothe cross-priming of CD8 + T cells for generating antimicrobial CTLs to efficiently eliminate pathogens, whereas they
suppressed antimicrobial CD4 + T cell-responses. Thus,
these findings reveal a critical role for CD205 + cDCs in theregulation of T cell immunity and homeostasis in vivo.

5. Fukaya T., Takagi H., Taya H., Sato K. DCs in immune
tolerance in steady-state conditions. Methods Mol.
Biol. 677, 113-126 (2010)101

Laboratory for

Cytokine Signaling
Group Director:

Masaru Taniguchi

Senior Research Scientist: Toshiyuki Fukada, Keigo Nishida
Research Scientist:

Satoru Yamasaki, Wakana Ohashi, Shintaro Hojyo

Technical Staff:

Ayumi Ito , Masami Kawamura, Mayumi Hara

Student Trainee:

Tomohiro Miyai

Despite their increasing prevalence in developed countries, the molecular mechanisms leading to allergy
and autoimmune diseases remain poorly understood. The
ultimate goal of the Cytokine Signaling Research Group is
to help elucidate the underlying molecular and immunological mechanisms of allergy and autoimmune diseases fromthe viewpoint of signal transduction within the immune
system. We have shown that heavy metal cations such as
Zinc ( Zn ) can act as intracellular signaling molecules, i.e.,
molecules whose intracellular status is altered in responseto an extracellular stimulus, and that are capable of transducing the extracellular stimulus into an intracellular signaling event. Zn is known to be important in the immune
system, although its precise roles and mechanisms havenot been resolved. Therefore, we are focusing our attention on the largely unknown universe of signaling throughZn transporter proteins, to reveal the role of Zn in immune
and other physiological systems.

Molecular mechanisms of the mast cell-dependent
allergic reaction
We are investigating the role of Zinc ( Zn ) in mast cells
in allergy. We previously showed that stimulation of thehigh-affinity IgE receptor ( FceRI ) rapidly released intracellular Zn2+ from the ER in mast cells and we named thisphenomenon the “Zn wave” ( Yamasaki et al., J Cell Biol,
2007) . However, the gatekeeper for the Zn wave and theroles of the Zn wave in allergic reactions remain unclear.
We showed that the a1 subunit of the Cav1.3 L-type calcium channel (LTCC, a1D ) is localized on the ER membranerather than on the plasma membrane in mast cells andhas a novel function as a gatekeeper for the Zn wave without affecting calcium influx. These results not only showedthat the a1D subunit is an authentic gatekeeper for the Zn
wave but also made it possible for us to answer the ques-tion of what are the roles of the Zn wave. Furthermore, we
showed that the Zn wave was involved in cytokine production without affecting degranulation of mast cells throughacting on the FceRI-mediated NF-kB signaling pathway.
Consistent with this finding, an LTCC antagonist inhibitedthe cytokine-mediated delayed-type allergic reaction inmice without affecting the immediate-type allergic reaction. Thus, the Zn wave regulates FceRI-mediated cytokine
production and is required for cytokine-dependent allergic
reaction ( Fig. 1, Yamasaki et al., PLoS ONE. 2012) .

Role of Zn and its transporters in immune and nonimmune tissues
We found that the Zn transporter Slc39a6/Zip6/Liv1 is a
STAT3 target gene and showed that it has a role in cell
migration during early zebrafish development. (Yamashita
et al., Nature. 2004). We also demonstrated that LPS-induced maturation of DCs is partly mediated through lowering the intracellular concentration of free Zn2+ by down
regulating Zn transporters, including Slc39a6 (Kitamura
et al., Nat Immunol. 2006), suggesting involvement of Zn2+
in MHC class II cell surface expression through regulating
endocytosis and membrane trafficking. In order to understand the role and function of Zn2+ and Zn transporters invivo, we have generated mice deficient in the Slc39/Zip
family of zinc transporters. We found that Slc39a13/Zip13
knockout mice (Slc39a13-KO ) show changes in connective
tissues reminiscent of the human disease Ehlers-Danlos
syndrome ( EDS ) , which is characterized by defects in thematuration of osteoblasts, chondrocytes, odontoblasts,
and fibroblasts. Impairment of bone morphogenic protein( BMP ) and TGF-b signaling was observed in the corresponding tissues and cells. A homozygous SLC39A13
loss of function mutation was identified in sibs affectedwith a unique variant of EDS that recapitulates the pheno-

Recent Publications
1. Yamasaki S., A. Hasegawa, S. Hojyo, W. Ohashi, T.
Fukada, K. Nishida, and T. Hirano. A Novel Role of the
L-Type Calcium Channel α (1D ) Subunit as a Gatekeeper
for Intracellular Zinc Signaling: Zinc Wave. PLoS ONE
7( 6 ): e39654, ( 2012 )102

2. 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 human ZIP13
protein: a homo-dimerized zinc transporter involved in
the Spondylocheiro dysplastic Ehlers-Danlos syndrome.
J Biol Chem. 286, 40255-40265 ( 2011)

3. Nishida K., S. Yamasaki, A. Hasegawa, A. Iwamatsu, H.
Koseki, and T. Hirano. Gab2, via PI-3K, Regulates ARF1
in Fc ε RI-Mediated Granule Translocation and Mast
Cell Degranulation. J Immunol. 187, 932-941 ( 2011)

Figure 1: The Zn wave participates in Fc e RI-mediated mast cell
activation.
FceRI stimulation induces the rapid elevation of intracellular
Zn2+ from the perinuclear region, around the ER area, and we
named this phenomenon the “Zn wave”. The a1D subunit of the
LTCC is expressed on the ER membrane and acts as the Zn wave
gatekeeper in mast cells. In addition, the LTCC-mediated Zn wave
is a positive regulator of the DNA-binding activity of NF-kB and is
involved in regulation of the allergic reaction.

type observed in Slc39a13-KO mice. Hence, our results
have revealed a crucial role of SLC39A13 in connective
tissue development, at least in part due to its involvementin BMP/TGF-b signaling pathways ( Fukada et al., PLoS
ONE, 2008 ) . We have found that the human ZIP13 protein
forms homo-dimers ( Bin et al., J. Biol. Chem, 2011) , andits further characterization by crystallography is underinvestigation. In addition, we have investigated the roleof Slc39a14 by generating deficient mice. The Slc39a14KO mice showed progressive systemic growth defects
and impaired signaling of a G-protein -coupled receptor( GPCR ) in the growth plate, pituitary grand, and liver. We
found that Slc39a14-mediated Zn2+ plays a role in control
of GPCR-cAMP response element-binding protein ( CREB )signaling via regulating cAMP levels by suppressing phosphodiestarase ( PDE ) activity ( Hojyo et al., PLoS ONE,2011) , suggesting a crucial role of Slc39a14 in control ofmammalian systemic growth. We also showed that Zn 2+
suppresses Th17-mediated autoimmune diseases by inhibiting the development of Th17 cells via attenuating STAT3
activation. In mice injected with type II collagen to induce

4. Fukada, T., S. Yamasaki, K. Nishida, M, Murakami, andHirano, T. Zinc homeostasis and signaling in health anddiseases: Zinc signaling. J Biol Inorg Chem. 16, 11231134 ( 2011)

Figure 2: “Zn signal axes” affect distinct signaling pathways
to promote and control cellular functions
Each Zn signal axis targets a specific molecule. Thismode of Zn action affects a variety of cellular functions
by selectively regulating distinct signaling pathways.
Therefore, the disruption of a Zn signal axis can bea pathogenesis factor for disease in the absence of
redundant systems.

arthritis, Zn2+ treatment inhibited Th17 cell development.
IL-6-mediated activation of STAT3 and in vitro Th17 cell
development were all suppressed by Zn 2+. Importantly,
Zn2+ binding changed the α-helical secondary structureof STAT3, disrupting its association with JAK2 kinase andwith a phospho-peptide that contained a STAT3-binding
motif from the IL-6 signal transducer gp130. Thus, we conclude that Zn2+ suppresses STAT3 activation, which is a
critical step for Th17 development ( Kitabayashi et al., Intnl
Immunol., 2010 ) . Together these results support the ideathat Zn2+ has roles in mediating and controlling intracellularsignaling events. We propose the term “Late Zn signaling”
for this type of signaling, in contrast to “Early Zn signaling”.
The former is dependent on changes in the transcriptionof Zn transporter genes, whereas the latter is not ( Hirano
et al., Adv Immunol., 2008 ) . We also proposed that a “Znsignal axis” composed of Zn transporters and target molecules determines the biological specificity of Zn signaling( Fig. 2, Fukada et al., J Bone Miner Metab., 2012) .

5. Hojyo, S.*, T. Fukada*, S. Shimoda, W. Ohashi, B-H. Bin,
H. Koseki, T. Hirano. The Zinc Transporter SLC39A14/
ZIP14 Controls G-Protein Coupled Receptor-MediatedSignaling Required for Systemic Growth. PLoS ONE
6 (3 ): e18059 (2011) (*Equally contribution )103

Laboratory for

Immunogenetics
Team Leader:

Hisahiro Yoshida
Research Scientist: Takuwa Yasuda
Technical Staff:
		
		
		
		

Hitomi Fukiage
Akimi Banno
Ayako Kobayashi
Mikiko Sato
Takeyuki GotoThe main activity of our team is the screening of a large
ethylnitrosourea ( ENU ) mutant mouse panel and is acollaborative effort with the RIKEN Genome Science Center (GSC). An important goal of this project for RCAI is thedevelopment of novel mouse models for various immunological disorders, notably, allergic and autoimmune disorders. Since ENU mutagenesis introduces approximately
3,000 point mutations in a genome, we can expect 100
coding region mutations in one pedigree. In parallel, we
are screening the mutant mice under environmental bias,
using a variety of approaches including immunization withallergens and adjuvants to identify allergy modifier genes.

ENU mutant panel study
In order to identify the genetic basis for immune disorders, we are screening a pool of mutant mice generated
by random chemical mutagenesis. ENU induces random
single-base pair changes in genomic DNA at approximately 3,000 sites throughout the entire genome, resulting
in approximately 100 sites in protein coding regions per
first-generation ( G1) mutant mouse.
During the first 4 years of this project we havescreened 7,600 recessive inheritances from 80 mouse
pedigrees. For basic phenotypic screening, we examined
the levels of immunoglobulins, cytokines, and autoantibodies, as well as expression of a panel of cell surface
markers on peripheral blood cells. We also examined tissue sections from animals at 16 weeks of age. We havethus analyzed the mutant phenotypes by pathological, cytological and molecular biological evaluations of affected
tissues, lymphoid organs and blood cells.
A phenotype identified in a mutant candidate line
is reexamined at a later developmental stage and, if thesame phenotype is confirmed, we begin mating the micefor phenotype inheritance tests. The responsible mutant
loci are mapped by backcrossing mutant individuals with
the C3H/HeJ strain for gene detection by single-nucleotide polymorphism analysis. After approximate mapping,candidate gene sequences are compared with wild typesequences in public databases by means of the PosMed
research system established by the RIKEN GSC. Candidate gene mapping has been done in collaboration with
the Phenome Informatic Team and Mouse Mutation Resource Exploration Team in the GSC, RIKEN.

Allergic disease mutant mouse model
By ENU recessive mutant screening, we identified andestablished a mutant mouse line with phenotypic features
reminiscent of a common human skin disease, atopic dermatitis, a Th2-mediated condition. The ear skin became
thicker and red and, as the mice aged, they started toscratch the ear skin and face. The serum IgG1 and IgElevels of these mice gradually increased at 3 weeks after
the dermatitis onset. Pathologically, the epidermal layer
was hypertrophic and many inflammatory cells were foundin the dermis of the lesion. Ultimately the mice developed

Recent Publications
1. Sugiyama M, Nakato G, Jinnohara T, Akiba H, Okumura
K, Ohno H, Yoshida H. Expression pattern changes andfunction of RANKL during mouse lymph node microarchitecture development. Int Immunol. 24, 369-378(2012 )

104

2. 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 T ( h ) 2- andT(h )17-cytokines. PLoS Biol. 10 (2 ):e1001255 (2012 )3. Tachibana M, Tenno M, Tezuka C, Sugiyama M, Yoshida
H, Taniuchi I. Runx1/Cbf β2 complexes are required for
lymphoid tissue inducer cell differentiation at two developmental stages. J Immunol. 186, 1450-1457 (2011)

Figure :An atopic dermatitis-like mutant mouse was established from the ENU screen.
The above scheme illustrates the stepwise progression of AD disease in the mutant animals. The responsible point
mutation in a single gene and the developmental mechanism(s) of this dermatitis have been clarified (manuscript
under submission).

chronic facial and ear skin inflammation and not only Th2
serum immunoglobulin but also Th1 serum immunoglobulin levels increased. These stepwise symptoms and findings are compatible with the diagnostic criteria for humaninfantile atopic dermatitis ( AD ) . We have mapped thephenotype-causing point mutation to a distinct gene anddirectly confirmed this causality by genetic manipulation
to introduce the same mutation into the gene of wild typemice. A bone marrow transplantation study demonstrated
that the disease is induced by mutant skin tissue and notby mutant bone marrow cells. We have clarified the disease onset mechanism and succeeded in disease onset
prevention based on the results of our studies. Interestingly, the causative mutation was found in a ubiquitously
expressed signal transduction gene and the responsible
tissue for dermatitis development was skin but not bonemarrow. In accordance with our findings, we have foundseveral ways to prevent the dermatitis onset ( manuscript
under submission ) . We are now further analyzing the
stepwise progression of this AD-like disease in the mutant
mouse to better understand the allergic disease progression in skin and the immune system.
By ENU recessive mutant screening, we have identified and established more than 145 mutant lines of

4. Nabeyama A, Kurita A, Asano K, Miyake Y, Yasuda T,
Miura I, Nishitai G, Arakawa S, Shimizu S, Wakana S,
Yoshida H, Tanaka M. xCT deficiency accelerates chemically induced tumorigenesis. Proc Natl Acad Sci U S A.
107,6436-6441 (2010 )immune or blood disease models. In keeping with our
expectation, more than 50 lines had phenotypes related toallergic diseases. From these mutant lines, we are now analyzing a mutant line with features of experimental allergic
asthma, rhinitis and conjunctivitis by OVA immunization.
We are now backcrossing some of the mice to the
C3H/HeJ strain in order to map the responsible loci. As
of December 2012, we have mapped thirty-two independent mutant-causing loci to distinct regions, and nine ofthem have been identified to be point mutations in independent genes. Along with allergic disease model mutant
screening, we have identified and characterized a few
other interesting phenotypes in mutant lines with/without
allergic defects. As listed in the publications section, we
have reported two independent mutant lines in which
there are point mutations in distinct genes. One mutation
is found in the Pla2g6 gene and may be a good model forthe human neurodegenerative disease infantile neuroaxonal dystrophy ( INAD ) ( Wada et al., Am J Pathol. 2009 ). The
other is caused by a point mutation in the Themis gene,which plays an important role in thymocyte development,
and this mutant line may be a good model for a primary
immunodeficiency disease ( Kakugawa et al., Mol Cell Biol.
2009 ) .

5. Yoshibumi M., Yoshiaki K., Toyoyuki T., Kunie M., Yuta
S., Hisahiro Y., Yoshiyuki M., Hajime K., and Hiromichi Y.
An Atopic Dermatitis-Like Skin Disease with Hyper-IgE–
Emia Develops in Mice Carrying a Spontaneous Recessive Point Mutation in the Traf3ip2 (Act1/CIKS ) Gene.
J Immunol. 185,2340-2349 (2010 )105Research Unit forAllergy

Unit Leader:

Toshiaki Kawakami
Research Scientist:
		 Jun-ichi Kashiwakura
Technical Staff:
		 Machiko Ishikawa

Histamine-releasing factor in allergyF

actors that can induce the release of histamine frombasophils and mast cells have been studied for morethan 30 years. A protein termed histamine-releasing factor( HRF ) was purified and molecularly cloned in 1995. HRF
can stimulate histamine release and IL-4 and IL-13 production from IgE-sensitized basophils and mast cells. HRF-like
activities were found in body fluids during the late phaseof allergic reactions, implicating HRF in allergic diseases.
However, definitive evidence for the role of HRF in allergic
diseases has been elusive. On the other hand, we foundeffects of monomeric IgE on the survival and activationof mast cells without any involvement of specific antigen,as well as heterogeneity of IgE molecules in their ability
to cause such IgE effects. The latter property of IgE molecules seemed to be similar to the heterogeneity of IgEs
in their ability to prime basophils in response to HRF. Thissimilarity led to our recent finding that ~30% of IgE andIgG molecules can bind to HRF via interactions of their
Fab with two binding sites within the HRF molecule ( Fig.
1) . Given the existence of a disulphide-linked dimeric formof HRF, this result can explain how co-stimulation with
HRF-reactive IgE and recombinant HRF can activate mast
cells and basophils in vitro ( Fig. 2 ) . Use of short IgE-bind-ing HRF-derived peptides that can block HRF-IgE interactions in a competitive fashion revealed an essential roleof HRF in promoting skin hypersensitivity ( i.e., passive cutaneous anaphylaxis ) and airway inflammation. Intranasal
injection of recombinant HRF induced macrophage-dominant lung inflammation in naïve mice, similar to thatobserved in transgenic mice expressing HRF in a Claracell-specific manner. We have begun to study the possible
involvement of HRF in human allergic diseases. In atopic
dermatitis ( AD ) , our data thus far indicate that HRF levels
are increased in sera of AD patients and some ( ~10 percent ) AD patients have measurable levels of HRF-reactive
IgE as measured by ELISA. By contrast, normal subjects
have undetectable levels of serum HRF-reactive IgE. Overall, our published studies strongly support the notion that
HRF secreted during allergic reactions promotes or amplifies allergic reactions by activating mast cells ( and basophils ) through cross-linking of HRF-reactive IgE boundto FceRI on these cells ( as well as through cross-linking
of HRF-reactive IgG-bound to FcgRs ) . Our unpublishedresults also suggest that HRF plays a proallergic role in amouse model of food allergy. We are currently measuringserum HRF and HRF-reactive IgG and IgE in food allergy
patients who have or have not benefited from rapid oral

Recent Publications
1. Kashiwakura, J.*, Ando, T.*, Matsumoto, K., Kimura,
M., Zajonc, D.M., Ozeki, T., Siraganian, R.P., Broide, D.,
Kawakami, Y., and Kawakami, T. Histamine-releasing
factor has a proinflammatory role in mouse models ofasthma and allergy. J Clin Invest. 122, 218-228 (2012)*equal contributions

106

2. Kawakami, T. Human FcγRIIA at the center stage of anaphylaxis. Blood 119, 2432-2433 (2012)

3. Kawakami, T., Ando, T., and Kawakami, Y. HRF-Interacting Molecules. Open Allergy J. 5, 41-46 (2012)

Figure 1: Interaction sites between HRF and IgG. Ig-binding sites were mapped to the N-terminal 19 residues(N19) and the H3 (residues 107-135) portion of HRF, while HRF binds to the Fab portion of Igs. The
top left and bottom left panels show the domain structure and the 3-D structure of human HRF (Protein
Data Bank, 1YZ1), respectively. The N19 and H3 portions are highlighted. The TCTP1 portion is omitted
from the model because it does not form interpretable electron density in the crystal structure. The rightpanel shows a cartoon of an IgG molecule.

Figure 2: Working model of HRF-mediated FceRI crosslinking. IgE binds the FceRIa chain via the interaction
between Ce3 and D2 domains. HRF can exist as a dimer and one HRF molecule can bind to twomolecules of IgE via interactions with the N19 and H3 regions of HRF. The top view (Top) of IgE at thelevel of Fab and the side view (Bottom) of IgE-bound FceRIa chain are shown on the left. After bindingof an HRF dimer, four FceRIa chain-nucleated complexes will be formed (Right). The cytoplasmic
portion of the FceRIa as well as β and γ chains of the FceRI are omitted for clarity.

tolerance induction clinical protocols. Such a study, together with global gene expression profiling and other experiments, will help clarify the role of HRF as a biomarker

4. Kashiwakura, J., Okayama, Y., Furue, M., Kabashima, K.,
Shimada, S., Ra, C., Siraganian, R.P., Kawakami, Y., andKawakami, T. Most highly cytokinergic IgEs have polyreactivity to autoantigens. Allergy Asthma Immunol
Res. 4, 332-340 (2012)

and/or pathogenic factor in food allergy and/or tolerance
induction or maintenance.

5. Xiao, W., Kawakami, Y., and Kawakami, T. Immune regulation by phospholipase C-β isoforms. Immunol Res.
56, 9-19 (2013)

107

Laboratory for

Vaccine Design
Team Leader:

Yasuyuki Ishii
Research Scientist: Hidetoshi Akimoto
Satoshi Komaniwa
Emi Fukuda
Visiting Scientist:

Kan Kaneko
Masako Ikemiyagi
Toshihito Hirai
Kenichi Masuda

Technical Staff:

Yoko Kaitsu
Risa Nozawa

Two major activities of invariant natural killer T ( iNKT )cells orchestrate the immune regulation between innate and acquired immunity. One is a host defense activity, including IFN-g production by iNKT cells stimulated with
CD11c + dendritic cells. The other is induction of immune
tolerance generated by multiple systemic administrationof a-galactosylceramide ( a-GalCer ) , a representative
exogenous ligand for iNKT cells. Although the tolerance
induction activity iNKT cells could potentially be applied toa variety of therapies for immune disorders, understandingof the detailed mechanism of action and development ofsuitable methods for its selective enhancement among
the multivalent activities of iNKT cells have remained
undiscovered. We previously found that a liposomal formof a-GalCer, but not an aqueous one, could predominantly
enhance the immunoregulatory functions of iNKT cells,such as IL-10 production. Based on these results, we attempted to suppress IgE antibody formation in a mouseallergy model by the administration of liposomal a-GalCer.
In the case of primary IgE antibody formation, a systemic
injection of liposomal a-GalCer prior to immunization withalum-absorbed antigen resulted in a remarkable suppression of both IgE and IgG antibody formation. By contrast,
both the secondary IgE antibody and total IgE response of
fully antigen-sensitized mice after antigen challenge weresignificantly suppressed, but the IgG antibody response

was not, even after repeated systemic injection of liposomal a-GalCer. The first aim is to clarify the mechanism of
IgE isotype-specific suppression induced by treatment
with liposomal a-GalCer.
Despite being a versatile delivery system, liposomes
have some disadvantages, including having a tendency
to clump upon storage and lacking sufficient stability fororal delivery. Stability in the presence of gastrointestinal(GI) tract bile salts can be improved through the use ofphospholipids with high transition temperatures, such as
dipalmitoyl-phosphatidylcholine ( DPPC ) . Two new liposomal a-GalCer formulations will be investigated in thisproject – silica and chitosan coated liposomes. Both theseformulations are still simple to produce but the liposomes
are stabilized by the coating process.

Mechanism for IgE isotype-specific suppression by
liposomal a-GalCer
In the course of our studies, we found that rhodamine-labeled liposomal a-GalCer was incorporated into splenic
B220 + CD1d high cells but not into B220 + CD1dlow cells, andmuch more than into CD11b + macrophages or CD11c +dendritic cells. Immunohistochemical observation clearly
showed that rhodamine-positive dots corresponding to
B220 + CD1d high cells were localized at the marginal zone
area in the spleen and that co-localization of iNKT cells

Recent Publications
1. Shimizu K, Mizuno T, Shinga J, Asakura M, Kakimi K,
Ishii Y, Masuda K, Maeda T, Sugahara H, Sato Y, Matsushita H, Nishida K, Hanada KI, Dörrie J, Schaft N, Bickham K, Koike H, Ando T, Nagai R and Fujii S, Vaccination
with antigen-transfected, NKT cell ligand-loaded, humancells elicits robust in situ immune responses by dendritic
cells. Cancer Res. 73, 62-73. ( 2012 )108

2. 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-versus-Leukemia Effect in Murine
Models. Biol Blood Marrow Transplant. 17, 11541168 (2011)

3. Ishii Y, Motohashi S, Shimizu K, Nakayama T, Taniguchi
M, and Fujii S. Application of NKT cells in immunotherapy, Curr Immunol Rev. 6, 109-115 ( 2010 )Figure 1 : Silica nanoparticle coated liposomes
Coating by colloidal silica (Ludox): Amorphous, nonporous, spherical particles with an average particle size of~40 nm. The coating was formed by the electrostatic interaction between the negatively charged silica nanoparticle
surface and the positively charged amine group of the phospholipid.

Figure 2 : Chitosan coated liposomes
Chitosan is biodegradable, mucoadhesive and can facilitate tight junction opening. Liposomes are coated by theelectrostatic interaction between the negatively charged phosphate of dicetyl phosphate and the positively charged
amine group of the chitosan.

after the rhodamine-liposome injection could be detected in the mice transferred with NKT-ES-V cells, in which
IRES-Venus was inserted downstream of the TCR Ca
stop codon in NKT-ES cells. This co-localization as well
as IL-21 mRNA expression by iNKT cells were completely inhibited by pre-treatment of the recipient mice with
anti-CXCL16 neutralizing mAb, suggesting that IL-21 production by iNKT cells was induced after their interaction
with the liposomal a-GalCer-pulsed B220 + CD1d high cells.
Indeed, IL-21 mRNA expression by iNKT cells was detected after in vitro co-culture of iNKT cells isolated fromnormal mouse spleen with splenic B220 + CD1d high cells
derived from mice injected with liposomal a-GalCer. Since
it has been reported that IL-21 is capable of inhibiting IgEclass switching, we wondered if IL-21 production by iNKT
cells might be involved in the IgE isotype-specific suppression generated by the liposomal a-GalCer. To explore
this possibility, we analyzed the expression of IgE, IL-21
receptor and Bmf ( Bcl-2 modifying factor) , which would
be activated by IL-21 in splenic B cells of IgE-producing
mice, after the injection of the rhodamine-labeled liposomal a-GalCer. The expression level of IL-21 receptor
mRNA in rhodamine-positive B cells was remarkably higher than that in rhodamine-negative B cells. Surprisingly,
there was substantial expression of IgE and Bmf mRNA in
rhodamine-positive B cells but not in rhodamine-negative
B cells. These results suggest that liposomal a-GalCer
was incorporated into the marginal zone B220-postive

cell population including IgE-producing Be cells, which
would express Bmf and IL-21 receptor. The Be cells would
be induced to undergo apoptosis by IL-21 derived fromiNKT cells after their interaction with a-GalCer-presenting
Be cells.

Manufacturing of liposomal -GalCer formulations
for oral administration
Oral tolerance has been expected to be utilized as oneof the key immunotherapies for allergic diseases. If oral
allergy vaccines were developed, allergy patients would
be more likely to accept such a non-invasive treatment.
However, oral immunotherapies still remain in the research
stage of development. In order to apply oral allergy vaccines to clinical studies, vaccine design based on solid
basic research is required. In an international researchcollaboration between RCAI and the University of Otago
in New Zealand, we began to combine our a-GalCer liposome technology with our extensive experience in theformulation of vaccines using not only liposome but also
silica, chitosan and emulsion based formulations for oral
administration (Fig. 1 and 2). Now we are screening to findthe best formulation of liposomal a-GalCer to induce oral
tolerance. Then we will perform studies to understand thedetailed mechanism of action, such as identification of target antigen-presenting cells and putative regulatory cells
in the GI-tract.

109

Laboratory for

Human Disease Models
Group Director:

Fumihiko Ishikawa
Senior Research Scientist: Yoriko Saito
Technical Staff: Nahoko Suzuki
		Mariko Tomizawa-Murasawa
Hiroshi Kajita
Ikuko Ogahara
Akiko Kaneko
Saera Fujiki
Student Trainee: Yuki Aoki (JRA)Shinsuke Takagi (JRA)Yuho Najima (JRA)To bridge mouse research and human research, wehave been creating humanized mouse models. Humanized mice developed by injecting human cord blood
HSCs into newborn NOD/SCID/Il2rgKO ( NSG ) have enabled us to analyze differentiation and function of thehuman immune system in vivo. In this fiscal year, we aim
to investigate the development of human innate immunity
in NSG recipients. However, in the current humanizedmouse models, human immune subsets differentiate frominjected human hematopoietic stem cells within a mousemicroenvironment. To better reconstitute human immunity
in mice, we have to develop immune-compromised micethat also have a humanized microenvironment.
To this end, we are currently creating humanizedthymic and bone marrow environments in NSG mice. To
date, we found that human CTL responses against virus-infected cells can be identified in an HLA-expressing
humanized mice. As another example, NSG with membrane-bound human stem cell factor supported the development of significantly higher levels of human myeloid
subsets. In collaboration with Dr. Leonard Shultz at theJackson Laboratory and Drs. Koseki and Ohara at RIKEN RCAI, we continue to investigate the significance ofhumanized mice with a humanized microenvironment andtry to apply these systems into understanding of humandiseases.

Development of human myeloid subsets inhumanized NSG mice
While physiological development of human lymphoid
subsets has become fairly well understood in humanized
mice, in vivo development of human myeloid subsets
has remained unevaluated. We have investigated in vivo
differentiation and function of human myeloid subsets in
NSG mouse recipients transplanted with purified human
cord blood hematopoietic stem cells. At four to six months
post-transplantation, we observed the development of
human neutrophils, basophils, mast cells, monocytes, aswell as conventional and plasmacytoid dendritic cells in
the recipient bone marrow. Frequencies of monocytes anddendritic cells in the humanized mouse bone marrow (BM)were similar to those seen in the primary human BM, while
the frequency of HLA-DR ( - ) granulocytes was lower in humanized mouse BM compared with human BM. Consistent with the expression of cytokine receptors and TLR by
the engrafted human myeloid cells, in vivo administrationof human G-CSF or LPS resulted in the recruitment of human myeloid cells into the recipient circulation or elevationof human inflammatory cytokines in the humanized mousesera. Human bone marrow monocytes and alveolar macrophages in the recipients displayed intact phagocytic
function. In particular, human BM-derived monocytes/
macrophages were further confirmed to be capable of

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 in vivo
human hematopoietic engraftment and myeloid differentiation. Blood 119, 2768-2777. (2012)110

2. Tanaka S, Saito Y, Kunisawa J, Kurashima, Y., Wake, T.,
Suzuki, N., Shultz, LD., Kiyono, H., Ishikawa, F. Development of mature and functional human myeloid subsets inhematopoietic stem cell-engrafted NOD/SCID/IL2rgKO
mice. J Immunol. 188, 6145-6155. (2012)

3. 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 HLAclass I expressing NOD/SCID/IL2rγnull humanized mice.
Proc Natl Acad Sci USA. 107, 13022-13027. (2010)

Figure :Development of human myeloid subsets in the BM of an NSG recipient(A)CD15+ neutrophils, HLA-DR+CD14+ monocytes, CD117+ mast cells, and CD203c+CD117- basohpils were present
in the BM of an NSG recipient transplanted with cord blood HSCs. (B) Engrafted human myeloid subsets express
TLR2 and TLR4 (white: isotype control). (C) In vivo administration of 15mg LPS into humanized NSG mice resulted
in rapid elevation of human inflammatory cytokines in the sera. (D) Humanized mouse BM-derived macrophages
phagocytized GFP-tagged Salmonella and inhibited Salmonella colony formation upon IFNg stimulation.

phagocytosis and killing of Salmonella Typhimurium upon
IFN-γ stimulation. In vivo human myelopoiesis establishedin the NSG humanized mouse system may facilitate theinvestigation of human myeloid cell biology, including
in vivo analyses of infectious diseases and therapeuticinterventions.

Human leukemia models
We are currently investigating two types of acute leukemia,
acute lymphoblastic leukemia with Mixed lineage leukemia(MLL) gene rearrangement and adult-onset acute myeloid
leukemia. For acute myeloid leukemia, we reported thattransplantation of CD34 + CD38 - leukemia cells into newborn NSG resulted in robust recapitulation of the patients’leukemic status in mice. In the recipient bone marrow,

4. Saito Y, Uchida N, Tanaka S, Suzuki N, Tomizawa-Murasawa 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)

CD34+ CD38- cells generated non-stem AML cells such as
CD34+ CD38 + cells or CD34- cells. By contrast, MLL-rearranged acute lymphoblastic leukemia ( ALL) shows a different hierarchy of leukemogenesis; i.e., leukemia developedin NSG recipients consisted of CD34-CD19 + ALL cells aswell as CD34+ CD38 + CD19 + cells. Furthermore, transplantation of CD34-CD19 + ALL cells resulted in the engraftmentof CD34 + CD38 + CD19 + cells and CD34-CD19 + cells in therecipient bone marrow.
Based on the distinct stem cell hierarchy in these two
types of leukemia, we are attempting to identify therapeutic targets in each one, through comparison of gene
expression profiles of leukemia stem cells and normal hematopoietic stem cells.

5. 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 for quiescent, chemotherapy-resistant human leukemia stem
cells. Sci Transl Med. 2, 17ra9. (2010)111

Research Unit for

Cellular Immunotherapy

Unit Leader :Shin-ichiro Fujii

Technical Staff : Jun Shinga
Student Trainee : Yusuke Sato (JRA)B

oth innate and adaptive immunity are crucial for cancer immune surveillance, but precise therapeutic
equations to restore such surveillance in cancer patients
have yet to be developed. The forms of antigen, adjuvant
and antigen-presenting cells may be crucial componentsof successful immunotherapy. Particularly, dendritic cells( DCs ) , as “nature’s adjuvants”, play a pivotal role in determining the character and magnitude of an immune response. The ex vivo loading of autologous patient DC withtumor specific antigens is one of the most promising current immunotherapeutic strategies. On the other hand, ex
vivo DC therapy requires the generation of large numbersof DCs from individual patients and the quality of the DCs
will likely depend on the patient’s condition at the time ofvenipuncture to harvest DC precursors.
We have studied the role of DCs in situ for tumor immunity by focusing on the link between innate and adaptive immunity. We evaluated another in vivo DC targetingstrategy that exploits the pro-inflammatory potential of

dying cells together with the adjuvant activity of invariant
natural killer T ( iNKT ) cells. We successfully developed astrategy in murine models for the induction of antigen-specific T cell responses using tumor-associated antigen expressing, a-GalCer-loaded allogeneic cells through in situ
DC maturation. Based on these findings, we have attempted to launch preclinical studies of human artificial adjuvant
vector cells ( aAVCs ) , in collaboration with Dr. Shimizu( Therapeutic Model Research Unit) .
We have been attempting to generate better strategies for immunotherapy, not only to establish vaccines andtherapeutic models, but also to elucidate the basic natureof the immune response in these systems.

Vaccination with antigen-transfected, NKT cell
ligand-loaded, human cells elicits robust in situ
immune responses by dendritic cells.
We previously demonstrated the in vivo effectivenessof allogeneic fibroblast cells loaded with a-GalCer andRecent Publications

112

1. Shimizu, K., Mizuno, T., Shinga, J., Asakura, M., Kakimi,
K., Ishii, Y., Masuda, K., Maeda, T., Sugahara, H., Sato,Y., Matsushita, H., Nishida, K., Hanada, K., Dorrie, J.,
Schaft, N., Bickham, K., Koike, H., Ando, T., Nagai, R.,
Fujii, S. Vaccination with antigen-transfected, NKT cell
ligand-loaded, human cells elicits robust in situ immune
responses by dendritic cells. Cancer Res. 73, 62-73(2013)

2. Fujii S., Shimizu K. DC-based immunotherapy targeting
NKT cells. In Terabe M and Berzofsky JA (eds ), Naturalkiller T cells: Balancing the regulation of tumor
immunity. Springer New York Dordrecht Heidelberg
London, 95-110 ( 2011)

3. 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)

Figure :The antigen presenting capacity of human DCs in NOG mice in response to aAVC
Immature DCs generated in vitro from human monocytes were transferred into NOG
mice and then iNKT cells and aAVC were transferred. Ten hours later, CFSE-labeled
HLA-A2+JKF6 cells (stable melanoma responding TIL cells) were adoptively transferred. Five days later, JKF6 T cell proliferation as a measure of human APC function
was evaluated by dilution of CFSE. The numbers indicate the percentages of divided
JKF6 T cells.

transfected with antigen-encoding mRNA, thus combining
the adjuvant effects of iNKT cell activation with deliveryof antigen to DCs ( Blood 2009 ) . In the current study, weestablished human artificial adjuvant vector cells ( aAVC )consisting of human HEK293 embryonic kidney cells
stably transfected with CD1d, loaded with a-GalCer andthen transfected with antigen-encoding mRNA. When administered to mice or dogs, these aAVCs activated iNKT
cells and elicited antigen-specific T cell responses with noadverse events. In parallel experiments, using the humandendritic cell ( hDC ) -transferred NOD/SCID/IL-2r c null immunodeficient (hDC-NOG ) mouse model, we also showedthat the human melanoma antigen MART-1 expressed bymRNA transfected aAVCs could be cross-presented toantigen-specific T cells by human DCs. Antigen-specific T
cell responses elicited and expanded by aAVC were verified as functional in tumor immunity. Our results support
the clinical development of aAVC to harness innate andadaptive immunity for effective cancer immunotherapy.

4. 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)

A strategy using in vivo DC maturation by NKT cells
to generate adaptive immunity with T cell memory
We have been examining an approach for inducing adaptive immunity based on the adjuvant effect of a-GalCer
and using in vivo DC maturation, which we have found to
be more effective than ex vivo manipulation of DCs. With
this strategy of using NKT cell ligand-loaded syngeneic
tumor antigen expressing cells, the activation of adaptive
immunity occurred through in vivo maturation of DCs andwas able to optimize tumor antigen delivery systems to
DCs. Moreover, we have found that DCs can play a rolein cross presentation of tumor antigen and glycolipid to
T cells and NKT cells, respectively. We also found that T
cells responding to the specific antigen can be maintained
as memory T cells, thus resulting in long-term protection.
We have recently been elucidating the mechanism for thismemory T cell response.

5. 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 )6. Fujii S, Goto A, Shimizu K. Antigen mRNA-transfected,
allogeneic fibroblasts loaded with NKT-cell ligand confer
antitumor immunity. Blood 113, 4262-4272 (2009 )113

Laboratory for

Immunogenomics
Group Director:

Osamu Ohara
Senior Research Scientist:		Takaho A. Endo
Research Scientist:		Takashi Watanabe, Yoko Kuroki,
		 Yoshitaka Shirasaki
Research Associate:		Atsushi Hijikata, Mai Yamagishi
Technical Staff:
		 Nobutake Suzuki, Atsuo Kobayashi,		Tomoko Hasegawa, Keiko Takahashi,		Naomi Inagaki, Fumie Yokoyama,		Noriko Utsumi, Emi Abe,		
Nobuyuki Goto
Student Trainee:		Nanako Shimura (JRA), Asahi NakaharaT

he very basic mission of the immunogenomics grouphas been to function as a “Gateway” to genomics for
immunologists. To achieve this mission at the center, wehave divided our research activities into three components
as follows: ( 1) central support activities; ( 2 ) strategic andcollaborative research activities; and ( 3 ) exploratory research activities aimed at new technology development.
The emergence of massively parallel DNA sequencing( MPS ) technology has considerably changed the genomics research style and more or less affects all research
activities. We have currently used MPS for various purposes and recently set up a high-throughput DNA sequencer( HiSeq1000, Illumina ) as a part of the central support activities. The advantage of MPS is not its high capacity fordata production: Because most conventional MPS methods are based on clonal amplification of a single DNA template, the generated data enable us to characterize a poolof heterogeneous DNA molecules at the single-nucleotide
resolution. In other words, MPS could directly analyze genetic heterogeneity of a cellular ensemble of immune cells
expressing different immunoglobulins or T-cell receptors ina quantitative manner. In this way, the MPS technology isparticularly useful for analysis of diversity and dynamics of
the immune system at single-cell resolution, which makes
single-cell analysis more accessible to immunologists

than before. Because we have also developed various single-cell analysis methods in the past, we consider that the
combination of analyses in a population context and in real-time will surely clarify unexplored aspects of the dynamics of the immune system. As examples of such approaches, 1) construction of the pipeline of MPS data processing
and 2) development of an inflammatory cytokine secretion
assay from single cells are described below.

Construction of the data analysis pipeline for MPS
The currently available high-throughput MPS machine has
a sequencing capacity of 300 x 10 9 bases ( 300 Gbase ) /run in a 100-base pair-end sequencing mode. Thus, it isobvious that we must establish an in house pipeline forroutinely managing such huge amounts of DNA sequencing data. However, the use of MPS varies widely, from DNA
sequence determination ( whole exon sequencing, conventionally termed “Exome sequencing” ) to measurement of
the occurrence frequencies of particular DNA fragments
mapped on the genome ( RNA sequencing and ChIP-sequencing ) . In a realistic situation, multiple samples labeledwith short identification tag sequences are simultaneously
run on the same machine and eventually separately processed according to their analysis purposes. Taking thesesituations into consideration, we first established a sample

Recent Publications
1. Shirasaki, Y., Yamagishi, M., Shimura, N., Hijikata, A.,
Ohara, O. Toward an understanding of immune cell sociology: Real-time monitoring of cytokine secretion at thesingle-cell level. IUBMB Life 65, 28-34 ( 2012 )114

2. Kaji, T., Ishige, A., Hikida, M., Taka, J., Hijikata, A.,
Kubo, M., Nagashima, T., Takahashi, Y., Kurosaki, T.,
Okada, M., Ohara, O., Rajewsky, K., Takemori, T. Distinct
cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory.
J Exp Med. 209, 2079-2097 ( 2012 )3. Izawa, K., Hijikata, A., Tanaka, N., Kawai, T., Saito, MK.,
Goldbach-Mansky, R., Aksentijevich, I., Yasumi, T.,
Nakahata, T., Heike, T., Nishikomori, R., Ohara, O. Detection of Base Substitution-Type Somatic Mosaicism of
the NLRP3 Gene with >99.9% Statistical Confidence by
Massively Parallel Sequencing. DNA Res. 19, 143-152( 2012 )Figure 1: Diagram of the automated data processing pipeline for MPS
Abbreviations shown in italics are names of publicly available
software for MPS data analysis. The pipeline is composed ofthese public software and the final data (FPKM data for RNA
sequencing; BAM/BED graph for ChIP-sequencing; VCF
for exome sequencing) are automatically generated without
human intervention.

Figure 2: Distribution of IL -1β secretion activities of humanmonocytes at the single-cell level.
IL-1β secretion was measured by the Microengraving method
at the single-cell level. As can be clearly seen, a considerable
number of human monocytes in the CINCA patient secrete
more IL-1β than those in the healthy donor without any
inflammatory stimulation.

annotation system that clearly describes the sample attribute. After the sequencing run, the data are automaticallytransferred and processed according to their attributes asshown in Fig. 1. Taking advantage of the automated dataprocessing pipeline thus constructed, it becomes possible
to fully utilize the power of MPS at the center.

duced aberrant IL-1b secretion from monocytes to sparkand to expand the inflammation, little is known about the
involvement of IL-1b secretion as a pathogenic mechanism.
Thus, we have tried to measure IL-1b secretion by humanmonocytes from healthy donors and CINCA patients at thesingle-cell level by the Microengraving method (Love et al.,Nat. Biotechnol. 2006). Fig. 2 shows the distributions ofamounts of IL-1b secretion from single monocytes purified
from a healthy donor and a CINCA patient and cultured
without any stimuli. Interestingly, human monocytes fromthe CINCA patient contain a larger fraction of actively IL1b secreting cells than those from the healthy donor evenunder these non-activating conditions, which suggeststhat the measurement of IL-1b from monocytes at the single-cell level might be used for rapid diagnosis of the CINCA syndrome. While more data must be obtained and statistical power calculated before using this assay for clinical
CINCA diagnosis, these data offer an interesting example
where the combination of MPS data and single-cell protein
measurements allows us to explore the etiology of a complex inflammatory disease.

Monitoring of IL-1β secretion from single humanmonocytes for diagnosis of CINCA syndrome
Chronic infantile neurological cutaneous and articularsyndrome ( CINCA ) , also known as neonatal-onset multisystem inflammatory disease ( NOMID ) , is a dominantly-inherited systemic autoinflammatory disease caused
by a heterozygous germline gain-of-function mutation in
the NLRP3 gene. As a response to our recent finding of ahigh incidence of NLRP3 somatic mosaicism in apparently
mutation-negative CINCA/NOMID patients (Tanaka et al.,Arthritis Rheum. 2011), we developed a rapid diagnosis
method for somatic NLRP3 mosaicism by using MPS (Izawa et al., DNA Res. 2012). However, the etiology of CINCA
syndrome has still remained unclear. Although several
investigators have suggested the possibility of mutation-in-

4. 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 in vivo human hematopoietic engraftment andmyeloid differentiation. Blood 119, 2768-2777 ( 2012 )5. 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. Plasmacytoid dendritic cells are crucial for the initiation of inflammation and T cell immunity in vivo. Immunity 35, 958971 (2011)115

Research Unit for

Immunoinformatics
Unit Leader:

S. Sujatha Mohan
Research Associate:
Subazini Thankaswamy Kosalai(until December, 2012)T

he main mission and focus of the RCAI Immunoinformatics research unit is to develop and maintain an
open access Bioinformatics platform and data resource inorder to gain insights into primary immunodeficiency diseases (PID ). Towards this end, we have developed and established a molecular disease database named RAPID—
Resource of Asian Primary Immunodeficiency Diseases(PID) ( http://rapid.rcai.riken.jp ) , a web-based informatics
platform that enables PID experts to easily mine collected genomic, transcriptomic, and proteomic data about
PID-causing genes. Our ultimate goal is to provide relevant, up-to-date and validated information on PID as per
global community standards in an easily decipherable andusable format.
At present, RAPID includes a total of 242 genes thatare confirmed to cause over 260 PIDs. There are over
5000 unique mutations annotated from over 1800 citations
as of January 2013.

Resource of Asian Primary Immunodeficiency
Diseases (RAPID ) phenotype ontology framework PhenomeR: Towards establishment of an e-clinicalsupport system for phenotype-based genetic
analysis of PID using semantic web technology
The main challenge for in silico genotype-phenotype
correlation for any genetic disease is to standardize
phenotype ontology terms and the genotype. We haveintroduced a newly developed PID ontology browser forsystematic integration and analysis of PID phenotype withthe genotype data from RAPID. Towards this end, we havedeveloped a user-friendly interface named, “PID PhenomeR” ( http://rapid.rcai.riken.jp/ontology/v1.0/phenomer.
php ) , which serves as a standardized phenotype ontology
resource to present ontology class structures and entities of all observed phenotypic terms in PID patients fromRAPID in standardized file formats - Web Ontology Language ( OWL) and Resource Description Framework ( RDF )using semantic web technology. PID PhenomeR consistsof 1466 standardized PID terms that are classified under
24 and 29 semantic types and categories respectively asof December, 2012 (The manuscript describing this project“PID PhenomeR- An integrated platform for developing
phenotype ontology structures for primary immunodeficiency diseases” has been submitted to Database, an Oxford University Press Journal ) . The standardization of PID
phenotype terms for addition of new terms is in progress,
using a unique semi-automated process including a logic
based assessment method. The home page and overall
work flow of PID Phenomer is shown in Fig. 1. In essence,
PID PhenomeR serves as an active integrated platform
for PID phenotype data, wherein the generated semantic framework is implemented in the integrated knowledge-base query interface i.e. SPARQL Protocol and RDF

Recent Publications
1. Telikicherla, D., Marimuthu, A., Kashyap, MK., Ramachandra, YL., Mohan, S., Roa, JC., Maharudraiah, J.,
Pandey, A. Overexpression of ribosome binding protein
1 (RRBP1) in breast cancer. Clin Proteomics. 9 (1): 7(2012 )

116

2. Telikicherla, D., Ambekar, A., Palapetta, SM., Dwivedi,
SB., Raju, R., Sharma, J., Prasad, TsK., Ramachandra, Y.,
Mohan, S., Maharudraiah, J., Mukherjee, S., Pandey, A.
A comprehensive curated resource for follicle stimulating
hormone signaling. BMC Res Notes. 4; 408 ( 2011)

3. Raju, R., Nanjappa, V., Balakrishnan, L., Radhakrishnan,A., Thomas, JK., Sharma, J., Tian, M., Palapetta, SM.,
Subbannayya, T., Sekhar, NR., Muthusamy, B., Goel,
R., Subbannayya, Y., Telikicherla, D., Bhattacharjee, M.,
Pinto, SM., Syed, N., Srikanth, MS., Sathe, GJ., Ahmad,
S., et al. NetSlim: high-confidence curated signaling
maps. Database (Oxford) bar032 (2011)

Figure 1 : Snapshot of PID Phenomer
A. Overview of PID PhenomeR work flow highlights various
stages of processes such as (A) Data collection, (B) Data
standardization and (C) Data storage and retrieval
B. Home page of PID PhenomeR presents term information
along with associated features, query options (simple andadvanced), term visualization, statistics and so on.

Figure 2 : Integrated Arthritis Database (IntARTdB)
A. Top page of IntARTdB (Formative stage of development)
B. IntARTdB schema shows four major compartments i.e.
User features - upload and feedback; OMICs; Other studies- Protein-protein interaction, Pathway, Localization, Posttranslational modification and Biomarker; therapeutics data
annotation and analyses, all shaded in different colors. This
schema is generated using the MySQL workbench

Query Language ( SPARQL ) endpoint for establishing awell-informed PID e-clinical decision support system. Thiskind of analysis should help bridge the gap between genotype and phenotype correlation, thereby improving phenotype-based genetic analysis of PID genes. Moreover, it
should aid clinicians in confirming early PID diagnosis andalso be helpful in implementing appropriate therapeuticinterventions.

ways, Biomarkers, Sub-cellular localization, etc. Once a
registered user inputs general information, the annotation
page will be subsequently accessed for loading, editing,viewing and updating Omics data according to individual
access privilege. The customized interface is also developed to facilitate uploading of bulk data in text or CSV
format files for direct storage in the database. Further, we
use Proteome Commons Tranche repository to manage
all high-throughput data file archives. Screenshots of thisdatabase top page and schema are shown in Fig. 2 (A & B ).

Inception of an integrated Arthritis KnowledgeBasefor high-throughput data storage and analysis
Integrated Arthritis database ( IntARTdB ) is a web based
compendium of high-throughput data on arthritis. It serves
as an integrated platform for arthritis-specific data collection, storage, annotation and analysis of genomics,
transcriptomics, proteomics, metabolomics and otheromics studies. The IntARTdB framework is based on theconstructed model-view-controller architecture. The data
annotation tool is made available to those who contributehigh-throughput analyzed data to IntARTdB, with controlled access privilege through a streamlined-registration.
The data entry and display of IntARTdB are performed using HTML front end using PHP interface as well as MySQL
database at the backend. Each Omic ( Genomics, Proteomics, Transcriptomics, Metabolomics, Lipidiomics andGlycomics ) input information consists of general, sample
and experimental annotation forms. Moreover, the database also hosts other studies such as PTMs, PPIs, Path-

4. Ramadoss, SK. and Mohan, S. In Silico Identification ofPrioritized Interacting Domains in Primary Immunodeficiency Disease Causing Genes. Intl J Biosci Biochem
Bioinforma. 1, 84-88 (2011)

Collaboration and funding
The PID project has been initiated in collaboration with
the Institute of Bioinformatics ( IOB, Bangalore, India )and the Immunogenomics research group at RIKEN
RCAI, Japan. This laboratory was supported by The Asia
S&T Strategic Cooperation Promotion Program, Special
Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Scienceand Technology ( MEXT ) between July, 2007 and March
2010. Now, it is being funded by a RCAI, RIKEN internalfunding source.
The Int ARTdB project has been commenced incollaboration with the Gobezie lab at Musculoskeletal
Proteomics, The Case Center for Proteomics and Systems
Biology, Case Western Reserve University - School of
Medicine, USA.

5. Hijikata, A., Raju, R., Keerthikumar, S., Ramabadran, S.,
Balakrishnan, L., Ramadoss, SK., Pandey, A., Mohan,
S., Ohara, O. Mutation@A Glance: an integrative web
application for analyzing mutations from human genetic
diseases. DNA Res. 17, 197-208 (2010 )117

Laboratory for

Cellular System Modeling

Team Leader:

Mariko Okada ( Hatakeyama)

Senior Research Scientist: Shinji Kondo (until May)

Technical Staff: Noriko Yumoto, Kaoru Takahashi, Ki Sewon

Research Scientist:

Student Trainee: Norihiko Inoue (JRA)Masaki Nomura
Hisaaki Shinohara
Koichi Saeki (JSPS)D

ysregulation of intracellular signaling activity isstrongly associated with a variety of diseases suchas cancer, diabetes, inflammation and immune diseases.
Our research interest is to identify important signaling
dynamics related to cellular phenotypes, and to integratesignal transduction and transcriptional data using mathematical models to understand mechanistic regulationof the signal-transcription network. This approach aims
to answer the question of how small quantitative changes in the initial signaling dynamics can induce qualitativechanges in transcriptional profiles or expression of marker
genes, changes that are often associated with diverse cellular phenotypes and disease states.

Spatiotemporal dynamics of NF-kB signaling in
B cell differentiation
Activity of the transcription factor nuclear factor-κB (NF-κB )is spatiotemporally controlled and displays particular dynamic behaviors in the process of cellular determination.
The current goal of our research is to identify the relationship between NF-κB dynamics and cell differentiation. Ourquantitative experimental and mathematical analyses indicated that a TAK1-IKK positive feedback loop enforced by
CARMA1 Ser-578 phosphorylation functions to produce
a switch-like response to set a threshold in NF-κB activity
and B cell proliferation. We are also trying to dissect interconnected signal-dependent networks in B cells by using
ligand dose-response analysis. Such an analysis allows
us to identify each signal-transcription relationship, howmultiple signaling pathways coordinately determine transcriptional output of a B cell, and the positive and negative

Recent Publications
1. Hiroshima M, Saeki Y, Okada-Hatakeyama M, and Sako
Y. Dynamically varying interactions between heregulin
and ErbB proteins detected by single-molecule analysis
in living cells. Proc Natl Acad Sci USA. 109, 1398413989 (2012 )118

2. Saeki Y, Nagashima T, Kimura S, and Okada-Hatakeyama M. An ErbB receptor-mediated AP-1 regulatory
network is modulated by STAT3 and c-MYC during
calcium-dependent keratinocyte differentiation. Exp
Dermatol. 21, 293-298 (2012 )3. 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)

Figure :Time-resolved integration of a signal-transduction network and its relationship with cellular and mouse phenotypes

roles of signaling pathways for B cell functions.

Integration of data
We have been attempting to connect signal transduction
pathways ( proteomics ) , transcriptional signatures ( transcriptome ) and phenotypes using cellular and mousemodels. Using this strategy, we identified a signal-early responsive gene network that potentially generates a tipping
point for cellular differentiation ( Cell, 2010 ), and predicted
and then validated with clinical samples a marker gene,
GSK3-b, for tamoxifen-resistant breast cancer (JBC, 2011).
Currently our several joint research studies focus on transcriptome analysis of single gene knockout mice and its
effect on intracellular signaling and metabolic pathways inmouse models. This strategy has successfully been filling
the gap between gene signatures and cellular and mouse
phenotypes.

Activation of membrane receptor-signaling pathways
is first triggered by extracellular ligand-receptor interactions, and differences in the dynamics of these interactions often evoke induction of distinct cellular phenotypes.
Inspired by the relationship between ligand-receptor interaction dynamics and cell fate decisions, we analyzed
single molecule binding kinetics of a growth factor-ErbB
membrane receptor interaction in living cells ( in collaboration with Dr. Yasushi Sako, RIKEN ASI ) . This and our earlier analyses indicated that binding parameters of the two
receptor-growth factor combinations are in a similar range,
whereas the resultant cellular phenotypes stimulated by
the growth factors are different. This analysis indicatedthat cytosolic reaction events rather than extracellular
ligand-receptor kinetics seemed to play an importantrole in cell fate decisions mediated by the ErbB receptor( Proc Natl Acad Sci USA, 2012) .

5. Nakakuki T, Birtwistle MR, Saeki Y, Yumoto N, Ide K,
Nagashima T, Brusch L, Ogunnaike BA, Okada-Hatakeyama M*, Kholodenko BN.* Ligand-specific c-Fos
expression emerges from the spatiotemporal control of
ErbB network dynamics. Cell 141, 884-896 ( 2010 ) *corresponding authors.

119Research Unit for

ThymicEnvironment
Unit Leader:

Willem van Ewijk

P referential generation of K5 + K8 + cTECs in thethymus following thymic injury caused by loss ofthymocytes.

T

he stroma of the thymus consists of distinct sets ofcortical and medullary thymic epithelial cells ( TECs ) .
Although early steps in the development of the thymic environments have recently been documented ( Immunity 36,
298–309, 2012) , the mechanism by which thymic environments are maintained postnatally is still unclear. We haveidentified a novel population of mature cortical ( c ) TECs
that emerge postnatally in the depth of cortex. Microscopically defined by co-expression of cytokeratin (K ) 5 and K8,
these cTECs eventually populate the entire thymic cortex
in aged mice.
We determined whether these K5 + K8 + cTECs emerge
as a consequence of K5 up-regulation in K5-K8 + cTECs as
a mark of senescence, or newly arise as a result of a tissue
repair process. For this purpose, a controlled perturbationof the thymic microenvironment was used to initiate tissue
repair mechanisms. We thus investigated the emergenceof K5 +K8 + cTECs in organ cultures of 15 dpc fetal thymic( FT ) lobes exposed to deoxyguanosine ( dGuo ) . dGuo
treatment depletes thymocytes and, consequently, ablates
cross-talk signals required for normal TEC development.
Subsequent transplantation of dGuo treated lobes under
the kidney capsule of wild type mice leads to reconstitution of thymocytes. Two months later, the thymic tissueof both untreated and dGuo-treated grafts dramaticallyincreased in size, the latter being slightly smaller. However,there was a clear difference with regard to the cTEC phenotype; a majority of cTECs in untreated FT grafts was K5K8 +, but almost all cTECs in the dGuo-treated FT grafts
displayed a K5 + K8 + phenotype ( data not shown ) .

The thymic cortex is replenished by K5 + K8 + cTECs
generated as a consequence of clonal expansion
during restoration of the injured thymic
environment.
The above finding does not completely preclude the possibility of an age-related phenotypic change, because a certain stress, such as loss of thymic cross-talk signals, might
accelerate senescence of cTECs. To determine whether
K5 + K8 + cTECs are newly generated by expansion of TEC
progenitors, we employed a genetic model that allows
clonal analysis of proliferating TECs. To this end, FT lobes
were isolated from mice carrying both a Rosa26-creER
allele and a loxP-stop-loxP EGFP transgene, and cultured
for 6 days in the presence of dGuo ( Fig. A ) . Subsequently,
4-hydroxy-tamoxifen ( 4-OHT ) was added to the cultures
for 6 hours at a low concentration. In a limited number ofTECs, this treatment results in the nuclear translocationof the Cre-ER fusion protein, which initiates expression of
GFP following Cre-mediated recombination. 4-OHT treatedthymic lobes were then grafted under the kidney capsuleof wild-type mice and reconstitution of thymopoiesis wasfollowed over time. Under these experimental conditions,clonally expanded TECs, if present, could be visualized as

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. 46, 1756-1767(2009)

120

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. 47, 1106-1113 (2010)

Figure :Thymic cortex injured by loss of thymocyte-derived cross-talk signaling is replenished by K5+K8+
cTECs, generated by clonal expansion.
A. A FT lobe from a 15 dpc fetus carrying both a Rosa26-CreER allele and a Lox-GFP transgene wasincubated with dGuo for 6 days and subsequently with a low dose of 4-OHT, for 6 hours. The lobe wasthen transplanted under the kidney capsule of a 10 wk old wild type recipient mouse, and the graft was
harvested 4 weeks later.
B. Rationale of the experimental system. By treatment of a low dose of 4-OHT, cre-mediatedrecombination is expected to occur randomly at a floxed site at very low frequency (lower than 1/100
cells in this case), resulting in the activation of GFP expression in cells separated from each other. If
cTECs are formed without any proliferation in the restored thymic lobes, GFP+ cTECs will appear as
individual cells, whereas if cTECs are formed by proliferation of precursor cells, GFP+ cTECs will beobserved as independent colonies consisting of multiple cells.
C. A cryostat section of the graft was stained with anti-GFP (green), and counterstained with DAPI (blue).
“C-clone” indicates a colony of clonally formed cTECs , whereas “M-clone” indicates clonally formed
mTECs. Bar; 100 mm.
D. The enlarged image of C-clone 6 in Fig. 1C. About 10 cTECs were estimated to be present in the planeof this section.
E. A cryostat section of the graft of the same experiment as in Fig.1 C was stained with anti-GFP (green)
and anti-K5 (red) antibodies, demonstrating the K5+K8+ TEC phenotype (yellow).

clusters of GFP-positive cells ( Fig. B ) . Clusters of ten or
more GFP+ cTECs were detected throughout the cortexof the rejuvenated thymic lobes, and the large majority of
GFP+ cTECs was organized in such clusters (Fig. C ) . Eachcluster was formed by several tenths of GFP+ cTECs, covering spherical areas larger than f100 mm and thought tocontain 500-1000 thymocytes ( Fig. D ) . It is quite unlikelythat these GFP+ cTECs represent a single cTEC, because
the size of one cluster is much larger than a so called“thymic nurse cell”, that usually contain fewer than 50 thy-

mocytes. All these GFP+ cTECs were found to co-express
K8 and K5 ( Fig. E ) . Hence, the restoration of the corticalepithelial compartment following dGuo-treatment occurs
predominantly via K5 + K8 + cTECs generated as a consequence of clonal expansion, and not by an age-related
phenotypic change of pre-existing K5 - K8 + cTECs. The
relation of these K5 + K8 + cTECs with thymic epithelial stem
cells remains to be elucidated.

121

Research Unit for

Immunoepigenetics
Unit Leader:

Miguel Vidal
Research Scientist: Yang LiPolycomb group ( PcG ) complexes function as transcriptional repressors of genes determining cell identity states and the transitions between them. In stem cells,
PcG complexes repress gene expression in ways that allow coordinate responses to inducing signals that promote
cell state conversions taking place during differentiation.
PcG complexes act both at the level of higher order chromatin structure and also locally. Their function depends, atleast in part, on their activities as histone modifiers ( mostly
trimethylation of histone H3 and monoubiquitylation ofhistone H2A ) . PRC1 complexes, the subset of PcG assemblies with ubiquitin ligase activity ( conferred by Ring1
protein ligases ) are a heterogeneous set of entities. Mutually exclusive binding of chromodomain proteins or RYBP/
Yaf2 proteins to Ring1 subunits specify two groups of
PRC1 complexes. Functional analysis of these complexes
is complicated by the presence of one or more paralogs
and the potential for redundant activities. We have initiatedwork to approach this issue for the RYBP, Yaf2 homolog
subunits.

Genetic interactions between RYBP and Yaf2
paralogs.
Our previous work showed prevalent association of RYBP
with canonical Polycomb targets in ES cells; however,RYBP inactivation did not alter expression of these genes.

Recent Publications
1. H is ada , K ., S ánchez, C ., E nd o, T., E nd o h, M .,Román-Trufero, M., Sharif, J., Koseki, H. and Vidal, M.
RYBP represses endogenous retroviruses, preimplantation- and germline-specific genes in mouse embryonicstem cells. Mol Cell Biol. 32, 1139-1149 ( 2012 )122

By contrast, RYBP was also bound to germ line genes andretrotransposon elements and these were effectively repressed. Because of the existence of a closely related homolog, Yaf2 ( Fig. 1, top ) , it was possible that the functional
outcome of RYBP inactivation in ES cells was being modulated by Yaf2 compensatory activity. Thus, we set out
to test this hypothesis by generating a compound RYBP,
Yaf2-mutant ES cell. Fig. 1 ( bottom ) depicts a floxed allele
designed ( Yaf2 f, Jun Shinga, Developmental Genetics ) for
conditional deletion. Mutant mice were mated to introduce
a tamoxifen-activated recombinase Cre and were alsomated to RYBP mutant mice, so that Yaf2 f and Yaf2 f ,RYBP f expressing an inducible Cre gene were generated.
Preliminary gene expression analysis of these cells
showed little Yaf2 activity ( although the gene is expressedin ES cells ) both alone and in combination with RYBP deficiency. Genome wide expression studies using RNA-Seq
are presently ongoing in order to determine a comprehensive picture that would confirm or refute the current notionthat whereas Yaf2 may control targets of its own, it does
not compensate for RYBP on Polycomb targets. The unexpected anti-synergism seen at RYBP targets (Fig. 2) willneed to be confirmed by biochemical studies that will befacilitated by the expression of a tagged-Yaf2 variant in our
ES cell lines

Figure 1: Murine RYBP, Yaf2 paralogs and Yaf2 allele for conditional inactivation.
Top: cartoons of RYBP and Yaf2 proteins depicting amino acid content, conserved regions andthe Zinc finger domain; bottom: wild type Yaf2 locus and the modified allele (Yaf2-biof ) andtheir protein products, respectively. Deletion of sequences flanked by loxP sites (pink arrows)
results in a null Yaf2 allele (Yaf2∆).

Figure 2: Gene expression in single RYBP, Yaf2 and compound mutant ES cells.
Relative expression in wild type (ethanol-treated) and mutPant (Tamoxifen, Tx, treated) cells,
measured by RT-qPCR, of representative genes among those repressed by RYBP (left panel) or
silenced by canonical Polycomb-repression (right panel).

123Research Unit for

ImmuneCrosstalk
Unit Leader:

Hilde Cheroutre

Defending the mucosal barrier against “the enemy”
invading from the outside and the inside.

Unlike lymphoid T cells, intestinal intraepithelial T cells
reside at epithelial surfaces and form the first line ofdefense against invading pathogens. Although indispensable for preventing the initial infection, epithelial T cells
need to balance their protective function with safeguarding
the integrity of the barrier and failure to do so compromises homeostasis of the organism.
These unique challenges drive specialized differentiation and regulation of epithelial T cells and the main goalsof our research are 1) to elucidate regulatory mechanismsthat preserve the mucosal barrier in the face of potentially destructive immune protection and 2 ) characterizing
unique processes that drive the generation of mucosal effector memory T cells that guarantee optimal pre-existing
protective immunity.
We have made milestone discoveries in both areasand showed that, for example, retinoic acid ( RA ) is a key
factor in controlling mucosal immune responses and under tolerogenic conditions. RA promotes the induction of
TGF-b-dependent Foxp3 iTreg cells whereas it suppresses the differentiation of inflammatory TGF-b-driven Th17
cells (Mucida et al., Science 2007, Mucida et al., Immunity
2009 ), .
We also showed that a novel affinity-based selective
process operates at the mucosal interface of the intestineand preserves the optimal effector cells to become long-

lived effector memory T cells ( TEM ) that establish pre-existing and heightened protective immunity ( Huang et al.,Nature Immunology 2011) . Unlike central memory T cells( TCM ) that reside in lymphoid tissues, TEM gain the capacity
to reside long-term in non-lymphoid tissues such as theintestine. TCM cells, which respond to antigen with robust
clonal expansion, are effective at protecting against infections by pathogens that replicate systemically, but they are
inadequate to prevent transmission of viruses, including
the human immunodeficiency virus ( HIV ) , or intracellularbacteria, which penetrate across mucosal epithelia. Effective resistance against transmission of such pathogens
requires the presence of local, highly efficient antigen-specific TEM prior to re-challenge. Nevertheless, because mostof the current knowledge of immune memory has been
gained from model systems that use systemic immunization routes for the generation of lymphoid TCM, the generation of TEM was very poorly understood.
We showed previously that the TCR repressor, CD
8aa, induced on activated CD8ab T cells, marks thoseprimary effector cells that preferentially differentiate intomemory cells ( Madakamutil et al., Science 2004 ) . The
expression level of CD8aa is controlled by signal strengthand it is most highly induced on those effector cells withthe highest antigen-affinity. We found that mucosal TEM
are highly enriched for CD8aa-expressing memory cells,
indicating that a selective mechanism might drive thepreferential accumulation of high affinity effector cells at

Recent Publications
1. Mucida D, Husain MM, Muroi S, van Wijk F, Shinnakasu
R, Naoe Y, Reis BS, Huang Y, Lambolez F, Docherty
M, Attinger A, Shui JW, Kim G, Lena CJ, Sakaguchi S,
Miyamoto C, Wang P, Atarashi K, Park Y, Nakayama
T, Honda K, Ellmeier W, Kronenberg M, Taniuchi I,
Cheroutre H. Transcriptional reprogramming of mature124

CD4 + helper T cells generates distinct MHC class
II-restricted cytotoxic T lymphocytes. Nat Immunol. 14,
281-289 (2013)

Figure :the mucosal interface of the intestine.
In an earlier study we had shown that the high affinity
ligand for CD8aa, the non-classical MHC class I molecule
thymus leukemia antigen ( TL) , is constitutively expressedon mucosal epithelial cells. Using oral infection with thefood borne pathogen Listeria monocytogenes (Lm), we
demonstrated the existence of an affinity-based selection
mechanism controlled by TL expressed on APCs, whichleads to the selective survival of high affinity, CD8aa+ CD8ab memory precursor cells. Furthermore, TL on the intestinal epithelial cells (IEC ) continues to impose selection
pressure and drives the affinity maturation of the resident
mucosal CD8ab TEM, guaranteeing the optimal protective
capacity of these memory cells.
Our findings represent a fundamentally new concept
for immune memory differentiation and indicate that anaffinity-based selective process operates in vivo that preserves the optimal effector cells to become long-lived TEM
that establish pre-existing and heightened protective immunity at mucosal borders.
Although unable to provide sterilizing protection, TEM
control the pathogen load and delay or prevent the initialinfection and spreading of the pathogen, as well as reduce
the potential for secondary transmission. Therefore, ourIntraepithelial lymphocytes protect the
mucosal barrier of the intestine from
pathogen- and inflammation-induced
pathology.
T cells that reside within the epithelium of theintestine are phenotypically heterogeneous
but they are all specialized to protect the
mucosal barrier against pathogen- and immune
cell-induced pathology. In contrast to the Tcells in the periphery, epithelial T cells are
antigen-experienced T cells that encountered
their antigen initially during selection in thethymus (agonist selected CD8αα TCRab and
TCRgd precursor cells) or as mature cells in
the periphery (CD8ab CTL and CD4 CTL).
Although the various epit hel i a l T cel l s
subsets display different antigen specificity
and MHC restriction and although they follow
different paths of effector differentiation, they
all acquire cytolytic and regulatory capacity.
The functional specialization of epithelial T
cells adapts them toprovide optimal protectionin the face of preserving the integrity of thedelicate mucosal barrier.

finding that an endogenous TCR quality-based mechanism selects for the most avid effector cells to form mucosal TEM has significant implications for the design of new
and improved strategies to induce effective pre-existing
protective immunity at the most vulnerable entry sites forpathogens.
Our research continues to elucidate mechanismsof mucosal immune protection and regulation and, in a
recent study, we uncovered an unexpected degree ofplasticity for CD4 T helper ( Th ) cells which, upon antigenic
stimulation, are able to terminate the expression of the Th
transcription factor, ThPOK, and differentiate into cytotoxic T lymphocytes ( CTL) . At steady state, these CD4 CTL
remain quiescent and express a self-regulated phenotype.
However under challenging conditions, these cells havethe potential to transform into potent inflammatory killer
effector cells (Mucida et al., Nat Immunol. 2013).
Overall, based on the insights we are gaining from ourresearch, a clear picture has begun to emerge showingthat the immune defense of the intestine adapts to the
local environment and specializes to provide the mostefficient and immediate protection in the face of preserving the integrity of the most critical mucosal barrier ofthe body.

125

Central Facilities

C

entral Facilities in RCAI provides all researchers in the Center with access tothe most advanced equipment and technologies. Central Facilities consist of
five sections; the FACS, Confocal, and Monoclonal 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 for nearly all immunological experiments. In 2012, the FACS Lab added three new machines,
two Aria III and one Canto II. One of the newly installed Aria III is equipped with 5 lasers ( blue, red,
yellow-green, violet and near UV ) , which enables detection of 14 colors.

Table 1: Instruments in the FACS Lab
FACS
FACS cell analyzer
FACS cell sorter

Number of machines

Calibur

4

CantoⅡ

2

AriaⅠ

2

AriaⅡ

1

AriaⅢ

2

Diva (digital vantage)

1

Imaging flow cytometer

Image Stream X

1Mass cytometer

CyTOF

1
Figure 1: Yellow-green laser in Aria III

FACS Lab provided various services for the FACS users as follows.
1.

Technical support and training
In 2012, the facility offered 15 technical courses (6 for cell sorting, 7 for cell analysis (2 for Calibur and
5 for Canto II) and 2 for CytoF ) . A total of 64 RCAI researchers took the courses in 2012.

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 2012, there were 426 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 staff, engineers from Becton Dickinson visit once a week to provide maintenance and technicalsupport.

Ikuo Ishige, Engineer from BM Equipment Co. Ltd., provided technical support for Image Stream X,
including training courses, consultations for experiments and operation and analysis services.

126

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 antibodies as diagnostics and drugs.
Two technical staff members, Tomomi Aoyama and Kazuyo Uchida, are engaged in producing, purifying and analyzing mAbs. In 2012, the Lab produced mAbs against 13 different antigens, which wererequested by 8 laboratories. The lab also prepared human chimeric antibodies, such as an anti-Cry j 1
human IgE antibody.

Confocal Lab
Visiting Scientist: Yasutaka Wakabayashi (Leica Microsystems Co. Ltd.)The Confocal Lab provides imaging equipment and technical support. The Confocal Lab is managed incollaboration with Leica Microsystems and there are 6 confocal microscope systems:
1.

Inverted system with visual and a multi-photon ( MP ) laser suitable for time-lapse imaging of living
cells and organs.

2.

Inverted system with a 405 nm laser suitable for a time-lapse imaging of living cells in a controlledenvironment (CO2, temperature, and humidity) .

3.

Leica SP5 confocal system, the successor of the SP2. Brighter and more striking images can beproduced because of the improved optical system.

4.

Upright system with visual and UV lasers suitable for standard fixed specimen observation.

5.

Intravital upright system with single visual laser, double MP lasers and high speed scanner that canbe used for in vivo imaging and for some other applications such as light stimulation.

6.

To increase the convenience of animal experiments, an intravital system with visual laser, MP and
OPO laser was relocated to the new animal area on the first floor.

During 2012, Confocal Lab provided training for 13 researchers. The total running time of the microscopes was over 2011 hours.

127Central Facilities
Animal Facility
Senior Research Scientist: Takanori Hasegawa
Technical Scientist:				

Shinobu Mochizuki
Masashi Matsuda

Technical Staff:		
Tomoyuki Ishikura		Yusuke Iizuka
		
Hiromi Tatsumi
		Yurie Kawamoto		Chie Yoshida		Takahisa Shimazu
Animal caretakers ( out sourced ): 21 people

Figure 2: New animal facility room on the first floor.

Over 9,000 cages of SPF mice are maintained in the Animal Facility. The facility provides the following
services for the users in the RIKEN Yokohama Institute.

128

1.

Maintenance, generation and cryostocks of genetic resources
The Animal Facility has been maintaining over 45,000 mice and 4 rats in the SPF area, 1,700 micein an isolated area and several germfree mice. They have newly introduced 623 mouse lines into the
SPF area by a combination of in vitro fertilization (IVF) and embryo transfer and generated cryostocks
of genetic resources for 512 lines. The facility also maintains relatively large colonies for several commonly used strains, such as NOD/SCID/CγKO mice, Rag1KO and cre deleters, and provides themto users on demand. They have also provided technical assistance to generate chimeras (136 lines)
and transgenic mice (10 lines) and to establish and maintain ES cells. They have also generated aninternally available database for genetic resources.

2.

Introduction of human BAC clones into NOD/SCID/CγKO miceThis work was undertaken in collaboration with Dr. Osamu Ohara ( RCAI Immunogenomics Group )and Dr. Fumihiko Ishikawa ( RCAI, Human Disease Model Unit) .
The Animal Facility launched a new activity to improve the efficacy of transplantation of human hematopoietic 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/CγKO mice. Up to now, they haveestablished 15 BAC transgenics and confirmed the expression of human genes on a C57BL/6 background and started back-crossing them onto the NOD/SCID/CγKO mice using the speed-congenic
method.

3.

Creation of germ-free mice, maintenance and management
This work is undertaken in collaboration with Dr. Hiroshi Ohno ( RCAI Epithelial Immunobiology
Group ).
The Animal Facility attempted sanitization of knockout mice and built a system that creates germ-freemice at the pace of 1 strain per month.

4.

Construction of two new rooms for Animal Maintenance
Two new rooms, fully equipped with autoclaves and cage washing systems, were added to the animal facility. These rooms were constructed on the first floor of the building, separately from the central animal facility on the 7th floor, and can be operated independently. In total, the facility has added
900 new cages where 4,500 animals can be kept. They have set up a multi-photon microscopy in
one room that can be used for imaging analysis. In the other room, we have constructed vinyl isolator
and bio-bubble facilities to breed and maintain ( immunologically) humanized mice (Fig. 2).

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 AbeFigure 3: TripleTOF5600 System

The Genomics Lab provides various services to the members of the Center: proteomics analysis, 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 ).

Table 2: Services provided by the Genomics Lab in 2012
Proteomics# of samples# of teams

Two-dimensional electrophoresis

42

Mass Spectrometry Analysis

58

1Multiplex suspension array# of samples# of teams

3,740

12

Affymetrix Genechip (Exon array, Gene array, miRNA array)# of samples# of teams

Human

103

5

Mouse

514

18

Total

617

21

# of samples# of teams

36cm capillary

13,9261650cm capillary

13,474

17

Total

27,40033# of clones# of teams

37

5# of sets# of teams

54

1Sanger DNA sequencing

cDNA clone delivery
Primer/labeled probe delivery

Because the speed of progress in genome technology is very fast, the Genomics Lab is keen to provide
the Center with the most up-dated technology on demand. We have already introduced two next generation sequencers, a GS Junior [ Roche ] and a HiSeq 1000 [Illumina], which enable us to obtain vast
amounts of massively parallel DNA sequencing data for genome DNA, transcripts, and so on. Both sequencers are already available in 2012. Additionally, we have recently introduced a mass spectrometer,a TripleTOF 5600 System [ AB SCIEX ] ( Fig. 3 ) . The system will make it possible to take quantitative proteomic approaches in various immunological studies. The members of the Center will be able to accessto the TripleTOF 5600 System in early 2013.

129

Administrative Coordination OfficeT

he Administrative Coordination Office (AdmCO) wasestablished in April 2010 to support the research activities of RCAI. It consists of assistants, the IT team andfacilities support staff.
The assistants support the Center and its laboratories

by providing secretarial assistance and serve as Secretariatsof the Center’s annual events, such as the International Symposium co-organized with JSI, the International Summer
Program, Harvard Summer School Program, Retreat, several

Table 3: Members of Administrative Coordination Office
2012

Position

Name

Office Manager

Ichiro Taniuchi

Administrative Staff
Chief Assistant

Hiroko Yamaguchi

joint workshops, the Graduate Students Tutorial Meeting, 21
seminars in the 2012 Seminar Series, etc. The IT teamdesigns and maintains RCAI’s unique server and databases,

Mari Kurosaki
Assistant

Hiromi Akita
Sachiko Dohisolves PC troubles and supports scientific meetings andChiaki Fukushima

conferences both inside and outside RCAI. The facilities staff

Sachiko Haraguchi

advises on RCAI facilities and arranges construction requiredin remodeling laboratories and installing large equipment,

Akiko Imaietc.

Akiko Imaizumi
Aiko Iyama

In the summer of 2012, RCAI resumed international programs after one year hiatus caused by the Great East Japan

Shihoko Kato

Earthquake. In April, 2013, a new center will be launched andReiko Kimura

RCAI started preparing for it. From December, 2012, remod-

Satomi Laweling and relocation of laboratories took place sequentially.

Rie Morita

Mr. Ogata of the facilities staff advised each moving labora-

Shoko Nishidatory about laboratory layout, and planned the moving sched-Kazuyo Nomura

ule. The IT team was busy virtualizing servers, which enabled

Yuko Ochi

RCAI members to acquire a more trustworthy IT environment

Rieko Okoshi

and greatly contributed to secure the precious research

Norie Takeuchi

data. Assistants helped laboratories move out and in, taking

Yuuki Yamada

charge of machine recycling, mouse transfer, and a large

Mio Yoshioka

number of MTAs, etc.

Motoko Yoshioka

Using the centralized approach, the AdmCO is now able
to provide smooth and efficient administrative support under
the same standards to all RCAI laboratories. For example,
even on the occasion of an assistant’s sudden leave or illness, another AdmCO member can replace her without any
interruption. The AdmCO, in cooperation with the Yokohama
Research Promotion Division (YRPD), has been successfully
coordinating and resolving various issues for the laboratories.
The strong spirit and constant efforts by the Administrative Coordination Office are now supporting the fundamentalbase of the RCAI activities and they will continue at the newcenter.

130

Hiroko Tanabe

IT team
Technical Scientist

Yasuaki Murahashi
Miho Izawa

Technical Staff

Aoi Ozawa

Facilities Staff
Technical Scientist

Toshihiko Ogata

2012Part 7

Data and Statistics

Publications (Apr. 2012 - Mar. 2013)
Table 1:RCAI publications FY2012
IF ( 2011)

FY201252.8

1Nat Rev Immunol.

33.3

1

Science31.2

1

Nat Immunol.

26.0

6

Cell Stem Cell

25.4

3

ROCKEFELLER UNIV

56.23

24,572422

OSAKA UNIV

54.00

55,021

1,019

Institutes with more than 300 papers

Citations/Citations
paper

PapersNat Med.

22.5

1

Immunity

21.6

4

RIKEN

46.13

22,326484

SCRIPPS RES INST

45.36

31,029684

Mol Cell.

14.2

1

J Exp Med.

13.9

4

KYOTO UNIV

44.87

27,731

618

Cell Host Microbe.

13.5

1

SLOAN KETTERING CANC CTR

44.79

16,797375

Immunol Rev.

11.1

1

YALE UNIV

44.16

57,4111,300

J Allergy Clin Immun.

11.0

1

WALTER & ELIZA HALL INST

43.25

15,051

348

UNIV CALIF BERKELEY

41.89

16,126

385

Blood

9.9

7

Proc Natl Acad Sci USA.

9.7

7

LA JOLLA INST ALLERGY & IMMUNOL

41.82

22,208531

41.63

43,004

1,033

41.39

32,614788

Curr Opin Immunol.

9.5

2

WASHINGTON UNIV

Circ Res.

9.5

1

BRIGHAM & WOMENS HOSP

EMBO J.

9.2

1

NEW YORK UNIV

38.62

26,303

681

Genome Biol

9.0

1

UNIV TEXAS HLTH SCI CTR HOUSTON

37.99

18,082

476

PLoS Genet.

8.7

1

KYUSHU UNIV

37.70

16,775445

Cancer Res.

7.9

1

UNIV CHICAGO

37.48

19,940532

Development

6.6

1

HARVARD UNIV

37.41

150,312

4,018

Cell Mol Life.

6.6

1

Philos Trans R Soc Lond B Biol Sci.

6.4

1

Semin Immunol.

6.4

1

J Immunol.

5.8

12

Mol Cell Biol.

5.5

1

J Virol.

5.4

2

DNA Res.

5.2

1

Eur J Immunol.

5.1

1

Mol Biol Cell.

4.9

1

J Biol Chem.

4.8

3

PLoS One

4.1

1

Dev Biol.

4.1

2

Int Immunol.

3.4

4

Other Journals

41

Total

119

Ato, M., Takahashi, Y., Fujii, H., Hashimoto, S., Kaji, T., Itamura, S., Horiuchi, Y.,
Arakawa, Y., Tashiro, M. & Takemori, T. Influenza A whole virion vaccine induces
a rapid reduction of peripheral blood leukocytes via interferon-alpha-dependent
apoptosis. Vaccine Vol. 31 2184-2190 (2013).

6

Farcas, A. M., Blackledge, N. P., Sudbery, I., Long, H. K., McGouran, J. F., Rose,N. R., Lee, S., Sims, D., Cerase, A., Sheahan, T. W., Koseki, H., Brockdorff, N.,
Ponting, C. P., Kessler, B. M. & Klose, R. J. KDM2B links the Polycomb Repressive
Complex 1 (PRC1) to recognition of CpG islands. Elife Vol. 1 e00205 (2012).

7Frank, T. D., Cheong, A., Okada-Hatakeyama, M. & Kholodenko, B. N. Catching transcriptional regulation by thermostatistical modeling. Phys Biol Vol. 9045007 (2012).

8

Fujii, Y., Shiota, M., Ohkawa, Y., Baba, A., Wanibuchi, H., Kinashi, T., Kurosaki, T.
& Baba, Y. Surf4 modulates STIM1-dependent calcium entry. Biochem Biophys
Res Commun Vol. 422 615-620 (2012).

9

Fukada, T., Nishida, K., Yamasaki, S. & Hojyo, S. Zinc signaling : a novel regulatory
system on bone homeostasis, and immune and allergic responses. Clin Calcium
Vol. 22 1707-1727 (2012).

10

Fukaya, T., Murakami, R., Takagi, H., Sato, K., Sato, Y., Otsuka, H., Ohno, M., Hijikata, A., Ohara, O., Hikida, M. & Malissen, B. Conditional ablation of CD205+ conventional dendritic cells impacts the regulation of T-cell immunity and homeostasis
in vivo. Proc Natl Acad Sci U S A Vol. 109 11288-11293 (2012).

11

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 Vol.
59 27-30 (2012).

2Blackburn, J., Ohazama, A., Kawasaki, K., Otsuka-Tanaka, Y., Liu, B., Honda, K.,
Rountree, R. B., Hu, Y., Kawasaki, M., Birchmeier, W., Schmidt-Ullrich, R., Kinoshita, A., Schutte, B. C., Hammond, N. L., Dixon, M. J. & Sharpe, P. T. The role of Irf6
in tooth epithelial invagination. Dev Biol Vol. 365 61-70 (2012).

12

Giacomin, P. R., Siracusa, M. C., Walsh, K. P., Grencis, R. K., Kubo, M., Comeau,
M. R. & Artis, D. Thymic stromal lymphopoietin-dependent basophils promote Th2
cytokine responses following intestinal helminth infection. J Immunol Vol. 189
4371-4378 (2012).

3

Dong, Z., Davidson, D., Perez-Quintero, L. A., Kurosaki, T., Swat, W. & Veillette, A.
The adaptor SAP controls NK cell activation by regulating the enzymes Vav-1 andSHIP-1 and by enhancing conjugates with target cells. Immunity Vol. 36 974-985(2012).

13

Glowacka, W. K., Alberts, P., Ouchida, R., Wang, J. Y. & Rotin, D. LAPTM5 protein
is a positive regulator of proinflammatory signaling pathways in macrophages.
J Biol Chem Vol. 287 27691-27702 (2012).

144Endoh, M., Endo, T. A., Endoh, T., Isono, K., Sharif, J., Ohara, O., Toyoda, T., Ito,T., Eskeland, R., Bickmore, W. A., Vidal, M., Bernstein, B. E. & Koseki, H. Histone
H2A mono-ubiquitination is a crucial step to mediate PRC1-dependent repressionof developmental genes to maintain ES cell identity. PLoS Genet Vol. 8 e1002774(2012).

Harada, Y., Tanaka, S., Motomura, Y., Ohno, S., Yanagi, Y., Inoue, H. & Kubo, M.
The 3’ enhancer CNS2 is a critical regulator of interleukin-4-mediated humoral
immunity in follicular helper T cells. Immunity Vol. 36 188-200 (2012).

15

Higuchi, K., Nakaoka, Y., Shioyama, W., Arita, Y., Hashimoto, T., Yasui, T., Ikeoka,
K., Kuroda, T., Minami, T., Nishida, K., Fujio, Y., Yamauchi-Takihara, K., Shirai, M.,
Mochizuki, N. & Komuro, I. Endothelial Gab1 deletion accelerates angiotensin II-dependent vascular inflammation and atherosclerosis in apolipoprotein E knockout
mice. Circ J Vol. 76 2031-2040 (2012).

5

132

Institutions ranking in immunology (Essential Science Indicators, The Thomson Coorporation, Sep., 2012)
When compared with institutions with more than 300 immunology papers, RIKEN’s citations per paper was ranked 3rd in theworld.

Annu Rev of Immunol.

Journals

1

Table 2:Engel, I., Zhao, M., Kappes, D., Taniuchi, I. & Kronenberg, M. The transcription
factor Th-POK negatively regulates Th17 differentiation in Valpha14i NKT cells.
Blood Vol. 120 4524-4532 (2012).

16

Hill, D. A., Siracusa, M. C., Abt, M. C., Kim, B. S., Kobuley, D., Kubo, M., Kambayashi, T., Larosa, D. F., Renner, E. D., Orange, J. S., Bushman, F. D. & Artis, D.
Commensal bacteria-derived signals regulate basophil hematopoiesis and allergic
inflammation. Nat Med Vol. 18 538-546 (2012).

17

Hiroshima, M., Saeki, Y., Okada-Hatakeyama, M. & Sako, Y. Dynamically varying
interactions between heregulin and ErbB proteins detected by single-molecule analysis in living cells. Proc Natl Acad Sci U S A Vol. 109 13984-13989 (2012).

18

Honda, K. IL-22 from T cells: better late than never. Immunity Vol. 37 952-954(2012).

19

Honda, K. & Littman, D. R. The microbiome in infectious disease and inflammation.
Annu Rev Immunol Vol. 30 759-795 (2012).

20

Hori, S. The Foxp3 interactome : a net work perspective of T( reg ) cells.
Nat Immunol Vol. 13 943-945 (2012).

21

Hori, T., Ohnishi, H., Teramoto, T., Tsubouchi, K., Naiki, T., Hirose, Y., Ohara, O.,
Seishima, M., Kaneko, H., Fukao, T. & Kondo, N. Autosomal-dominant chronic
mucocutaneous candidiasis with STAT1-mutation can be complicated with chronic
active hepatitis and hypothyroidism. J Clin Immunol Vol. 32 1213-1220 (2012).

22

Hotta, K., Nashimoto, A., Yasumura, E., Suzuki, M., Azuma, M., Iizumi, Y., Shima,D., Nabeshima, R., Hiramoto, M., Okada, A., Sakata-Sogawa, K., Tokunaga, M., Ito,
T., Ando, H., Sakamoto, S., Kabe, Y., Aizawa, S., Imai, T., Yamaguchi, Y., Watanabe,
H. & Handa, H. Vesnarinone Suppresses TNFalpha mRNA Expression by Inhibiting
Valosin-containing Protein. Mol Pharmacol (2013).

23

Ishibashi, D., Atarashi, R., Fuse, T., Nakagaki, T., Yamaguchi, N., Satoh, K., Honda,
K. & Nishida, N. Protective role of interferon regulatory factor 3-mediated signaling
against prion infection. J Virol Vol. 86 4947-4955 (2012).

24

Ishikawa, F. Use of rHuG-CSF for the Treatment of Myeloid Leukemia and in
Targeting Leukemia Stem Cells. Twenty Years of G-CSF Clinical and Nonclinical
Discoveries (eds G. Molineux, M. Foote, & T. Arvedson) 169-182 (Springer, 2012).

25

26

Isoda, T., Takagi, M., Piao, J., Nakagama, S., Sato, M., Masuda, K., Ikawa, T.,
Azuma, M., Morio, T., Kawamoto, H. & Mizutani, S. Process for immune defectand chromosomal translocation during early thymocyte development lacking ATM.
Blood Vol. 120 789-799 (2012).
Ito, H., Yan, X., Nagata, N., Aritake, K., Katsumata, Y., Matsuhashi, T., Nakamura,M., Hirai, H., Urade, Y., Asano, K., Kubo, M., Utsunomiya, Y., Hosoya, T., Fukuda, K.
& Sano, M. PGD2-CRTH2 pathway promotes tubulointerstitial fibrosis. J Am Soc
Nephrol Vol. 23 1797-1809 (2012).

27

Ivanov, II & Honda, K. Intestinal commensal microbes as immune modulators.
Cell Host Microbe Vol. 12 496-508 (2012).

28

Izawa, K., Hijikata, A., Tanaka, N., Kawai, T., Saito, M. K., Goldbach-Mansky, R.,
Aksentijevich, I., Yasumi, T., Nakahata, T., Heike, T., Nishikomori, R. & Ohara, O.
Detection of base substitution-type somatic mosaicism of the NLRP3 gene with
>99.9% statistical confidence by massively parallel sequencing. DNA Res Vol. 19
143-152 (2012).

29

30

31

32

33

Jiang, Y., Arase, N., Kohyama, M., Hirayasu, K., Suenaga, T., Jin, H., Matsumoto, M.,
Shida, K., Lanier, L. L., Saito, T. & Arase, H. Transport of misfolded endoplasmic
reticulum proteins to the cell surface by MHC class II molecules. Int Immunol Vol.
25 235-246 (2013).
Kagiyama, Y., Kitaura, J., Togami, K., Uchida, T., Inoue, D., Matsukawa, T., Izawa,
K., Kawabata, K. C., Komeno, Y., Oki, T., Nakahara, F., Sato, K., Aburatani, H. &Kitamura, T. Upregulation of CD200R1 in lineage-negative leukemic cells is characteristic of AML1-ETO-positive leukemia in mice. Int J Hematol Vol. 96 638-648(2012).
Kaji, T., Ishige, A., Hikida, M., Taka, J., Hijikata, A., Kubo, M., Nagashima, T.,
Takahashi, Y., Kurosaki, T., Okada, M., Ohara, O., Rajewsky, K. & Takemori, T.
Distinct cellular pathways select germline-encoded and somatically mutatedantibodies into immunological memory. J Exp Med Vol. 209 2079-2097 (2012).
Kajikawa, M., Li, P. C., Goto, E., Miyashita, N., Aoki-Kawasumi, M., Mito-Yoshida,M., Ikegaya, M., Sugita, Y. & Ishido, S. The intertransmembrane region of Kaposi’s
sarcoma-associated herpesvirus modulator of immune recognition 2 contributes to
B7-2 downregulation. J Virol Vol. 86 5288-5296 (2012).
Kamae, C., Nakagawa, N., Sato, H., Honma, K., Mitsuiki, N., Ohara, O., Kanegane,
H., Pasic, S., Pan-Hammarstrom, Q., van Zelm, M. C., Morio, T., Imai, K. & Nonoyama, S. Common variable immunodeficiency classification by quantifying T-cell
receptor and immunoglobulin kappa-deleting recombination excision circles.
J Allergy Clin Immunol (2012).

34

Kanaya, T., Hase, K., Takahashi, D., Fukuda, S., Hoshino, K., Sasaki, I., Hemmi, H.,
Knoop, K. A., Kumar, N., Sato, M., Katsuno, T., Yokosuka, O., Toyooka, K., Nakai,K., Sakamoto, A., Kitahara, Y., Jinnohara, T., McSorley, S. J., Kaisho, T., Williams, I.
R. & Ohno, H. The Ets transcription factor Spi-B is essential for the differentiation ofintestinal microfold cells. Nat Immunol Vol. 13 729-736 (2012).

35

Kato, L., Begum, N. A., Burroughs, A. M., Doi, T., Kawai, J., Daub, C. O., Kawaguchi,T., Matsuda, F., Hayashizaki, Y. & Honjo, T. Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin
genes. Proc Natl Acad Sci U S A Vol. 109 2479-2484 (2012).

36

Kato, L., Stanlie, A., Begum, N. A., Kobayashi, M., Aida, M. & Honjo, T. An evolutionary view of the mechanism for immune and genome diversity. J Immunol Vol.
188 3559-3566 (2012).

37

Kawamoto, S., Tran, T. H., Maruya, M., Suzuki, K., Doi, Y., Tsutsui, Y., Kato, L. M.
& Fagarasan, S. The inhibitory receptor PD-1 regulates IgA selection and bacterial
composition in the gut. Science Vol. 336 485-489 (2012).

38

Kawano, Y., Ouchida, R., Wang, J. Y., Yoshikawa, S., Yamamoto, M., Kitamura, D.
& Karasuyama, H. A novel mechanism for the autonomous termination of pre-B cell
receptor expression via induction of lysosome-associated protein transmembrane 5.
Mol Cell Biol Vol. 32 4462-4471 (2012).

39

Kawasaki, Y., Toyoda, H., Otsuki, S., Iwasa, T., Iwamoto, S., Azuma, E., Itoh-Habe,
N., Wada, H., Fujimura, Y., Morio, T., Imai, K., Mitsuiki, N., Ohara, O. & Komada,
Y. A novel Wiskott-Aldrich syndrome protein mutation in an infant with thrombotic
thrombocytopenic purpura. Eur J Haematol Vol. 90 164-168 (2013).

40

Kimura, S., Matsumura, K. & Okada-Hatakeyama, M. Inference of S-systemmodels of genetic networks by solving linear programming problems and setsof linear algebraic equations. WCCI 2012 IEEE World Congress on Computational
Intelligence (eds J. Liu et al.) 1-8 (Springer, 2012).

41

Koseki, H. An interview with Haruhiko Koseki. Interviewed by Eva Amsen.
Development Vol. 139 3469-3470 (2012).

42

Koyasu, S. Natural helper cells and TH2-type innate immunity. Cornea Vol. 31
Suppl 1 S20-24 (2012).

43

Koyasu, S. & Moro, K. Natural “helper” cells in the lung: good or bad help?
Immunity Vol. 36 317-319 (2012).

44

Koyasu, S. & Moro, K. Role of innate lymphocytes in infection and inflammation.
Front Immunol Vol. 3 101 (2012).

45

Ku, M., Jaffe, J. D., Koche, R. P., Rheinbay, E., Endoh, M., Koseki, H., Carr, S. A.
& Bernstein, B. E. H2A.Z landscapes and dual modifications in pluripotent andmultipotent stem cells underlie complex genome regulatory functions. Genome
Biol Vol. 13 R85 (2012).

46

Kubota, S., Miyaji, K., Shimo, Y., Shimakura, H., Takase, Y., Okamoto, N., Kiuchi, A.,
Fujimura, M., Fujimura, T., DeBoer, D. J., Tsukui, T. & Sakaguchi, M. IgE reactivity
to a Cry j 3, an allergen of Japanese cedar (Cryptomeria japonica) pollen in dogs
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47

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135

Invited Presentations

136

Month and
year

Name of the speaker

Title of the presentation

Name of the meeting

Place of the meeting(City and country)

Apr-12

Fagarasan, S.

Mucosal Immunology. Role of PD-1 in IgA synthesis in gut.

Korean Immunology Conference

Seoul, Korea

Apr-12

Taniguchi, M.

NKT cell system as a new therapeutic target

The 112th Annual Congress of Japan Surgical Society

Chiba, Japan

Apr-12

Wang, J-Y.

The IgM Fc receptor positively regulates B cell survival andantibody production

Innovation Summit Tokyo 2012 - Chronic Inflammation andAutoimmune Diseases

Tokyo, Japan

Apr-12

Sato, K.

Control of immune response by dendritic cells

Seminar on Hiroshima Liver Conference

Hiroshima, Japan

Apr-12

Ohno, H.

M cells, a unique subset of intestinal epithelial cells
specialized for mucosal antigen-uptake

The 2012 Spring Conference of the Korean Association of
Immunologists

Seoul, Korea

Apr-12

Ohno, H.

Gut microbiota and host protection - prevention of0157-infectious death of mice by bifidobacteria

The 22nd Tokyo Alcoholic Clinica Forum

Tokyo, Japan

Apr-12

Ohno, H.

Role of Commensal Microbiota in Intestinal Immunity andHost Defense

The 3rd International Forum, The 98th General Meeting of theJapanese Society of Gastroenterology

Tokyo, Japan

Apr-12

Saito, T.

CRTAM-mediated cell adhesion regulates development ofpathogenic effector T cells.

ICAD Innovation Summit Tokyo 2012

Tokyo, Japan

Apr-12Cheroutre, H.

Mucosal Memory CD8 T Cells: Saving the Best for (to) Last

Southwestern Medical Center at Dallas Excellence in Immunology
Lecture Series

Dallas, USA

Apr-12

Taniuchi, I.

Transcriptionaland epigentic control of thymocytes
development

Seminar at NIH

Bethesda, USA

Apr-12Cheroutre, H.

Mucosal Memory CD8 T Cells: Saving the Best for (to) Last

Wake Forest University School of Medicine, Microbiology and
Immunology Research Seminar

Winston-Salem, USA

May-12

Koseki, H.

Polycomb repression in mammalian development

Seminar, Dulbecco Telethon InstituteRome, Italy

May-12

Koseki, H.

Polycomb repression in mammalian development

Seminar, European School of Oncology, European Institute of
Oncology

Milan, Italy

May-12

Koyasu, S.

Natural helper cells and Th2-type innate immunity

99th Annual Meeting: The Americal Association of Immunologists

Boston, USA

May-12Ishikawa, F.

Creation of Humanized Mouse Model for Human Immunity
and Diseases

Workshop on Improving Animal Models for Regenerative Medicine,
NIH

Bethesda, USA

May-12

Fujii, S.

Licensing by invariant NKT cells harness the power of thedendritic cells (DCs)A Scientific Symposium in Memory of Ralph M. Steinman

New York, USA

May-12

Koyasu, S.

Masters Lecture: Type 2 innate immune responses mediatedby natural helper cells

5th FIMSA Congress

New Delhi, India

May-12

Fukada, T.

Zinc signal in growth and bone homeostasis

The 66th Annual Meeting of Japan Society of Nutrition and Food
Science

Sendai, Japan

May-12

Kurosaki, T.

Activation Mechanisms of B Lymphocytes

Freiburg Institute for Advanced Studies Seminar Series

Freiburg, Germany

May-12

Koyasu, S.

Natural helper cell, a novel innate lymphocyte producing
Th2 cytokines

Xth Latin-American Congress of Immunology-ALAI2012

Lima, Peru

May-12Moro, K.

IL-33-dependent eosinophilia in the lung mediated bynatural helper cells.

20th International Symposium on Molecular Cell Biology of
Macrophages 2012

Tokyo, Japan

May-12Moro, K.

The role of Natural helper cells in helminth infection andallergy77th Japan Congress of Interferon and cytokine

Kobe, Japan

May-12Moro, K.

The role of Natural helper cells during allergy

Rochester conferenceTokyo, Japan

May-12

Kurosaki, T.

Activation Mechanisms of B Lymphocytes

Seminar, Max Planck Institute of Immunology and Epigenetics

Freiburg, Germany

Jun-12

Takemori, T.

Memory B cells require memory T cells for their terminal
differentiation.

Immunological memory in Health and Diseases joint HKS andIMPAM meeting

Berlin, Germany

Jun-12

Taniuchi, I.

Transcriptionaland epigentic control of thymocytes
development

Seminar at Medical University of Viena

Viena, Austria

Jun-12

Saito, T.

Spatiotemporal regulation of T cell activation and costimulation.

Gordon Research Conferences—Immunochemistry &Immunobiology

Les Diablerets, Switzerland

Jun-12

Ohno, H.

Integrated ‘omics’ approach reveals that bifidobacteria
protect from enterohaemorrhagic Escherichia coli infectious
death through production of acetate.

GI Research Academy

Tokyo, Japan

Jun-12

Ohno, H.

Antigen delivery to the gut immune system throughepithelium: M cells vs FAE?

Gordon Research Conference, Lysosomes & Endocytosis

Andover, NH, USA

Jun-12

Okada, T.

Imaging of lymphocyte dynamics during the germinal center RIKEN RCAI International Summer Program 2012
formation

Yokohama, Japan

Jun-12Cheroutre, H.

Mucosal T cells: Shaped by the challenge

Yokohama, Japan,

Jun-12

Taniguchi, M.

NKT cell-mediated adjuvant activity on antitumor responses. FOCIS2012

Vancouver, Canada

Jun-12

Fukada, T.

“Zinc Signal”: A new signal mechanistic
regulating systemic growth and bone homeostasis

12th Scientific Meeting of the JapaneseSociety of Anti-Aging Medicine

Yokohama, Japan

Jun-12

Fujii, S.

NKT cell-mediated licensing of dendritic cells (DCs) in vivo

RCAI-JSI International Symposium on Immunology

Yokohama, Japan

Jun-12

Koyasu, S.

Critical role of GATA3 in natural helper cell differentiationand function

RCAI-JSI International Symposium on Immunology 2012

Yokohama, Japan

Jun-12

Ohno, H.

Function and differentiation of M cells, a unique subset ofintestinal epithelial cells specialized for mucosal antigenuptake

RCAI-JSI Interntional Symposium on Immunology 2012

Yokohama, Japan

Jun-12

Nishida, K.

Critical role of zinc transporter ZnT5/Slc30a5 in cytokinemediated delayed-type allergic reaction

The 23rd Annual Scientific Meeting of the Japanese Society for
Biomedical Research on Trace Elements

Tokyo, Japan

Jul-12

Fukada, T.

Biochemical characterization of human ZIP13
protein: A homo-dimerized zinc transporter
involved in the Spondylocheiro dysplastic
Ehlers-Danlos syndrome

The 23rd Annual Scientific Meeting of the Japanese Society for
Biomedical Research on Trace Elements

Tokyo, Japan

Jul-12

Fukada, T.

Zinc signal study and Metallomics

The 23th Annual Scientific Meeting of the Japanese Society for
Biomedical Research on Trace Elements

Tokyo, Japan

Jul-12

Ohno, H.

Differentiation and function of M cells, a unique subset
of intestinal epithelial cells important for the induction ofintestinal immunity

The 2nd Dietary Sicence Forum

Tokyo, Japan

Jul-12

Kurosaki, T.

Molecular Basis for Humoral Memory Response

JSI Immunolgy Summer School 2012

Nasu, Japan

Jul-12

Kurosaki, T.

Development and activation mechanisms of B lymphocytes

JSI Immunolgy Summer School 2012

Nasu, Japan

Jul-12

Saito, T.

Dynamicregulation of T cell activation

JSI Immunolgy Summer School 2012

Nasu, JapanRIKEN RCAI International Summer Program 2012

Jul-12

Kawamoto, H.

Lineage restriction process from hermatopoietic stem cells
to T cell progenitors

JSI Summer School Memorial Lecture for JSI Award

Nasu, Japan

Jul-12

Fujii, S.

Development of artificial adjuvant vector cells targeting invivo DCs

The 6th Annual Meeting of Japanese Association of Cancer
Immunology

Sapporo, Japan

Jul-12

Kawamoto, H.

A novel model of hematopoiesis

Kitano Hospital Core Lecture

Osaka, Japan

Aug-12

Fujii, S.

Cross-talk between innate lymphocytes and dendnritc cells

8th Lecture on Transplantation Immunology

Nagoya, Japan

Aug-12

Fujii, S.

Discovery of dendritic cells by Prof. Steinman

Men-eki Fushigi Mirai 2012

Tokyo, Japan

Aug-12

Taniuchi, I.

Transcriptional and Epigenetic Views
of CD4/CD8 Lineage Commitment in Thymus

Seminar at Cancer Science InstituteSingapore, Singapore

Aug-12

Taniuchi, I.

Transcriptional Control of
CD4/CD8 Lineage Decision in Thymus

Semiar at Walter and Eliza Hall Institute

Melbourne, Australia

Aug-12

Okada, T.

Imaging of cellular dynamics during the adaptive immune
response

14th International Congress of Histochemistry and Cytochemistry,
Kyoto, Japan

Kyoto, Japan

Aug-12

Sakata-Sogawa, K. &Tokunaga, M.

Quantitative analysis of single molecule imaging ofphysicochemical field for genetic activities

The 50th Annual Meeting of the Biophysical Society of Japan,
Symposium

Nagoya, Japan

Sep-12

Koyasu, S., Furusawa,
J.-I., Moro, K.

Critical role of GATA3 in natural helper cell differentiationand function

Cold Spring Harbor Asia Conferences “Frontiers of Immunology in
Health and Diseases”

Suzuhou, China

Sep-12

Honda, K.

Treg-inducing commensal Clostridium species derived fromthe human microbiome

Cold Spring Harbor Asia Conferences “Frontiers of Immunology in
Health and Diseases”

Suzuhou, China

Sep-12Ishikawa, F.

Humanized mouse research

INSERM Necker Hospital

Paris, France

Sep-12

Taniuchi, I.

Transcriptional Control of
CD4/CD8 Lineage Decision in Thymus

Seminar at Weatherall Institute of Molecular Medicine

Oxford, UK

Sep-12

Ohno, H.

M cells, a unique subset of intestinal epithelial cells
specialized for mucosal antigen-uptake

The 11th Awaji International Forum on Infection and Immunity
ProgramAwaji, Japan

Sep-12

Hase, K.

Intestinal microbiota shapes intestinal T cell balance via
epigenetic modifications

The 71st Annual Meeting of the Japanese Cancer Association

Sapporo, Japan

Sep-12

Koseki, H.

Polycomb repression in mammalian development

University of Luxembourg, Luxembourg Center for Systems
Biomedicine team reatreat

Nennig, Germany

Sep-12

Ohara, O.

Integra1ve Medical Sciences at RIKEN RCAI: to understandthe process of disease development

Australia-Japan workshop on biomedical breakthroughs andsystems biology

Tokyo,Japan

Sep-12Moro, K.

Critical roles for natural helper cells in IL-33 dependent lung The 12th Annual International and Innate Immunity Society Meeting
eosinophilia upon helminth infection

Tokyo, Japan

Oct-12

Fujii, S.

Artificial cellular vaccine as front-line immune therapy
against cancer

RIKEN cutting edge symposium

Tokyo, Japan

Oct-12

Okada, T.

Imaging of cellular dynamics underlying the immune tissueorganization

The 25th Annual Mouse Molecular Genetics Conference

Pacific Grove, USA

Oct-12

Koseki, H.

Polycomb repression in mammalian development

Seminar, Life Sciences Institute, University of British Columbia

Vancouver, Canada

Oct-12

Okada, T.

Imaging of cross-presenting dendiritic cells in the lymph
node

University of California, San Francisco, Immunology Seminar Series

San Francisco, USA

Oct-12Cheroutre, H.

A transcriptional switch reprograms mature CD4 T helper
cells to cytotoxic T lymphocytes.

International Symposium on Immune Tolerance and Mucosal
Immunology. Sichuan University

Chengdu, China

Oct-12Ishikawa, F.

Targeting chemotherapy-resistant AML stem cells

74th Japan Society of Hematology Meeting, Asian Joint Symposium Kyoto, Japan

Oct-12

Ohno, H.

The role of M cells in intestinal immunity

International Endotoxin & Innate Immunity Society Meeting 2012,
Homeostatic Inflammation Symposium, Japan Endotoxin and Innate
Immunity Society 2012

Tokyo, Japan

Oct-12

Ohno, H.

Function and differentiation of M cells, a unique subset ofintestinal epithelial cells specialized for mucosal antigenuptake

The 12th International Symposium on Dendritic Cells

Daegu, Korea

Oct-12

Ohno, H.

Multi-omics approach reveals that bifidobacteria protectfrom enterohemorrhagic Escherichia coli infection throughproduction of acetate

The 34th Naito Conference, Infection, Immunityand their Control
for Health

Sapporo, Japan

Oct-12

Hori, S.

Resolving the controversy over regulatory T cell plasticity

The 3rd International Conference on Regulatory T Cells and Th
Subsets and Clinical Application in Human Diseases

Shanghai, China

Oct-12

Shiroguchi, K.

System-wide digital counting of nucleic acid molecules
using next generation sequencer with optimized singlemolecule barcodes

International meeting of “Spying Minority in Biological Phenomena”, Taipei, Republic of China
Grant-in-Aid for Scientific Research on Innovative Areas, MEXT,
Japan

Oct-12

Taniguchi, M.

NKT cells. Target for the treatment of intractable diseases
based on a new concept.

Joint Symposium on Tsukuba University/Tokyo University of
Science, Research Institute for Biomedical Sciences.

Tokyo, Japan.

Oct-12

Hase, K.

Epithelial immune functions at the interface between self
and non-self

IEIIS2012 Homeostatic Inflammation Satellite Symposium Infection, Inflammation and Immunity

Tokyo, Japan

Oct-12

Tanaka, T.

Negative regulation of inflammatory responses by LIM
proteins.

The 12th Biennial International Endotoxin & Innate Immunity Society Tokyo, Japan
Meeting, Symposium L3, Chronic inflammationOct-12

Fujii, S.

Development of artificial adjuvant vector cells as new typeof cancer vaccineTokyo University of Pharmacy and Life Sciences’ Seminar

Hachioji, Japan

Oct-12

Ohara, O.

Towards an understanding of immune cell sociology basedon single-cell analysis

International Symposium on Bio-Interactomics

Sevilla, Spain

Oct-12Moro, K.

Development of an innate Th2 producer ‘natural helper cell

International Symposium on genetic and epigenetic control ofcell fate

Kyoto, Japan

Nov-12

Koseki, H.

Mechanisms underlying Polycomb repression

Grants-in-Aid for Scientific Research on Innovative Areas: “Genetic
and Epigenetic Control of cell Fate”

Kyoto, Japan

Nov-12

Takemori, T.

Immunological Memory consists of multiple layers.

International Symposium: from Cell sorting to Immunlogical Memory Berlin, Germany

Nov-12

Kawamoto, H.

Molecular mechanisms for the production and maintenanceof the T cell lineage

The Intternational Symposium on Genetic and Epigenetic Controlof Cell Fate

Kyoto, Japan

Nov-12

Fujii, S.

Development of vaccine linking innate and adaptive
immunity

Hoshima University Seminar Series

Tokyo, Japan

Nov-12

Kurosaki, T.

B Cell Intrinsic and Extrinsic Mechanisms for Rapid
Responsiveness of IgG1 Type Memory B Cells

Keystone Symposia: B Cell Development and Function

Keystone Resort, USA

Nov-12

Hori, S.

Control of autoimmunity by Foxp3+ regulatory T cells

International Symposium on Glyco-minded Biology of Diseases as a
Basis of Pharmaceutical Sciences

Tokyo, Japan

Nov-12

Sato, K.

Control of immune response by dendritic cells

The 70th Special Seminar on Clinical Allergy Society

Tokyo, Japan

137

138

Nov-12

Kawamoto, H.

Lymphocyte Development

Hong Kong University Pasteur Institute Immunology Course Lecture

Hong Kong, China

Nov-12

Kurosaki, T.

Challenging for Mysteries of Immune Memory

The second WPI joint outreach symposium “Let’s enjoy the world’s
advanced science!”

Tsukuba, Japan

Nov-12

Kurosaki, T.

Activation Mechanisms of Memory B cells

Graduate School Seminar, University of Occupational andEnvironmental Health

Kitakyushu, Japan

Dec-12

Fagarasan, S.

IgA synthesis: a form of adaptation extending beyond gut.

42nd Annual Scientific Meeting Australasian Society for
Immunology.

Melbourne, Australia

Dec-12

Kawamoto, H.

Myeloid-based model of hematopiesis: revison of theclassical myeloid-lymphoid dichotomy concept

Osaka University Medial Tissue Engeneering Forum

Osaka, Japan

Dec-12

Kawamoto, H.

Epigenetic mechanisms maintaining the T cell and thymic
epithelial cell lineage

2012 Annual Meeting of the Japanese Society for Immunology

Kobe, Japan

Dec-12

Ohara, O.

Systems immunology: Tackling the complexity of theimmune system

2012 Annual Meeting of the Japanese Society for Immunology

Kobe, Japan

Dec-12

Watarai, H.

Invariant natural killer T cells mediate airway inflammation
through IL-25 and IL-18.

2012 Annual Meeting of the Japanese Society for Immunology

Kobe, Japan

Dec-12

Hase, K.

Epithelial Notch signaling secures lymphoid organogenesis
and immune homeostasis in the gut

2012 Annual Meeting of the Japanese Society for Immunology

Kobe, Japan

Dec-12

Hori, S.

Genetic and environmental control of regulatory T cell
fitness in tissues

2012 Annual Meeting of the Japanese Society for Immunology

Kobe, Japan

Dec-12

Taniguchi, M.

NKT cells as an ideal target for anti-tumor immunotherapy.

2012 Annual Meeting of the Japanese Society for Immunology

Kobe, Japan

Dec-12

Wang Ji-Yang

The Fc receptor for IgM (FcμR) positively regulates B cell
survival and activation

The 42nd Annual Meeting of The Molecular Biology Society ofJapan

Kobe, Japan

Dec-12

Okada, T.

Two-photon imaging of cellular dynamics during adaptive
immune responses

The 42nd Annual Meeting of The Molecular Biology Society ofJapan

Fukuoka, Japan

Dec-12

Hase, K.

Roles of intestinal M cells in mucosal infection andimmunity

The 85th Annual Meeting of the Japanese Biochemical Society

Fukuoka, Japan

Dec-12

Fukada, T.

Zinc signal: A new regulatory system in bone
and immunity

The 85th Annual Meeting of the Japanese Biochemical Society

Fukuoka, Japan

Dec-12

Ohno, H.

Understanding the host-gut microbiota interaction withmulti-omics approach

The 85th Annual Meeting of the Japanese Biochemical Society

Fukuoka, Japan

Jan-13

Saito, T.

Dynamic and cytoskeletal regulation of T cell activation

RCAI-Michigan Joint Workshop

Ann Arbor, USA

Jan-13

Koseki, H.

Polycomb potentiates Meis2 activation in midbrain bymediating precursory interaction of promoter withtissue-specific enhancer

RCAI-Michigan Joint Workshop

Ann Arbor, USA

Jan-13Ishido, S.

Novel immune regulation by MHC II ubiquitination

The 2nd Innovative Area Symposiumy

Kobe, Japan

Jan-13

Koyasu, S., Moro, K.

Natural helper cell, a Th2-type innate lymphocyte

IFReC-SIgN Winter School 2012 on Advanced Immunology

Osaka, Japan

Jan-13

Fagarasan, S.

Regulation of IgA synthesis and bacteria in the gut by PD-1

International Symposium on Immunological Self

Kyoto, Japan

Jan-13

Okada, M.

A switch in NF-kappaB immune signaling

First Annual Winter q-bio Meeting

Honolulu, USA

Feb-13

Kawamoto, H.

A revised model of hematopoiesis

Five hub-Institute Meeting

Kobe, Japan

Feb-13

Kawamoto, H.

Thymic epithelial cells and thymic cyst

Special Lecture at the Annual meeting of Japanese Association for
Research on the Thymus

Sapporo, Japan

Feb-13

Fagarasan, S.

New roles of PD-1 in immune homeostasis

New Horizons in the Immune System

Tokushima, Japan

Feb-13

Fagarasan, S.

IgA synthesis: A form of functional immune adaptation
extending beyond gut

Keystone symposia. X1 2013

Colorado, USA.

Feb-13

Saito, T.

Dynamic imaging analysis of T cell activation regulation

2nd New Zealand-RIKEN-Chiba Joint Workshop

Auckland, New ZealandFeb-13

Fujii, S.

iNKT cell-triggered in vivo DC targeting immunotherapy

2nd New Zealand-RIKEN-Chiba Joint Workshop

Auckland, New ZealandFeb-13

Fukada, T.

Zinc biology as medical science: why zinc isessential for us?

The 4 th Kinki Zinc-Nutritional Research
Meeting

Osaka, Japan

Feb-13Cheroutre, H.

Selective Differentiation of Mucosal Effector Memory T cells: Department of Immunology, Mayo Clinic College of Medicine.
Saving the Best for (to) Last

Rochester, USA

Feb-13Cheroutre, H.

Autoimmunity: New Players, Old Faces

Benaroya Research Institute at Virginia Mason, Wilske Lecture
Series, Science & Medicine

Seattle, USA

Mar-13

Honda, K.

Microbiota’s Influence on Immunity

3rd Schloss Elmau Meeting on Resistance and Disease promoting
Principles of Innate Immunity

Elmau, Germany

Mar-13

Hori, S.

Stability and adaptability of Foxp3+ regulatory T cells

The 7th International Symposium on the Clinical Use of Cellular
Products

Erlangen, Germany

Mar-13Ohno, H.

Multi-omics approach reveals that Bifidobacteria protectfrom enterohaemorrhagic Escherichia coli infection throughproduction of acetate: Importance of sugar transporter

3rd TNO Beneficial Microbes Conference: international conference
on the health impact andfuture potential of beneficial microbes

Noordwijkerhout, The
Netherlands

Mar-13Ohno, H.

Gut microbiota and healtch care focusing on the
immunological analysis with lactic acid bacteria andbifidobacteria

Metropolitan Bio Network Japan Seminar

Tokyo, Japan

Mar-13

Fukuda, S.

Integrated omics approach identified acetate produced
by probiotic bifidobacteria to protect host fromenteropathogenic infection

The 3th Stage Surface Barrier Immunology Study Group

Osaka, Japan

Mar-13

Honda, K., Atarashi, K.,
Tanoue, T., Morita, H.,
Hattori, M.

Induction of intestinal regulatory T cells

World Immune Regulation Meeting-VIII

Davos, Switzerland

Mar-13

Mohan, S.

A peek overview of emerging informatic tools for mutation
analyses in general and PID perspectives

1st National conference on Primary Immunodeficiency Diseases,
The Postgraduate Institute of Medical Education and Research(PGIMER)

Chandigarh, India

Mar-13

Mohan, S.

The current status and emerging trends in Asian primary
immunodeficiency network

1st National conference on Primary Immunodeficiency Diseases,
The Postgraduate Institute of Medical Education and Research(PGIMER)

Chandigarh, India

Mar-13

Honda, K.

Intestinal Commensal Bacteria-Mediated Treg Induction

Keystone Symposia, The Microbiome

Keystone, USA

Mar-13

Fukada, T.

Zinc signal: the action of Zn ion controlling
cellular and physiological events

The 86th Annual Meeting of the JapanesePharmacological Society

Fukuoka, Japan

Mar-13

Tanaka, T.

Negative regulation of T-helper cell differentiation by LIM
proteins.

The 132th Annual Meeting of the Pharmatheutical Sciety of Japan,
Special Symposium OS06, Frontiers in Researches of Immune
Regulators

Sapporo, Japan

RCAI Seminars 2012
Date

Speaker

Title

Affiliation

Country

Apr 16th

Dr. Seishi Ogawa

Novel pathway mutations in myeloid dysplasia

Project associated professor, Cancer
Genomics,Project, University of TokyoJapan

Apr 25th

Dr. Stephen
Schoenberger

Cellular and Molecular Determinants of CD8+ T
Cell Memory

La Jolla Institute for Allergy & Immunology

USA

May 17th

Dr. Toshiaki Kawakami

Atopic Dermatitis due to the impairment ofphospholipase C-β3

La Jolla Institute for Allergy & Immunology

USA

May 21st

Dr. Yoichi Gondo

The new utilization system of the RIKEN ENU
mutant mouse library (in Japanese)

RIKEN BioResource CenterJapan

May 24th

Dr. Hodaka Fujii

Locus-specific biochemical epigenetics byinsertional chromatin immunoprecipitation(iChIP)

Research Institute for Microbial Diseases, Osaka
University

Japan

May 28th

Dr. Yoshio Kodera

Comprehensive approach to develop diagnostic
biomarker peptides in serum. -Establishment of
discovery and validation methods targeting
low-abundance peptides-

Director, Center for Disease Proteomics
Associate Professor, Department of Physics
Kitasato University School of ScienceJapan

May 29th

Dr. Motonari Kondo

Roles of bone marrow microenvironment inlymphoid and myeloid lineage choice bylymphoid-specified multipotent progenitors

Associate Professor, Department of Molecular
Immunology Toho University School of MedicineJapan

Jun 5thDr. Akihiro Fujimoto

Whole genome sequence analysis of a humangenome and cancer genome

Researcher, Riken Center for Genomic MedicineJapan

Jun 11th

Dr. Hans Clevers

Lgr5 stem cells in self-renewal and cancer

Professor and Director, the Hubrecht Institute in
Utrecht,University Medical Centre Utrech

Netherlands

Jun 11th

Dr. Yin Chun Lin

Global analysis of long-range genomic
interactions in developing B cells

Assistant Project Scientist, Department of Biological
Sciences, University of California, San DiegoUSA

Jun 12th

Dr. Ryo Yoshida

Bayesian statistical modeling and computational
technologies in systems biology andchemoinformatics

Associate Professor, The Institute of Statistical
Mathematics

Japan

Jun 14th

Dr. Makoto Nakanishi

Regulation of maintenance DNA methylation
through novel histone modification

Professor, Nagoya City University

Japan

Jun 20th

Dr. Martin FrithReliable large-scale sequence comparison

Senior Researcher,Computational Biology ResearchCenter, National Institute for Advanced Industrial
Science and Technology (AIST)Japan

RIKEN RCAI
Seminar Series
2012

June 25th

Dr. Hisataka Kobayashi

Molecular cancer imaging; New optical
diagnostic technologies and beyond

National Cancer Institute, National Institutes of
HealthUSA

RIKEN RCAI
Seminar Series
2012

Jul 2nd

Dr. Atsushi Iwama

Role of the polycomb group proteins in themaintenance of hematopoietic stem cells andrestriction of tumor development

Professor, Graduate School of Medicine, Chiba
University

Japan

Jul 10th

Dr. Todd Taylor

Metagenomics: an introduction and applications
to immunology

Team Leader, RIKEN Quantitative Biology CenterJapan

Jul 11th

Dr. Shunsuke Chikuma

Regulation of T cell function by epigenetic
TRIM28Assistant Professor, Graduate School of Medicine,
Kyoto University

Japan

Jul 12th

Dr. Masakatsu Yamashita

Menin-Bach2 axis controls senescenceassociated secretory phenotype (SASP) in CD4
T cells.

Head of the Laboratory, Lab. of Med. Genomics,
Kazusa DNA Research InstituteJapan

Jul 17th

Dr. Jun Sese

Revisiting Multiple Testing – Statistics for
Genome-Wide Experiments and Discovery ofCombinatorial Regulations of Transcription
Factors

Associate Professor,Dept. of Computer Science,
Tokyo Institute of Technology

Japan

RIKEN RCAI
Seminar Series
2012

Jul 24th

Dr. Hideya Kawaji

Computational approaches in data-driven
transcriptome

Unit Leader, RIKEN Omics Science Center

Japan

RIKEN RCAI
Seminar Series
2012

Jul 26th

Dr. Takehiko Kamijo

Role of Polycomb in Tumorigenesis

Head of Division of Biochemistry and Molecular
Carcinogenesis, Chiba Cancer Center ResearchInstituteJapan

Jul 31st

Dr. Keisuke Nagao

Langerhans cells and the mammalian hair inskin immunology

Assistant Professor, Keio University School of
MedicineJapan

Aug 9thDr. Mari Sato

Born/Bone this way (in Japanese)

Division of Hematology, Department of Internal
Medicine, Kobe University Graduate School of
MedicineJapan

Aug 10th

Dr Takuji YamadaEnterotypes of the human gut microbiome

Lecturer, Tokyo Institute of TechnologyJapan

Sep 18th

Dr. Satoru Miyano

Uncovering Molecular Networks in Cancer bySupercomputer

Professor, Human Genome Center,
The Institute for Medical Science, The Universityof TokyoJapan

RIKEN RCAI
Seminar Series2012

Sep 25th

Dr. Hiroyuki Kurata

Integration of omics data into metabolic andgene regulatory networks

Professor, Kyushu Institute of Technology

Japan

RIKEN RCAI
Seminar Series2012

Sep 25th

Dr. Min-Tze Liong

Probiotics &Hypercholesterolemia: Trials,
Challenges & Personalized Nutrition

Associate Professor,Bioprocess Technology Division
School of Industrial Technology, Universiti Sains
Malaysia

Malaysia

The fourth RCAICGM Joint Seminar

RIKEN RCAI
Seminar Series
2012

RIKEN RCAI
Seminar Series
2012

RIKEN RCAI
Seminar Series
2012

RIKEN RCAI
Seminar Series
2012

RIKEN RCAI
Seminar Series
2012

139

140

Sep 27th

Dr. Hitomi Mimuro

Crosstalk between Helicobacter pylori and Host
Cells

Associate Professor, Department of Infectious
Diseases Control,International Research Center for
Infectious Diseases,The Institute of Medical Science,
The University of TokyoJapan

Oct 9thDr. Hiroyuki Aburatani

Epigenome dynamics

Professor, Research Center for Advanced Scienceand Technology , The University of TokyoJapan

Oct 18th

Dr. Matteo Barberis

Timing in systems biology: the cell cycle

Senior Post-doctoral Research Associate,
Theoretical Biophysics, Institute for Biology,
Humboldt University Berlin Post-doctoral Research
Associate, Max Planck Institute for Molecular
Genetics

Germany

Oct 16th

Dr. Tatsuo Shibata

Basal dynamics of signal transduction system inchemotaxis cells

Unit Leader, RIKEN Center for Developmental Biology

Japan

RIKEN RCAI
Seminar Series2012

Oct 23rd

Dr. David Schlessinger

Genetic variants associated with age-related
traits and diseases in the Sardinian population

Chief, Laboratory of Genetics, National Institute on
Aging, National Institutes of Health

USA

RIKEN RCAI
Seminar Series2012

Oct 23rd

Dr. Françesco Cucca

Finding genetic factors in the Sardinian
population for both levels of immune cells andmolecules, and correlated autoimmune disease
pathogenesis

Director, Institute of Genetics and Biomedical
Research, National Research Council

USA

RIKEN RCAI
Seminar Series
2012

Oct 30th

Dr. Yuichi Wakamoto

Dynamic persistence of bacteria against
antibiotic drugs

Research Center for Complex Systems Biology, The
University of TokyoJapan

RIKEN RCAI
Seminar Series2012

Nov 2ndDr. Cornelis Murre

Global analysis of long-range genomic
interactions and the establishment oflymphocyte cell fate

Professor, Division of Biological Sciences, Universityof California, San DiegoUSA

Nov 5thDr. Shuji Ishihara

Phase map analysis of complex cell morpholigy

Assistant Professor, Graduate School of Arts andSciences, The University of TokyoJapan

Nov 8thDr. Kristian Helin

Roles of TET and Polycomb Group Proteins in
Stem Cells and Cancer

Director and Professor, Biotech Research &Innovation Centre, University of Copenhagen

Denmark

Nov 9thDr. François Karch

Long-distance regulatory interactions in the
Drosophila bithorax complex: from boundaries,
PREs and ncRNAs to the female post-mating
response

Associate Professor, Department of Genetics andEvolution, University of Geneva

Switzerland

Nov 13th

Dr. Ichiro Hiratani

Developmental regulation of nuclear genome
organization and DNA replication timing

Assistant Professor, National Institute of Genetics

Japan

Nov 20th

Dr. Toshiro Sato

Intestinal stem cells in homeostatic andregenerative state

Project Assistant Professor, Department of
Gastroenterology, Keio University School of Medicine

Japan

Nov 22nd

Dr. Frederic Geissmann

Development and functions of myeloid cells

Professor, Center for Molecular and Cellular Biology
of Inflammation King’s College London

UK

Nov 27th

Dr. Neil Brockdorff

Targeting epigenetic systems in a changing
landscapeGroup Leader, Department of Biochemistry,
University of Oxford

UK

Dec 4thDr. Yui-Hsi Wang

Novel IL-9-producing innate helper cells
promote oral antigen-induced anaphylaxis.

Division of Allergy and Immunology, Cincinnati
Children’s Hospital Medical CenterUSA

Dec - 10th

Dr. Harry W. Schroeder,
Jr.

Immune Deficiency, Autoimmunity and Control of
the Antibody Repertoire

University of Alabama at Birmingham
Division of Clinical Immunology and RheumatologyUSA

Dec - 11th

Dr. Paul Wilmes

Eco-Systems Biology of the human microbiome
highlights the potential importance of small
RNAs in human-microbe interactions.

ATTRACT Research Fellow, Luxembourg Centre for
Systems Biomedicine, University of Luxembourg

Germany

Dec - 18th

Dr. Masahiro Morita

RNA biology through the 5’ cap structure and 3’
poly (A) tail

Postdoctoral Fellow, Dr. Nahum Sonenberg’s lab,
Department of Biochemistry and Goodman Cancer
Research Centre, McGill University

CanadaDec - 18th

Dr. Daron M. Standley

A structural view of immune function

Immunology Frontier Research Center (IFReC),
Osaka University

Japan

Jan - 23rd

Dr. Shinichi Nishikawa

Development of Hematopoietic Stem Cell: A final
scenario

Group Director, Center for Developmental Biology,
RIKEN

Japan

Jan - 28th

Dr. Becca Asquith

Efficiency of the CD8+ T cell response topersistent viral infection

Senior Lecturer in Within-Host Dynamics, Imperial
College London

UK

Feb - 15th

Dr. Wilfred T.V.
Germeraad

Development of new Immunotherapies forcancer

Associate Professor, Department of Internal
Medicine, Division of Haematology, GROW Schoolfor Oncology and Developmental Biology
Maastricht University

Netherlands

Mar - 7th

Dr. Denis Duboule

Long range regulation of HoxD genes during
mammalian development

Professor, Department of Genetics and Evolution,
University of Geneva

Italy

Mar - 7th

Dr. Yusuke Miyanari

Reprogramming and nuclear dynamics

Postdoctral fellow, Institute of Genetics andMolecular and Cellular Biology (IGBMC)

FranceMar - 13th

Dr. Eugene Oltz

Defining the Malignant Epigenome in Follicular
Lymphoma

Professor of Pathology & Immunology, Washington
University School of Medicine

USA

RIKEN RCAI
Seminar Series
2012

RIKEN RCAI
Seminar Series
2012

RIKEN RCAI
Seminar Series
2012

RIKEN RCAI
Seminar Series
2012Budget, Personnel and Patents
RCAI Budget ( JPY 100 Million)

RCAI Budget FY2001-2012( JPY 100 Million )70

Note: Budgets for FY2001-2003 include construction
expenses for RCAI facility

100 million yen

60

RCAI Budget (JPY 100 Million )

50
40
30
20
10
0
2000

2002

2004

2006

2008

2010

2012

Fiscal Year

400
350

RCAI Personnel

300
250
200
150
100
50
0
2002

2004

2006
Fiscal Year

RCAI Personnel

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

2012

31.04

RCAI staff composition(as of Mar. 2013)

RCAI Personnel

450

2000

2001

2008

2010

2012

Category

NumberDirector

1

Senior Advisor

2

Group Director

6Team Leader

14

Unit Leader

12

Coordinator

2

Senior Scientist

8

Research Scientist

50

Special Postdoctoral Researcher

5

Foreign Postdoctoral Researcher

1

Research Associate

7

Junior Research Associate

16International Program Associate

7Student Trainee

41

Research Fellow

2

Research Consultant

2

2001

84

Visiting Scientist

49

2002

156

Senior Technical Scientist

1

2003

191

Technical Scientist

5

2004

328

Technical Staff I

31

2005

363

2006

366

Technical Staff II

66

2007

339

Assistant

22

2008

326

Temporary Employment

4

Total

354

2009

380

2010

400

2011

404

2012

354

RCAI Patents ( as of Mar. 2013)
There were 16 patents filed from April 2012- March 2013.

141