RIKEN
Research Center for Allergy and Immunology ( RCAI )Organization

1

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

Group
Lab. for Cell Signaling Takashi Saito, Ph.D.
Lab. for Lymphocyte Differentiation Tomohiro Kurosaki, M.D., Ph.D.

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

Lab. for Immunogenomics Osamu Ohara, Ph.D.
Lab. for Developmental Genetics Haruhiko Koseki, M.D., Ph.D.
Lab. for Immune Regulation Masaru Taniguchi, M.D., Ph.D.
Lab. for Cytokine Signaling Toshio Hirano, M.D., Ph.D.

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

Lab. for Immunological Memory Toshitada Takemori, M.D., Ph.D.

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

Yasuyuki Ishii, Ph.D.

Lab. for Cellular Systems Modeling

Mariko Okada-Hatakeyama, Ph.D.

Unit
Deputy Directors
Takashi Saito, Ph.D.
Toshio Hirano, M.D., Ph.D.

Unit for Molecular Systems Immunology Makio Tokunaga, Ph.D.
Unit for Human Disease Model Fumihiko Ishikawa, M.D., Ph.D.
Unit for Immune Homeostasis Shohei Hori, Ph.D.
Unit for Cellular Immunotherapy Shin-ichiro Fujii, M.D., Ph.D.
Unit for Immunoinformatics S. Sujatha Mohan, Ph.D.

Coordination Office

Unit for Lymphocyte Cloning Andrei Rybouchkin, Ph.D.

Research Coordination Office

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

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

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

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

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

Peter Burrows, Ph.D.

Unit for Immunodynamics Takaharu Okada, Ph.D.

Administrative Coordination Office
Takashi Saito, Ph.D.

International Research Unit
Unit for Thymic Environments

Willem van Ewijk, Ph.D.

Unit for Immunoepigenetics Miguel Vidal, Ph.D.

i

C on tents
Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ii

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

iv

Part 1Creation of New Paradigms

Bacterial Guests Double as Bodyguards. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

Sensing of Environmental Stimuli Promotes IgA Generation in the Gut . . . . . . . . . . . . . . . . . . . . . . . . . .

3

Crossing the Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4Maintaining Independence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5Fighting Their Way to the Middle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6

The Long and Short of Cell Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7

Ready and Waiting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

Awaiting Orders to Retaliate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9Creating a Life-saving Killer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10Giving Tumor Vaccines a Proper Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11

Holding Fire on the ‘Good Guys’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12

Hirano Receives the Japan Prize 2011 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

Outstanding Contribution of the Year 2010 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15

Other Prize Winners 2010 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

19

Excellent Paper of the Year 2010 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21

Excellent Poster Award 2010 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

22Part 2

Technology and Innovation

RCAI and Torii Collaborate for the Allergy Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

24

Open Laboratory for Allergy Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

25

iPS Technology Development for Anti-cancer Immune Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

26

Development of Immune Cell Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27

Network for Primary Immunodeficiency Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

28

Development of a Humanized Mouse Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29

Drug Discovery Antibody Platform Unit is Established in RCAI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30

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

30Part 3

Nurturing Young Scientists

RCAI International Summer Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

32

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

34

RCAI Retreat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

34

RIKEN Joint Retreat. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

35

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

36

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

36

RIKEN Joint Graduate School Program International Program Associate . . . . . . . . . . . . . . . . . . . . . . . . .
RIKEN Junior Research Associate (JRA ) Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

37

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

39

RCAI Administrative Coordination Office . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40Part 4

ii

i

37
38

Collaborative Networks

INSERM/Pasteur-RIKEN Joint Workshop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

42

LIAI-RCAI Joint Workshop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

44

New Zealand-RIKEN-CHIBA Joint Workshop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

46

The 17th International RUNX Workshop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48

Systems Biology Open Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49

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

49

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

Part 550

Outreach Activities

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

52

Tokyo Metropolitan Assembly Members Visited RCAI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

52

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

52

Science Frontier Seminar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53

Science Café . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53

RCAI Participated in the Annual Festival of the Yokohama Science Frontier High School . . . . . . . . . . . . . . .

54

Celebration of RIKEN Yokohama Institute 10th Anniversary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

54

Part 6

Laboratory Activities

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

56

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

58

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

60

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

62

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

64

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

66

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

68

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

70

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

72

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

74

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

76

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

78

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

80

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

82

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

84

Laboratory for Immune Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

86

Laboratory for Dendritic Cell Immunobiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

88

Laboratory for Immunochaperones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

90

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

92

Laboratory for Cytokine Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

94

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

96

Laboratory for Immunogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

98

Laboratory for Vaccine Design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Research Unit for Human Disease Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Research Unit for Cellular Immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Laboratory for Cellular Systems Modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Laboratory for Immunogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Research Unit for Immunoinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Research Unit for Immunodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Research Unit for Molecular Systems Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Research Unit for Thymic Environments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Research Unit for Immunoepigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Central Facilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Part 7

Data and Statistics

Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Invited Presentations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
RCAI Seminars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Budget, Personnel and Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Cover and Section Heading Photo Legends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

iii

Director's Report

Earthquake
FY2010 was a turbulent period. The disastrous magnitude 9.0 Tohoku-Pacific Ocean Earthquake hit the North Easternregion of Japan on March 11, 2011. It was the most powerful earthquake we have ever experienced in Japan and thefifth largest in the world. It shifted the earth’s axis by 10 cm and moved the mainland of Japan ~2.5 m. The zone plateboundary between the Pacific and North America plates was lifted up along a 500 km long stretch, which caused a
massive Tsunami more than 15 m high. Currently, there are 12,157 confirmed deaths and 15,496 have been reported
missing, and the numbers are still growing. I feel great sympathy for those who lost their lives, their surviving family
members and friends, including RCAI members who lost their families.
On Friday, Mar. 11, we felt two strong quakes at 2 : 46 p.m. and 3 : 15 p.m. at our Center. Soon after the first quake,the electric power went out. The trains were stopped and no traffic signals were working, which caused major traffic
jams. Most RCAI staff had to stay overnight in the Center. The Group Directors discussed how to maintain emergency
power as long as possible, especially for the SPF animal facility. The blackout continued for 10 hours, which was wellbeyond our expectation. It was very fortunate that all RCAI members were safe, and there was no major damage toresearch samples or mice.
My thoughts go out to the people now suffering. There are around 1.1 million people in the shelters. Many ofthem cannot return home because of the severe damage resulting from the earthquake, tsunami and/or the Fukushima
nuclear accident. Hoping to be of assistance in rebuilding the educational and research environment in the region, RCAI,in collaboration with the Japanese Society for Immunology ( JSI ) has been requesting information about the damage andneeds of the affected laboratories. RCAI together with immunology researchers in the Max Planck Institute Germany, La
Jolla Institute for Allergy and Immunology and the NIH USA, and INSERM/Pasteur Institute France also offer support toimmunology researchers there. Based on the responses, we are instituting targeted strategies to meet these needs.

Policies for innovation
According to the Science and Technology Policy for FY2011-2015 drafted by the Council for Science and Technology
Policy, the government will shift their policies from "Science and Technology" to "Science, Technology and Innovation".
Social contributions resulting from scientific research are now more expected in the society. According to Dr. Noyori,iv

RIKEN's president, RIKEN will also shift its focus to innovative science or solution research. RCAI has been making
important social contributions since its establishment, e.g., development of an allergy vaccine, novel immune cell therapy
for cancer, humanized mouse models and artificial lymph nodes, etc. However, I am very concerned that too rapid a shift
to solution research will reduce the researchers' motivation and the attractiveness of the institute, which will result in aweaker scientific base and lower performance, not only in basic science but also in social contributions.
The Science and Technology Policy FY2011-2015 was scheduled to be approved by the cabinet by the end of
March, 2011. However, the cabinet decided on a reconsideration of the policy because of the earthquake and thenuclear accident.

BudgetDuring FY2010, we faced a government plan for severe and steady cuts in the research budget. In June 2010, theJapanese cabinet decided to fix "general expenditures" of the government at 71 trillion yen/year for FY2010- FY2013.
The general expenditure category includes social security, education, science, public works, national defense, etc.
Because of the projected steady increase in social security spending of 1.3 trillion yen per year during this period, thispolicy required reductions in the budgets for other areas including science. Based on this cabinet decision, the Ministry
for Education, Science and Technology ( MEXT ) decided to reduce RIKEN's budget by 3.5% per year, and RIKEN decided
to reduce each Center's budget 9% per year. The only rescue plan suggested by the government was a so-called "Policy
Contest" to revitalize Japan. The selected policies would be supported by an extra special budget ( total 1 trillion yen) .
RCAI was fortunate that the allergy vaccine project was selected and funded with 300 million yen as a part of a healthinnovation policy. However, when we think of the current government situation, it is inevitable that our research budget
will be cut in the very near future. We have to seek different funding mechanisms.

Research achievements
In FY2010, the creativity of RCAI scientists continued to yield important research achievements. I would like to mention
especially the advances in gut immunity. Two achievements in this area were awarded the Outstanding Contribution of
the Year.
It is widely believed that probiotics, i.e. “good” intestinal bacteria such as bifidobacteria and lactobacilli, benefit
health, although scientific evidence in support of this notion is largely non-existent. Drs. Shinji Fukuda and Hiroshi Ohno
took advantage of a simplified gnotobiotic mouse model, in which one or two well-studied bacterial species are used
to colonize germfree mice, to tackle this issue. Combining metabolomic, transcriptomic and genomic analyses, they
have identified for the first time the molecular mechanisms by which bifidobacteria protect mice from death caused
by infection with enterohemorrhagic E. coli O157. Although this must be just the tip of iceberg for a wide range ofprobiotic functions, the multiple ‘omics’ approach they established here can be applied to understand other mechanisms
involved in these functions, studies that will contribute to the improvement of public health. ( Fukuda, et al., Nature,
469:543, 2011 )IgA is the major antibody of the humoral immune response in the gut. The mechanisms of gut IgA synthesis and therole of secreted IgA on mucosal surfaces are critical issues, whose understanding will aid in the design of oral vaccines
and therapy of inflammatory bowel diseases. In this field, Drs. Keiichiro Suzuki and Sidonia Fagarasan have shown that
intestinal IgA plays a critical role in the maintenance of a “healthy” gut microbiota. They have further contributed to
our understanding of the induction and regulation of gut-IgA responses, and recently showed that Foxp3+ T cells andfollicular dendritic cells in Peyer’s patches have unique characteristics that promote IgA generation in the gut. Together
these findings provide new insights into the mechanisms of intestinal IgA synthesis, and into the dynamic interactions
between host immune cells and gut-environmental factors. ( Suzuki K, et al., Immunity, 33 : 71, 2010 )The following publications received this year’s Excellent Paper Award:
Drs. Yuichi Aiba and Tomohiro Kurosaki showed that, contrary to textbook models, long-lived memory B cells are localizedadjacent to contracted germinal centers in the spleen ( PNAS, 107 : 12192, 2010 ) . Drs. Kenichi Asano and Masato Tanakav

identified the APC responsible for crosspresentation of tumor antigens to CD8+ T cells; CD169+ macrophages phagocytose
dead tumor cells transported via lymphatic flow ( Immunity, 34 : 85, 2011) . Mr. Tomohiro Fukaya, Mr. Hideaki Takagi and
Dr. Katsuaki Sato showed a crucial role for the B7 family of costimulatory molecules in generating regulatory T cells toestablish oral tolerance ( Blood, 116 : 2266, 2010 ) . Drs. Tomokatsu Ikawa and Hiroshi Kawamoto identified the earliest
known checkpoint during T cell development in the thymus and showed that is dependent on the Bcl11b transcription
factor ( Science, 328 : 93, 2010 ) . Drs. Noriko Yumoto, Takashi Nakakuki, Mariko Okada-Hatakeyama and collaborators
were able to show how a spatially distributed, signaling-transcription cascade robustly discriminates between transientand sustained ERK activities at the c-Fos system level ( Cell, 141: 1, 2010 ) . Drs. Shinya Tanaka, Yasutaka Motomura
and Masato Kubo identified a critical enhancer element for regulating GATA-3-mediated IL4 transcription in Th2 cells( Nature Immunology, 12:77, 2011) . Drs. Hiroshi Watarai, Daisuke Yamada, Shin-ichiro Fujii, Haruhiko Koseki and Masaru
Taniguchi demonstrated the feasibility of expanding functionally competent NKT cells via an induced pluripotent stem cell
( iPSC) phase, an approach that may be adapted for NKT cell-targeted therapy in humans ( Journal of Clinical Investigation,
120 : 2610, 2010 ) . Drs. Tomoharu Yasuda, Kohei Kometani and Tomohiro Kurosaki showed that Erk kinases control plasma
cell differentiation by regulating expression of a key transcriptional repressor Blimp-1 ( Science Signaling, 4 (169 ): ra25,2011) . Drs. Tadashi Yokosuka and Takashi Saito used high resolution imaging to demonstrate the spatiotemporal basis of
the negative regulation of T cell activation by the CTLA-4 costimulatory molecule ( Immunity, 33 : 326, 2010) .

Nurturing research scientists
I believe that it is RIKEN's role and mission to create new research paradigms. For this purpose, I think it very importantto nurture young investigators, who have the fresh power and mental flexibility to challenge new concepts. Unfortunately,in Japan the situation seems to be evolving in the wrong direction. The career path is becoming narrow. There are fewer
tenured positions, and the safest shortcut to publication is to pursue the research already ongoing in the laboratory,
rather than breaking new ground. In view of this problem, we established a new program to provide a career path foryoung investigators who conduct multidisciplinary research that will bridge immunology with other research fields. In
this program, the selected Young Chief Investigators ( age below 40) will head independent research laboratories but will
be mentored by multiple specialists in related research fields, i.e. the leaders in various RIKEN Institutes. At the endof FY2010, we selected the first three investigators: Dr. Hayato Naka-Kaneda - aging of hematopoietic stem cells, Dr.
Shinji Nakaoka - mathematical modeling of immune responses, and Dr. Koji Hase - epigenetic regulation of the hostenvironment interface. I hope that these individuals will grow under the mentorship from multiple fields and will become
the pioneers in new research fields.
In the end of FY2010, we also appointed Dr. Hilde Cheroutre ( LIAI ) as the leader of RCAI's International Research
Unit, "Research Unit for Immune Crosstalk". She will conduct collaborative research on CD8αα with Dr. Ichiro Taniuchi in
RCAI. I expect her research will impact not only the biology of CD8αα but also the field of central tolerance and thymic
selection and T cell development, and promote our understanding of protective immunity and the design of effective
vaccination strategies.

The future
We are at the juncture for a real revitalization of Japan. Big changes must occur after this tragedy. New ideas andstructures will be necessary in the social system, energy policies, economics, politics, technology and science. It will
be a long way to go, but I believe that we Japanese, especially the young generations, have the strength of mind andthe toughness of spirit that will be needed to overcome any difficulties that we may encounter.

Mar. 31, 2011
Masaru Taniguchi

vi

2010

Part 1Creation of New Paradigms

Bacterial Guests Double as
Bodyguards

Hiroshi Ohno

‘Good citizens’ in the human gut bacterial community produce
protective compounds that help prevent the onset of food poisoningI

nfection with the O157: H7 strain of E. coli can cause various symptoms
that range from diarrhea to blood cell loss, encephelopathy and kidney
failure. The causative factor is Shiga toxin (Stx), a bacterially secreted
compound that makes its way from the intestine into the bloodstream,and ultimately binds to target receptors on cells in the kidney and brain.
According to Hiroshi Ohno of RIKEN RCAI, the acquisition of protection against O157: H7 appears to be also related to diet. In addition
to nutrition, mother’s milk from breast-feeding helps to establish thecommunity of gut bacteria. These include the bifidobacteria, whichhave subsequently been associated with conferring O157: H7 resistance. A recent study from a team led by Ohno and Masahira Hattoriat The University of Tokyo has revealed the defining characteristics ofShinji Fukuda (left) and Yumi Chiba (right)
these defenders of human health.
To characterize the protective properties of several different strains of bifidobacteria, the researchers used mice that lacked gut bacteria of their own. Completely ‘germ-free’ mice typically died within a week of exposure to O157: H7, but
all animals pre-colonized with a protective strain of Bifidobacterium longum ( Fig.) ,termed ‘BL’ survived. Mice carrying BL also showed considerably lower levels of
Stx in their bloodstream, and the lining of their intestines appeared healthier.
Ohno, Hattori and their colleagues examined additional preventive and nonpreventive bifidobacteria strains, and the researchers learned that the preventive
strains tended to generate higher levels of acetate as a metabolic byproduct.
Acetate, in turn, switches on the activity of a group of anti-inflammatory genes.
Experiments with human colon epithelial cells showed that acetate treatment wasprotective against the negative effects of Stx, which otherwise induced the formaFigure :tion of ruptures in layers of cultured cells. This suggests that acetate-producing
Certain strains of Bifidobacterium longum
bacteria prevent poisoning by actively preserving the integrity of the intestinal walland other bifidobacterial species residing
and keeping Stx out of the bloodstream.
within the gut can help fight off theill effects of E. coli food poisoning.
A comparison of the complete genomic sequences of five different bifidobacterial strains revealed a subset of genes encoding proteins involved in uptake andmetabolism of the sugar fructose that appear to be exclusively present in preventive strains. Accordingly, disabling these genes drastically undermined the protective qualities of such strains. They also
determined that it was possible to cut out the bacterial ‘middleman’, and demonstrated that considerable protection against
Stx lethality could be achieved by simply feeding mice a diet that was enriched in acetylated starch.
The team points out that all bifidobacteria strains produce acetate during the process of glucose metabolism, protecting theupper stretches of the colon. However, the selectively expressed fructose-metabolizing pathway is likely to provide important
protection in the lower region of the colon, a portion of the digestive tract where glucose is likely to be largely depleted.
Ohno and his colleagues see their bacterial detective work as a powerful proof of concept for understanding the health implications of the close relationship between microbes and their hosts, and for advancing the development of microorganism-mediated‘probiotic’ therapeutic strategies in the future. “The beneficial effects of probiotics arediverse, and acetate alone could not explain everything; we would therefore like to
ORIGINAL RESEARCH PAPER
elucidate the mechanisms underlying other probiotic effects,” he explains, “and we
Fukuda, S., Toh, H., Hase, K., Oshima, K.,
would also like to apply the ‘multi-omics’ approach we took here for analyzing more
Nakanishi, Y., Yoshimura, K., Tobe, T.,
complex gut ecosystems.”
Clarke, J.M., Topping, D.L., Suzuki, T. et al.
Bifidobacteria can protect from enteropathogenic infection through production of
acetate. Nature 469, 543-547 (2011).

2This article is abstracted from RIKEN RESEARCH with permission.

http: //www.rikenresearch.riken.jp/eng/hom/6585

Sensing of Environmental
Stimuli Promotes IgAGeneration in the Gut

Sidonia Fagarasan

I

mmune responses occur in organized lymphoid structures such as Peyer’s
patches ( PPs) in the gut and lymph nodes in the peripheral tissues ( pLNs ) .
Upon antigen stimulation, both PPs and LNs undergo extensive remodeling. T
cell accumulation and extensive B cell proliferations result in the developmentof germinal centers ( GC ) . However, several characteristics distinguish GCs
in gut PPs from GCs in pLNs. First, GCs are always present in PPs, as they
are induced through the constant stimulation by commensal bacteria. By contrast, GCs in pLNs develop only upon systemic infections or after deliberate
immunization. PP GCs also differ from pLN GCs in regard to the type of antibodies produced. B cells in PP GCs produce IgA, whereas activated B cells
Mikako Maruya (left) and Keiichiro Suzuki ( right )in pLN GCs mainly generate IgG antibodies. IgA secreted into the gut lumen
joins innate immune defenses to provide mucosal protection by regulating bacterial communities in the gut.
The molecular and cellular mechanisms underlying the continuous presence of GCs in PPs and the almost exclusive generation ofIgA were incompletely understood. Keiichiro Suzuki and Sidonia
Fagarasan, RIKEN RCAI, speculated that follicular dendritic cells( FDCs ) are critical in B cell activation and class switching from IgM
to IgA. To evaluate the hypothesis, they isolated PP and pLN FDCs
from immunized mice and investigated their gene expression profiles. They found that the PP FDCs had features that were distinct
from pLN FDCs. Genes highly expressed in PP FDCs encoded proteins important for lymphocyte recruitment and retention ( CXCL13) ,Figure:B cell survival ( BAFF) , and activation of TGF-β, a cytokine important for IgA class
Reduced expression of CXCL13 by PP
switching.
FDCs in the absence of vitamin A.
Suzuki and Fagarasan reasoned that these differences are due to FDCs
Immunofluorescent microscopy analysis of
PPs horizontal sections from the WT mice andrecognition of specific gut environmental stimuli that modifies their functions in
VAD mice, stained for FDCs in green (CD21),
CXCL13 in red and naïve B cells (IgD) in blue.
PP GCs, thus contributing to IgA production. In PP, bacterial antigens continuously
activate innate immunity through Toll-like receptors ( TLRs) and food components
such as retinoic acid ( RA) constantly activate RA receptors ( RARs) . To test thishypothesis, they stimulated pLN FDCs with TLR ligands and RA in vitro. Surprisingly this synergistic stimulation enhanced the transcription of PP signature
genes such as Cxcl13, Tnfsf13b, which encodes BAFF, and TGF-β1 activators in
pLN FDCs. Next, the effect of these environmental factors in vivo was also
confirmed. In both Myd88-/- mice, which are defective in TLR downstream signaling,
and in mice kept on a diet deficient in vitamin A, the RA precursor, the expressionof CXCL13 and BAFF by PP FDC was severely reduced. Thus, FDCs are critical for
production of CXCL13 and BAFF upon gut environmental stimulation. They
further showed that TLRs and RARs regulate the conversion of latent TGF-β1 to
its active form, which enhanced IgA class switching of B cells.
ORIGINAL RESEARCH PAPER“The pathway supported by FDC is quite efficient. FDCs sense the bacterial
Suzuki, K., Maruya, M., Kawamoto, H.,
components and food components in the gut lumen, and secrete CXCL13 to
Sitnik, K., Kitamura, H., Agace, W., andFagarasan, S. The sensing of environmental
recruit B cells and T cells. They also secrete TGF-β1 that help B cell switch from IgM
stimuli by follicular dendritic cells promotes
to IgA, and BAFF that enhances the survival of these switched cells and their difimmunoglobulin A generation in the gut. Immunity 33, 71-83 (2010).
ferentiation into plasma cells,” Suzuki said. “However, this is not the only way togenerate IgA in the gut. Actually, there is another stromal cell type, and there aremany other interesting regulatory factors, studied by the members of Sidonia’s lab.
This field is becoming more and more interesting.”3

Crossing the Line
Understanding of blood cell lineages advances withthe discovery of a transcription factor crucial to T celldifferentiation

Hiroshi KawamotoAmaster gene that underpins the development of specificblood cell lineages has been identified by a research teamled by Hiroshi Kawamoto at RIKEN RCAI. The team has published its findings in the journal Science.
Precursor cells in the immune system, known as hematopoietic progenitor cells, can give rise to multiple immune cell types.
Kawamoto and his team cultured multipotent progenitor cells
from mice that could become T cells that shape the immune
response, B cells that generate antibodies, or myeloid cells thatcan engulf pathogens. Their special culture system could stimulate the Notch signaling pathway, which is required for progenitor
cell renewal, and included immune system regulators such as
interleukin-7 ( IL-7) .
The researchers found they could induce the immune
progenitor cells to lose their ability to become B cells underthese conditions. However, this halted development of thecells past this stage, as the progenitors were unable tocease proliferating and mature into either T cells or myeloid
cells.
Kawamoto and colleagues then observed that removing IL-7 from the cell culture medium was sufficient to drive
the progenitors to mature into T cells. They found that withdrawing IL-7 induced the expression of the transcription
factor Bcl11b, which is known to be expressed in T cells.
Interestingly, even when IL-7 was present in the cell culture
medium, they could push immune progenitor cells into
becoming T cells by forcing Bcl11b to be expressed in thecells. This suggested to the researchers that this transcription factor drives this step in the commitment of these
immune progenitor cells to the T cell lineage.
The team also showed that progenitor cells lacking the
Bcl11b gene were unable to mature into T cells, and could
continue to proliferate ( Fig.) . This is consistent with previous
findings by other research teams that disruption of thefunction of Bcl11b is linked to leukemia and lymphoma,
which may be caused by the inability of the progenitor cells
to mature properly into T cells, and to instead continue toproliferate. Kawamoto and his colleagues think that Bcl11b
may drive progenitor cells to take on the T cell fate bysuppressing the genes that characterize the myeloid celllineage.
“Our findings may facilitate the study of the molecularmechanisms of T cell lineage commitment by elucidating theexact timing for this commitment,” explains Kawamoto,“and by identifying a master gene for the establishment of
the T cell lineage.”

4

Tomokatsu Ikawa

Figure :The transcription factor Bcl11b regulates the formationof T cells from immune progenitor cells.

This article is reproduced from RIKEN RESEARCH with permission.

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

http: //www.rikenresearch.riken.jp/eng/research/6380

Maintaining Independence
A set of neighboring immune-system genes each receive
separate activation instructions despite being controlled
by a common factor

Masato Kubo

As part of the immune response to foreign antigens, naïve T
cells mature into different types of helper T cells. TH1 cells
and TH17 cells, for example, secrete a subset of signaling factors
known as cytokines that promote inflammatory responses to viral
infections, while TH 2 cells secrete cytokines that promote antibody secretion by B cells and drive allergic reactions.
The GATA-3 protein is known as a ‘master switch’ for TH 2
differentiation, stimulating production of cytokines such as interleukin ( IL ) -4 and IL-13, but new findings from a team led by
Masato Kubo at RIKEN RCAI have revealed an unexpected
degree of complexity in this activation process.
“The idea that genes encoding TH2 cytokines are
Yasutaka Motomura
coordinately regulated … has been widely accepted,”says Kubo. Many of these genes are situated in thesame chromosomal neighborhood, and some scientists
believe that the chromosome physically loops so that
DNA-bound GATA can regulate multiple sites simultaneously. However, Kubo and colleagues found that GATA
appears to independently bind multiple, distinct sitesthat each confer regulatory control over individual TH 2associated genes.
One of these sites, HS2, specifically governs IL-4
expression, and GATA binding at this site induceschemical modification of the DNA segment containing
the Il4 gene, leading to increased cytokine production.
Naïve T cells from mice lacking this chromosomal region
Figure:give rise to TH 2 cells that generate normal levels of mostNaïve T cells (left) begin to mature into TH2 cells within 12–24
hours of being stimulated by the presence of a foreign antigen
cytokines, but fail to produce IL-4 ; these animals also
(middle). As a component of this process, a ‘master switch’show fundamental defects in their allergic response.
protein drives the production of TH2 cytokines IL-4 andIL-13 by binding to and promoting chemical modification in
In parallel, the researchers identified a second
the vicinity of the HS2 or CGRE sites, respectively (right).
GATA-binding site, CGRE, which specifically regulates
production of IL-13. Like HS2, GATA interaction withthis site is associated with targeted chemical modification of a nearby stretch of DNA containing the Il13 gene, and disruption of CGRE
essentially eliminates production of this cytokine while leaving IL-4 production unaffected ( Fig.) . “These results came as a surprise,” says Kubo. “They indicate that theindependent recruitment of GATA-3 to locus-specific regulatory elements controls
the expression status of individual genes encoding TH2 cytokines.” These findings
also parallel previous data suggesting that GATA coordinates expression of IL-5,
another TH 2 cytokine, independently of IL-13.
ORIGINAL RESEARCH PAPER
Other types of immune cells also secrete TH 2 cytokines, and Kubo and colTanaka, S., Motomura, Y., Suzuki, Y., Yagi,
leagues now hope to determine whether their findings represent a broadly used
R., Inoue, H., Miyatake, S. & Kubo, M. The
enhancer HS2 critically regulates GATA-3mechanism for regulating production of these cytokines. “Our next priority will be
mediated Il4 transcription in TH 2 cells. Nature
exploring the relative contribution of these discrete elements to transcriptional reguImmunology 12, 77- 85 (2011).
lation of IL-4 and IL-13 among these cell types,” he says.

This article is reproduced from RIKEN RESEARCH with permission.

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5

Fighting Their Way to the Middle
Immune cells get switched off by the accumulation ofdense clusters of inhibitory proteinsTakashi Saito

F

oreign entities within the body get chopped
into pieces by antigen-presenting cells( APCs) , which display the resulting chunks ontheir surface. These antigens can subsequently
be recognized and bound by T cell receptors( TCRs ) , and the interaction between a T celland an antigen-bearing APC eventually triggers
the onset of an immune response against the
antigen.
Inappropriate responses by this system,
Masako Takamatsu (left), Wakana Kobayashi (center) and Tadashi Yokosuka (right)
however, can give rise to disastrous medical
consequences, and there is a keen interest in
developing more sophisticated ways to modulate how T cells react to perceived threats. “Ultimately, we would
like to regulate T cell function and activation in order to overcome
autoimmune diseases, allergy, or infectious diseases,” explains
Takashi Saito, RIKEN RCAI in Yokohama.
New findings from a team led by Saito and RCAI colleague
Tadashi Yokosuka could ultimately prove valuable for such efforts,
by revealing insights into the mode of action of cytotoxic T-lymphocyte-associated protein 4 ( CTLA-4) , an inhibitor of TCR signaling. When T cells associate with APCs, they form what is
known as an ‘immunological synapse’, a juncture where numerous proteins assemble into elaborate complexes, such as thecentral supramolecular activation cluster ( cSMAC ) . By establishing experimental conditions that simulate this cellular interaction,
the researchers were able to monitor the dynamic rearrangements that take place at the cSMAC and its environs.
CD28, a T cell protein that promotes TCR signaling, typicallyassembles at a narrowly defined region of the cSMAC, where it
Figure :interacts with CD80, a protein expressed on the surface of APCs.
Over time, clusters of CTLA-4 molecules (yellow) gather
alongside TCRs (red) within the cSMAC; in doing so, they
Saito, Yokosuka and colleagues determined that CTLA-4 gradudisplace CD28 (green) and suppress TCR signaling.
ally gathers at the immunological synapse and subsequently
forms into clusters within the exact same area of the cSMAC as
CD28, where it directly competes to bind CD80 ( Fig.) . “Positive
regulation by CD28 and negative regulation by CTLA-4 are
induced at the same place in the cell,” says Saito. “Without accumulating at thecSMAC, CTLA-4 cannot inhibit T cell activation.”
In addition to illuminating a mechanism by which T cell responses get finetuned, these findings could ultimately yield benefits for patients suffering from a
ORIGINAL RESEARCH PAPER
variety of conditions. “Anti-CTLA-4 antibody therapy has been utilized for cancer
Yokosuka, T., Kobayashi, W., Takamatsu,
patients, enhancing tumor immunity by inhibiting regulatory T cells, and CTLA-4M., Sakata-Sogawa, K., Zeng, H., Hashimoto-Tane, A., Yagita, H., Tokunaga, M. &based fusion proteins have been used to block autoimmune diseases such as
Saito, T. Spatiotemporal basis of CTLA-4
arthritis,” says Saito. “Our findings will enable us to explore new therapeutic concostimulatory molecule-mediated negative
regulation of T Cell activation. Immunity 33,
cepts based on the inhibition of the dynamic movement of regulatory molecules
326-339 (2010).
such as CTLA-4.”

6

This article is reproduced from RIKEN RESEARCH with permission.

http://www.rikenresearch.riken.jp/eng/research/6462

The Long and Short of Cell Signaling
By bolstering a sophisticated computational model withquantitative experimental data, researchers begin to
decipher the workings of a complex signaling network

Mariko OkadaHatakeyamaL

ike a telegraph transmission, the significance of a cellularsignal can change greatly depending on whether it arrives
as a brief ‘dot’ or a sustained ‘dash’ ( Fig.) . For example, transient activation of extracellular receptor kinase 1/2 ( ERK ) by
epidermal growth factor ( EGF) causes cells to divide, while
prolonged ERK activation induced by heregulin ( HRG) instructs
these same cells to differentiate.
Cell biologists have struggled to untangle the relationshipbetween this signaling network and cell fate, but a collaborative effort between Mariko Okada-Hatakeyama at RIKEN RCAI
and Boris Kholodenko at University College Dublin in Ireland
has achieved an important breakthrough by pairing quantitative experiments with computational modeling.
Okada-Hatakeyama’s team previously examined theexpression of c-fos, a so-called ‘immediate early gene’ whose expression is induced
shortly following ERK activation, and obtained somewhat contradictory findings.
”Early gene expression time-course profiles were the same regardless of whether
the upstream ERK signal is transient or sustained,” she says. “However, levels of
c-Fos protein were ‘all or none’ for sustained and transient signals, respectively.”
Based on an initial interpretation of their computational model of this pathway,
Okada-Hatakeyama, Kholodenko and colleagues proposed that the effects of both
HRG and EGF on c-fos expression were modulated purely by dual-specificity phosphatases ( DUSPs) , enzymes that inhibit ERK’s capability to induce c-fos. However,experiments with a forced reduction of DUSP in cultured cells did not fully replicate
these predictions. “There were long and serious discussions about whether our
experiment was working properly or the model was wrong,” says Okada-Hatakeyama.
This reassessment led to experiments that enabled the researchers to demonstrate the existence of a second, previously unknown mechanism for repression ofc-fos expression that is triggered only in response to HRG. Together, these two
ERK-activated ‘negative feedback’ systems appropriately control c-fos transcription in the process of cellular differentiation.
In parallel, their model also revealed how the combination of ERK-induced
c-fos expression and sustained signaling activity by ERK outside the nucleus leadto steady production of c-Fos protein. This system architecture results in a highly
stable signaling arrangement that filters out extraneous background noise andinduces all-or-none output, according to Okada-Hatakeyama. “We learned fromthis study that cells possess very simple but robust system structures that can fightagainst unwanted perturbations,” she says. “This cellular signaling network is still a‘black box’ and what we can do with computational modeling is very much limited… but we hope to untangle the cell decision program someday.”Figure:Like dots and dashes from a Morse codetransmission, brief and sustained cell signals
can have different meanings for cell fate.

ORIGINAL RESEARCH PAPER
Nakakuki, T., Birtwistle, M.R., Saeki, Y.,
Yumoto, N., Ide, K., Nagashima, T., Brusch,
L., Ogunnaike, B.A., Okada-Hatakeyama, M.
& Kholodenko, B.N. Ligand-specific c-Fos
expression emerges from the spatiotemporalcontrol of ErbB network dynamics. Cell 141,884-896 (2010)

RELATED ARTICLE
Nagashima, T., Shimodaira, H., Ide, K., Nakakuki, T., Tani, Y., Takahashi, K., Yumoto,N., & Hatakeyama, M. Quantitative transcriptional control of ErbB receptor signaling
undergoes graded to biphasic response forcell differentiation. The Journal of Biological
Chemistry 282, 4045-4056 (2007).

This article is reproduced from RIKEN RESEARCH with permission.

http://www.rikenresearch.riken.jp/eng/research/6350

7

Ready and Waiting
A subset of immune cells remain ideally positioned
to respond quickly to the reappearance of previously
encountered pathogens

Tomohiro Kurosaki

Like a burglar tripping an alarm, infectious threats within the
body set off a chain reaction of signaling events that enable
the immune system to mount a proper defensive response. Oncethe crisis is averted, populations of target-specific ‘memory’ B
cells ensure that any return visit by the same pathogen will be
dealt with promptly and harshly.
Memory B cells initially arise within germinal centers in spleen
and lymph nodes, but it has proven challenging to determine
whether they continue to reside there or circulate throughout the
body. By applying sophisticated cellular imaging techniques, ateam led by Tomohiro Kurosaki of RIKEN RCAI has now resolved
this question for at least one major subset of these cells.
B cells are primarily categorized based on the
immunoglobulin protein chains they incorporate into
their antibodies, and Kurosaki’s team primarily focused
their attention on immunoglobulin G-expressing ( IgG+)

Yuuichi Aiba (left) and Kohei Kometani (right)

cells. Using a variety of fluorescent labeling strategies,
they were able to determine that IgG+ memory cells
remain clustered close to the germinal centers longafter the initial immune response in mice injectedwith the immunostimulatory molecule nitrophenol. By
comparison, immunoglobulin M-expressing ( IgM+)
memory cells are found scattered at discrete sites
throughout the spleen.
Subsequent experiments with a fluorescent indicator of cell division showed that IgG+ cells replicate
Figure :rapidly in response to a secondary challenge with
A fluorescent indicator of cell division (green) reveals that
nitrophenol ( Fig.) , and that this process is dependent
IgG memory B cells (purple) are largely quiescent during an
on direct interaction with helper T cells, which are also
initial antigen challenge (left), but undergo activation andproliferation in response to a secondary challenge (right).
located in close proximity to germinal centers. The
communication between these two cell types appears
to directly contribute to elevated production of antigen-specific antibodies. “Although preliminary, ourdata suggest that IgG+ memory B cells are more prone to differentiate into antibodyproducing plasma cells than IgM+ memory B cells, which may contribute to regeneration of the memory pool after a secondary antigen challenge,” says Kurosaki.
The memory cell-mediated immune response is generally faster and morerobust with regard to target recognition than the ‘first encounter’ with a given
pathogen, and Kurosaki believes that these findings represent an important step
toward understanding the efficiency of the memory cell response. “Before our study,
ORIGINAL RESEARCH PAPER
people believed that memory B cells leave the germinal centers and are recirculated
Aiba, Y., Kometani, K., Hamadate, M.,
all over the body by the lymphatic system and blood,” he says. “However, our study
Moriyama, S., Sakaue-Sawano, A., Tomura,M., Luche, H., Fehling, H.J., Casellas, R.,
clearly demonstrates that some IgG — but not IgM — memory B cells reside
Kanagawa, O. et al. Preferential localizacontinuously near germinal centers and thus enable rapid activation after antigen
tion of IgG memory B cells adjacent tocontracted germinal centers. Proceedings
re-challenging.”of the National Academy of Sciences USA
107, 12192-12197 (2010).

8This article is reproduced from RIKEN RESEARCH with permission.

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Awaiting Orders to Retaliate
Signaling proteins that help immune cells develop alsoenable those cells to mount an effective counterattack
against infections

Tomohiro KurosakiWhen immune system B cells are alerted to the presence ofa threat within the body, they form structures called germinal centers, which serve as ad hoc headquarters for marshalinga targeted immune response. These cells subsequently differentiate into plasma cells, which produce antibodies directed against
foreign entities, or memory cells, which retain the capacity to
become plasma cells if the same threat reappears in the future.
The extracellular signal-regulated kinase proteins ( ERK1/2)
are integrally involved in the early stages of this process, making
it a challenge to assess their subsequent contributions. “If wedelete both ERK genes entirely, differentiation of B cells is
impaired and we cannot analyze the function of ERKs during theimmune response,” explains Kohei Kometani, a researcher in
Tomohiro Kurosaki’s group at RIKEN RCAI.
To address this challenge, Tomoharu Yasuda andKometani developed transgenic mice in which ERK expression is lost only after the initial differentiation of B cells. Theirinitial results were striking; following vaccination with ahighly immunogenic antigen, ERK-deficient mice showed a
10- to 40-fold reduction in antibody production ( Fig.) . This
selective deletion of the two ERK genes led to a sharp
decrease in the number of antigen-specific plasma cells but
had little effect on memory cell counts. The researchersdetermined that these signaling factors appear to directly
facilitate plasma cell maturation. “It was surprising that
ERKs regulate differentiation but do not affect cell proliferation, because many people think of the ERKs as important
molecules for cell growth,” explains Kometani.
Several proteins known as transcription factors contribute to
the maturation of plasma cells by turning key genes on or off.
Blimp-1 is among the most important of these, as it also helps toinhibit transcription factors that maintain germinal center B cells.
Yasuda and Kometani determined that the gene encoding
Blimp-1 is a primary target of ERK signaling. They also identified
another protein, Elk-1, which appears to be an important intermediary in this process.
As their findings also indicate that other signaling pathways
are likely to intersect with ERK signaling in this developmental
process, Yasuda and colleagues hope to explore this complexity
in the future. “Harmful or excess antibody production are
sometimes the cause of autoimmunity and allergy,” he says,“and from this point of view, it may be interesting to check the
involvement of not only the ERKs, but also the molecules
upstream and downstream.”This article is reproduced from RIKEN RESEARCH with permission.

Kohei Komtani (left) and Noriko Takahashi (right)

Figure:Fluorescently labeled spleen tissue from wild-type(left) and ERK-deficient (right) mice reveals a
dramatic difference in the production of antibodies(green) in response to an antigen challenge. Blue
indicates expression of B220, a B cell specific marker,
while red indicates labeling for a marker commonly
found in germinal centers. Scale bars, 200 μm.

ORIGINAL RESEARCH PAPER
Yasuda, T., Kometani, K., Takahashi, N.,
Imai, Y., Aiba, Y. & Kurosaki, T. ERKs induce
expression of the transcriptional repressor
Blimp-1 and subsequent plasma cell differentiation. Science Signaling 4, ra25 (2011).

http://www.rikenresearch.riken.jp/eng/research/6634

9

Creating a Life-saving Killer
Cancer may be kept in check by a method
for generating patient-specific immune
cells with antitumor activity

Shin-ichiro Fujii

Haruhiko Koseki

Masaru TaniguchiU

pon receiving the appropriate
activating signal, natural killer T( NKT ) cells live up to their name,releasing a torrent of molecules thattrigger the protective immune response
necessary to eliminate pathogens oreven thwart tumor growth.
These cells represent a promisingclinical tool, as demonstrated in a
Daisuke Yamada
Yuko Nagata (left), Hiroshi Watarai (center) and Sakura Sakata (right)
recent clinical trial in which lung cancer
patients received NKT-stimulating
injections of dendritic cells that had been pretreated with α-galactosylceramide( α-GalCer) . Some 60% of treated patients exhibited a striking seven-foldimprovement in their median survival time relative to their untreated counterparts. “The effects are superior to other molecular-targeted cancer drugs,”
says Masaru Taniguchi of RIKEN RCAI, whose team participated in this study.
“However, this therapy is not applicable to two-thirds of patients because of
their limited number of NKT cells.”
To solve this problem, Taniguchi teamed up with RCAI colleagues Haruhiko Koseki and Shin-ichiro Fujii to develop a method for generating transplantable NKTs. They derived these from induced pluripotent stem cells( iPSCs ) , embryonic-like cells that are typically generated via virus-mediateddelivery of ‘reprogramming genes’ into skin cells. However, NKT maturation
involves a complex genomic rearrangement event, making them difficult toderive from conventional iPSCs. As such, Taniguchi and Koseki devised a
Figure :novel approach for generating mouse iPSCs from existing NKTs ( Fig.) , which
A cluster of undifferentiated iPSCs derived from
reprogrammed NKTs with high expression levels of
have already undergone this rearrangement. They used these iPSCs to generthe Oct3/4 pluripotency factors (green fluorescence)
ate large numbers of new NKTs in vitro.
(DAPI (red), scale bar, 10 micrometers).
Their method efficiently produced mature NKTs, which rapidly establisheda stable population within the liver upon transplantation into mice. To theresearchers’ pleasant surprise, these new NKTs displayed typical activation
behavior in response to α-GalCer-treated dendritic cells and proved capable
ORIGINAL RESEARCH PAPER
of coordinating an effective immune response. “In general, cells generated
Watarai, H., Fujii, S., Yamada, D., Rybouchkin,from in vitro culture die quickly in vivo or are killed by host immune cells …
A., Sakata, S., Nagata, Y., Iida-Kobayashi,
M., Sekine-Kondo, E., Shimizu, K., Shozaki,
however, this was not the case here,” says Taniguchi. “We detected iPSCY. et al. Murine induced pluripotent stem cells
derived NKT cells with adjuvant activity and tumor-eradicating effects two
can be derived from and differentiate into
natural killer T cells. The Journal of Clinicalweeks after cell transfer.”
Investigation 120, 2610-2618 (2010).
Taniguchi, Koseki and colleagues are now keen to begin working withhuman cells. This transition will involve many new challenges, but the researchRELATED ARTICLE
ers see great clinical potential in their approach — particularly in the US, where
Motohashi, S., Nagato, K., Kunii, N., Yamathe Food and Drug Administration has approved development of cell-based
moto, H., Yamasaki, K., Okita, K., Hanaoka,
H., Shimizu, N., Suzuki, M., Yoshino, I., ettherapies. “NKT cell-targeted adjuvant cell therapy is applicable to any type ofal. A phase I-II study of alpha-galactosylcecancer patient, because it can overcome their immunodeficient status andramide-pulsed IL-2/GM-CSF-cultured peripheral blood mononuclear cells in patientsenhance antitumor responses,” says Taniguchi. “At present, the delivery ofwith advanced and recurrent non-small cellpatient dendritic cells is the main limiting factor.”lung cancer. Journal of Immunology 182,2492-2501 (2009).

10This article is reproduced from RIKEN RESEARCH with permission.

http://www.rikenresearch.riken.jp/eng/research/6361

Giving Tumor Vaccines a
Proper Introduction
Therapies that target specialized cells residing within thelymph nodes may help to rally tumor-killing immune responses

Masato TanakaGiven how effectively the immune system can eliminate
foreign threats such bacteria and viruses, hopes are highfor the development of strategies that might turn these same
defense mechanisms against cancerous targets. However,attempts to train the immune system to recognize malignancies
via the intravenous injection of vaccines that present tumorderived antigens have fallen short.
According to Kenichi Asano, a researcher with Masato
Tanaka’s group at RIKEN RCAI, this is the result of ‘tolerance’
mechanisms that protect against autoimmune disease. “Billions
of cells die every day, and cell corpses must be removed swiftlyfrom our body in order not to induce detrimental effects,” hesays. In this scenario, macrophage cells in the spleen clean
house by devouring such debris in a process known as phagocytosis, thereby preventing dead cells from triggering an inflammatory response.
Tumor cells delivered into the lymphatic system via subcutaneous injection, however, can successfully elicit a strong immune
response, and new research from Asano and colleagues explains
why this is the case. In order to rouse an effective reaction,
phagocytic cells must present recognizable chunks of thosedead cells to tumor-killing cytotoxic T lymphocytes ( CTLs ) . The
researchers identified a very specific subset of macrophages
within the lymph nodes that perform this task.
Intriguingly, these cells, which are distinguishable by theirexpression of the cell-surface protein CD169, are non-migratory
and reside stably within the sinuses of the lymph node, awaiting
their prey like spiders in a web. Dead cancer cells delivered tothese sinuses via the lymphatic system are rapidly digested by
the macrophages ( Fig.) , which in turn cross-present the resulting
antigens to CTLs. By selectively killing off these macrophages
with diphtheria toxin, the researchers were able to essentially disable the immune response. “Without CD169 macrophages,
tumor-directed T cells were no longer activated — that means
these cells dominate anti-tumor immunity after tumor cell death,”says Asano.
These findings help explain why the dead cells that slough
off of tumors into the lymphatic system during radiation or chemotherapy are sometimes sufficient to provoke an immune
response, and could provide the foundation for far more effective
cancer immunotherapy strategies. “I believe it is very promising
to mount anti-tumor immunity in patients with solid tumors bydelivering tumor antigens specifically to CD169 macrophages,”says Asano. “It's my dream to invent artificial materials that possess the characteristics of dead cells and are safe for administration to patients.”

This article is reproduced from RIKEN RESEARCH with permission.

Kenichi Asano

Figure:CD169-positive macrophages (green) residing within
the lymph sinuses rapidly consume the dead tumor
cells (red) that make their way through the lymphatic
system following subcutaneous injection.

ORIGINAL RESEARCH PAPER
Asano, K., Nabeyama, A., Miyake, Y., Qiu,
C.-H., Kurita, A., Tomura, M., Kanagawa,O., Fujii, S.-I. & Tanaka, M. CD169-positive
macrophages dominate antitumor immunity by crosspresenting dead cell-associatedantigens. Immunity 34, 85-95 (2011).

http://www.rikenresearch.riken.jp/eng/research/6560

11

Holding Fire on the ‘Good Guys’
Tolerating the foreign materials in food that mice andhumans need hinges on the presence of B7 proteins

Katsuaki SatoAn international team of molecular biologists led by RIKEN
researchers has unraveled key details of the molecularmechanism whereby the body’s immune system determines
what to attack among the organisms and food taken into themouth, and what to leave alone or tolerate. The researchers haveshown the pivotal role of two proteins found on the surface ofcells that stimulate the immune system into action, the dendritic
antigen-presenting cells ( APCs) . The work may lead to newtherapies for immune disorders, and to ways of boosting theeffectiveness of oral vaccines.
Hideaki Takagi (left) Tomohiro Fukaya (center)
Animals could not survive without the nutrients and benefiand Yumiko Sato (right)
cial micro-organisms they ingest when feeding. But the foreignmaterial taken in through the mouth and passing through the gut also contains harmful substances and organisms. So the immune system
must balance active protection against pathogens and toxins with a non-responsiveness tofood and commensal bacteria known as oral
tolerance. In the past, researchers have proposed two mechanisms for oral tolerance —reducing the numbers of effector T cells, theimmune-system foot soldiers that move against
foreign material and suppressing their action
Figure :by means of specialized regulatory T cells.
Antigen-specific de novo conversion of CD4+Foxp3-T cells into CD4+Foxp3+inducible
The triggering of effector T cells depends
regulatory T cells (iTregs) in mesenteric lymph nodes during oral tolerance. KJ126+Foxp3 EGFP-T cells were transferred into BALB/c mice, and the animals wereon interaction with two distinct types of prosubsequently fed PBS (A) or OVA protein (B) one day later. Cryosections from mesenteric
teins on the surface of the APCs, antigens thatlymph nodes on day 11 after the adoptive transfer were stained for KJ1-26 (red) and B220
(blue). Foxp3 was detected by green fluorescence from the Foxp3EGFP knock-in allele.
are markers of foreign material and co-stimulatory proteins of the B7 family, which regulate
the response. Both must be present, however, to initiate any action.
Using mice deficient in B7 co-stimulatory proteins Katsuaki Sato and colleagues
from RIKEN RCAI, together with researchers from other laboratories in Japan, andin the US and France, found that oral tolerance demanded the presence of B7-H1
and B7-DC proteins. In fact, without these proteins the immune response wasenhanced. Of the APCs, the dendritic cells of the mesenteric lymph nodes, in themembranes surrounding the digestive system, display higher levels of these proteins.
When the researchers investigated the role of the two B7 proteins, again using
B7-deficient mice, they discovered the proteins induced the generation of regulaORIGINAL RESEARCH PAPER
tory T cells rather than normal effector cells (Fig.). These regulatory T cells then
Fukaya, T., Takagi, H., Sato, Y., Sato, Kaori,
damp down the immune response promoting tolerance. During inflammation, howEizumi, K., Taya, H., Shin, T., Chen, L.,
Dong, C., Azuma, M., Yagita, H., Malissen,
ever, their action is swamped.
B. & Sato, K. Crucial roles of B7-H1 and B7The research group wants to continue analyzing the role of different groups of
DC expressed on mesenteric lymph nodedendritic cells in the generation of antigendendritic cells in live mice, Sato says. “In particular, we wish to identify the molecuspecific CD4+Foxp3+ regulatory T cells in thelar basis of the regulation of the function of these cells.”
establishment of oral tolerance. Blood 116,
2266-2276 (2010).

12This article is reproduced from RIKEN RESEARCH with permission.

http://www.rikenresearch.riken.jp/eng/research/6457

Hirano Receives
the Japan Prize 2011D

r. Toshio Hirano received the Japan Prize 2011 in the field of "Bioscience and Medical Science",
together with Dr. Tadamitsu Kishimoto. The award was given for their discovery of interleukin-6 ( IL-6)
and its application in treating diseases.
They originally purified IL-6 protein, which plays a vital role in the production of antibodies, and thensucceeded in cloning the gene in 1986. In addition, they have identified a wide range of other IL-6 functions and their research has contributed to the progress of biomedical science and the development oftherapeutic drugs for inflammatory diseases.

Discovery of interleukin-6
Hirano was born in 1947 in Osaka. He entered the Faculty of Medicine at Osaka University in 1966.
In his 5th year of medical school, he heard a lecture by the late Yuichi Yamamura, a pioneer in immunological research in Japan. Yamamura always made the students think about the real causes of a disease.
Yamamura’s attitude, to consider the actual nature of a phenomenons intrigued Hirano, leading him toconsider autoimmune disorders, where the immune system revolts and attacks our own bodies.
In 1973, Hirano graduated from the Faculty of Medicine at Osaka University and went on to study
immunology at the National Institute of Health ( NIH) in Baltimore, Maryland, U.S., where he becameacquainted with Kishimoto. When he arrived at the airport, Kishimoto, who was already studying at Johns
Hopkins University in Baltimore, came to pick him up. It was the start of their long-term relationship.
At that time, immunology was a field in which cutthroat competition was already underway. Researchers around the world were competing to understand the nature of interleukins. In 1975, Kishimoto began
studies with Robert Good at the Memorial Sloan-Kettering Cancer Center in New York City, and showed
the presence of a new candidate interleukin. Meanwhile, Hirano returned to Japan in 1976, and continued his own research at Habikino Hospital and the School of Medicine at Kumamoto University to identify
a novel interleukin acting on B cells to induce immunoglobulin production, eventually culminating in the
discovery of interleukin 6 together with Kishimoto at 1986.
However, this was only the beginning of research on the interleukin. One characteristic of thesemolecules, which came to be called cytokines, is that one type can have many functions, and there aremany companion molecules that can have similar, overlapping functions. Because of this redundancy,
even if the biological activities of the discovered substance were studied, it would be difficult to prove thatit really was a new substance. The academic society at that time only acknowledged a substance as new
if the encoding gene was successfully cloned.
In 1982, Kishimoto became a professor of the Institute for Molecular and Cellular Biology established13

Back row: Chairman Yoshikawa and his wife, President Itoand his wife. Front row: Hirano and his wife, Kishimoto andhis wife

at Osaka University. In the following year, Kishimoto welcomed Hirano as his research partner.
They adopted leading-edge genetic engineering
techniques and continued in their gene hunt.
Initially, they were unsuccessful and continued intheir low-profile experiments for days and thenfor years, often struggling with frustration. Hirano
recalls the period of the long dark night when
only the people who believe could go forward.
However, in May 1986, they finally succeeded in capturing the long-sought gene. Withso much joy, Hirano and colleagues jumped intoa swimming pool with their clothes on during thelab’s summer camp. In 1988, at an international
conference, the name interleukin 6 ( IL-6) was
given to the new cytokine.

Opening the way from basic research
After the successful gene cloning, a highly pure IL-6 was obtained by means of genetic modification
techniques, and progress was also seen in the clarification of the mechanism of IL-6 activity. The researchgroup with both doctors as core members also discovered the structure of the IL-6 receptor. In addition,
they identified the intracellular signals that transmit IL-6 information to the cell nucleus.
What proved fortunate was that as the research progressed, it became evident that IL-6 not only
enhances antibody production, but also has a wide variety of other functions. For example, it can stimulate the production of a liver protein, CRP, when acute inflammation occurs in the body. It also increases
production of platelets, blood components that coagulate blood in the event of an injury. The more
research is done, the more functions are discovered, thus contributing to the progress of medicine.
Particularly noteworthy was the discovery that IL-6 is involved in rheumatoid arthritis, which is a representative autoimmune disorder. The two doctors had already noted from an early stage of their researchthat IL-6 is deeply involved in inflammatory reactions within the body, but it was newly discovered that alarge quantity of IL-6 exists in the joint fluid of rheumatoid arthritis patients. Based on this discovery,
important insight was obtained about its pathogenic mechanism. Research originating from the discovery of IL-6 has broadened its horizons from leading-edge medicine to bioscience. Based on basic
research, Kishimoto has jointly developed, with a pharmaceutical company, an antibody drug
Tocilizumab that inhibits IL-6 action. After being approved in Japan in 2008, the drug has been approvedin 70 countries worldwide including Europe and the U.S.
At a press-conference by the Japan Prize 2011, Hirano spoke.
“With a firm belief in the importance of attaining the true nature of things, I have been challenging andconfronting difficulties one by one as if I was climbing up mountains and cliffs to reach the extreme summit.
Through these fundamental research efforts, we were blessed with the discovery of IL-6, which opened up
the way for the development of therapeutic medications for diseases like rheumatoid arthritis. It is something I could never have imagined when I graduated from the medical department of Osaka University 39
years ago. Today, I am determined as ever to keep contributing to the society through medical researchand developing the next generation of researchers.”“I believe that Japan, for the sake of its future, must contribute to the peace and prosperity of
mankind by acknowledging the importance of fundamental science, and engaging in scientific andtechnological development. I sincerely hope that this prize will reaffirm the importance of fundamental
scientific research and set a vision and dream for the young people.”
Hirano gave a lecture at RCAI on May 13, 2011, and RCAI members celebrated his receipt of
the award.

Photos and the information courtesy of The Japan Prize Foundation
http: //www.japanprize.jp

The Japan Prize is awarded to honor the achievements of people throughout the world who have contributed to the progress ofscience and technology and the advancement of world peace and prosperity. In principle, the Prize is given for work done in any
field of science and technology, but each year two particular fields are designated based on trends within these areas and otherconsiderations. Laureates receive "Japan Prize" certificates of merit, prize medals and a cash award of 50 million yen for each field.

14

Outstanding Contribution of the Year 2010
K

eiichiro Suzuki ( Photo 1) and Sidonia Fagarasan ( Photo 2) in the
Laboratory for Mucosal Immunity, and Shinji Fukuda ( Photo 3)
and Hiroshi Ohno ( Photo 4) in the Laboratory for Epithelial Immunobiology received RIKEN RCAI’s Outstanding Contribution of the YearAward 2010 at a ceremony held on April 22, 2011 for their recent
contributions to advancing the field of mucosal immunology. Thisaward was established in 2006 to recognize RCAI investigators whomake outstanding contributions to the Center. The awardees, whowere selected by the Director, received a plaque and a monetary
prize.
Suzuki and Fagarasan received the award for their studies on
gut-IgA synthesis and interactions between host and environmental
factors in the gut. They recently showed that Foxp3+T cells and follicular dendritic cells in Peyer’s patches have unique characteristics
that promote IgA generation ( Tsuji et al. 2009; Suzuki et al. 2010). At
the ceremony, Suzuki presented the part of their work in which hemade major contributions.
The starting point of their studies was the analysis of activationinduced cytidine deaminase ( AID) knockout mice ( Fagarasan et al.
2002; Suzuki et al. 2004) , which lack IgA because of a defect in isotype switching and are also unable to undergo somatic hypermutation of their Ig variable region genes. When the composition of gut
commensal flora in these mice was analyzed, it was quite different
from that in normal mice. Sticky anaerobic organisms called segmented filamentous bacteria were expanded and had penetrated into
the gut epithelial cells, causing strong stimulation to the host immune
system. As a result, there was hyper-proliferation of germinal center
B cells and the mice had larger Peyer's patches and isolated lymphoid follicles ( ILF) . The expansion of germinal center B cells wasseen not only in the gut but also in systemic immune organs, e.g.,spleen, indicating that IgA is critical for the regulation of both gut andsystemic immune systems.
To address where IgA is generated, these investigators used aly/
aly mice, which have no lymph nodes, Peyer's patches, ILF, or follicular structures, and no IgA+ cells in the gut because of functional
defects in lymphocytes and stromal cells. Transfer of normal bonemarrow did not normalize IgA production. However, to their surprise,bone marrow cells together with stromal cells, which were reconstituted by injection of normal lamina propria cells, partially restored
IgA+ cells. Thus, lamina propria stromal cell, were necessary for thegeneration of IgA in the absence of organized follicular structures likethe Peyer’s patches ( Fig. 1) ( Suzuki et al. 2005) .
However, in the normal situation, most of the IgA+ cells arederived from Peyer's patches, and without them, IgA levels declined
significantly, indicating that Peyer’s patches are important for the efficient generation of IgA. So, Suzuki and Fagarasan next studied how
IgA+ cells are generated in Peyer's patches. To address the importance of stromal cells for IgA generation in Peyer’s patches, theydecided to study follicular dendritic cells ( FDC) , a specialized stromal
cell in the germinal center ( Fig. 2) . The isolation of these cells wasquite difficult, however, and Suzuki had to struggle for one year justfor the FDC purification.

Photo 1 : Keiichiro Suzuki

Photo 2 : Sidonia Fagarasan

Photo 3 : Shinji Fukuda

Photo 4 : Hiroshi Ohno

Figure 1: LP stromal cells support gut IgA generation

15

A comparison of gene expression profiles revealed that
FDCs of Peyer's patch and lymph node are quite different( Fig. 3) . Suzuki speculated that FDCs in the Peyer’s patch
sense environmental cues, such as gut bacteria or food components. To test this idea, they stimulated lymph node ( LN)
FDC with bacterial components ( TLR ligands) and/or foodcomponents ( retinoic acid) in vitro. Many PP FDC signature

Figure 2: PP and pLN FDCs have distinctive gene expression profiles

Figure 3: In vitro TLR and RA stimulations partially
recapitulate the “PP FDC signature”

Figure 4: Gut IgA synthesis within and outside of Peyer’s patches

16

genes were recapitulated in LN FDC when they were co-stimulated, indicating that FDCs induce IgA generation throughthese molecules, which include CXCL13 for lymphocyte
recruitment, BAFF for lymphocyte survival, and TGFβ activators ( Suzuki et al. 2010) .
To summarize ( Fig. 4) , the FDCs in PPs sense bacterial
and food components in the gut lumen, and secrete CXCL13
to recruit B cells and T cells. They also secrete TGF-β1 thatinduces IgM+ B cells to switch to IgA, and BAFF that enhances
the survival of the switched cells and their differentiation into
plasma cells in the lamina propria.
Suzuki moved to Kyoto Univ. in April, 2011 to set up his
own lab. It was almost 10 years ago that he joined Fagarasan’slaboratory. “I was a Ph.D. student from a clinical department atthat time, and didn't know anything about immunology orexperiments. All experimental skills, knowledge and criticalway of thinking were provided by Sidonia,” he said. “Now, I
have to work on my own feet. I am pretty sure that all of theexperiments I performed in Sidonia's lab will help me a lot. I
also would like to thank all of you for a lot of help, many discussions, chatting, and sometimes drinking. Everything was a very
good experience. This is the real treasure, and in my future, I
am pretty sure that this experience will help me a lot as well,”Suzuki said at the ceremony.
Shinji Fukuda and Hiroshi Ohno received the award fortheir multi-omics-based approach to study host-gut microbe
interactions ( Fukuda et al. 2011) .
It is widely believed that probiotics such as bifidobacteria
and lactobacilli are good for health, although scientific evidence
for their mechanisms of action is largely nonexistent. Enterohemorrhagic Escherichia coli ( EHEC) causes illnesses ranging
from mild diarrhea to severe diseases such as hemorrhagic
colitis and hemolytic uremic syndrome. EHEC O157 produces
the lethal Shiga toxin and it is responsible for public healthproblems worldwide.
To study the probiotic effects against EHEC O157 infection, Fukuda and Ohno took advantage of a simplified gnotobiotic mouse model, in which germ-free mice were colonizedwith individual strains of bifidobacteria. Initial experiments
revealed striking differences between strains. They found thatmice colonized by one bifidobacterium subspecies, B. longum,
were able to survive when fed E. coli O157, while mice without
the bifidobacteria died of infection within 7 days. However,another bifidobacterium strain, B. adolescentis, had no suchprotective effect ( Fig. 5) .

Figure 5: Effect of the presence of bifidobacteria
against toxicity of EHEC O157:H7

Neither the concentration of Shiga toxin in thegut, nor the number of E. coli O157 was different
between the mice colonized with the protective or
non-protective bifidobacteria. However, the bloodconcentration of Shiga toxin was markedly lower inthe surviving mice colonized by probiotic B. longum.
Histological analysis showed a slight inflammation
only in the distal colon of the dying mice, but not in
the surviving mice, suggesting that probiotic bifidobacteria protect epithelial cells and prevent systemic
infiltration of Shiga toxin.
By analysis of fecal metabolic profiles using a
combination of techniques from genomics, transcriptomics and metabolomics, Fukuda and Ohno
succeeded in pinpointing the source of this difference as the production of acetate, which enhanced
Figure 6: Non-preventing bifidobacteria overexpressing probiotic
intestinal epithelial defenses. The key player in thistransporter prevents O157 lethal infection
protection was a carbohydrate transporter present
in protective strains of bifidobacteria such as B.
longum, but absent from the non-protective strains. The carbohydrate transporter enabled these bacteria to utilize fructose to produce sufficient amounts of acetate in the distal colon to promote an antiinflammatory effect. However, non-probiotic bifidobacteria could not produce sufficient amounts ofacetate due to the lack of this transporter. After enteropathogenic infection, inflammation developed andthe toxin infiltrated into the body.
To confirm this idea, the investigators knocked-out the carbohydrate transporter genes in B. longum,
and colonized mice. The mice colonized with the mutant strain had a significantly reduced capacity tocatabolize fructose and a reduction of acetate production. More importantly, survival after infection with
E. coli O157 was significantly less ( Fig. 6) .

17

Figure 7: Disruption of the genes encoding probiotic transporter
lacks the preventive effect against O157 lethal infection

They also generated a strain of B. adolescentis thatexogenously expressed the carbohydrate transporters
and, when mice were colonized with this strain, they survived longer after infection with E. coli O157 ( Fig. 7) .
Finally, they asked whether simply increasing theamount of acetate in the distal colon could protect miceagainst E. coli O157-induced death. They colonized themice with B. adolescentis, and then fed them a dietsupplemented with acetylated starch, which is graduallyhydrolyzed, releasing acetate in the intestinal tract.
Administration of the acetylated starch significantly
increased the amount of acetate in the feces and improved
the survival of B. adolescentis-associated mice infectedwith E. coli O157, suggesting that the levels of acetate
are crucial for this protection. (Fig. 8)
Shinji Fukuda started to work at RIKEN as a researcher
in 2006. In collaboration with Prof. Itoh at the Universityof Tokyo, who had a germ-free facility and had establishedthis enteropathogenic infection model, they discovered
these important findings in the subsequent three years
from 2006 to 2008. “We submitted the paper to Nature
in Sep. 2009. After a long, long struggle with Nature editors and four reviewers, the paper was accepted finally in
November, 2010. It was really tough work for me,”
Fukuda said at the ceremony. After the publication of the
Nature paper, many newspapers and TV stations featured
their findings, reflecting the lay society’s interests in probiotic effects and EHEC O157 infection. “I strongly believethat our findings can open up a new field in the area ofmucosal immunology. I will make every effort to progress
this research and keep our position as the front runner inthis field,” said Fukuda.

Figure 8: Carbohydrate transporters confer the
probiotic effect to bifidobacteria

References
Fagarasan, S., M. Muramatsu, K. Suzuki, H. Nagaoka, H. Hiai and T. Honjo. Critical roles of activation-induced cytidine deaminase in
the homeostasis of gut flora. Science 298: 1424-1427 (2002).
Fukuda, S., H. Toh, K. Hase, K. Oshima, Y. Nakanishi, K. Yoshimura, T. Tobe, J. M. Clarke, D. L. Topping, T. Suzuki, T. D. Taylor, K.
Itoh, J. Kikuchi, H. Morita, M. Hattori and H. Ohno. Bifidobacteria can protect from enteropathogenic infection through production of
acetate. Nature 469: 543-547 (2011).
Suzuki, K., M. Maruya, S. Kawamoto, K. Sitnik, H. Kitamura, W. W. Agace and S. Fagarasan. The sensing of environmental stimuli byfollicular dendritic cells promotes immunoglobulin A generation in the gut. Immunity 33: 71-83 (2010).
Suzuki, K., B. Meek, Y. Doi, T. Honjo and S. Fagarasan. Two distinctive pathways for recruitment of naive and primed IgM+ B cells to
the gut lamina propria. Proc Natl Acad Sci U S A 102: 2482-2486 (2005).
Suzuki, K., B. Meek, Y. Doi, M. Muramatsu, T. Chiba, T. Honjo and S. Fagarasan. Aberrant expansion of segmented filamentous bacteria
in IgA-deficient gut. Proc Natl Acad Sci U S A 101: 1981-1986 (2004).
Tsuji, M., N. Komatsu, S. Kawamoto, K. Suzuki, O. Kanagawa, T. Honjo, S. Hori and S. Fagarasan. Preferential generation of follicular
B helper T cells from Foxp3+ T cells in gut Peyer's patches. Science 323: 1488-1492 (2009).

18

Other Prize Winners 2010
Hiroshi Kawamoto (photo 1), Leader of the Laboratory for Lymphocyte

Photo 1: Hiroshi Kawamoto

Photo 2: Koji Hase

Photo 3: Keiichiro Suzuki

Photo 4: Tomokatsu Ikawa

Development, received the 13th Japanese Society for Immunology Award for his
validation of a new hematopoietic cell lineage model. Before his discovery, theclassical model included a common progenitor cell that would give rise to
B lymphocytes or T lymphocytes, but no other hematopoietic cell. However,Kawamoto discovered that no progenitors could differentiate only into B
lymphocytes and T lymphocytes, though there were progenitors that would
differentiate into T lymphocytes and phagocytes or into B lymphocytes andphagocytes. In his myeloid-based model, he proposed that progenitors retain
the ability to differentiate into phagocytes until their fate is sealed by differentiation
into erythrocytes, B lymphocytes, or T lymphocytes. He later discovered that thetranscription factor Bcl11b regulates the formation of T cells from progenitor
cells.
His work was highly recognized to receive the award because of its
originality and the fact that it overturned the conventional wisdom. The JapaneseSociety for Immunology Award is given to a researcher, age of 50 or below,
whose contribution to the advancement of immunology was original andextraordinary and from whom further research achievements are expected in
the future.

Koji Hase (photo 2), Researcher of the Laboratory for Epithelial Immunobiology,
received the Research Encouragement Award of the Japanese Society for Immunology for his elucidation of M-cell functions. He developed a method to screen forgenes expressed specifically in M-cells and, by analyzing the molecular functions ofthese genes, discovered glycoprotein 2 ( GP2) as a transcytotic receptor for mucosal
antigens. Later, he also discovered a mammalian protein, M-Sec, which induces
formation of numerous membrane protrusions extending from the plasma membrane. These protrusions tether onto adjacent cells and subsequently form tunneling nanotubes, which can transmit calcium signals or viruses between cells.
This award is given to young investigators, age 40 or younger, who have
performed distinguished immunological research.
RIKEN Research and Technology Incentive Awards 2010 were presented to two
RCAI researchers, Keiichiro Suzuki ( photo 3 ) and Tomokatsu Ikawa( photo 4 ) . Suzuki, Researcher of the Laboratory for Mucosal Immunity, received
the award for his study on mechanisms of IgA generation and its functions.
Tomokatsu Ikawa, Researcher of the Laboratory for Lymphocyte Development,
received the award for his study on molecular mechanisms of T cell lineage developmental decisions.
The RIKEN Research and Technology Incentive Award was established in
FY2009 to recognize young researchers and technicians under age 40 who havecontributed to furthering RIKEN’s ideals by achieving exemplary results in theirresearch or research support activities.

Hiroshi Watarai ( photo 5 ), Researcher of the Laboratory for Immune Regula-

Photo 5: Hiroshi Wataraition, received the Best Researcher Award from the Young Researchers’ Study
Group of Hematology, headed by Dr. Mineo Kurokawa. The study group selects
excellent papers published by Japanese researchers in the journal Blood every
year, and invites the first authors for discussions. Watarai’s presentation “Reprogramming of natural killer T ( NKT ) cells to induced pluripotent stem cells and theirdevelopment into functional NKT cells in vitro” was awarded among those selected
researchers.

19Toshiyuki Fukada ( photo 6 ), Researcher of the Laboratory for Cytokine Signaling, received the excellent poster award at the annual meeting of the Japan Transporter
Research Association for his poster presentation on “The loss of function of SLC39A13/
ZIP13 cases a novel type of Ehlers-Danlos Syndrome”. He also received the Research
Encouragement Award at the Frontiers in Oral Medical Science meeting for his oralpresentation on “Zinc transporter Zip13: its involvement in tooth development andinherited dental disease.” His recent review featured the front cover of Metallomics.

Masahiro Kitano ( photo 7 ), Researcher of the Research Unit for Immunodynam-

Photo 6: Toshiyuki Fukada

ics, received an Excellent Poster Award at The 16 th Takeda Science Foundation Symposium on Bioscience “Casting Light on Life” for his poster presentation on “BCL6
Expression Dynamics in the Lymph Node during the Antibody Response”.

Mariko Okada-Hatakeyama (Laboratory for Cellular Systems Modeling,
RCAI ) , in collaboration with Junya Yamagishi, Noriaki Okimoto, Atsushi Suenaga and
Makoto Taiji (RIKEN ASI ) , received an Excellent Poster Award at the annual meeting of
The Chem-Bio Informatics Society for their poster presentation on “Strategy for Structure-Based Peptide Design”.

Photo 7: Masahiro Kitano

Saya Moriyama ( photo 8 ), Junior Research Associate in the Laboratory for Lymphocyte Differentiation, received a Biology/Medical Science Prize at the RIKEN Noyori
Summer School 2010. Her poster presentation "BCL6 Expression Dynamics in the
Lymph Node during the Antibody Response" was the fruit from an RCAI internal collaboration with Masahiro Kitano and Takaharu Okada, Research Unit for Immunodynamics.
Seven graduate students studying in RCAI received Young Investigators' Travel Awards
from the Japanese Society for Immunology for the 14 th International Conference of
Immunology. The awardees were Masashi Ebisawa ( photo 9 ) , Daisuke
Takahashi ( photo 10 ), Yuhki Obata ( photo 11 ) and Hideaki Shima( photo 12 ) of the Laboratory for Epithelial Immunobiology, Hideaki Takagi ( photo13 ) and Tomohiro Fukaya ( photo 14 ) of the Laboratory for Dendritic Cell Immunobiology and Izumi Sasaki ( photo 15 ) of the Laboratory for Host Defense.

Photo 8: Saya Moriyama

20

Photo 9: Masashi Ebisawa

Photo 10: Daisuke Takahashi

Photo 11: Yuhki Obata

Photo 12: Hideaki Shima

Photo 13: Takagi Hideaki

Photo 14: Tomohiro Fukaya

Photo 15: Izumi SasakiExcellent Paper of the Year 2010T

he RCAI Award for Excellent Paper was originally established in 2004 with donations from Dr. Masaru
Taniguchi and Dr. Toshio Hirano. The annual award aims to recognize exceptional publications by RCAI
scientists. Although the funds were depleted by 2008, RCAI’s strategic committee decided to provide thefunding to continue this prize, as because there was great value in awarding young researchers' excellent
achievements and encouraging their efforts.
In 2010, 10 excellent papers were selected from 13 candidates for this award.

Shinji Fukuda and Hiroshi Ohno
Bifidobacteria can protect from enteropathogenic infection through production of acetate
Nature, Vol. 469, 543-547, 2011

Noriko Yumoto, Takeshi Nagashima and Mariko Okada-Hatakeyama
Ligand-Speciﬁc c-Fos Expression Emerges from the Spatiotemporal Control of ErbB Network Dynamics
Cell, Vol. 141, 884-896, 2010

Tomokatsu Ikawa and Hiroshi Kawamoto
An essential developmental checkpoint for production of the T cell lineage
Science, Vol. 329, 93-96, 2010

Keiichiro Suzuki and Sidonia Fagarasan
The Sensing of Environmental Stimuli by Follicular Dendritic Cells Promotes Immunoglobulin A Generation in the Gut
Immunity, Vol. 33, 71-83, 2010

Shinya Tanaka, Yasutaka Motomura and Masato Kubo
The enhancer HS2 critically regulates GATA-3-mediated Il4 transcription in TH 2 cells.
Nature Immunology, Vol. 12, 77-85, 2011

Tadashi Yokosuka and Takashi Saito
Spatiotemporal Basis of CTLA-4 Costimulatory Molecule-Mediated Negative Regulation of T Cell Activation
Immunity, Vol. 33, 326-339, 2010

Kenichi Asano and Masato Tanaka
CD169-positive Macrophages Dominate Anti-tumor Immunity by Crosspresenting Dead Cell-associated Antigens
Immunity, Vol. 34, 85-95, 2011

Yuichi Aiba and Tomohiro Kurosaki
Preferential localization of IgG memory B cells adjacent to contracted germinal centers
Proc Natl Acad Sci USA., Vol.107, 12192-12197, 2010

Hiroshi Watarai, Daisuke Yamada, Shin-ichiro Fujii,
Haruhiko Koseki and Masaru Taniguchi
Murine induced pluripotent stem cells can be derived from and differentiate into natural killer T cells
J Clin Invest., Vol.120, 2610-2618, 2010

Tomoharu Yasuda, Kohei Kometani and Tomohiro Kurosaki
Erk kinases control plasma cell differentiation by regulating expression of Blimp-1
Science Signaling, Vol. 4, p. ra25

Tomohiro Fukaya, Hideaki Takagi and Katsuaki Sato
Crucial roles of B7-H1 and B7-DC expressed on mesenteric lymph node dendritic cells in the generation of antigen-specificCD4+Foxp3+regulatory T cells in the establishment of oral tolerance

Blood, Vol. 116, 2266-2276, 201021

Excellent Poster Award 2010On Nov. 29th, RCAI held two poster presentation sessions during its retreat meeting in Chiba. Among
104 posters presented, nine posters were selected for “Excellent Poster Award 2010” by vote oflaboratory heads.

Awardees of Excellent Poster Award 2010 :22

Koji Hase

Masahiro Kitano

Yu-ichi Fujimura

Lab. for Epithelial Immunobiology

Research Unit for Immunodynamics

Lab. for Developmental Genetics

Satoru Yamasaki

Akiko Hashimoto-Tane

Masashi Tachibana

Lab. for Cytokine Signaling

Lab. for Cell Signaling

Research Unit for Immune Homeostasis

Hideaki Takagi

Yu KatoShinji Fukuda

Lab. for Dendritic Cell Immunobiology

Lab. for Immunochaperones

Lab. for Epithelial Immunobiology

2010Part 2

Technology and Innovation

RCAI and Torii Collaborate for the Allergy
VaccineJ

apanese cedar ( Cryptomeria japonica) pollinosis is a common allergy in Japan, with aprevalence estimated to be 26% in a nationwide
survey conducted in 2008. Impaired performancedue to pollinosis and medications used for treating pollinosis is thought to be an important causeof the loss of concentration and productivity ofpatients in the workplace. Antigen-specific immunotherapy ( SIT ) is considered to be the only curative therapy for allergy; however, SIT has not been
a common treatment in Japan due to the risk ofside effects and poor clinical outcomes. Cry j 1, amajor allergen of Japanese cedar pollen, is one of
the candidates for a pollinosis vaccine. Vaccines
using allergoids and modified Cry j 1, for example,
T cell epitopes, pathogen-related molecular pattern molecule-conjugated allergen, and others,
have been developed and used for pre-clinical trials; however, none of them has been commerciallyavailable for medical use either due to poor clinical outcomes of late stage clinical trials or the
failure to find a cooperative pharmaceutical comFigure : Overview of the exchange zone to introduce the vaccine into the market.
pany to introduce the vaccines into the market.
Development of a safe and more effective vaccine
is eagerly anticipated by both physicians andpatients with Japanese cedar pollinosis. There is aogy is used to conjugate two major allergens from Japanesesocial need to develop a recombinant SIT vaccine with highcedar pollen, namely Cry j 1 and Cry j 2. The fusion protein
safety, tolerability and efficacy, and to introduce the vaccine
is then further modified with polyethylene glycol to prevent
into the Japanese market.
binding with immunoglobulin E ( IgE ) . Prophylactic and therEstablishment of the Joint Research Team
apeutic treatment with the vaccine was shown to prevent
To fill the critical gap between basic research and later stage
the increase of serum Cry j 1-specific IgE following chaldrug development, RCAI offered a program called thelenge of mice with Cry j 1. In order to introduce the vaccine‘exchange zone’ where RCAI, universities, hospitals, andinto the market, it is necessary to test the therapeutic effipharmaceutical companies work together for drug developcacy of the vaccine using mouse models of allergic rhinitis
ment, including the allergy vaccine for Japanese cedar
and conjunctivitis and to perform research on its mechapollinosis. The exchange zone is based on the concept thatnisms of action. It is also necessary to develop a simple
sprinters in a relay race run together in the exchange zone tomethod for the purification of the recombinant vaccine
give/receive the baton firmly and smoothly. The idea here isexpressed in prokaryote or eukaryote systems, based onthat RCAI creates the baton ( drug candidate) and passes it
considerations of cost-effectiveness and purity.
firmly to the next runner, namely a pharmaceutical company
Development of a mouse model of Japanese cedar
to develop the drug for the market. Based on this program,pollinosis
in December 2009 RIKEN and Torii pharmaceutical Co., Ltd.
To evaluate the safety and therapeutic effects of the recomagreed to collaborate for the development of a vaccine forbinant vaccine, the team is developing a mouse model ofJapanese cedar pollinosis ( Fig.) .
allergic rhinitis and conjunctivitis by Cry j 1 sensitization.
Torii and RIKEN set up a joint research laboratory in
Their aim is to establish a model where there is high Cry j
RCAI in May 2010 and began research and development of
1-specific IgE in serum as the primary endpoint and sneezthe recombinant vaccine considering GLP and GMP guideing, conjunctivitis, itching of the nose and/or eyes, or infiltralines. The vaccine was originally developed at RCAI as safer
tion of inflammatory cells as secondary endpoints. Therefore,
SIT vaccine, and is less likely to induce dangerous adverse
they injected Cry j 1 adsorbed with aluminum hydroxide gel
events after injection. For the vaccine, recombinant technol-24

( Alum ) into mice intraperitoneally. After this primary sensitization, they challenged the mice with Cry j 1 adsorbed with
Alum or Cry j 1 alone intraperitoneally or subcutaneously.
The serum Cry j 1-specfic IgE and IgG titers were increased
in all cases, and the titer from the mice injected with Cry j 1/
Alum intraperitoneally was highest compared to the othergroups; however, the individual titers varied widely even
within the same treatment group.
To establish a model of allergic rhinitis and conjunctivitis, they first sensitized the mice with Cry j 1 as above, andthen challenged them with Cry j 1 via the nose and eye
once every day for seven consecutive days. Conjunctivitis
and sneezing were not evident in any of the treatmentgroups; however, after intranasal and ocular challengewith Cry j 1, the mice in the Cry j 1 subcutaneously sensitized group were observed to be scratching their noses.
Inflammatory cells such as eosinophils and mast cells infiltrated the nasal mucosa after intranasal challenge withCry j 1. They will test the therapeutic activity of the vaccine
using this mouse model with allergic inflammation.

Members of RIKEN-Torii Joint Research Team
Team Leader Masaru Taniguchi
Senior Research Scientist : Takashi Fujimura
Visiting Scientists: Hiroyuki Miyazaki
		 Yasushi Okumura
		 Koji Fujinami
		 Masao Matsuda
Technical Staff: Kyounga Seo

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

Open Laboratory for Allergy ResearchR

CAI has launched an open laboratory for allergy
research where researchers from external institutes,
universities or hospitals can have access to the Center’s
resources and conduct collaborative allergy research proj-

ects. Taeko Dohi ( National Center for Global Health andMedicine) newly joined the open laboratory program in 2010.
Four projects are on-going under this program.

1. Masato Kubo ( Research Institute for Biological Science, Tokyo University of Science )"Understanding the principles of IgE production"
2. Noriko Tsuji ( Biomedical Research Institute, Advanced Industrial Science and Technology )“Elucidating mechanisms of oral tolerance”
3. Kenji Matsumoto ( National Research Institute for Child Health and Development ) and Naoki Shimojo (Chiba University)“Cohort study of allergy in infants and the generation of a humanized mouse model”
4. Taeko Dohi (National Center for Global Health and Medicine)“Intestinal flora and epigenomic imprinting”

25

iPS Technology Development for
Anti-cancer Immune Therapy
Induced pluripotent stem ( iPS)
cells possess tremendous
therapeutic potential not only inthe field of regenerative medicine but also for immune therapy. RCAI has initiated an
activity to apply iPS technology
to mouse and human immunology research and therapeutic
development. The core facility
for iPS research is engaged indeveloping efficient protocols to
reprogram various lymphocytes
and induce differentiation of iPS
cells into lymphoid lineage cells.
These initiatives are being doneon a collaborative basis withindividual research activities in
RCAI and are partially supported
by CREST from JST.

Identification of genesthat resist reprogramming(Yamada et al., in progress)

Figure :Strategy for generating a large number of NKT cells from
iPS cells in vitro for use in anti-cancer therapy.

Reprogramming of mature lymphocytes to generate iPS
cells is a less efficient process compared to that of fibroblasts. The iPS group hypothesized that certain genes inlymphocytes are difficult to fully reprogram. To test this, they
established 20 and 5 independent iPSC clones from mature
B- and NKT cells, respectively. They have analyzed the gene
expression profiles of 6 B cell-derived and 2 NKT cell-derived
iPS cell lines and compared them with those of ESCs. They
identified a group of genes in lymphocytes that reproducibly
failed to be reprogrammed.

Reprogramming of natural killer T ( NKT ) cells to
induced pluripotent stem cells and their development into functional NKT cells in vitro. ( Watarai etal., 2010 )Although iPS cells hold tremendous potential in cell-replacement therapy, the efficacy of cells with desired
functions differentiated in vitro from iPSCs has not been
rigorously assessed. The iPS group established iPSCs
derived from natural killer T ( NKT ) cell cloned mice ( NKT
mice ) , which harbor functional NKT cell-specific rearranged
T cell receptor loci in their germline. These iPSCs could be
efficiently differentiated into NKT cells in vitro, moreover,upon adoptive transfer into NKT cell-deficient mice, these
iPSC-derived NKT cells recapitulated the known adjuvant
effects of natural NKT cells and suppressed tumor growthin vivo. This study showed for the first time that fully func-

26

tional immune cells can be generated from iPSCs and also
demonstrates the feasibility of expanding functionally competent NKT cells via an iPSC phase in a mouse model. Thesefindings point to the clinical potential of this approach for
NKT cell-targeted adjuvant therapy in humans.

Development of human iPS-derived hematopoietic
stem & progenitor cells (Ishikawa et al., in progress)
The aim is to induce hematopoietic stem/progenitor cells
from human iPS cells. After culturing human iPS cells on
OP9 or OP9/DLL1, hCD45-CD34+ cells appeared at day 7
and hCD45+CD34+ cells appeared at day 14. The iPS group
found that the efficiency of inducing CD34+ cells from iPSCs
is not affected by Notch signaling. This year, they analyzed
marrow repopulating capacity in NOD/SCID/IL2rgKO mice,in vitro colony forming capacity, and expression of transcription factors by the iPS-derived CD34+ cells. These cells wereable to generate granulocyte-monocyte and erythroid colonies in methylcellulose semi-solid agar cultures. Throughtranscriptome analysis, they found that NANOG, OCT3/4,
and SOX2 are nicely down-regulated and several HOX genes
are up-regulated in the iPS-derived CD34+ cells. They are
currently trying to transduce HSC-associated signature
genes into iPS cells for generating self-renewing human
HSCs.

Functional difference between ES and iPS cells
revealed by B cell induction in vitro (Kawamoto etal., in progress)
The iPS group investigated whether murine iPS cells havethe same features as ES cells by comparing their developmental potential to produce hematopoietic cells. They
tested a total of 20 iPS clones including those derived frommature B cells, from NKT cell and from mouse embryonic
fibroblasts ( MEF) , in comparison with several ES lines. All ES
cell clones produced all types of hematopoietic cells in in
vitro culture systems, including erythrocytes, myeloid lineage
cells, T cells and B cells. By contrast, eight out of nine iPS
cell clones did not produce B cells, although the other typesof hematopoietic cells were generated. This can not be due
to insufficient reprogramming, because the same observations were seen in several iPS cell clones derived frommature B cells. These results clearly show that iPS cells are
not identical to ES cells in regard to their B cell potential.

Members of iPS Group
Leaders		

: Haruhiko Koseki

			
			
			

Hiroshi Kawamoto
Fumihiko Ishikawa
Osamu Ohara
Senior Research Scientist : Hiroshi Watarai
Research Scientists: Daisuke Yamada, Kyoko Masuda
Technical Staff: Momoko Okoshi-Sato,		 Genta Kitahara, Masako Fujita,		 Chieko Tezuka, Sakura Sakata,		 Yuko Nagata, Mariko Tomizawa,
		 Yuri Suzuki

Recent publications
1. Watarai H, Rybouchkin A, Hongo N, Nagata Y, Sakata S, Sekine E,
Dashtsoodol N, Tashiro T, Fujii SI, Shimizu K, Mori K, Masuda K,
Kawamoto H, Koseki H, and Taniguchi M. Generation of functional
NKT cells in vitro from embryonic stem cells bearing rearranged
invariant Va14-Ja18 TCRa gene. Blood 115:230-7 (2010)
2. Watarai H, Fujii S, Yamada D, Rybouchkin A, Sakata S, Nagata Y, Iida-Kobayashi
M, Sekine-Kondo E, Shimizu K, Shozaki Y, Sharif J, Matsuda M, Mochiduki S,
Hasegawa T, Kitahara G, Endo TA, Toyoda T, Ohara O, Harigaya K, *Koseki H,*Taniguchi M. Murine induced pluripotent stem cells can be derived from anddifferentiate into natural killer T cells. J Clin Invest. 120:2610-2618 (2010)

Development of Immune Cell TherapyIn collaboration with Chiba University ( Prof. Toshinori
Nakayama, Prof. Takehiko Fujisawa and Associate Prof.
Shin-ichiro Motohashi ) , RCAI has been developing a Phase
I/IIa clinical study of the application of NKT cell-targeted
therapy for advanced lung cancer ( stage IIIB, IV or recurrence) . α-GalCer-pulsed APCs ( 1x109/m2 PBMC-derived
DCs) were intravenously administered four times. In thisstudy, the MST ( median survival time) of the respondergroup, which was defined by an increased number of IFN- γ
producing NKT cells compared to the non-responder group,
was longer than the non-responder group ( 31.9 months versus 9.7 months) . Thus, the increased IFN- γ production by
NKT cells upon α-GalCer stimulation showed a significant
association with clinical outcome. These phase I/IIa trial
results are encouraging and warrant further evaluation of
the survival benefit of this immunotherapy.
For the next step, RCAI applied in 2011 for the advanced
medical care assessment system established in 2008 by

the Japanese Ministry of Health, Labor and Welfare ( MHLW ) ,by which the patients’ immunotherapy treatment will becovered in part by health insurance. It usually takes approximately 10 -12 months to obtain approval by the MHLW
review board to use this system. The system was established because advanced medical technologies using
medical devices or pharmaceuticals, such as immune celltherapy, that are yet to be approved under the Pharmaceutical Affairs Law ( PAL) , are not covered by health insurance inJapan. However in response to recent rapid progress inmedical technology and the patients’ need to have safe andlower cost treatment with such technologies, this system
was introduced to allow health insurance payments. It isalso the case that the collection of appropriate clinicalresearch data is facilitated by this system, and thus can thenlead to approval of the advanced medical technologies
under the PAL.

27

Network for Primary Immunodeficiency
Diseases
R

28

1200

Growth of PIDJ
963

1000

879

Number of Entries

800

777
671

600

573
495
418

400
348
279

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191

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20

IKEN RCAI is establishing a strong presence in the
research community of Primary Immunodeficiency Diseases ( PID) in Japan and the surrounding region. In keepingwith its mission of improving immune health, RIKEN RCAI
has been tightening and expanding a collaboration networkwith the clinical study group for PID from 13 universities andthe Kazusa DNA Research Institute ( Chiba) to serve as amodel demonstrating synergy between basic and clinicalresearch. At the beginning, the approach taken by RCAI wasthought to potentially have two main risks: The first was thatclinicians would not want to be involved because they could
not foresee any long-term benefits for their PID patients; thesecond risk was that the basic immunologists at RCAI might
be too far removed from the PID clinics to participate in suchstudies of human immunology because they have a tendency to focus on mouse models. It is now evident that
RCAI has avoided both risks by properly managing this project. In fact, the number of entries deposited in a PID clinicalarchive established by RCAI, named PID Japan ( PIDJ ) , hascontinuously increased and now exceeds 1000 ( Fig. 1) . The
first risk thus proved unfounded, probably because many
clinicians, at least in the field of PID research, have been
seeking such an opportunity to closely work with genome
scientists and basic immunologists. As for the second risk,
basic immunologists at RCAI are in general very keen tocollaborate with clinicians. This is because they have astrong motivation to demonstrate the relevance of theirmouse models to human diseases; a recent RIKEN focus is
on “Creation of Social Wisdom” to solve the critical issues
that confront humanity today. For example, we have started
to examine the functionality of newly characterized immune
genes that were first found to be important in mouse models
in PID patients. In particular, the RCAI “humanized mouse”
project and the immune iPS cell project will offer key
technologies to fill the gap between studies of human andmouse immune systems. Such a course of collaboration,
extending from basic immunologists to clinicians, has justbegun and is expected to bear fruit in the near future on thebasis of the PID network.
Initially, there was a serious concern about how to unify
a network in which clinicians, genome scientists, and basic
immunologists are separated not only by geography but
also by disciplines. However, a strong unifying force for the
PID network was developed by providing clinicians andbasic immunologists with genomic analysis data performed
by RCAI and the Kazusa DNA Research Institute. The
capacity to share clinical, immunological, and genetic data
played a pivotal role in tightening the network. On the other
hand, although the number of PID genes is continuously
increasing and now exceeds 200, many PID patients still

Figure 1: Growth of PIDJ

remain genetically undiagnosed because they do not havemutations in any of the known PID genes. To address thisproblem, we have tackled common variable immunodeficiency, one of the most common but complex PIDs, throughintegrating efforts by clinical and basic immunologists andgenome scientists. Recent advances in genomics ( e.g.,
next-generation DNA sequencing) have drastically changed
the strategy for exploration of genetic causes of humandiseases and RCAI is one of the major workhorses for
introducing these new technologies into the PID researchcommunity.
Another key component to enhance the network activity
originates from exploiting the power of the internet andbioinformatics. We have already established PID-related
informational platforms such as PIDJ ( a secured clinicalarchive, http://pidj.rcai.riken.jp/ ) , RAPID ( an integrated bioinformatics platform for PID, http://rapid.rcai.riken.jp) , andMutation@A Glance ( a mutation annotation server, http://
rapid.rcai.riken.jp/mutation/ ) . The latter two platforms weredeveloped through a collaboration with the Institute of
Bioinformatics in India. Notably, the function of Mutation@A
Glance has now been enhanced to cover all known humandisease genes. Furthermore, because RIKEN has a wealthof information potentially useful for analyses of humandiseases, we have begun to integrate all these lines of
information in RIKEN to tackle human diseases. Towards
this final goal, we have worked together with the RIKEN
Bioinformatics and Systems Engineering division to fully

utilize SciNetS ( Scientist’s Networking System) in PID. The
PID network, including interdisciplinary researchers such asthese, is steadily growing.
As an activity of the RIKEN Jeffrey Modell Diagnostic
and Research Center for PID, which was launched in 2008,
a Japanese-version of the poster “10 Warning Signs of Primary Immunodeficiency” ( Jeffrey Modell Foundation) was
prepared and distributed to the clinical community ( Fig. 2) .
In addition, we have continued our efforts in networking the
PID community in Asia. For this purpose, the 3rd PID meeting
was held as a session of the 7th Pediatric Academic Societies
and Asian Society for Pediatric Research Joint Meeting in
Denver, CO, USA on May 1, 2011.

Figure 2: Poster on the signs of primary
immunodeficiency diseases

Development of a Humanized Mouse ModelR

CAI’s Research Unit for Human Disease Model ( Unit
Leader: Fumihiko Ishikawa), Laboratory for Developmental Genetics ( Group Director: Haruhiko Koseki) andLaboratory for Immunogenomics ( Group Director: Osamu
Ohara ) have aimed to develop the next generations ofhumanized mice carrying essential human genes, including
MHC class I and II, cytokines and adhesion molecules. Theirgoal is to develop humanized mice in which the immune
responses to various pathogens and environmental stresses
will precisely recapitulate that seen in humans. These next
generation mice will enable us to create useful models of
individual human diseases.
By creating humanized mice carrying the human leukocyte antigen ( HLA ) through collaboration with the JacksonLaboratory, they showed that the expression of human genes
is essential to fully recapitulate human lymphopoiesis in

immunodeficient NOD/SCID/IL2r γ KO mice. In the humanized mice they created ( HLA class I A02 transgenic NOD/
SCID/IL2r γ KO mice) , human cytotoxic T lymphocytes ( CD8+
T cells) developed successfully and these CD8+ T cells
expressed effector molecules such as granzyme, in accordance with their phenotypic maturation from naïve to central
memory and effector memory cells. These human CD8+ T
cells were functional in terms of cytokine production andcytotoxicity. They recognized Epstein-Barr virus ( EBV ) infected cells and virus-associated peptides, and showed
cytotoxicity in an HLA-restricted manner. Moreover, thisantiviral cytotoxic T lymphocyte response was inhibited by
the addition of anti-HLA class I antibody ( Shultz et al., Proc
Natl Acad Sci U S A, 2010) . ( Fig.)
The next generation humanized mice is expected tocreate various models of individual human diseases.

HLA-restricted cytotoxicity

Lymphoproliferative lesion
HE staining

DAPI

CD3 ( T cell )% specific lysis

40

LMP1 ( EBV )CD19 ( B cell )Merge (B & T )30

Figure :At 5-6 weeks post-EBV infection,formation of lympho-proliferative
disease was identified in the lungof HLA class I Tg humanized
mice (left). Human CD8+ T
cells developed in the HLA
class I expressing humanized
mice exhibited HLA-restricted
cytotoxicity against EBVinfected cells (right).

20
10+class l Ab

(-)

(+)

(-)

+class ll Ab

(-)

(-)

(+)

29

Drug Discovery Antibody Platform Unit is
Established in RCAII

n order to conduct research and development of novel
drugs and medical technologies, RIKEN established theDrug and Medical Technology Basic Program ( DMP) in
December 2009. Since then, DMP has selected drug discovery themes and projects proposed by scientists at RIKEN
research centers. According to DMP guidelines, the selected
projects focus on addressing intractable diseases.
To achieve effective progress in each of the projects,
DMP needs to increase technological excellence and skilled
personnel at each research center. Hence, DMP required
various technological platform units to manage the program
through close cooperation with the Drug Discovery Basic
Unit established at each research center. In this context, aDrug Discovery Antibody Platform Unit ( APU) was established in RCAI ( Unit leader: Toshitada Takemori ) , in order todevelop monoclonal antibodies that can be used as therapeutic drugs for treatment of cancer and other diseases.
The APU is a collaboration with the Crystallographic Drug
Discovery Platform Unit, which possesses powerful technol-

ogy for preparing recombinant transmembrane type antigens, for example signaling receptors expressed by tumor
cells, for use both as immunogens and screening reagents.
In collaboration with theme leaders, the APU prepares
monoclonal antibodies by hybridoma technology and selects
antibodies with the desired properties. The APU has
attempted to establish a novel approach for preparing highaffinity monoclonal antibodies by use of artificial lymph nodetechnology which was created at RCAI. RCAI has also
developed humanized mice, in which the human hematopoietic system is reconstituted into immunocompromised mice.
A future plan of the APU is to develop this system to create
completely human monoclonal antibodies in mice. The activity of this Unit is supported by RCAI Leaders, Takashi Saito,
Hiroshi Kawamoto, and Fumihiko Ishikawa, who are experts
in monoclonal antibody production, artificial lymph nodes,
and humanized mice, respectively.

RCAI Innovation Projects in Drug
Discovery and MedicineT

he government changed its science policy recently
from “Science and Technology” to “Science, Technology and Innovation”, and RIKEN launched “RIKEN Research
Cluster for Innovation” in April, 2010. In this cluster, “platform for drug discovery and medicine” was newly established. Considering this direction, RCAI decided to support

limited number of projects with the aim of innovation in drug
discovery and medicine.
RCAI launched Innovation Projects in Drug Discovery
and Medicine and selected three projects from five applications.

1. Yasuyuki Ishii“Monoclonal antibody specific to malignant lymphoma that induces apoptosis ”
2. Shin-ichiro Fujii“Development of artificial adjunvant vector cells”
3. Takashi Tanaka“Development of a novel treatment for wounds using siRNA against PDLIM2”

30

2010

Part 3

Nurturing Young Scientists

RCAI International Summer Program

T

August 17-27, 2010

he RCAI International Summer Program (RISP) was held on August 17- 27, 2010. This was the fifth
annual RISP and was co-sponsored by the Global Center of Excellence at Chiba University. There were
141 applicants to the program and forty-four graduate students and postdoctoral fellows from ten countries
were accepted to participate in RISP 2010. The first part of the program was held in Yokohama at RCAI.
There were morning oral presentations by the students, and their posters were available for viewing anddiscussion throughout the week. The afternoon sessions featured lectures by invited experts from RCAI,Japanese universities, and abroad. There was also a presentation by Kevin Da Silva, an editor for Nature
Medicine, who described the journal’s submission and review process. This year’s RISP was unique – forthe second part of the program the participants traveled to Kobe where they attended the 14th International
Congress of Immunology. A prerequisite for their acceptance into RISP was that they present their researchin poster or oral format at this meeting. After the Kobe meeting, three of the RISP participants returned to
Yokohama for a month-long laboratory internship at RCAI in the laboratories of Takashi Saito, Hiroshi Ohno
and Masato Tanaka.
The RISP lectures at RCAI were very diverse. Topics included lymphocyte development differentiation andphysiology, as well as the function of microRNAs in these processes (Qi-jing Li, photo1), dendritic cells, epigenetic regulation of gene expression, signaling, tolerance, mucosal immunity, and the innate immune system.
Because of their breadth, these lectures provided an expansive overview of the immune system, withapproaches ranging from single molecule to whole animal and “humanized” mouse analyses. The invited
speakers incorporated introductory material as well as recent highlights from their own research into theirtalks. The research interests of the participants were similarly varied, making for a unique opportunity forcross-fertilization among immunology subdisciplines during the oral and poster presentations as well as inmore informal settings. The question periods following talks by the invited lecturers and participants werespirited and stimulating. Awards for best RISP posters were presented at a reception held at RCAI the evening
before the students departed for Kobe on August 21st. All of the participants expressed their appreciation tothe organizers for inviting them to RISP, and many of them established collaborations as a result of the RISP
that are likely to be long lasting.
The RISP 2010 students had the opportunity to explore both the Yokohama /Tokyo and Kobe areas. Witha small meeting atmosphere at the RCAI venue and a large international meeting flavor at Kobe, this provided
an exceptional experience from both scientific and cultural perspectives. In a survey completed after the RISP,
the participants unanimously agreed that they would recommend the program to colleagues. Half indicatedthat they would consider a postdoctoral position at RIKEN and nearly all would consider returning to RCAI fora short period of collaborative research. The success of this unique program was due to the efforts of theOrganizing Committee, chaired by Hiroshi Ohno ( photo 2 ) and Tomohiro Kurosaki, and the RISP Secretariat,
Mari Kurosaki, Yuko Ochi (photo 3 left), and Sachiko Dohi ( photo 3 center), who kept the entire operation
running smoothly, as well as to the efforts of the outstanding participants.

32

1

2

3

Table 1 : Lectures
Lecturer

Title

Paul W. Kincade, Oklahoma Medical Research Foundation, USA

Producing immune effector cells from diverse stem and progenitor cells

Qi-Jing Li, Duke University Medical Center, USA

MicroRNA and its micromanagement during T cell development and antigen
responses

Toshinori Nakayama, Chiba University Graduate School of Medicine,JAPAN

Epigenetic regulation of GATA3 expression in Th2 cells

Shigeo Koyasu, Keio University School of Medicine, JAPANNatural helper cell: a newly identified Th2 type innate lymphocyte

Balbino Alarcón, Consejo Superior de Investigaciones Científicas,
SPAIN

Multiple roles of RRas GTPases in the immune system

Fumihiko Ishikawa, RIKEN RCAI, JAPANInvestigating normal & diseased human immune systems using the humanizedmouse

Masaru Taniguchi, RIKEN RCAI, JAPANDiscovery of NKT cells with adjuvant activity and their clinical application

Hiroshi Ohno, RIKEN RCAI, JAPANInteraction of host, commensal microbiota, and pathogens

Diane Mathis, Harvard Medical School, USA

Immunological tolerance, centrally

Shohei Hori, RIKEN RCAI, JAPAN

Dominant immunological tolerance

Ifor R. Williams, Emory University School of Medicine, USA

The genesis of antigen-sampling intestinal M cells: elucidating the relative
contributions of RANKL, the enteric microflora, and B lymphocytes

Klaus Rajewsky, Harvard Medical School, USA

B cell physiology, Epstein-Barr-Virus and immune surveillance of EBV-infected
and -transformed B cells

Tsuneyasu Kaisho, RIKEN RCAI, JAPANHow dendritic cells sense and respond to nucleic acid adjuvants

Ruslan Medzhitov, Yale University School of Medicine, USA

Innate immune system

Kevin Da Silva, Nature Medicine, USA

Publishing in Nature Medicine

Table 2 : ParticipantsName (country)
Cindy Banh (USA)Joseph Barbi (USA)Tyani Chan (Australia)

Pheh Ping Chang (Malaysia)

Jean-Enno Charton (Germany)

Luisa Cimmino (Australia)

Nicholas Clarkson (UK)

Jonathan Clingan (USA)

Sumit Deswal (India)

David DiLillo (USA)

Georg Gasteiger (Germany)

Eliver Ghosn (Brazil)

Stephanie Grabow (Germany)

Stephan Halle (Germany)

Robert Johnston (USA)Steven Josefowicz (Canada/ USA)

Justin Killebrew (USA)

Byung-Seok Kim (South Korea)

Kathryn Knoop (USA)

Dmitriy Kolodin (Russia)

Ryan Larson (USA)Monica Leung (Canada)

Yinming Liang (China)

Jing Shan Lim (Singapore)

Xi Liu (China)

Lisa Ma (China)

Mona Mashayekhi (Iran)

Kendle Maslowski (Australia)

Frederick Masson (France)

Melba Munoz-Roldan (Colombia)

Hae-Young Park (South Korea)

Carolina Prado (Chile)

Priyanka Sathe (Australia)

Olga Schulz (Germany)

Vera Schwierzeck (Germany)

Fernando Sepulveda (Chile)

Alice O. Kamphorst Silva (Brazil/ France)

Santi Suryani (Australia)

Anastasia Tikhonova (Russia)

Annick van de Ven (Netherlands)

Catharina Van Elssen (Netherlands)

Eduardo Villablanca (Chile)

Annie Xin (China)

John Yi (USA)33

Harvard Summer School at RCAI

June 14-August 27, 2010

R

CAI offers a summer internship program for undergraduate students from Harvard University. This program
consists of a research internship in the laboratory of an RCAI PI as well as courses in basic immunology andJapanese. The participants receive course credit from Harvard. Three students, Michael Wang, Liang Cheng and
Chinwe Madubata, stayed at RCAI from June 14 to August 22, 2010.
Wang conducted his research project in the Research Unit for Human Disease Model, Cheng in the Laboratory for Immunological Memory, and Madubata in the Laboratory for Immune Regulation. During the internship,
the students had numerous discussions with RCAI researchers and, at the end of the program, they gave oralpresentations describing their projects. In addition to the scientific aspects of the program, there was a cultural
exchange at the Science Frontier High School, where the Harvard students learned Shodo (Japanese caligraphy)
and Sado (tea ceremony) from the local high school students. At the farewell party, they played beautiful classic
ensembles and dedicated the music to Director Masaru Taniguchi.

Photo 1:

Immunology Lecture. From left :Masaru Taniguchi (Director),
Chinwe Madubata, Liang Cheng, Michael Wang, and Yoshiro
Kawaoka (Guest Lecturer from The Univ. of Tokyo)

Photo 2: Cultural Exchange at Science
Frontier High School

RCAI Retreat November 29-30, 2010T

his year’s RCAI Retreat was held on Nov. 29-30 at Kazusa Academia Park in Chiba prefecture. The aim of
the RCAI Retreat is to provide the RCAI scientists with the opportunity for open communication and in depth
discussion of ongoing research projects by gathering the members at a quiet place with few distractions. One
hundred and fifty one RCAI members and two editors, John F. Foley of Science Signaling and Zoltan Fehervariof Nature Immunology, participated in the Retreat. ( photo 1 right and left, respectively )Two poster sessions were held on the first day. One-hundred four posters were presented, and nine posters
were selected for awards by vote of the laboratory heads ( See Excellent Posters on page 22). Between thesessions, eighty-seven participants took a short walk to the nearby Kazusa DNA Research Institute ( KDRI ),where Michio Ohishi ( photo 2 ), Director of KDRI, greeted them at the entrance and conducted a tour. The participants were all impressed by the achievements and the state-of-the-art equipment in the institute.
The second day began with talks by the two Editors, which were followed by ten oral presentations byselected young researchers. Their stimulating presentations provoked active discussions.
The great success of this year’s retreat was due to the dedicated efforts of the organizers, Ji-Yang Wang and
Hisahiro Yoshida ( photo 3 right and left, respectively ), and the support of their lab members (Lab. for Immune
Diversity and Lab. for Immunogenetics) and the secretariats (Mizuki Shimura, Norie Takeuchi and Hiroko Iwamoto). There were several changes from the past Retreats; the organizers decided to hold the meeting in autumn,
searched for the best venue on a limited budget, offered a tour of KDRI, and discontinued the on-site duties ofthe secretariats. Based on responses to the post-retreat questionnaire, more than 80% of the participants hadvery good impressions and many hoped to join the meeting in Kazusa again.
Wang noted that it was very difficult to select the best posters, and that a common standard for the selection
and the decision making process must be reconsidered. Active participation by all researchers is also essential
for the success of Retreat. Therefore, this year’s efforts for change brought great success and satisfaction, andeven inspired further ideas for improvement of future Retreats.

1

34

2

3

RIKEN Joint Retreat

January 31 - February 1, 2011

A

RIKEN 2011 Joint Retreat
was held at YAMAHA Resort
Tsumagoi in Kakegawa, Shizuoka
prefecture on Jan. 31 and Feb. 1,
2011. This year’s meeting wascoordinated by RCAI, and 117
1
2
researchers gathered from 7
RIKEN centers: Advance Science
Institute ( ASI ), BioResource Center (BRC), Brain Science Institute (BSI), Center for Developmental Biology (CDB),
Omics Science Center ( OSC ), Research Center for Allergy and Immunology ( RCAI ) and Spring-8.
This meeting aims to stimulate synergy within RIKEN by gathering and thinking together about common
biological topics in a relaxed atmosphere. In the opening remarks, Tomohiro Kurosaki ( RCAI ) ( photo 1), the chief
meeting organizer, said “The meeting will provide valuable opportunities to get to know the researchers from3other fields and create synergistic collaboration.”
The theme of this year’s meeting was evolution. Although evolution has been a common theme in biology
for a long time, there remain many questions. The recent spectacular advances in molecular sciences and theassociated research techniques should provide keys to solve the remaining evolutionary questions, however, notmany RIKEN labs really focus on them. During the sessions, participants revisited the evolutionary themes andthought together on the molecules and methods that are common in various biological fields.
The first speaker, Shigeru Kuratani (CDB ) ( photo 2 ) introduced his recent discoveries on the formation of theturtle shell (carapace), which is a specialized variation in the more familiar rib growth seen in other vertebrates.
Hisaya Kakinuma (BSI) then talked about the evolution of the cerebellum in vertebrates. During development, themidbrain-hindbrain boundary (MHB) region acts as a signaling center for cerebellar development. Specific geneenhancers conserved in jawed vertebrates promote this development, but are absent in the jawless lampreys,
which have a very small cerebellum. The third speaker, Shigeharu Moriya ( ASI ), discussed strategies to understand the evolution of the complex ecosystem. Although phylogenetic analysis has been used as the conventional approach to track the evolutionary trail, a large part of the biosphere is easily rearranged by lateral genetransfer, generation of organelles and /or symbiosis. He introduced a range of omics approaches to analyze thesymbiotic protists of termites and then presented an integrated multi-omics analysis of marine microbial communities, which may help us to understand the effect of environmental changes. Hiroshi Ohno ( RCAI ) thenintroduced his recent meta-genomics research on gut bifidobacteria. They found that mice colonized by bifidobacterium subspecies B. longum survived when fed the pathogenic bacteria E. coli O157, and they were able topinpoint acetate as the protective molecule. The last speaker was Akihiko Nakano ( ASI ). He discussed evolutionary aspects of post-Golgi and endocytic trafficking. He introduced unique plant molecules involved in membrane trafficking and indicated that land plants have evolved a unique and complex membrane trafficking
mechanism.
That evening, 86 posters were presented, giving the participants the opportunity for informal discussions
that should ultimately increase communication among the centers. ( photo 3 )On the second day, six investigators from various centers introduced the most recent research highlights. In
the closing remarks, Carina Hanashima (CDB ) said, “this meeting is still in the process of evolution. The next
meeting is planned two years from now, and more feedback signals are appreciated.”Thanks to the participants, the organizers, Tomohiro Kurosaki ( RCAI ), Masato Tanaka (RCAI) ( photo 4 right),
Satoshi Ishido (RCAI) ( photo 4 left), Atsushi Mochizuki ( ASI ) ( photo 5 ), Yoshihiro Yoshihara (BSI ), Carina Hanashima( CDB ) ( photo 6 ) and the secretariat office, especially Kazunari Fukushima ( Yokohama RIKEN ), Yasuaki
Murahashi (RCAI ), Aiko Iyama (RCAI ) and Rie Morita (RCAI ), the meeting was constructive and a great success.
“It was my first time to participate in this meeting, but it was a great opportunity to get to know researchers fromother Centers,” said a young postdoc from RCAI.

4

5

6

35

Young Chief Investigator ProgramR

CAI established a new program, Young Chief Investigator Program, to provide a career path for young
investigators who conduct multidisciplinary research that will bridge immunology with other research
fields. In this program, the selected Young Chief Investigator (age below 40) will head an independent
research laboratory but will have structured access to mentoring by multiple senior specialists in related
research fields, i.e. the leaders in various RIKEN Institutes. Mentors provide guidance for experimental
design, preparation of papers and presentations, promotion of international visibility, and obtaining researchfunding. The laboratory will also share space, equipment and facilities with a host laboratory in RCAI
(Groups, Teams or Units). (Fig.)
There will be an initial 5-year appointment, with the possibility of extending for an additional 2 years
after evaluation by the Director and an internal committee. At that point, a Young Chief Investigator canleave to take a position at another institution or be promoted to another type of position within RCAI.
At the end of FY2010, three researchers were selected for the Young Chief Investigator Program,
Hayato Naka-Kaneda (stem cells and aging reversal ), Shinji Nakaoka (development of interface between
integrative biology and mathematics), and Koji Hase (mucosal flora and epigenetic analysis).

RIKEN
Other Center

RCAI
Host Lab.
Space &
equipmentPI
mentorOther Center

Young
Investigator Lab:

mentorPI

Facilities &experimental
supportmentorPI

CentralFacility

Other Centers : all the research centers in RIKEN including PSC, CGM, SSBC, OSC, BASE, CRNID, ASI, BSI, CDB, CMIS, BRC, Spring-8, XFEL, Nishina Center, Sendai
Facility, Nagoya Facility, Center for Computational and Quantitative Life Science, etc.

Figure:Scheme of Young Chief Investigator Program

RIKEN Foreign Postdoctoral Researcher

T

he RIKEN Foreign Postdoctoral Researcher ( FPR) program offers aspiring young foreign researchers with creative ideas who show promise of
becoming internationally active in the future the opportunity to pursue innovative research at RIKEN under the direction of a RIKEN laboratory head.
The FPR program is one of RIKEN's initiatives to open up its facilities andresources to the world and create a stimulating research environment thatwill place RIKEN at the forefront of global science and technology.
In 2010, Jafar Sharif ( Photo) studied in the Lab. for Developmental
Genetics as a RIKEN FPR.

36

RIKEN Special Postdoctoral Researcher (SPDR) Program

01

02

03

04

05

06

07

08

R

IKEN’s program for Special Postdoctoral
Researchers was instituted to provide young
and creative scientists the opportunity to be
involved in autonomous and independent researchin 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 toconduct their research at one of its laboratories.
This year, eight postdocs conducted theirresearch at RCAI through the SPDR program.

Kenichi Asano ( Lab. for Innate Cellular Immunity ) ( 01)
Kohei Kometani ( Lab. for Lymphocyte Differentiation ) (02 )Yoshitaka Shirasaki ( Lab. for Immunogenomics ) (03 )Nayuta Yakushiji ( Lab. for Developmental Genetics ) ( 04 )Yuki Horisawa-Takada ( Lab. for Developmental Genetics ) (05 )Takashi Kanaya ( Lab. for Epithelial Immunobiology ) (06 )Mizuho Kajikawa ( Lab. for Infectious Immunity ) (07 )Masahiro Kitano ( Research Unit for Immunodynamics ) ( 08 )RIKEN Joint Graduate School Program
International Program Associate

01

02

03

04

05

R

CAI accepted four international students as RIKEN International Program Associates ( IPA ). Under this IPA
program, RCAI lab heads host 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 studying at RCAI in 2010 were
Yingqian Li (Nanjing University, China) studied in the Lab. for Immune Diversity ( Photo 1)
Sebastian Nieke (Tubingen Univ, Germany) studied in the Lab. for Transcriptional Regulation ( Photo 2 )Shuyin Li (China Agriculture University, China) studied in the Lab. for Immune Diversity ( Photo 3 )Yue Ren (Jilin University, China ) studied in the Lab. for Immune Regulation ( Photo 4)( The Univ. of Tokyo) from Germany studied in the Lab. for Cellular
Felix Reinhard Helmut Jonas
Systems Modeling ( Photo 5)

37

RIKEN Junior Research Associate (JRA) ProgramT

he Junior Research Associate program was launched in 1996 to encourage young scientists with fresh ideas
and youthful enthusiasm to collaborate with, and learn from, veteran scientists with years of experience. This
program provides 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 alsoenergizing RIKEN with their innovative thinking.
This year, seventeen JRA students studied in RCAI.
Minoru Sawaguchi ( Lab. for Cell Signaling ) Photo ( 01)
Hideaki Takagi ( Lab. for Dendritic Cell Immunobiology ) Photo (02)Tomohiro Fukaya ( Lab. for Dendritic Cell Immunobiology ) Photo (03)Machiko Sugiyama ( Lab. for Immunogenetics ) Photo ( 04)
Izumi Sasaki ( Lab. for Host Defense ) Photo (05)
Chihiro Yamazaki ( Lab. for Host Defense ) Photo (06)Toshi Jinnohara-Yuyama ( Lab. for Epithelial Immunobiology ) Photo (07)Shinsuke Takagi ( Research Unit for Human Disease Model ) Photo (08)Takanori Sawaguchi ( Lab. for Developmental Genetics ) Photo (09)Yuuhou Najima ( Research Unit for Human Disease Model ) Photo (10)Masanaka Sugiyama ( Lab. for Host Defense ) Photo (11)Yuki Aoki ( Research Unit for Human Disease Model ) Photo (12)Beum-Ho Byn ( Lab. for Cytokine Signaling ) Photo (13 )Nanako Shimura ( Lab. for Immunogenomics ) Photo (14 )Kazunori Kadokura ( Lab. for Epithelial Immunobiology ) Photo (15 )Daisuke Takahashi ( Lab. for Epithelial Immunobiology ) Photo (16 )Saya Moriyama ( Lab. for Lymphocyte Differentiation) Photo (17 )

01
04

05

06

07

08

09

10

11

12

15

16

17

02

03

13

38

14

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 nine domestic university graduate schools. The total number of adjunct professors/
associate professors in RCAI is now 29 ( Table ). Fifty-eight students were studying at RCAI in2010.
On May 23, RCAI held a briefing session on the Adjunct Graduate School Program ( AGSP )for potential applicants. Eighteen participants, including 4 from companies, gathered from allover Japan - Kumamoto, Tokushima, Hyogo, Osaka, Toyama, Nagano, Aichi, Shizuoka, Iwate,
Kanagawa and Tokyo. The session began with an introduction of RCAI and a summary of the
AGSP by the organizer, Hisahiro Yoshida. Next, five RCAI PIs, Haruhiko Koseki, Hiroshi Ohno, Masato
Kubo, Hei-ichiro Udono and Katsuaki Sato, briefly introduced their research programs. After the oral session, the individual laboratories held poster presentations
and then each participant had the opportunity to visit thelaboratory of his/her interests.
All sixteen participants indicated that they are thinking positively about entering the adjunct graduate school.
One participant noted that it was very good to get
acquainted with the atmosphere of RCAI and that he wasimpressed by the facilities, but he suggested that thissession be held in different places in Japan because it wasa long way to come.

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)
Tsuneyasu Kaisho (Professor)
Toshiyuki Fukada (Visiting Associate Professor)

Graduate School of Medicine,
Chiba University

Takashi Saito (Visiting Professor)
Haruhiko Koseki (Visiting Professor)
Hiroshi Ohno (Visiting Professor)
Shinichiro Fujii (Visiting Associate Professor)
Yasuyuki Ishii (Visiting Associate Professor)
Fumihiko Ishikawa (Visiting Associate Professor)

Graduate School of Biomedical Science,
Tokyo Medical and Dental University

Takashi Saito (Visiting Professor)
Tomohiro Kurosaki (Visiting Professor)
Masato Kubo (Visiting Professor)
Mariko Okada-Hatakeyama (Visiting Professor)
Sidonia Fagarasan (Visiting Associate Professor)

International Graduate School of Arts and Sciences,
Yokohama City University

Hiroshi Ohno (Visiting Professor)
Tsuneyasu Kaisho (Visiting Professor)
Satoshi Ishido (Visiting Associate Professor)

Research Institute of Biological Sciences, Tokyo University of
Science

Masato Kubo (Professor)
Osamu Ohara (Visiting Professor)
Shohei Hori (Visiting Associate Professor)
Tadashi Yokosuka (Visiting Associate Professor)

Graduate School of Frontier Sciences, The University of Tokyo

Mariko Okada-Hatakeyama (Visiting Associate Professor)

Graduate School of Medicine, Kyoto University

Fumihiko Ishikawa (Visiting Associate Professor)

Graduate School of Medicine, Kobe University

Masaru Taniguchi (Adjunct Lecturer)

39

RCAI Administrative Coordination Office

Table : Members of AdmCO
Position

Name

Office Manager

Takashi Saito

Administrative Staff

Chief Assistants

Hiroko Tanabe
Hiroko Yamaguchi
Mari Kurosaki

Assistants

Sachiko Dohi
Sachiko Haraguchi
Akiko Imai
Akiko Imaizumi
Hiroko Iwamoto
Aiko Iyama
Shihoko Kato
Satomi Law
Ryoko Moriizumi
Rie Morita
Toshiko Nakamura
Kazuyo Nomura
Yuko Ochi
Mizuki Shimura
Norie Takeuchi
Yuuki Yamada
Mio Yoshioka

IT Team

Technical Scientists

Yasuaki Murahashi
Miho Izawa

Technical Staff

Aoi Ozawa

Facilities Staff

Technical Scientist

40

Toshihiko OgataR

CAI reorganized the administrative staff and establishedthe Administrative Coordination Office ( AdmCO) in April,2010. The AdmCO, which coordinates and manages a wide
range of tasks, consists of not only assistants, but also the ITteam and facilities support staff. Assistants support the Centerand its laboratories by providing secretarial assistance andserve as Secretariats of the Center’s annual events, such as
the International Summer Program, Harvard Summer School
Program, Retreat, Joint Workshops, Graduate Students Tutorial Meeting, etc. The IT team designs and maintains the RCAI’s
unique server and databases, fixes PC troubles and supports
scientific meetings and conferences both inside and outside
RCAI. The facilities staff gives advice on RCAI facilities andarranges construction required in remodeling laboratories andinstalling large equipment, etc.
Owing to the tremendous efforts by Chief Assistants and
AdmCO members, the reorganization has made great progress. A new data sharing system has been installed, whichenables each member to share information, creating an environment where they can learn from each other. Using thiscentralized approach, the AdmCO is now able to provide
smooth and efficient administrative support under the same
standards to all RCAI laboratories. Among the AdmCO’s contribution to the Center, the highlight of this year is the development of an RCAI Order 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. It has provena useful system to prevent fraud and to manage various budgets simultaneously. The AdmCO, in cooperation with the
YRPD, also manages and coordinates the issues between thelaboratories and the YRPD. The strong spirit and constantefforts by the AdmCO are now supporting the fundamentalbase of the RCAI activities.

2010Part 4

Collaborative Networks

INSERM/Pasteur-RIKEN Joint Workshop August 30-31, 2010

01

T

02

03

04

he second INSERM/Pasteur-RIKEN Joint Workshop “Frontier in Immunology – from basic to clinics” washeld on August 30-31, 2010 at RCAI. The first of these workshops was held in 2008 at the La Ferme desVallees ( The Farm of the Valleys ) in Auffargis, France. Participating scientists at the 2010 meeting were fromRIKEN RCAI in Yokohama, the Pasteur Institute in Paris, and several INSERM-affiliated research institutes
throughout France. The French scientists had been attending the 14 th International Congress of Immunology inKobe before traveling to Yokohama for this small focused workshop, which was organized by Takashi Saito( RIKEN, RCAI, photo 1), Marc Bonneville ( INSERM, Nantes, photo 2 ), and Bernard Malissen ( INSERM, Centred’Immunologie de Marseille-Luminy ( CIML ), photo 3 ).
The goal of these joint workshops is to promote collaboration between Japanese and French researchers
and also between investigators in France, all of whom had the opportunity for in depth discussions at RCAI.
Many common interests became apparent in each of the different workshop sessions. The first session on“Hemopoiesis and lymphocyte development” featured talks on lymphoid progenitors and lineage commitment at
both cellular and molecular levels ( Cumano ( photo 4 ), Kawamoto, Taniuchi ), and included a spirited discussion
on whether the “common” lymphoid progenitor, a cell restricted in its potential to the generation of B and T cells,
actually exists. Next was a large session on “Lymphocyte activation and function”. The first two presentations
dealt with innate type lymphocytes, the NK cell ( Vivier ) and the γδ T cell ( Bonneville ). The next series of talks wereon various aspects of conventional T cell activation by dendritic cells ( Amigorena, Ishido ) and the signaling
events in the activated T cell ( Saito, Malissen ). In the last talk in this session, Kurosaki provided new insights into
the physical location of memory B cells in spleen, adjacent to contracted germinal centers ( GC ), their birthplace.
The final session of the day focused on regulation of allergic and inflammatory responses by intracellular signaling
molecules ( Kubo ) and an intranuclear ubiquitin E3 ligase, PDLIM2 ( T. Tanaka ).
The second day of the workshop began with three talks highlighting new developments in the area of themucosal immune system. IgA is the major mucosal antibody isotype, and Fagarasan (photo 5) described how thefollicular dendritic cells in the GC of Peyer’s patches are particularly well-equipped to promote IgA class switch-42

ing. Eberl (photo 6) described unusual innate lymphoid cells that mirror all shades of conventional helper T cells
and are highly enriched in the intestinal lamina propria, where they promote intestinal homeostasis. Ohno has
been studying the mechanisms by which so-called probiotic bacteria, in this case Bifidobacterium longum,
protect against the harmful effects of pathogenic strains.
The final session moved to the “clinics”, as promised in the workshop title. Taniguchi (photo 7) describedvery promising clinical trials of NKT cell-targeted adjuvant therapy for human non-small cell lung cancer as wellas efforts to generate NKT cells from iPS cells in vitro for potential clinical application. Chatenoud discussed
translational studies using antibodies to CD3 to promote immune tolerance in type 1 diabetes, and Fischer
described two diseases affecting lymphocyte homeostasis, including new forms of inherited hemophagocytic
lymphohistio-cytosis and somatically acquired defects in Fas-mediated apoptosis that can cause autoimmune
diseases in some cases. In a strategy combining human and animal models, Ishikawa described the use of“humanized” mice, which can be reconstituted with human immune cells, to study to pathogenesis and potential
treatment of acute myeloid leukemia.
The second INSERM/Pasteur-RIKEN Joint Workshop ended with a renewed commitment to fostering collaboration between RCAI and the French institutes, as well as plans to hold a 3 rd such workshop in the future.
Table: Program of INSERM/Pasteur-RIKEN Joint Workshop 2010
August 30
Session 1: Hemopoiesis and Lymphocyte development

Ana Cumano (Pasteur)

Heterogeneity of the common lymphoid progenitors in fetal liver and bone marrow

Hiroshi Kawamoto (RIKEN RCAI)

Termination of myeloid potential in T progenitors: an essential developmental checkpoint for
production of the T cell lineage

Ichiro Taniuchi (RIKEN RCAI)

Regulation of thymocyte developments by the silencers

Session 2 : Lymphocyte activation and function

Eric Vivier (INSERM, CIML)

Regulation of natural killer cell function

Marc Bonneville (INSERM, Nantes)

Key role played by a butyrophilin family gene product in human γδ T cell activation

Sebastian Amigorena (INSERM, Institut Curie)

Imaging FoxP3+ regulatory T cells in vivo: insights into their mechanisms of action

Satoshi Ishido (RIKEN RCAI)

MARCH-I: a new regulator of dendritic cell function

Takashi Saito (RIKEN RCAI)

Imaging of positive/negative T cell co-stimulation

Bernard Malissen (INSERM, CIML)

How T cells keep protein tyrosine kinase activity in check?

Tomohiro Kurosaki (RIKEN RCAI)

Localization of IgM and IgG memory B cells

Jean Claude Weill (INSERM, Necker)

Multiple layers of B cell memory with different effector functions

Session 3 : Immune regulation and inflammation

Masato Kubo (RIKEN RCAI)

Role of IL-6-STAT3-SOCS3 in psoriatic skin inflammatory disease

Takashi Tanaka (RIKEN RCAI)PDLIM2, a nuclear ubiquitin E3 ligase, negatively regulates inflammatory responses

August 31
Sidonia Fagarasan (RIKEN RCAI)

New aspects of IgA synthesis in gut Peyer’s patches

Gerard Eberl (INSERM/Pasteur)

Symbiotic microbiota and the development of the immune system

Hiroshi Ohno (RIKEN RCAI)

Multi-omics analysis for probiotic effect of bifidobacteria in a gnotobiotic mouse model ofO157 infection

Session 4 : Immune diseases and therapy

Masaru Taniguchi (RIKEN RCAI)NKT cell-mediated adjuvant activity

Lucienne Chatenoud (INSERM, Necker)

Modalities of immune tolerance induction in primed hosts

Alain Fischer (INSERM, Necker)

Inherited diseases of lymphocyte homeostasis

Fumihiko Ishikawa (RIKEN RCAI)

Creating therapeutic strategies targeting human leukemia stem cells

05

06

07

43

LIAI-RCAI Joint Workshop November 11-12, 2010

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04

CAI and the La Jolla Institute for Allergy and Immunology ( LIAI ) held a Joint Workshop "New Horizon in
Immune Regulation: Towards Disease Intervention" on November 11-12, 2010 at RCAI. RCAI and LIAI
established a collaborative agreement in 2004, and since then have held a series of joint workshops to facilitate
research collaboration. The first meeting was held at LIAI in 2006 and the second at RCAI in 2008. In this thirdworkshop, eleven researchers from LIAI and nine from RCAI presented their recent findings.
The first day began with the topics of developmental lineages of lymphocytes, transcriptional control andlineage plasticity. Kawamoto ( RCAI ) explained that reduction of IL-7 triggers upregulation of Bcl11b and enforcestransition from DN2 to DN3 stage. Taniuchi ( RCAI ) discussed transcriptional control of CD4/CD8 lineage choice
and described interesting candidates for the regulator of ThPOK silencer. Cheroutre ( LIAI, photo 1) explained
how gut-specific stimulation reverses ThPOK activity and gives rise to CD4CD8αα T cells in the gut. Hori ( RCAI )discussed the lineage stability and plasticity of Foxp3+ T cells, and Rao ( LIAI, photo 2 ) described how DNA
domain-swap by FOXP3 dimers is important for suppressor activity. Kurosaki ( RCAI ) described his studies ofmemory B cells and suggested that the localization of these cells adjacent to the germinal center ( GC ) is important for their survival and quick reactivation during a memory response.
The second day started with topics related to allergy. Kawakami ( LIAI ) reported the development of an
atopic dermatitis-like skin inflammation in PLCβ3KO mice. Mast cells were abundant in the skin due to Stat5dependent IL-3 mediated proliferation of mast cells. SNP analysis revealed significant association of PLCb3,
STAT5A and STAT5B genes with human atopic dermatitis. Croft ( LIAI ) described how LIGHT, a member of thetumor necrosis factor receptor family, controls asthmatic airway remodeling and Th2 persistence, and Nishida( RCAI ) introduced ARF1 as the regulator of granule translocation during mast cell degranulation. The topic thenmoved to signaling and cellular dynamics. T. Okada ( RCAI ) and von Herrath ( LIAI, photo 3 ) described theirstudies using two-photon microscopy. Okada analyzed BCL6 expression in the GC and found that upregulationof BCL6 in pre-GC B cells contributed to their interactions with helper T cells and their entry into GC clusters.
After the development of Tfh cells, BCL6 was gradually downmodulated and the IL-7 receptor was upregulated.

44von Herrath showed that the IL-21R was required for pancreatic DCs
to express CCR7 after antigen uptake and then to provide the antigen,
MHCII and CD86 to autoreactive effector cells in draining lymph nodes.
Thus he speculated that IL-21R deficiency causes defects in CD4 T
cells and antigen presentation, which prevents autoreactive CD8T cells
from entering the pancreatic islets and causing type1 diabetes.
During the meeting, Mitchell Kronenberg ( photo 4 ), President of
LIAI, talked about how LIAI and RCAI share a few things in common. Both names contain "Allergy and Immunology", and, as the names indicate, they share the same mission. Both institutes are about the same size, andboth have an important relationship with Kimishige Ishizaka, who discovered IgE. Ishizaka is the founder of LIAI
and the Special Advisor of RCAI. "It is so natural that both institutes collaborate with each other to achieve theirsame mission," said Kronenberg. Cheroutre and Taniuchi continue a collaboration that began in 2005, when
they received RCAI's International Collaboration Award. Aside from them, most researchers from the two institutes already knew each other, and in the resulting friendly atmosphere there were many questions and enjoyable
overtime discussions. The joint workshop provided a good occasion for the researchers to expand and deepen
their collaborative relationships.
The period of the joint workshop coincidentally overlapped with the Asia-Pacific Economic Cooperation( APEC ) meeting in Yokohama. No hotels were available in Yokohama and traffic was under strict control for
security reasons. Thanks to the efforts of the organizer ( Takashi Saito ) and the secretariats ( Hiroko Yamaguchi,
Toshiko Nakamura, and Sachiko Haraguchi ) this joint workshop went smoothly despite this unusual situation.

Table : Program of LIAI-RCAI Joint Workshop 2010
November 11
Session 1. Development & Selection of Lymphocytes

Hiroshi Kawamoto (RIKEN RCAI)

Chair: Hilde Cheroutre & Shohei Hori

Retention and termination of myeloid potential in T cell progenitors: An essential developmental
checkpoint for production of the T cell

Ichiro Taniuchi (RIKEN RCAI)

Transcriptional control of CD4/CD8 lineage choice

Hilde Cheroutre (LIAI)

Stretching plasticity: mature CD4 effector T cells switch from the Th- to the CTL-lineage

Tomohiro Kurosaki (RIKEN RCAI)

Heterogeneity of memory B cells of different isotypes

Session 2. Immune Homeostasis & Mucosal Immunity

Chair: Hilde Cheroutre & Shohei Hori

Shohei Hori (RIKEN RCAI)On the origin and nature of Foxp3+ T cells that exhibit developmental plasticity

Anjana Rao (LIAI)

Structure of a domain-swapped FOXP3 dimer on DNA and its function in regulatory T cells

Hiroshi Ohno (RIKEN RCAI)

Spontaneous colitis developed in mice deficient in AP-1B, the epithelium-specific polarized sorting
factorNovember 12
Session 3. Mechanism & Intervention of Allergy

Chair: Joel Linden & Takashi Saito

Toshiaki Kawakami (LIAI)

Phospholipase C-b3 deficiency predisposes to atopic dermatitis-like skin inflammation in a mastcell-dependent manner

Michael Croft (LIAI)

LIGHT interactions control asthmatic airway remodeling and Th2 cell survival

Keigo Nishida (RIKEN RCAI)The adaptor molecule Gab2, via PI-3K, regulates ARF1 in Fc epsilon RI-mediated granuletranslocation and mast cell degranulation

Session 4. Dynamics & Signaling of Immune Response

Chair: Joel Linden & Takashi Saito

Patrick Hogan (LIAI)

Calcium signalling via the CRAC channel and the calcineurin-NFAT pathway

Amnon Altman (LIAI)

The molecular basis for PKCθ function and recruitment to the immunological synapse: A novel drug
target opportunity?

Takaharu Okada (RIKEN RCAI)BCL6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell
heterogeneity

Klaus Ley (LIAI)

New macrophage phenotypes

Session 5. Innate & Infection Immunity

Chair: Chris Benedict & Masato Tanaka

Chris Benedict (LIAI)

Cytomegalovirus suppression of TRAIL death receptors attenuates persistent infection

Tsuneyasu Kaisho (RIKEN RCAI)

Molecular mechanisms for dendritic cell responses against nucleic acid adjuvants

Mitchell Kronenberg (LIAI)

Recognition of microbial and environmental antigens by invariant Natural Killer T cells

Session 6. Disease Regulation

Chair: Chris Benedict & Masato Tanaka

Matthias von Herrath (LIAI)

IL-21R deficiency causes defects in CD4 T cells and antigen presentation preventing autoreactive
CD8 T cells from entering pancreatic islets and causing type 1 diabetes

Mariko Okada-Hatakeyama (RIKEN RCAI)

Modeling cellular signaling functions for cell differentiation

Joel Linden (LIAI)

Engagement of A 2A adenosine receptors suppresses innate and adaptive immune responses

45

New Zealand-RIKEN-CHIBA Joint Workshop June 14-16, 2010

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mmunity is one of our fundamental physiological systems whose normal task is to combat pathogens (e.g.
viruses, bacteria and fungi ) and maintain our health. On the other hand, dysregulated or excessive immunity
may cause serious immune-mediated diseases (e.g. allergy and autoimmune disease). Therefore, it is very
important to understand how the immune system is regulated, and this knowledge will help us to develop
strategies for the treatment of diseases, including vaccines for infectious disease. All countries share this vision
for the future direction of medical science. In order to strengthen the relationship between immunologists ofJapan and New Zealand (NZ) and thus hasten the realization of this goal, RIKEN, Chiba University and the NZ
Ministry of Research, Science and Technology (MoRST ) held the NZ-RIKEN-CHIBA Joint Workshop on June
14-16 at RCAI and Chiba University. The goals of the meeting were threefold: 1) to bring together leading experts
in immunology and translational research to exchange current state-of-the-art research findings, 2) to identify
difficulties in terms of applying current basic knowledge to the clinic and discuss how they can be overcome,and 3) to identify opportunities for interactions and collaborations at the level of individual investigators.
In the first session, the initial step of many immune responses, i.e. the interaction between T cells andantigen-presenting cells ( APC ), was discussed. Since dendritic cells (DCs) are potent antigen-presenting cells, it
is important to understand how their development and function are regulated. RCAI members, Sato and Kaisho
described their studies on the molecular basis of DC development, the importance of DC-mediated immune
regulation and possible clinical application of DCs. Specifically, they presented data about the discovery of a newtranscription factor important for DC development, novel immune regulation by specific subset of DCs andestablishment of DC-mediated immunotherapy for cancers. T cells are stimulated not only by DCs but also bymacrophages. Macrophage-mediated immune regulation was discussed by M. Tanaka (RCAI) and La Flamme(NZ, photo 1). The discussions on APCs were extended to T cell-mediated immune regulation by Painter, Hermans and Bogle ( NZ ); how the function of T cells can be regulated and what is the most effective strategy for T
cell regulation by Rod Dunbar (NZ, photo 2). From the clinical point of view, the molecular basis and possible
treatment of allergic disorders were intensively discussed by Nakayama (Chiba Univ., photo 3), Okamoto (Chiba

46

Photo 4: From left; Shigeyuki Yokoyama (Director, RIKEN Systems and Structural Biology Center), Hiroshi Ikukawa (Director,
RIKEN Yokohama Institute Promotion Division), Kenji Okuma (Director, RIKEN Yokohama Institute), Kenji Takeda(RIKEN Executive Director), Hon. Dr. Wayne Mapp (Minister for Research Science and Technology, New Zealand), Ian
Kennedy (New Zealand Ambassador to Japan), Masaru Taniguchi (Director, RIKEN RCAI) and Takashi Saito (Deputy
Director, RIKEN RCAI)

Univ.) and Fujii (RCAI). Last, on the agenda was a discussion of bacterial pathogenesis and vaccine development.
During this meeting, there were constructive and valuable exchanges on both sides. Based on this interaction, Hook (NZ ) initiated the preparation of collaborative studies with Ishii (RCAI ). In addition, Hon. Dr. Mapp,
Minister for NZ MoRST ( photo 4) visited RIKEN Yokohama Institute and RCAI on Oct. 13, 2010. Thus, this joint
meeting successfully fulfilled its aims, and hopefully the collaborations between NZ and Japan will result in great
contributions to basic science and translational research in immunology.

Table: Program of New Zealand-RIKEN-Chiba Joint Workshop
I : Immune activation and regulation

Chair : Takashi Saito

Gavin F. Painter, Industrial Research Limited

Forodesine: a rationally designed enzyme inhibitor blocking human T-cell proliferation

Ian F. Hermans, Malaghan Institute of Medical
Research

Strategies to enhance CD8+ T cell responses to vaccination

Gib Bogle, Auckland Bioengineering InstituteMobilizing the T cell army - agent-based simulation of T cell activation and proliferation in a lymph
node

Katsuaki Sato, RIKEN RCAI

Crucial role of plasmacytoid dendritic cells for the regulation of inflammation and T cell immunity invivo

Hiroshi Nakajima, Chiba University

Regulation of IL-21 production in CD4+ T cells

II : Infection, autoimmune, allergy and vaccine

Chair : Tsuneyasu Kaisho

Anne Camille La Flamme, Victoria University of
Wellington

Regulating inflammation by altering macrophage activation

Sarah Hook, University of Otago

Modifying immune responses to vaccines through formulation

Tsuneyasu Kaisho, RIKEN RCAI

Molecular mechanisms for dendritic cell heterogeneity

Masato Tanaka, RIKEN RCAI

Immune regulation by apoptotic cell clearance

Masafumi Arima, Chiba University

A critical role of the IL-4 intron enhancer in chromatin remodeling of Th2 cytokine gene loci andallergic response

III : Immunotherapy

Chair : Toshinori Nakayama

John Fraser, University of Auckland

Mechanisms of staphylococcal virulence and pathogenicity

Rod Dunbar, University of Auckland

Programming human T cells

Toshinori Nakayama, Chiba University

iNKT cell-based immunotherapy for cancer

Yoshitaka Okamoto, Chiba University

Development of effective mucosal immunotherapy for allergic rhinitis

Shin-ichiro Fujii, RIKEN RCAIiNKT cell-triggered in vivo DC targeting immunotherapy

47

The 17 th International RUNX Workshop July 11-14, 2010

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he 17 th International RUNX Workshop was held at The Oriental Hotel Hiroshima, Hiroshima City in Japan on
July 11-14, 2010. The meeting was very successful, with about 100 attendees including 61 scientists from
12 overseas countries ( Israel, France, Germany, United Kingdom, Scotland, USA, India, Thailand, Singapore,
China, Taiwan and Korea ).
The RUNX gene family was identified in 1993 based on studies from multiple fields including virology, oncology and developmental biology. This brief history of discovery already indicates the variety and complexity of thebiological functions of the RUNX proteins, a situation that stimulated researchers in the field to set up a joint
interdisciplinary RUNX workshop. The 1st RUNX workshop was held in 1994, with just a few participating laboratories, and has been held annually since then. The International RUNX Workshop has become well recognized
all over the world because of the rapid growth of this research area as well as a meeting spirit of sharing unpublished results, which has led to several epoch-making presentations at these workshops .
The International RUNX Workshop came back to Japan this year, for the first time since 2000, when Yoshiaki
Ito, Kyoto University organized the 7 th RUNX workshop in Kyoto. Ichiro Taniuchi ( photo 1) from RIKEN, ResearchCenter for Allergy and Immunology, who is a president of The 17th International RUNX Workshop, selected
Hiroshima as the meeting venue. Hiroshima suffered the disaster of the Atomic Bomb attack in 1945 and thepeople of Hiroshima have been afflicted with many diseases, mainly leukemias. Considering that one of the major
topics in the RUNX workshop was leukemogenesis by RUNX1/AML1 dysfunction, as well as in keeping withthe contemporary international movement for the abolition of nuclear weapons, Taniuchi decided to havethe meeting at Hiroshima. Indeed, almost all participants visited the Hiroshima Peace Memorial Museum during
the meeting.
The meeting program started with a talk by Yoshiaki Ito ( photo 2 ), who is recognized as “a father of RUNX”
for his pioneering studies on the function of RUNX3 in preventing gastrointestinal tract (GIT ) epithelium carcinogenesis. Other topics discussed during the meeting included hematopoietic stem cell generation, lymphocyte
development, bone and cartilage formation, neuronal development, and leukemogenesis. As in other fields,
genome wide analyses incorporating novel technologies such as ‘omics’ studies, ChIP-sequence and RNAilibrary based screening, are widely used in the RUNX field. Reflecting the close relationships that have developed
among regular participants in this workshop, which has been ongoing for nearly two decades, many issues weredeeply and, to some extent emotionally, discussed again in Hiroshima. Historically, this workshop has highlighted
different model organisms, from C. elegans, Drosophila, all the way to mouse. This year, the octopus was shownby a chairperson of the model animal session (photo 3), but it soon turned out to be an expression of sympathy
for two disappointed attendees from the Netherlands. The final of the FIFA World Cup, in which Spain defeatedthe Netherlands after extra time, fell on the second day of the workshop and the octopus on the screen was theworld-renowned psychic octopus Paul, who successfully predicted the winners of multiple soccer matches.
The 18th international RUNX Workshop will be held next in San Diego, USA.

48

Systems Biology Open LaboratorySystems biology is a newly emerging field of biology and thus its definition still
remains multivalent. However, immunologists consider that the immune system
is one of the most attractive fields in biology from a systems biological viewpoint
because of its high complexity and dynamics. To uncover the secrets of the immune
systems, we think that multidisciplinary approaches beyond conventional immunology
will be indispensable. With this in mind, the Systems Biology Open Lab has beenlaunched to function as a gateway for researchers outside of immunology. At theoutset, researchers in mathematics and systems engineering have been invited to join the Open Lab. However, it is
not generally straightforward to exchange ideas among multidisciplinary researchers (e.g., mathematicians andimmunologists) mainly because the vocabulary used in their discussions is not shared at all, even when describing
the same biological events. To solve this problem, we invited young researchers in mathematics and systems
engineering to RCAI and set up opportunities for free discussion with them about some current problems in immunology (Photo). Three main topics were discussed in this framework: Modeling of CD4 T cell proliferation and differentiation, granulopoiesis, and the ecology of gut microbiota and the immune system. These discussions withmathematicians have given us valuable lessons in how to make interdisciplinary collaboration work. In this regard,
collaboration with mathematicians and systems engineers marks only the beginning of the systems biology initiativeat RCAI and is just a part of what will become the “systems immunology” collaborative network. Given that themission of RCAI is to prevent and/or regulate immune diseases, the direction of the systems biology initiative at RCAI
should be the same as its mission. To borrow a phrase from the systems biological lexicon, what we ultimately
expect from the systems biology initiative is the ability to fix a global immune system by applying a local action. This
capacity requires knowledge of how the entire system operates, which is very difficult to obtain using strictly reductive scientific approaches. While the systems biology initiative at RCAI has just begun, the Systems Biology OpenLab will keep serving as an incubator for multidisciplinary collaboration, not only for theoretical/mathematical
approaches but also in the development of quantitative measurement technology to grapple with the problems ofcomplex human immune diseases.

Multidisciplinary Research ProjectsW

ith the aim of developing new paradigms in immunology, RCAI launched a program in 2009 to support alimited number of multidisciplinary collaborative projects led by RCAI researchers. The program provides
5-10 million JPY/year to each collaborative research projects for up to five years.
In FY2010, eight projects were selected and received the support.

Table : Awardees of RCAI Multidisciplinary Research Projects 2010
Year

Name

Project Title

Collaborators

2009-

Hiroshi Ohno

Genomic, transcriptomic and metabolomic
analysis of the human ulcerous colitis

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

2009-

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

Creation of the artificial immune cells

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

2009-

Satoshi Ishido

Modeling of antigen presentation2009-

Hisaaki Shinohara

Modeling of NF-kB activation signals

Yuji Sugita (RIKEN Advanced Science Institute)Mariko Okada-Hatakeyama (RIKEN RCAI /RIKEN Advanced Science Institute)

2009-

Ichiro Taniuchi

Regulation of gene expressions for thymic
development

Atsushi Mochizuki (RIKEN Advanced Science Institute)

2010-

Tsuneyasu Kaisho

Generation of antibodies against chemokine
receptor XCR1 and elucidation of its crystal
structure

Shigeyuki Yokoyama
Tomomi Someyama (RIKEN Systems and Structural Biology Center)

2010-

Haruhiko Koseki

Elucidation of the lymphocyte differentiation
mediated by alternative splicing

Yoshinori Naoe (National Center for Geriatrics and Gerontology)

2010-

Takashi Saito

Elucidation of the structure and function of
the immune receptor complex

Shigeyuki Yokoyama
Mikako Shirouzu (RIKEN Systems and Structural Biology Center)

49

International Research Collaboration AwardT

he RCAI International Research Collaboration Award is a unique program supporting researchers outside ofJapan 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 to each 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, 15 projects have been funded ( Table ) and the collaborations have resulted
in several important papers. 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 ).
Dustin and Saito published their work on T cell microclusters in Nat. Immunol. (2005) and Immunity (2006). Ewijk
and Kawamoto’s work on thymic progenitor cells resulted in three papers in Development (2006) and Mol.
Immunol. ( 2009 and 2010 ). Bix and Kubo published their work on the IL4 repressor in Nat. Immunol (2009), andEllemeier and Taniuchi published their study on transcription factor network for CD4/CD8 lineage choice inNat. Immunol. ( 2010 ).

Table : Awardees of RCAI International Collaboration Award Program

50

Year

Host Lab.

Title of ResearchAwardee20042006

Takashi
Saito

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

Dr. Michael DUSTIN
New York University School of Medicine

20042006

Hiroshi
Kawamoto

Regulatory role of lymphoid progenitors during development ofthymic microenvironments

Dr. Willem van EWIJK
Leiden University Medical Center

20042006

Haruhiko
Koseki

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

Dr. Miguel VIDAL
Centro de Investigaciones Biologicas, CSIC

20042005

MasaruTaniguchi

Role of NKT cells in TSLP-mediated allergic inflammation

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

20042006

Ji-Yang
Wang

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

Dr. Peter D. BURROWS
University of Alabama at Birmingham

20052007

IchiroTaniuchiStudy of T cell differentiation mediated by regulated expressionof CD8 genes

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

20052007

Masato Kubo

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

Dr. Mark BIX
University of Washington, Seattle, Washington

20052006

Yasuyuki
Ishii

Gene-array analysis and proteomics of Th2 tolerance

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

20052007

Osami
Kanagawa

Visualization of STAT protein in the cytokine mediated signaling
at a single molecular level

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

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

20062008

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

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

20072008

TakeshiWatanabe

A study on the spleen and lymph nodes mesenchymal cells
that participate in the assembly of artificial secondary lymphoidorgansDr. Andrea BRENDOLAN
Cornell University Medical Center, Department of Cell andDevelopmental Biology

20072009

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

Dr. Motomi OSATO and Dr. Yoshiaki ITO
Institute of Molecular and Cell Biology, National University ofSingapore20072009

Sidonia
Fagarasan

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

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

2010-

MarikoOkadaHatakeyama

Proteomics based-quantitative analysis of signal-transcriptional
networkDr. Boris KHOLODENKO
University College Dublin, Ireland
Dr. Richard JONES
University of Chicago, USA

2010Part 5

Outreach Activities

RIKEN Yokohama Open
Campus July 3, 2010

R

IKEN Yokohama Institute Open Campus was held on
July 3, 2010. There were 2,629 visitors, including
1,757 adults and 872 children. Fumihiko Ishikawa, Leaderof the Research Unit for Human Disease Model, gave aseminar “Humanized Mouse: Challenges for the New
Medical Care and the Drug Development.” He first introduced what humanized mice are, and then described how
these mice can contribute to new medical care for incurable diseases. The audience spanned many generations,students from high schools and universities, young
couples, families and retired folks, and they were surprised
to learn that such new technology is becoming a reality.
The seminar can be viewed on the RIKEN Channel ofYouTube ( in Japanese only ) < http: //www.youtube.com/
watch?v=Qrq-g230M0o >.
Twenty RCAI teams exhibited posters and theresearchers explained their projects to the visitors. Hiroshi
Kawamoto and the members of the Laboratory for Lymphocyte Development gave hands-on practice to isolate
lymphocytes using the fluorescence activated cell sorter.
After learning the basics of lymphocyte development andthe principles of the cell sorter, the twenty participants
were divided into groups and isolated CD4+CD8+T cells
from mice. “I took this course before, but came backagain. To me, this is one of the highlights of this Open
Campus,” one participant explained.

Tokyo Metropolitan
Assembly Members
Visited RCAI July 15, 2010

C

edar pollinosis afflicts an estimated more than 20% of
the Japanese population, and it is said to cause
serious economic losses every year. Twenty-five members of
the Tokyo Metropolitan Assembly, mainly members of
the Committee for Countermeasures against Pollinosis,
visited RCAI on July 15, 2010. After hearing the presentation
about RCAI’s strategies for allergy research by Masaru Taniguchi, Director of RCAI, they had many questions and active
discussions on the development of cedar pollinosis vaccines. Because RCAI is the only research institute for immunology and allergy in Japan, there is a tremendous social
expectation for the development of allergy vaccines (Seealso page 24 in the Technology and Innovation section ).

Immunology Workshop forHigh School Students
July 30, 2010E

very year, RCAI holds one-day workshop on immunology for high school students in Kanagawa prefecture.
The program includes lectures by RCAI researchers, handson experiments, group discussions and presentations. The
sixth of these workshops was held on July 30, 2010.
Twenty-three students and four teachers participatedfrom Yokohama Science Frontier High School, Hakuyo
High School, Yokosuka High School and Kibogaoka High52

School. In the morning session, there were two lectures,"Influenza virus and immunity" by Tomohiro Kurosaki and"Basics of immunology" by Hisahiro Yoshida. After they
enjoyed lunch together with RCAI researchers, the students
experienced hands-on practice of mouse dissections in theafternoon. Then the students were divided into four groups
and given a discussion question "Imagine that a virus pandemic occurred in Asia. What kind of virus is that, and how
should we react?" Each group made a short presentationon the question, and the first and second place winners
were announced at the end of the program. Nine researchers, technicians and graduate students from RCAI volunteered to help with the program this year. "It was very
difficult to trim each organ. I'd like to thank the staff forteaching me so well during the dissection. I could understand well by dissecting real organs, and I also realized theimportance of life," a student said. "It was a stimulating day
and I am now really thinking of my future path," anotherstudent said.

research in an easily understood manner. The second talkwas "The day we were defeated by dinosaurs" by Harukazu Suzuki (OSC ). He described how his gene transcription-network project was published in Cell, but the news
did not appear in newspapers or on TV, because, unfortunately on the same day, a discovery concerning the extinction of dinosaurs was published in Science. He discussed
how outreach activity is becoming important in order tofacilitate the people's understanding of scientific research.
The third talk was "Allergy and Immunology" by Yasuyuki
Ishii ( RCAI ). He explained the current status of allergy
therapeutics and his allergy vaccine project. After the presentations, emcee ( Haruka Iwano, RCAI ) led the panel
discussion by the three speakers, who answered questions from the floor. "I could ask my long-time questions. I
hope this kind of event will be repeated," one participant
commented.

Science Café
September 4, 2010

Science Frontier Seminar
August 8, 2010

R

IKEN RCAI, OSC (Omics Science Center ) and GSC( Genome Science Center ) jointly held a public seminarat Yokoyama Science Frontier High School, a public school
nearby the RIKEN Yokohama Institute. This seminar was
one of a series of events planned to celebrate the 10th
anniversary of the RIKEN Yokohama Institute, and there
were 52 participants. Such a joint outreach activity by the
RIKEN Centers was the first of its kind apart from the annual
open campus. The first talk by Yoichi Gondo (GSC) wasentitled "Mouse tells the genome : What are the genome,
DNA and genes?" He explained the importance of genomeR

CAI held a Science Café on Saturday, Sep. 4 at Kanagawa Prefectural Kawasaki Library. Hiroshi Kawamoto( Leader, Laboratory for Lymphocyte Development) introduced a story “Making iPS cells from lymphocytes.” Fiftyfive participants, from high school students to retired folks,
learned about iPS cells that Kawamoto and colleagues
generated from NKT cells and their possible use for cancer
therapies. Kawamoto’s original illustrations, a video of hisamateur rock band “Negative Selection,” and lots of humorous stories helped the participants understand the topic
easily and feel more familiar with researchers. A high school
girl said that she was worrying about what she should
major in at the university, and Kawamoto told her that she

53

doesn’t have to be worried too much because immunology
is related to various scientific fields, and he invited her tovisit his laboratory.
This was the first Science Café organized by RCAIwith the support of the RIKEN Yokohama Institute. It wasplanned as one of a series of events to celebrate the 10th
anniversary of the RIKEN Yokohama Institute, but becauseof its success, the Yokohama Institute is considering holding these kinds of outreach events continuously.

RCAI Participated in the
Annual Festival of the
Yokohama Science FrontierHigh School Oct. 2-3, 2010T

he Yokohama Science Frontier High School is a new
public high school established in 2009, and locatedin Tsurumi-ono near the RIKEN Yokohama Institute. The
school emphasizes the teaching of advanced science andtechnology, and RIKEN Yokohama Institute closely provides support for the students’ education. Akiyoshi Wada,
the first Director of the RIKEN Genome Science Center, is
now the Super Advisor of the high school, and RCAI’s Hiroshi Kawamoto (Leader, Laboratory for Lymphocyte Development) is also an advisor there.
On Saturday and Sunday, Oct. 2nd and 3rd, Kawamoto participated in the high school’s annual festival andused a poster to explain the generation of NKT-iPS cells forcancer therapy. There were 4,586 visitors to the entire
school festival, mainly high school students, teachers,
neighbors, junior high school students who are interested inentering the school and their families. Other research institutes and companies also prepared booths at the festival.
“It was good to talk with researchers on this occasion
and get to know RIKEN’s activities,” one student said. “The
number of visitors was nearly twice that of RIKEN Yokohama Institute’s Open Campus. This may be a good occasion to communicate with young students and help them todevelop interests in science,” a RIKEN staff member noted.

54

Celebration of RIKEN Yokohama
Institute 10 th Anniversary
Nov. 25, 2010T

he RIKEN Yokohama Institute celebrated the 10th
anniversary of its establishment on Nov. 25 at the
Yokohama Kaikou Memorial Museum. There were 270 participants including 93 invited guests. After the opening
remarks by RIKEN President Noyori and speeches by theguests, there was a two-hour panel discussion on “Scienceand Society”. The discussants were Mitsuru Miyata (chief
editor of Nikkei BP publishing ), Yuji Kagohashi ( Torii pharmaceutical ), Satoshi Kawakami ( DOWA eco-system ),Yoshifumi Nishimura ( Yokohama City University ) and Directors of RIKEN Yokohama Institutes; Masaru Taniguchi( RCAI ), Kazuo Shinozaki ( PSC ), Naoyuki Kamatani ( CGM ),Yoshihide Hayashizaki ( OSC ), Shigeyuki Yokoyama ( SSBC ),Tetsuro Toyoda ( BASE ) and Yoshiyuki Nagai ( CRNID ).
During 2010, RIKEN Yokohama Institute researchers
had continued a series of brainstorming discussions about
the future direction of the institute and how they can collaborate together. From these discussions, a common keyword “Life and Environment” emerged, and they started aconsideration of actual collaboration schemes. To expand
this discussion, they decided to use the occasion of theanniversary event to reconsider the relationship between
science and the society, and the functions of RIKEN.
According to the new policy drafted by the Council for
Science and Technology Policy, the government will shift theiremphasis from "Science and Technology" to "Science, Technology and Innovation." Social contributions resulting fromscientific research are now more expected by the society.
The participants discussed the crucial importance of basic
science for true innovation, and how a bridging framework
can be built between basic science and solution science.
The Science and Society discussion provided thepeople an opportunity to think about the future direction ofscience in Japan. “Foundations for creative research is thekey, I thought. Japan will be respected by other countries
in that context, and it will be our pride,” a participant said.
“Preventive medicine will be important for the future Japan.
I am impressed that therapeutic medicine is only one aspectof medical sciences,” another remarked.

2010Part 6

Laboratory Activities

Laboratory for

Developmental
Genetics
Group Director

Haruhiko Koseki

Research Scientists : Yaman-Deveci, Yixin Dong, Mitsuhiro Endo, 				Yûichi Fujimura, Kyôichi Isono,
		Osamu Masui, Daisuke Yamada,		 Yuki Takada (SPDR), Jafar Sharif (SPDR), 		
		 Nayuta Yakushiji (SPDR)
Technical Staff: Hiroshi Kajita, Iyo Kataoka,
		Kayoko Katsuyama, Fuyuko Kezuka,		Yôko Koseki, Tamie Morisawa,		Naoko Ohnaga, Naomi Otsuka, Rie Suzuki
Student TraineeT

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 the maintenance of a high-standard mouse facility at RCAI. Throughthe Animal Core Facility, the group is also responsible for thegeneration 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. Particular emphasis has been put
on epigenetic regulation mediated by the combinatorial
actions of Polycomb group ( PcG) gene products and DNA
methylation mechanisms in development and various stem
cell functions.

The role of epigenetic regulators during
development and differentiation
PcG proteins mediate heritable silencing of developmental
regulators by forming two distinct multimeric protein complexes, Polycomb repressive complexes -1 ( PRC1) and -2( PRC2) , during development and differentiation. PRC2 is: Takanori Sawaguchi (JRA)shown to mediate histone H3K27 trimethylation ( H3K27me3)
while PRC1 recognizes H3K27me3 and mediates monoubiquitination of histone H2A ( HaAub1) . We have been investigating how PcG proteins regulate cellular differentiation. A
significant part of our study is focused on embryonic stem
cells ( ESCs) and trophectodermal stem cells ( TSCs ) , both ofwhich represent the first cell lineage diversification that
occurs during pre-implantation development. We havereported that PcG proteins Ring1A/B are indispensable forsuppressing ESC differentiation programs ( Endoh et al.,2008) . We have recently demonstrated an essential requirement of H2Aub1 for Polycomb-mediated silencing, which ismediated by the convergence of PRC2-dependent and-independent pathways ( Endoh et al., submitted ) . In parallel,
surprisingly, we found that Ring1A /B were dispensable formaintaining TSCs, whilst their differentiation into trophectoblastic giant cells ( TGCs ) required Ring1A/B by repressing
key transcription factors including Cdx2, Eomes and Sox2,
in a differentiation-dependent manner. These observations
indicate the critical involvement of PcG proteins in cell lineage choice and progressive differentiation. During the last

Recent publications
1. 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 of56

PRC2 that can differentially modulate
Polycomb activity at both the Hox
gene cluster and at Cdkn2a genes.
Mol Cell Biol. 31, 351-364 (2011)

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

Figure :Subnuclear colocalization of α and β tubulin
with Lamin-B in mouse ES cells.

few years, we have expanded this study to other cell lineages and have revealed a critical requirement of PcG proteins for neural differentiation, lymphocyte development,
spermatogenesis, and limb development. Studies to elucidate the molecular functions of PcG proteins in these lineages are currently under way.
Our previous studies have also suggested functional
interactions between PcG proteins and regulatory mechanisms for DNA methylation, and identified the SRA protein
Np95, which interacts with PcG proteins and is also a key
component of the DNA methylation maintenance machinery( Sharif et al., 2007 ) . We are focusing on the mechanisms
underlying repression of the endogenous retrotransposon
associated IAP ( intercisternal A particles ) loci, at which
Dnmt1, Np95 and PcG-related proteins are converged. Our
results suggest that Np95 may have a basal activity to
induce IAP transcription in WT cells, which can then be
much more amplified or suppressed upon receiving developmental/epigenetic cues. At present we are working toelucidate the molecular mechanisms.

Regulation of large scale chromatin structures by
epigenetic regulators
Compartmentalization in eukaryotic nuclei is important fordivision and coordination of various events that proceed
simultaneously in a single nucleus. We have shown that
PRC1 components exhibit focal accumulations, which aredesignated the “PcG body”, in primary cells and in developing embryos and identified a role for PRC1 in condensing the

3. Harada M, Murakami H, Okawa A,
Okimoto N, Hiraoka S, Nakahara T,
Akasaka R, Shiraishi Y, Futatsugi
N, Mizutani-Koseki Y, Kuroiwa
A, Shirouzu M, Yokoyama S, Taiji
M, Iseki S, Ornitz DM, Koseki H..
FGF9 monomer-dimer equilibrium

regulates extracellular matrix
affinity and tissue diffusion. Nat
Genet. 41, 289-298 (2009)

Hoxb cluster into a single Polycomb body tomediate repression. We further identified the roleof SAM domains of polyhometic homologues tomediate Polycomb body formation by linking multiple PRC1 molecules via homophilic interactions.
Polycomb body formation was further suggested
to be a process to enhance recruitment of PRC1
to polycomb target genes. This study therefore
identified an additional layer of Polycomb repression, and potentially provides new insight into
epigenetic regulation of cellular differentiation.
We have previously reported the interactionof PRC1 with various components of the spliceosome and their regulation of Polycomb repression. We have identified a critical region for thisinteraction within the AT-hook domain of Cbx2.
We further found that this region was essential
for Hox repression by using an AT-hook domain
point mutant allele. Notably, we have found thatHox-derepression in this mutant was not accompanied
by defects in local recruitment of PRC1. We are currently
investigating the interaction of PRC1 with the spliceosome
in this mutant.
In the process of affinity purification of PRC1, we haverepeatedly co-purified α and β-tubulins, proteins know
historically as primal components of microtubules. By
immunofluorescence analysis of ESCs after extraction
by detergents and high concentration of salt, we identified
a fraction of tubulins that persistently associated to the
nuclear lamina and exhibited a filamentous structure. ChIPchip analysis for tubulins and Lamin-B, a major componentof nuclear lamina, revealed significant co-occupancy of theirtarget genes in ESCs and thus confirmed the interaction of
tubulins with the nuclear lamina. By using Vinblastin, which
depolymerizes tubulins, we found that this is a highly polymerized form of tubulins. Immuno-FISH analysis for several
genes that were bound by both tubulins and Lamin-B,
showed their relocalization from the nuclear periphery to the
nuclear interior. This was accompanied by dissociation ofthese genes from Lamin-B as shown by ChIP. These observations indicated the role of nuclear tubulins is to link theirtarget to the nuclear lamina. Interestingly, tubulins extensively colocalized with HDACs at nuclear lamina and thisaccumulation of HDACs depends on tubulin polymerization.
Our data suggest that tubulins act as a scaffold for the convergence of chromatin-HDAC-nuclear lamina, and are necessary for maintaining the hypoacetylated status of genesthat associate with the nuclear lamina.

4. Endoh M., Endo T.A., Endoh T.,
Fujimura Y., Ohara O., Toyoda T.,
Otte A.P., Okano M., Brockdorff N.,
Vidal M., Koseki H. Polycomb groupproteins Ring1A/B are functionallylinked to the core transcriptional
regulatory circuitry to maintainES cell identity. Development
135,1513-1524 (2008)

57

Laboratory for

Lymphocyte
Development
Team Leader

Hiroshi Kawamoto

Research Scientists : Tomokatsu Ikawa
		 Kiyokazu Kakugawa
		 Kyoko Masuda
Research Associate : Rumi SatohTechnical Staff: Raul Vizcardo Sakoda
		 Asako Shibano
		 Chiho Matsuura
Visiting Scientists: Nagahiro Minato
		 Yoshimoto KatsuraThe major aim of our team is to elucidate the molecularmechanisms that regulate cell fate decisions in the process of lineage restriction from multipotent hematopoietic
stem cells to unipotent progenitors. A series of studies fromour laboratory on early hematopoiesis have led to a fundamental redefinition of lymphoid progenitors and the ontogeny and phylogeny of T- and B-cell development. We thus
have proposed our new model of hematopoiesis, the myeloid-based model, in which myeloid potential is retained
along with specification towards erythroid, T, and B cell lineages.

Lineage commitment of hematopoietic stem cells
progressing toward the unipotent T cellprogenitor stage
We have recently revealed that early thymic T cell progenitors that have lost B cell potential are still able to generate
myeloid cells ( Wada et al, Nature, 2008 ) . Therefore, themost critical step for construction of the T cell lineage is now
thought to be at the point where myeloid potential is terminated. We have also shown that such T cell lineage determi-

nation occurs in the midst of the intrathymic DN2 stage.
We then addressed the issue of whether such a determination step serves as a developmental checkpoint. Developmental checkpoints can be visualized when progenitors
exhibit developmental arrest at a certain stage under particular culture conditions or as a result of genetic manipulation.
We recently found that when murine hematopoietic progenitors were cultured on immobilized Notch ligand DLL4
protein in the presence of a cocktail of cytokines includinginterleukin-7, progenitors developing toward T cells werearrested and the arrested cells entered a self-renewal cycle,
while maintaining myeloid potential. Reducing the concentration of interleukin-7 promoted T cell lineage determination. A similar arrest and self-renewal of progenitors wereobserved in thymocytes of mice deficient in the transcription
factor Bcl11b. Our study thus identifies the earliest
checkpoint during T cell development and shows that it is
Bcl11b-dependent ( Ikawa et al, Science, 2010) .

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

58

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

3. Moro K., T. Yamada, M. Tanabe, T.
Takeuchi, T. Ikawa, H. Kawamoto,J-I. Furusawa, M. Ohtani, H. Fujii,
and S. Koyasu. Innate production ofTh2 cytokines by adipose tissueassociated c-Kit+Sca-1+ lymphocytes.
Nature 463: 540-544, (2010).

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

Figure1: An essential developmental checkpoint for
production of the T cell lineage
A. Immobilized DLL4 with a combination of cytokines induces
self-renewing expansion of immature thymocytes. Lin-ckit+Sca1+(LKS) progenitor cells (200 cells) from 13 dpc murine fetalliver mice were cultured on TSt-4/DLL4 cells [feeder (+)] or
immobilized Fc-DLL4 supplemented with 10 ng/ml of SCF, IL-7
and Flt3L [feeder (-)] for 14 days. Profiles for CD4 vs CD8 andc-kit vs CD25 gated on Lin- cells are shown. The cells are arrested
at the c-kit+CD25+ (DN2) stage in the feeder-free culture.
B. Bcl11b is essential for T lineage determination. Fetal liver cells from
Bcl11b+/+ or Bcl11b-/- mice (Ly5.2) were transferred into lethally
irradiated mice (Ly5.1). Adult thymocytes derived from Bcl11b-/progenitors exhibited a developmental arrest at the DN2 stage.
C. Model of normal T cell lineage determination. Non-T lineage
potential, including that for myeloid, DC, and NK lineages,of T cell progenitors is terminated during the DN2 stage,
subdividing this stage into DN2mt and DN2t stages. Bcl11b is
required for the DN2mt cells to pass through this checkpoint.
Sustained IL-7 signaling or Bcl11b deficiency induces
developmental arrest at the DN2mt stage. The arrested DN2mt
cells acquire self-renewal ability and maintain the potential
to generate macrophages, NK cells and dendritic cells.

Figure2: Arrest-restart culture system of the B
cell lineage determination step
A. LKS cells from murine fetal liver were transduced with
ER-Id3 and cultured under B cell inducing conditions in thepresence or absence of Tamoxifen. Cells are arrested at theB220+CD19- prepro-B cell stage in the Tamoxifen cultures.
B. When Tamoxifen is removed from the cultures,
cells restart B cell differentiation.

Arrest-restart in vitro system of cell-differentiation
capable of manipulating lineage commitment ofhematopoietic progenitors
The ideal experimental system for studies on the mechanisms of lineage commitment and differentiation is one inwhich commitment and differentiation can be synchronously
controlled. We succeeded in establishing such systems
where the arrest and restart of development of progenitors
can be induced at a stage prior to the critical step of early T
cell or B cell development. The merits of these systems inbasic research are ( i ) cells are homogenous, ( ii ) cells arenormal ( unlike immortalized cell lines ) , ( iii ) time course
analysis can be performed, ( iv ) a large number of cells areavailable, making it possible to analyze genome-wide gene
expression profiles and epigenetic status. Another important
potential merit is that these systems may become applicablein a clinical setting as a method to expand hematopoietic
progenitors.

In this context, the culture system in which the T-lineage
determination step can be controlled will be a useful method
for further study of the mechanisms of T cell lineage determination.
We also applied this system in the study of early B cell
development. It has been known that the developmental
arrest of cells at the multipotent pre-proB cell stage, as wellas the self-renewal of arrested progenitors, occurs in E2A
deficient progenitors. We expanded on this observation toestablish a controllable culture system in which Id protein, anatural dominant negative factor for E2A, was conjugatedwith the estrogen receptor ( ER ) and retrovirally overexpressed in murine hematopoietic progenitors. By adding
Tamoxifen to the culture medium, we were able to expand
multipotent progenitors retaining T, B and myeloid potential,
and then to restart differentiation by withdrawing Tamoxifen.
This system can be used not only for the study of B cell
development, but also for other hematooietic cells.

5. Wada H, Masuda K, Satoh R,
Kakugawa K, Ikawa, T, Katsura
Y, Kawamoto H. Adult T cellprogenitors retain myeloid potential.
Nature, 452: 768-772, (2008).

59

Laboratory forTeam Leader

Transcriptional Regulation

Ichiro Taniuchi

Research Scientists : Hirokazu Tanaka, Taku Naito, Wooseok Seo, Mari Tenno
Technical Staff: Sawako Muroi, Chizuko Miyamoto, Risa Chihara
Student Trainees: Sebastian Nieke (IPA), Hisashi WadaO

ne of the major questions in developmental biology is
how the fate of progenitor cells differentiating into
divergent lineages is determined. Even as we learn more
about cell fate determination, other questions arise, namely,
how genetic programming after lineage specification functions to establish cell identity and then how cell identity, once
established, is maintained in differentiated cells. Research
projects in my laboratory are directed toward understanding( a) how progenitor cells sense external or intrinsic stimuli
and turn on a genetic program for regulating lineage choiceand ( b) how a specific gene expression pattern is establishedduring the commitment process to become fully differentiated cells. We are addressing these questions by studying
transcription factor networks regulating T lymphocyte development. In particular, we have been studying the transcriptional regulation of lineage choice by CD4+CD8+
double-positive ( DP) thymocytes differentiating into either
CD4+ helper- or CD8+ cytotoxic-lineage T cells. Expressionof the ThPOK transcription factor is essential for development of helper-lineage cells. Our previous studies have identified a transcriptional silencer ( the ThPOK silencer ) in the
ThPOK locus and have shown that this silencer is essential
to restrict ThPOK gene expression in cells expressing an
MHC class II-restricted TCR, and thereby is necessary to

induce a cytotoxic fate in MHC class I-restricted cells. We
have shown that the Runx1/Cbfβ transcription factor complexes are essential to activate silencers in both the ThPOKand the Cd4 loci, and that ThPOK has an antagonistic function against these silencers.
We are expanding our findings to understand how theactivity of these silencers is regulated at a molecular level
and how Runx complexes are involved in immune systemdevelopment.

Mechanism regulating ThPOK gene expression
Both CD4/CD8 co-receptor expression and the specificity ofTCRs for MHC molecules correlate well with the lineage
decision outcome of CD4+CD8+ DP thymocytes. Cells
expressing MHC class I-restricted TCR differentiate into thecytotoxic-lineage and terminate Cd4 gene expression,
whereas cells expressing class II-restricted TCR give rise to
the CD4+ helper-lineage and lose Cd8 gene expression.
However, it remains unclear how differences in TCR signals
are sensed and integrated into developmental programmingin the cell nucleus of post-selection thymocytes. It has been
assumed that factors regulating Cd4/Cd8 gene expression
must also be involved in regulation of lineage choice. The
BTB/POZ family transcription factor MAZR has been identi-

Recent publications
1. Sakaguchi S., Hombauer M., Bilic
I., Naoe Y., Schebesta A., Taniuchi I.
Ellmeier W. The zinc finger protein
MAZR is part of the transcription
factor network controlling CD4/CD8
cell fate decision of DP thymocytes.
Nat. Immunol. 11, 442-48 (2010).

60

2. Setoguchi R., Tachibana M., Kitoh
A., Ono M., Naoe Y., Ohkura N.,
Yamaguchi T., Yaguchi H., Kitabayashi
I., Tsukada T., Nomura T., Miyachi Y.,
Taniuchi I. Sakaguchi S. Indispensablerole of the Runx1-Cbfβ transcription
complex for in vivo-suppressivefunction of FoxP3+ regulatory T cells.

Immunity 3, 609-20 (2009).

3. Collins A., Littman D.R., Taniuchi
I. RUNX proteins in transcription
factor networks that regulate
T-cell lineage choice. Nat. Rev.
Immunol. 9, 106-15 (2009)

Figure 1 : Cis-regulatory elements and trans-acting factors regulating expression of theThPOK gene. The expression of the ThPOK gene during T cell development isregulated mainly by combined activation and inactivation of the two enhancers and thesilencer. Binding of Runx and MAZR to the silencer is essential to exert silencer activity.
Gata3 functions as an upstream factor for ThPOK expression by activating enhancers.

fied as an important regulator of Cd8 expression. By analyzing gene targeted mice, we have shown that loss of MAZR
led to partial redirection of MHC class I-restricted thymocytes into CD4+ helper-like T cells, a response that correlated with de-repression of ThPOK. MAZR binds to the
ThPOK silencer and interacts with Runx1 protein. Thus, wedemonstrated that MAZR is part of the transcription factor
network that regulates CD8 cytotoxic lineage differentiationof DP thymocytes ( Figure 1) .
However, we still do not understand how lineage specific activity of the ThPOK silencer is regulated. In order tofind crucial molecule( s) or mechanisms that act as a switch
to reverse the ThPOK silencer activity for inducing ThPOK
expression upon engagement of class II-restricted TCRs, weare isolating novel proteins that bind to the ThPOK silencer.
In addition to neutralization of the ThPOK silencer, activation of positive regulatory elements, such as enhancers
and promoters, is required for efficient induction of ThPOK
expression. Previously, we have identified a proximalenhancer that is necessary to maintain ThPOK expression atlater developmental stages. Recently we have mapped
another enhancer in the 5’ region, in close proximity to thesilencer. Our results indicate that this enhancer, designateda thymic enhancer, plays an essential role in an efficientinduction of ThPOK expression, in part via erasing preexisting repressive epigenetic marks.

4. Naoe Y., Muroi S., Akiyama K.,
Tezuka C., Okuda T., Taniuchi I.
Repression of the Transcription
Factor Th-POK by Runx Complexes
in Cytotoxic T Cell Development.
Science 319, 816-19 (2008)

Figure 2 :		Model for possible developmental pathway of
LTi cells in fetal liver. Runx1/Cbfβ2 complexes
play important roles in regulating differentiationof α4β7− IL-7Rαmid cells into α4β7+IL-7Rαmid cells.
This is followed by Id2 and Rorγt dependent
developmental program that directs α4β7+IL7Rαmid cells to become α4β7+IL-7Rαhigh LTi cells.

Further studies on the molecular control of these regulatory elements, as well as epigenetic regulation at the ThPOK
locus, will advance our understanding of how cellular signaling initiated by external stimuli is converted into genetic
programs in the cell nucleus and is imprinted for stabilizing
cell identity.

Role of Runx complexes in lymphoidorganogenesis
Lymphoid tissue inducer ( LTi) cells are essential for thedevelopment of secondary lymphoid tissues such as lymph
nodes and Peyer’s patches. It remains unknown how specification toward an LTi-lineage is programmed in multipotent
hematopoietic progenitors. By using mice in which the function of Runx1/Cbfβ complexes was attenuated by loss ofeither the P1-Runx1 or Cbfβ2 variant protein, we found that
Runx1/Cbfβ2 complexes are necessary for development ofearly LTi precursors in fetal liver. In addition, loss of Cbfβ2,
but not P1-Runx1, resulted in an inefficient upregulation of
Rorγt in residual LTi cells in the secondary lymphoid tissue
anlagen. Our results thus revealed that Runx1/Cbfβ2 complexes regulate the differentiation of LTi cells at two stages,
an early specification toward the LTi-lineage in hematopoietic progenitors and subsequent activation of the Rorγt genein the anlagen ( Figure 2) .

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

61

Laboratory forGroup Director

Cell Signaling

Takashi Saito

Senior Research Scientist : Tadashi Yokosuka
Research Scientists : Takayuki Imanishi, Arata Takeuchi, Akiko Hashimoto-Tane, Shin-ichi Tsukumo, Zijin Guo
Technical Staff: Wakana Kobayashi, Machie Sakuma, Masako Takamatsu, Akiko Takumi, Midori Unno

Student Trainee: Minoru Sawaguchi (JRA)Visiting Scientists: Ikuo Ishige, Noriko M. Tsuji, Yasutaka Wakabayashi, Sho Yamasaki, Hu Zeng

RCAI Central Facility FACS Laboratory: Hanae Fujimoto, Yukiko Hachiman

RCAI Central Facility Confocal Laboratory : Yasutaka Wakabayashi
RCAI Central Facility mAb Laboratory(Leica): Tomomi Aoyama, Mayuko MatsudaThe long-term goal of the Cell Signaling group is to determine the molecular mechanisms of activation, functional
differentiation and homeostasis of T cells in order to be able
to modulate T cell activation/function in immunological disorders. Therefore, our group is involved in a range of projects
related to the basic mechanisms of antigen recognition, activation, differentiation, and functional regulation of T cells
from the viewpoint of signal transduction. Particularly interms of early T cell activation, the group has been using
real-time imaging analysis to analyze the dynamic regulationof the assembly of the TCR signaling complex at the immunological synapse and related downstream pathways uponantigen recognition. These analyses of regulatory mechanisms of T cell activation include the roles of co-stimulation
signals, innate-related signals and the cytoskeleton. The
group also analyzes regulation at later phases of T cell activation for cell migration and establishment of peripheral
antigen-specific effector functions.

Dynamic regulation of T cell activation andco-stimulation
We have studied the dynamic movement of signaling molecules in the process of the formation of the immunologicalsynapse ( IS ) and T cell activation upon antigen recognitionat the single-cell level. Using a combination of imaging techniques of model planar bilayers and TIRF, we could visualize
the behavior of signaling molecules during the dynamic process of T cell activation. We have identified TCR-microclusters ( MCs) as the signalsome to assemble signaling
molecules and induce activation signals, which translocate
into the center of the IS to generate the cSMAC.
In addition, we have examined the dynamic features ofco-stimulation signals by analyzing the positive co-stimulatory receptor CD28 and its negative counterpart CTLA-4
and their relationship with TCR-MCs. CD28 is initially colocalized with TCR-MCs and then accumulates into thecSMAC together with PKCθ for sustained co-stimulation.

Recent publications
1. 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-mediatednegative regulation of T cell activation.
Immunity 33, 326-339 (2010)62

2. Hashimoto-Tane A., Yokosuka T.,
Ishihara C., Sakuma M., Kobayashi
W. and Saito T. TCR-microclusters
critical for T-cell activation are formed
independently of lipid raft clustering.
Mol. Cel. Biol. 30, 3421-3429 (2010)

3. Takeuchi A., Itoh Y., Takumi A., Ishihara
C., Arase N., Yokosuka T., Koseki H.,
Yamasaki S., Takai Y., Miyoshi J.,
Ogasawara K., and Saito T. CRTAM
confers late-stage activation of
CD8+ T cells to regulate retention
within lymph node. J. Immunol.
183, 4220-4228 (2009)

4. Yokosuka T. and Saito T. Dynamic
regulation of T-cell costimulation
through TCR-CD28 microclusters
Immunol. Rev. 229, 27-40 (2009)

Figure :Spatially distinct regions of the cSMAC for positive and negative co-stimulation
Upon interaction with an antigen-presenting cell, a T cell generates TCR-microclusters (MC), which contain signaling molecules
to induce T cell activation. TCR-MCs move to the center of the interface to form the cSMAC (green). The co-stimulatory receptor
CD28 accumulates in a sub-region of the cSMAC (light green; signaling cSMAC) together with PKCθ and CARMA1 to induce
sustained co-stimulation signals. Later after the initial activation, the negative co-stimulatory receptor CTLA4, already present
in secretary granules, moves toward the membrane and accumulates in the same region as CD28-PKCθ, where it competes
with CD28 for binding to their common APC ligand, CD80/86. CD28 is then displaced from the cSMAC and T cell activationceases (leftmost). On the other hand, CTLA-4 chimera that cannot enter into the cSMAC due to its large size (rightmost), fails to
mediate suppression, indicating that the localization of CTLA-4 at the cSMAC is necessary for inhibition of T cell activation.

Later on, CTLA-4 also accumulates in the same area of thecSMAC as CD28, where it competes with CD28 for ligandbinding. Thus, we have clarified the spatio-temporal dynamic
regulation of positive/negative co-stimulation and how they
are quantitatively fine-tuned.

Cytoskeletal regulation of T cell activation
TCR-MCs generated at the periphery of the IS move to thecenter to form the cSMAC. This translocation of TCR-MCs
thus regulates TCR activation by reducing the number offunctional TCR-MCs through their degradation at thecSMAC. We analyzed the mechanism of TCR-MC translocation and found that the minus-end microtubule-associatedmotor protein, dynein complex is co-localized with TCRMCs and co-precipitated with the TCR complex. We haveshown that TCR-MCs are translocated along microtubules
to form the cSMAC. As predicted by our model, reduction of
the amount of the dynein complex or its function resulted inenhanced T cell signals and activation.

Regulation of T cell responses by innate signals
We have analyzed the interconnected activity of signaling
molecules of the innate and acquired immune systems. Particularly, after we identified the critical roles of IRAK4 in
T cell activation, we analyzed the function of TLRs expressedby T cells and found that TLR2/3/9 mediate co-stimulatory
functions whereas TLR2 directly activates Th1 cells to induce
cytokines independently of TCR stimulation. T cell co-stimulation through TLR2 is completely MyD88-dependent, similar
to situation in innate cells, however, surprisingly, theco-stimulation function of TLR3 and TLR9 was found to
be independent of MyD88/TRIF. We have identified the
optimal structure of nucleic acids for this T cell co-stimulatory activity, but the recognition of these nucleic acids
appears to be mediated by an as yet unknown novel T cellspecific sensor.

5. Yamasaki S., Matsumoto M., TakeuchiO., Matsuzawa T., Ishikawa E., Sakuma
M., Tateno H., Uno J., HirabayashiJ., Mikami Y., Takeda K., Akira S.,
and Saito T. C-type lectin Mincle isan activating receptor for pathogenic
fungus, Malassezia. Proc. Natl. Acad.
Sci. USA. 10, 1897-1902 (2009)63

Research Unit forSingle Molecule Imaging
Unit Leader		

Kumiko Sakata-Sogawa

Technical Staff : 		 Yoshiyuki SasakiT

he focus of our research is to understand immune
responses and signaling processes using the techniqueof single molecule imaging and quantification. Single molecule approaches enable us to capture transient intermediates and heterogeneous behavior, avoiding ensemble
averaging. This ability is powerful in elucidating mechanismsof cellular functions at multiple levels: 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. To this end we installed amicroscope system and we have focused on the development of a novel type of fluorescence microscopy ( HILO) for
use in single cell/single molecule studies. HILO has anadvantage for making observations inside cells, with a highsignal to noise ratio of 7.6. Further, this microscope system
has been optimized to observe immunological responses
ranging from the plasma membrane to the nucleus by introducing a system for computer control of optical devices.
For quantitative analysis of the interaction of signaling
proteins, it is important to observe multiple molecules simul-

taneously. It is also necessary to prepare many different celllines harboring target genes for high throughput data collection. To this end, we have established methods to obtain
isogenic cell lines expressing two fluorescence tagged proteins at a homogenous and low level, which is essential forthis type of analysis. We can expand this technique for multicolor imaging of single molecules.

NF-κB activation mechanism regulated by IκBα
NF-κB is an important transcription factor that activates
expression of inflammatory genes in response to a variety ofstimuli. In resting cells, NF-κB associates with IκBα and issequestered in the cytoplasm. Stimulation leads to phosphorylation of Ser residues of IkBa and its subsequent ubiquitination and proteasomal degradation. The liberated NF-κB
translocates to the nucleus and activates expression of multiple target genes, one of which is the IκBα gene. The
nascent IκBα binds to nuclear NF-κB and exports it backinto the cytoplasm, resulting in negative feedback loop.
We aim to directly confirm molecular mechanism of themultiple functions of IκBα by single molecule fluorescence

Recent publications
1. Yokosuka T., Kobayashi W., Takamatsu
M., Sakata-Sogawa K., Zeng H.,
Hashimoto-Tane A., Yagita H.,
Tokunaga M., Saito T.: Spatiotemporalbasis of CTLA-4-mediated negative
regulation of T-cell activation.
Immunity. 33 (3) 326-339 (2010)64

2. Miletic AV., Graham DB. Sakata-Sogawa
K., Hiroshima M., Hamann HJ.,
Cemerski S., Kloeppel T., Billadearu
DD., Kanagawa O., Tokunaga M. andSwat, W: Vav Links the T Cell Antigen
Receptor to the Actin Cytoskelton and
T Cell Activation Independently of
Intrinsic Guanine Nucleotide Exchange
Activity. PLoS One. 4 (8), e6599 (2009)

3. Yokosuka T., Kobayashi W., SakataSogawa K., Takamatsu M., HashimotoTane A., Dustin ML., Tokunaga
M., and Saito T.: Spatiotemporal
Regulation of T Cell Costimulation
by TCR-CD28 Microclusters andProtein Kinase C θ Translocation.
Immunity 29, 589-601 (2008)

4. Tokunaga, M., Imamoto, N.,
Sakata-Sogawa, K.: Highly inclined
thin illumination enables clear
single-molecule imaging in cells.
Nat Methods. 5, 159 -161 (2008)

Figure :Response of NF-κB and IκBα after TNF-α stimulation.
HeLa cells expressing both NF-κB-RFP and IκBα-GFP were stimulated withhTNF-α. (A) Before stimulation. (B) 30 min after stimulation. Bar: 20µm.

microscopy. For this purpose, we established HeLa cell lines
expressing GFP- IκBα and RFP-NF-κB ( p65 subunit) fusion
proteins and confirmed their co-localization in the cytoplasm.
We visualized single molecule movements of IκBα both in
the cytoplasm and nucleus, suggesting the shuttling of
NF-κB / IκBα complexes. After stimulation with TNFα, IκBαremained in the cytoplasm and disappeared, whereas p65
translocated into the nucleus. We also tested a dominantnegative IκBα mutant that lacks the phosphorylated Ser
residues. In this case there was no IκBα degradation or p65
nuclear translocation. We are currently investigating the
movements of IκBα molecules in the cytoplasm using single
molecule imaging analysis and comparing the behavior ofIκBα wild type and mutant proteins.

Dynamics of spatial-temporal regulation of NF-κB
inactivation

Tanaka and Dr. Kaisho in RCAI found that the E3 ubiquitin
ligase PDLIM2 terminates NF-κB activation by intranuclear
sequestration and degradation. However detailed mechanisms of PDLIM2 function remain unclear.
Aiming to elucidate PDLIM2 regulatory mechanisms, weestablished dual gene expressing cell lines with GFPPDLIM2 and RFP- NF-κB ( p65 ) fusion proteins. Using single
molecule fluorescence microscopy, we visualized the dynamics of PDLIM2 function. In resting cells, both proteins werelocalized in the cytoplasm. Upon stimulation, they translocated to the nucleus and colocalized. A PDLIM2 mutant
protein ( DLIM ) , which lacks the ubiquitin ligase domain, alsotranslocated to the nucleus but did not co-localize with p65
in stimulated cells. These results demonstrate that the LIM
domain is important for the control of NF-κB localization,suggesting that this domain is responsible for regulating thetranscriptional activity of NF-κB.

Inflammatory response-mediated NF-κB activation plays animportant role in the immune system. As excessive inflammatory responses cause massive damage to host cells, theregulation of NF-κB activation is indispensable. Recently Dr.

65

Laboratory for

Lymphocyte
Differentiation
Group Director

Tomohiro Kurosaki

Research Scientists : Yuuich Aiba
		 Hisaaki Shinohara
		 Kohei Kometani
Visiting Scientists: Yoshihiro Baba
		 Masanori Matsumoto
		 Rinako Nakagawa
Technical Staff: Yoko Fujii
		 Shiori Maeda
		 Miwako Tochigi
Student TraineeBcells play an essential role in the regulation of immune
responses. Upon encountering their cognate antigen, B
cells assume multiple functions including antibody production, antigen-presentation, and induction of T cell differentiation. In addition to protective roles against pathogens, B
cells also have regulatory roles, e.g. serving as negative
regulators of autoimmunity by secreting anti-inflammatory
cytokines such as interleukin-10 ( IL-10) and transforming
growth factor-β ( TGF-β ) . In order to exert such multiple B
cell functions, signals propagated through the B cell
receptor ( BCR ) , Toll-like receptors ( TLRs ) , TNF-family receptors, cytokine receptors, and chemokine receptors areimportant. The quality and quantity of the signals are dictated by multiple factors including the receptors themselves
and intracellular signaling molecules and transcription factors. Our laboratory has focused on a molecular understanding of the signaling pathways that lead to crucial B cell fatedecisions such as memory versus plasma cell differentiation.

: Saya Moriyama (JRA)Function of calcium sensors Stim1 and Stim2 in
B cells
The calcium flux triggered by BCR engagement occurs in
two waves, first a rapid but transient release of Ca 2+ fromendoplasmic reticulum ( ER) stores, and then a sustained
influx of extracellular Ca 2+ across the plasma membrane( PM ) . Initiation of this second wave is thought to result
directly from the emptying of ER Ca 2+ stores, which activates
Ca 2+ channels located in the PM, a process referred to as
store-operated Ca 2+ ( SOC) influx. Until recently, the mechanism of SOC influx was a mystery, but the identification ofseveral molecules including Stim1 ( sensor stromal interaction molecule 1) has provided long-awaited insight.
Calcium signaling is thought to be critical for multiple B
cell functions; however, supportive in vivo evidence has
been lacking. Thus, discovery of Stim1 and a sister molecule,
Stim2, has allowed us to approach this question by taking a
genetic approach. We generated mice with B cell-specificdeletions of both Stim1 and Stim2 and found that, althoughboth molecules are critically required for BCR-induced SOC
influx and proliferation in vitro, they were dispensable for B

Recent publications
1. Yasuda T., Kometani, K., Takahashi,N., Imai, Y., Aiba, Y., and Kurosaki,
T. Erk kinases control plasma
cell differentiation by regulating
expression of Blimp-1. Sci.
Signal. 4(169): ra25 (2011)

66

2. Aiba, Y., Kometani, K., Hamadate, M.,
Moriyama, S., Sakaue-Sawano, A.,
Tomura, M., Luche, H., Fehling, H.J.,
Casella, R., Kanagawa, O., Miyawaki, A.,
and Kurosaki, T. Preferential localizationof IgG memory B cells adjacent tocontracted germinal centers. Proc. Natl.
Acad. Sci. U.S.A. 107, 12192-7 (2010)

3. Hawkins, B.J., Irrinki, K.M., Lien, Y-C.,
Wang, Y., Bhanumathy, C.D., Ramasamy,S., Mallilankaraman, K., Ritchie, M.F.,
Soboloff, J., Baba, Y., Kurosaki, T.,
Joseph, S.K., Gill, D.L. and Madesh
M. S-glutahionylation activates STIM1
and alters mitochondrial homeostaisis.
J. Cell Biol. 190(3):391-405 (2010)

4. Kurosaki, T., Shinohara, H., and Baba,
Y. B Cell Signaling and Fate Decision.
Annu. Rev. Immunol. 28, 21-55 (2010)

Figure :cell development and antibody responses in vivo. However,
the ablation of Stim1 and Stim2 in B cells caused defects in
NFAT activation, B cell intrinsic IL-10 production, and suppression of an EAE model of autoimmune diseases. Thus
our results provide genetic evidence for the significance ofStim-dependent Ca 2+ signaling in B cell regulatory functions.

CIN85 regulates the canonical NF-κB pathway in
B cells
By recruiting signaling proteins into complexes, adaptor
molecules create nodes of regulation and activity. CD2AP,
the founding member of the CD2AP/CIN85 family of adaptor
proteins, was initially isolated in a yeast interaction screen asa binding partner of CD2 expressed on T cells. Subsequently,
its mammalian homologue CIN85 ( Cbl interacting protein of
85 KDa) was identified as a partner of the E3 ubiquitin ligase
Cbl. Based on coimmunoprecipitation experiments, colocalization studies, and in vitro protein-protein interaction assays
using fibroblasts, it has been proposed that CIN85 primarily
functions in endocytosis to down-regulate receptor tyrosine
kinase activity. According to this model, CIN85 constitutively
associates with endophilin and, on stimulation with growth

Simplified model of STIM-dependent
IL-10 production by B cells
B cell receptor stimulation causes PLC-γ/IP3/
IP3 receptor (IP3R)-mediated Ca2+ release fromendoplasmic reticulum (ER) Ca2+ stores. A
reduction of the Ca2+ concentration within the ER
is ‘sensed’ by STIM proteins, which in turn induce
an influx of extracellular Ca2+ across the plasma
membrane through Orai channels. The main source
for Ca2+ entry in B cells is the STIM-dependent
Ca2+ influx, which activates the calcineurin-NFAT
pathway. Nuclear translocation of NFAT promotes
expression of the anti-inflammatory cytokine
IL -10 by a subset of B cells and thereby suppresses
the development of autoimmune diseases.

factors such as epidermal growth factor ( EGF ) , complexes
with Cbl to mediate receptor down-regulation.
The same mechanism also appears to operate in mast
cells. CIN85 overexpression in the RBL-2H3 rat mast cellline accelerated the distribution of engaged FcεRI complexes, their sorting into early endosomes, and their delivery
to a lysosomal compartment for degradation. In contrast to
observations in mast cell lines, a positive role for CIN85 in
pre-TCR signaling has been recently suggested. Thus,
CIN85 may mediate distinct biological outcomes, which
depend on the type and developmental stage of each
immune cell. To determine the role of CIN85 in B cells, wehave used the mb1-Cre line to generate mice with a B cellspecific deletion of CIN85. These mice manifested impaired
T-independent type II antibody responses in vivo and diminished IKKβ activation and cellular responses to BCR crosslinking in vitro. Furthermore, introduction of a constitutively
active IKKβ corrected the defective antibody responses aswell as the cellular responses in the mutant mice. Thus, ourresults suggest that CIN85 participates in linking BCR to
IKKβ activation, thereby contributing to T-independent
immune responses.

5. Kurosaki, T., Aiba, Y., Kometani, K.,
Moriyama, S., and Takahashi, Y.
Unique properties of memory B cells ofdifferent isotypes.
Immunol. Rev. 237;104-16 (2010)67

Laboratory forTeam Leader

Immune Diversity

Ji-Yang Wang

Research Scientist: Rika OuchidaStudent Trainees: Chie Kano, Yingqian Li (IPA), Shuyin Li (IPA)

Technical Staff: Hiromi MoriDuring an adaptive immune response, resting B cells canbe activated by a variety of external stimuli including
antigen stimulation, CD40 ligation and cytokine stimulation.
The activated B cells form germinal centers ( GC) where they
expand enormously. These rapidly dividing GC B cells
undergo somatic hypermutation ( SHM) and class switch
recombination ( CSR) of their immunoglobulin ( Ig ) genes andultimately differentiate into antibody-producing plasma cells
or memory B cells. Abnormalities in B cell activation andterminal differentiation can lead to immunodeficiency, autoimmune diseases and B cell malignancies. My laboratory
focuses on the molecular mechanisms that control B cell
activation and differentiation. Specifically, we have two
research aims. The first is to understand how somatic mutations at A:T base pairs are induced in the Ig V genes of GC
B cells. The second is to identify genes highly expressed in
B and GC B cells and elucidate their physiological function
by genetic approaches.

Mechanism of A:T mutations in the GC B cells
The activation-induced cytidine deaminase ( AID) initiates
SHM by catalyzing the deamination of cytosine ( C) to uracil( U) , which generates a U : G lesion. Yet half of the somatic
mutations are induced at A : T base pairs, which are not a
direct target of AID. Using a sensitive lacZ-transgenic system to detect spontaneous genome mutations, we haveshown that GC B cells have an intrinsic propensity to generate A : T mutations. Moreover, using an in vitro mutagenesis
system that closely mirrors SHM in GC B cells, we foundthat the induction of A : T mutations is dependent on the GC
B cell environment but independent of the target gene.
Studies thus far indicate that A : T mutations are generated
by an atypical “error prone” mismatch repair ( MMR) pathwaythat utilizes the low-fidelity DNA polymerase η ( POLH) for
repair synthesis. We are now addressing the following three
non-exclusive hypotheses to explain the efficient inductionof A : T mutations in GC B cells. 1) Rapid cell division may
predispose GC B cells to utilize POLH for repair synthesis as
the high-fidelity POLδ is fully devoted to DNA replication. 2) .
GC B cells express a specific factor ( s ) that function toactively recruit POLH during DNA repair. 3) MMR components and POLH undergo specific modifications in GC B
cells, resulting in an atypical repair process not observed inother cell types. Preliminary results suggest that rapid cell

Recent publications
1. Li, Y., Gao, X. and Wang, J.-Y.
Comparison of two POLQ mutants
reveals that a polymerase-inactive
POLQ retains significant function
in tolerance to etoposide andγ-irradiation in mouse B cells.
Genes Cells 16: 973-983 (2011).

68

2. Kano, C., Ouchida, R., Kokubo, T.
and Wang J.-Y. Rapid cell divisioncontributes to efficient inductionof A/T mutations during Ig gene
hypermutation. Mol. Immunol.
15-16: 1993-1999 (2011).

3. Masuda, K., Mori, H., Ohara, O.,
Nakayama, M., Wang, J.-Y. andBurrows, P. Defining the immunologicalphenotype of Fc receptor-like B(FCRLB) deficient mice: confoundingrole of the inhibitory Fcγ RIIb. Cell.
Immunol. 266 : 24-31 (2010).

4. Ouchida, R., Kurosaki, T. and Wang,
J.-Y. A role for lysosomal-associatedprotein transmembrane 5 in thenegative regulation of surface BCRlevels and B cell activation.
J. Immunol. 185: 294-301 (2010).
(Featured in “In This Issue”)

Figure 1: Model of LAPTM5-mediated BCR degradation. Upon
antigen (Ag) stimulation, surface BCR is rapidly internalized
into the endosomes. The internalized BCR/Ag complex
can be recycled back to the surface, translocated tothe specialized late endosomal compartment rich in MHC
class II (MIIC) for Ag presentation, or transported to thelysosomes for degradation. Our results indicate that LAPTM5
functions to promote the lysosomal degradation of theinternalzied BCR and prevent its recycling to the cell surface.

division is one of the conditions that promote efficient induction of A : T mutations.

Identification and characterization of genes highly
expressed in B and GC B cells
To better understand the diverse and complex processes
regulating B cell activation and differentiation, we havefocused on genes that are highly expressed in resting B and
GC B cells. LAPTM5 is a lysosomal protein highly expressedin resting B cells but downregulated in GC B cells. We foundthat LAPTM5 negatively regulates cell surface expression of
the B cell antigen receptor ( BCR) by promoting its lysosomal
degradation ( Fig. 1) . The absence of LAPTM5 leads toFigure 2: Increased levels of serum IgM and autoantibodies inaged Laptm5-/- mice. A, Levels of serum IgM and B antidsDNA IgM and IgG. *p < 0.05; **p < 0.005 (unpaired
t-test). C, Presence of anti-nuclear antibodies in thesera of Laptm5-/- mice. Hep2 cells were stained with sera
(100-fold dilution) from WT or Laptm5 -/- mice.

increased levels of serum IgM and autoantibody production
in aged mice ( Fig. 2 ) . BCP1 is an intracellular molecule thatis specifically expressed in B cells and upregulated in GC B
cells. Analysis of BCP1-deficient B cells revealed that thismolecule negatively regulates signaling through CD19, an
important co-stimulatory molecule for B cell activation. Morerecently, in collaboration with other groups, we are analyzing
the function of the Fc receptor for IgM, which is exclusively
expressed in B and GC B cells in mice. Through further
analysis of these mice, we aim to uncover new pathways
that control the activation and terminal differentiation of B
cells during adaptive immune responses.

5. Shima, H. Takatsu, H., Fukuda, S.,
Ohmae, M., Hase, K., Kubagawa,
H., Wang, J.-Y., and Ohno, H.
Identification of TOSO/FAIM3
as an Fc receptor for IgM. Int.
Immunol. 22 : 149-156 (2010).

69

Laboratory for

Immunological
MemoryGroup Director

Toshitada Takemori

Research Scientist: Tomohiro Kaji

Technical staff: Akiko Sugimoto
		 Mitsue Hanami,
		 Natsumi Yoneda
		 Natsuki Kobayashi
		 Aika Sawada
Guest Scientists: Yoshimasa Takahashi
		 Masaki Hikida
Student TraineeM

emory B cells are antigen ( Ag ) experienced cells thatare sustained for a long period after immunization in
the absence of the immunizing Ag. These memory cells
respond to antigen stimulation more rapidly than naive B
cells, and possess efficient APC activity to antigen-primed
helper T cells ( unpublished data) . After a secondary challenge, memory cell-derived plasma cells produce large
amounts of high affinity antibodies, implying that GC B cells
are precursors to high-affinity memory B cells. However,class-switched memory B cells develop by both GC-independent and –dependent pathways ( Kaji et al. submitted) .
Our recent analyses suggest that the orphan receptor,
EBV-induced molecule 2 ( EBI2) , guides activated B cells tointerfollicular and outer follicular niche ( s) , where EBI2 ligand
is expressed. Once Bcl6 is upregulated in B cells after activation, GC-committed B cells repress the expression of
EBI2 and move to the center of the follicle where the GC
reaction takes place, generating high-affinity memory B cells
and plasma cells ( Reviewed by Pereira et al. Int. Immunol.) .
We observed that activated B cells also differentiate into IgGmemory B cells early after stimulation, prior to GC formation: Naoka Itoh

( Kaji et al. submitted) . Consistently, single cell PCR revealed
that EBI2 was highly expressed in most of these GC-independent memory B cells, but was barely detected in GC B
cells. This GC-independent memory pathway requires T celldependent B cell activation by antigen and subsequent proliferative expansion of the cells. The cells entering this pathway undergo class-switch recombination ( CSR) , but not
somatic hypermutation ( SHM) .
Thus, B cell memory sustains two classes of antibody
repertoire, one with V genes in a germline configuration andthe other accumulating SHM as a consequence of selectionin the GC. The generation of high-affinity somatic mutants isadvantageous for the system to rapidly and efficiently
exclude the initiating pathogen, although, in the process,this narrows-down cross-reactivity to any variants or related
pathogens. Of importance, the germ line encoded antibody
memory repertoire, with its cross-reacting specificities, may
provide a rapid response to both the initial pathogen and its
variants, followed by production of large amount of antibodies that are useful as a first-line of defense against common
pathogens.

Recent publications
1. Fujimura T, Yonekura S, Taniguchi Y,
Horiguchi S, Saito A, Yasueda H,
Nakayama T, Takemori T, Taniguchi
M, Sakaguchi M, Okamoto Y.
The Induced Regulatory T Cell
Level, Defined as the Proportionof IL-10Foxp3(+) Cells among70

CD25(+)CD4(+) Leukocytes, Is a
Potential Therapeutic Biomarker
for Sublingual Immunotherapy: A
Preliminary Report. Int Arch Allergy
Immunol. 2010;153(4):378-87.

2. Kishi Y, Aiba Y, Higuchi T, Furukawa
K, Tokuhisa T, Takemori T,
Tsubata T. Augmented antibody
response with premature
germinal center regressionin CD40L-transgenic mice. J
Immunol. 2010, 185(1):211-9.

3. Hikida M, Casola S, Takahashi
N, Kaji T, Takemori T, Rajewsky
K, Kurosaki T. PLC-gamma2
is essential for formation andmaintenance of memory B cells. J
Exp Med. 2009 206(3):681-9.

Figure :IgG memory B cell-specific genes
We compared the gene expression profiles in three replicates of day 7 and day 40 NP-specific/IgG1+ memory B cells,
GC B cells, plasma cells, and naïve B cells with or without multiple types of in vitro stimulation. Ninety-four genes
were identified as memory B cell-specific using two distinct statistical methods, Tukey’s multiple comparison test and
ROKU. We identified 106 probe sets corresponding to 94 genes that are highly expressed in the IgG1 memory B cells.

In the context of the role of the immune system in
defense against infection, it seems reasonable to proposethat the germ line encoded antibody repertoire is constantly
evolving to provide optimal protection.
Because the secondary response is characterized byhigh-affinity antibody production, affinity selection in the GC
reaction has been suggested to determine the fate of B cells
to differentiate into memory B cells. However, the existenceof GC-independent memory B cells excludes this as a possibility. We do not know the intrinsic signals that elicit memory B cell formation, perhaps influenced by signals received
from local niche cells, and regulate the quality and quantityof the memory cells, as distinct from other types of B cells.
To address this issue, we utilized Affymetrix GenChip technology and determined the intrinsic differences that distinguish memory and other types of B cells by comparison of
the transcriptional profiles of highly purified populations ofthese cell types isolated from immunized mice. We purified
NP-specific memory B cells from WT mice seven days after
immunization ( mostly GC-independent memory B cells) andat day 40 ( GC-independent and -dependent memory B cells)
and from immunized mice without formation of GC by invitro treatment with anti-CD40L mAb.
After qRT-PCR validation of the microarray results, we
identified 30 genes that are highly expressed in NP-specific/
IgG1+ memory B cells compared to other type of B cells.
Nine genes encode transcriptional activators or repressors,

five encode receptors, and the rest cytoplasmic proteins
involved in cell signaling and protein sorting. GC-independent day 7 and day 40 memory B cells shared the expression of a group of these transcripts, whereas the level ofcertain transcripts increased or decreased in GC-independent memory B cells over time. These results suggest kinetic
changes of gene expression in GC-independent memory
cells, perhaps in response to environmental cues.
To understand the role of these candidate genes inmemory B cell development and function, we first established a screening system prior to the gene targeting step.
We knocked down the candidate genes in purified B cells
from B1-8 high mice ( CD45.1+) , in which the frequency of NP
specific naïve B cells is greatly increase through the knock-in
of a rearranged, mutant V186.2 gene segment ( Shih et al.
2002) . After gene knockdown, B cells are transferred intosyngeneic CD45.2 recipients. Immunization of recipient micewith NP-CG causes synchronous activation of many NPspecific donor B cells, allowing us to dissect their early
response in vivo. Several pilot experiments haven shownthat this system works well.
Up until now, by using gene targeting technology wehave identified two genes that play roles in memory B cell
development in mucosal tissues and their differentiation
into plasma cells during the secondary antibody response( unpublished) .

71

Laboratory for

Epithelial
Immunobiology
Team Leader

Hiroshi Ohno

Senior Research Scientist : Koji Hase
Research Scientists : Shinji Fukuda, Shunsuke Kimura,		Gaku Nakato, Takashi Kanaya (SPDR)
Technical Staff: Yumi Chiba, Yumiko Fujimura, Kumiko Nakai,
		 Masumi Ohmae, Sayuri Sakakibara,
		 Ayako Yamashita
Student Trainees: Yumiko Nakanishi (JRA), Daisuke Takahashi (JRA),		 Masashi Ebisawa (JRA), Toshi Jinnohara (JRA),		 Kazunori Kadokura (JRA), Tamotsu Kato (JRA),		 Chikako Uetake, Hideaki Shima,
		 Misato Hanazato, Akemi Fujiwara, Keiko Kato,		 Yuuki Kitahara, Yuuki Obata, Takao Sato,		 Ai Takahashi, Yoshiko Usami, Shunji YamadaThe mucosal epithelium that lines the inner surfaces ofthe body, especially within the intestine, is exposed to awide 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 vital for maintaining
immune homeostasis. Epithelial cells that overlay the gutassociated lymphoid tissue ( GALT ) , such as Peyer’s patches( PPs) and isolated lymphoid follicles, are distinct fromabsorptive epithelial cells of the villi and are termed follicleassociated epithelium ( FAE ) . The FAE contains a specialized
subset of epithelial cells, the M cells, which are thought toplay a pivotal role in immune surveillance by delivering luminal microorganisms to the underlying lymphoid cells via
transcytosis. One of the primary aims of our laboratory is tounderstand the mechanisms that underlie the differentiationand function of FAE and M cells. Our research team is also
investigating the interaction of commensal microbiota with
the intestinal epithelium and its influence on mucosal andsystemic immunity. These studies may lead to the development of novel and more efficient mucosal vaccination proto-cols/drug delivery systems as well as functional foods/
preventive medicine based on host-microbiota interactions.

Identification of CCR6hi CD11c int B cells as a promoter of M-cell differentiation in the Peyer’s patch
M cells are atypical epithelial cells specialized for uptake ofluminal antigens, including the phagocytosis of viruses andbacteria, to deliver them to dendritic cells that accumulatebeneath the FAE. More than three decades after their initialdescription, however, molecular mechanisms of M-cell differentiation are not well understood. The differentiation of M
cells is thought to be induced by interactions between FAE
and PP cells; however, the identity of the types of immune
cells that function as M-cell inducers has remained elusive.
Therefore, we attempted to identify the cells that serve as an
M-cell inducer(s) in PP. To this end, we focused on the mice
deficient in CCR6, the sole receptor for a chemokine CCL20.
We previously found that CCL20 is constitutively expressedby FAE, thus implicating its involvement in FAE-PP cellinteractions. We found that a unique B-cell subset, characterized as CCR6hiCD11c int, resided in the subepithelial dome

Recent publications
1. Fukuda S., Toh H., Hase K., OshimaK., 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, 543-7 (2011)72

2. Nakanishi Y, Fukuda S, Chikayama E,
Kimura Y, Ohno H, Kikuchi J. Dynamic
Omics Approach Identifies NutritionMediated Microbial Interactions. J.
Proteome Res. 10, 824-36 (2011)

3. Shima H., Takatsu H., Fukuda S.,
Ohmae M., Hase K., Kubagawa H.,
Wang J.-Y., Ohno H. Identification of
TOSO/FAIM3 as an Fc receptor for IgM.
Int. Immunol. 22, 149-156 (2010)

4. Hase K., Kawano K., Nochi T., Pontes
G. S., Fukuda S., Ebisawa M., KadokuraK., 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

Figure :Schematic representation of the mechanisms of O157 lethal infection and protection bybifidobacteria. Both B. longum (BL) and B. adrescentis (BA) possess glucose transporter(s) and can
produce acetate from glucose in the proximal colon, where glucose is thought to be present; this acetate
exerts an anti-apoptotic effect on colonic epithelium. In the distal colon, by contrast, only fructose isavailable and thus only BL, with its fructose transporters, is able to produce acetate in this location. BA
lacks the fructose transporters, and thus fails to produce enough acetate to prevent epithelial apoptosis by
O157. This results in translocation of Shiga toxin into the bloodstream, leading to death of the mice.

( SED) of the mouse PP. CCR6 hiCD11c int B cells showedchemotactic migration in response to CCL20. Furthermore,this unique B-cell subset was substantially decreased in the
PP of CCR6-deficient mice, indicating that the SED localization of CCR6 hiCD11c int B cells is most likely regulated by the
CCL20-CCR6 system. Concomitantly, CCR6 deficiency
caused a remarkable decrement of M cells. Moreover, adoptive transfer of CCR6hiCD11c int B cells from wild-type mice
partially restored the M-cell defect in the CCR6-deficientmice. Collectively, our data indicate that CCR6 hi CD11c int B
cells possess an M-cell inducing capacity, and that the spatial regulation of CCR6hiCD11c int B cells via the CCL20-CCR6
system may play a vital role in M-cell differentiation in mice.

Bifidobacteria protect from enteropathogenic
Escherichia coli infection through production of
acetate: Importance of a sugar transporter
Intestinal microbiota impact 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 most distinctive benefits of these bacteria are the modulation of host

initiates mucosal immune response.
Nature 462, 226-230 (2009)

defense responses and protection against infectious diseases. Nevertheless, the molecular mechanisms underlying
these beneficial effects have barely been elucidated. To
investigate these mechanisms, we used a simplified modelof lethal infection with enterohaemorrhagic Escherichia coli
O157:H7 ( O157 ) of mice associated with certain bifidobacterial strains, together with an integrated ‘omics’ approach.
We showed that genes encoding an ATP-binding-cassette( ABC) -type carbohydrate transporter present in certain bifidobacteria contribute to protecting mice against death
induced by O157. We found that this effect was attributed,
at least in part, to increased production of acetate. Our datastrongly suggest that acetate produced in large amounts by
the bifidobacteria possessing the genes encoding the ABCtype transporter exerts its action on the colonic epitheliumby inducing anti-inflammatory and/or anti-apoptotic effects,
which prevents translocation of the O157 Shiga toxin fromthe gut lumen into the blood. We therefore propose that
acetate produced by protective bifidobacteria improves
intestinal defense mediated by epithelial cells and thereby
protects the host against lethal infection.

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 withRal and the exocyst complex. Nature
Cell Biol. 11, 1427-1432 (2009)73

Laboratory forTeam Leader

Mucosal Immunity

Sidonia Fagarasan

Research Scientists : Keiichiro Suzuki, Tran Huy Thinh, Michio Miyajima
Research Associate : Mikako Maruya
Technical Staff: Yasuko Doi, Yumi Tsutsui
Student Trainee: Shimpei Kawamoto ( received PhD from Kyoto University in December 2010 )A

daptive coevolution of mammals and bacteria has led
to the establishment of mutualistic and symbiotic relationships that have contributed to the development of ourimmune system and maintenance of homeostasis. In spiteof having available a wealth of immune sensing and effector
mechanisms capable of triggering inflammation in responseto microbial intrusion, we can live together with our body’s
bacteria without any adverse effects. This is made possible
by a continuous dialog between bacteria and host cells thatgenerates finely tuned signaling programs ensuring a stateof ‘hypo-responsiveness’ against dietary antigens and commensal bacteria. At the same time and somewhat paradoxically, these signaling programs generate a state of active
readiness that allows efficient and prompt immune responses
against pathogens.
Our long-term goal is to understand the host-microbial
relationship in the gut and to apply the knowledge for healthand disease. Current vaccine strategies and immune therapies could be vastly improved through further advances inmucosal immunology.

A unique gut signature provided by vitamin A andpattern-recognition receptors that modulates theadaptive immune response

Secretion of immunoglobulin A ( IgA ) is critical for immune
homeostasis. We have provided evidence that mucosal IgA
secretions need to be continuously produced in order tomaintain a highly diverse bacterial community in the gut( Fagarasan et al, Science, 2002, Suzuki et al, PNAS, 2004) .
The presence of highly mutated IgA is apparently required toprevent expansion of certain bacteria species ( i.e. segmented
filamentous bacteria, SFB) that can cause over-activation of
the mucosal and systemic immune systems. Intestinal synthesis of highly mutated IgAs occurs mainly in gut lymphoidstructures, such as Peyer’s patches. More specifically, mostof the intestinal IgAs are generated in specialized microenvironments of germinal centers ( GC) in the presence of follicular helper T cells ( TFH ) ( Tsuji et al, Science, 2009) .
GC formation requires the presence of special stromalcells called follicular dendritic cells ( FDCs) . These radioresistant cells are well known for their ability to retain immune
complexes. Although FDCs play a central role in GC development and function, their role in the specialized function of
PP to produce high amounts of IgA was not known. In otherwords, whether the FDC may help partition mucosal andsystemic immune compartments was unresolved.
We demonstrate that FDCs are equipped with surface,cytoplasmic and nuclear receptors capable of recognizing

Recent publications
1. Maruya M, Suzuki K, Fujimoto H,
Miyajima M, Kanagawa O, Wakayama
T, Fagarasan S. Vitamin A-dependenttranscriptional activation of the nuclear
factor of activated Tcells c1 (NFATc1)
is critical for the development andsurvival of B1 cells. Proc Natl Acad
Sci U S A. 2011, 108(2), 722-7.

74

2. Suzuki K, Maruya M, Kawamoto S, Sitnik
K, Kitamura H, Agace WW, Fagarasan
S. The sensing of environmental stimuli
by follicular dendritic cells promotes
immunoglobulin A generation in thegut. Immunity. 2010, 33(1), 71-83.

3. Fagarasan S, Kawamoto S, Kanagawa
O, Suzuki K. Adaptive immune regulationin the gut: T cell-dependent and T
cell-independent IgA synthesis. Annu
Rev Immunol. 2010, 28, 243-73.

4. Tsuji M, Komatsu N, Kawamoto S,
Suzuki K, Kanagawa O, Honjo T, Hori S,
Fagarasan S. Preferential generationof follicular B helper T (TFH ) cells from
Foxp3+ T cells in gut Peyer’s patches.
Science, 2009, 323, 1488-1492.

Figure1: In vivo and ex vivo images of FDCs
Left panel: A horizontal section of the Peyer’s patches
showing a B cell follicle (B220 showed in magenta) built
on FDCs (B-cell chemoattractant CXCL13 producedby FDC stained in blue). Right panel: A micrograph of
FDCs sorted from the Peyer’s patches, spun on a glass
slide and stained with May-Grunwald Giemsa.

Figure 3 : Whole mount staining of adipose tissues associated withperitoneum. Photomicrographs showing peritoneal B1 cells
stained for IgM present in the omentum ‘milky’ spot (left) and B1
cell -derived plasmablasts along the mesenteric vessels (right).

The role of dietary vitamin A in bridging the innate
and adaptive immune responses

Figure 2 : FDCs promote IgA generation in PPs upon
deciphering the gut environmental code.
FDCs express surface and cytoplasmic TLRs and retinoic acidreceptors (RARs). Among RARs, RARb is highly expressed in
PP FDCs and is induced by RA. Continuous and combined
stimulation by bacterial components (delivered by subepithelia
dome DCs) through TLR and by RA [produced mainly by theepithelial cells (ECs))] through RARs, up-regulate the expressionof chemokines (i.e.CXCL13), adhesion molecules (i.e. VCAM-1,
ICAM-1), survival factors (i.e. BAFF) and molecules involvedin activation of TGF-b1 (i.e. MMPs, LTBPs). Latent TGF-β1(LAP- TGF-β1) present on the surface of FDCs can be producedby FDCs or by other local cells including ECs. However, FDCs
have the capacity to activate TGF-β1. The factors secreted by
PP FDCs facilitate recruitment of B and T cells to the GCs,
direct preferential class switching of B cells from IgM to IgA,
enhance survival of highly proliferating AID-expressing cells
or those recently switched IgA+ B cells and help differentiationof IgA+ B cells into plasmablasts that will home to the LP.

gut signal profiles such as vitamin A and bacteria. Furthermore, FDCs convert such signals into effector molecules( CXCL13, BAFF, clusterin, MadCAM-1, matrix modulating
and TGF-β1 activating proteins) that are then conveyed toimmune cells. Hence, in the GC microenvironment, immune
cells are educated by FDCs to respond in a ‘mucosal manner’, namely to preferential switch to the production of IgA( instead of IgG ) . ( Suzuki K. et al, Immunity. 2010, Comment
by Deshane J and Chaplin DD., Immunity, 2010)

Adaptive immune responses require days to weeks for activation, expansion and differentiation of conventional B2 cells
and T cells into effector cells. The optimal transition between
early innate responses and the adaptive immune responses
is made by special subsets of B cells called B1 cells. These
innate-like cells produce the first wave of antibodies required
for antigenic clearance. Peritoneal B1 cells are also known
to be the source of many gut IgA plasma cells. The mucosal
IgA response to commensal bacteria by B1 cells is regarded
as part of a primitive mechanism that bridges innate andadaptive immunity in the gut.
We have previously shown that the sensing of bacteria
by peritoneal B1 cells ( through PRRs) induces complex
changes that lead to re-localization of B1 cells to the gut andgeneration of IgA plasma cells ( Ha et al, J.Exp.Med, 2006) .
Whether peritoneal B1 cells sense other environmental
cues from the gut and how these gut signals would impact
on the B1 cell biology remained unresolved.
We found that B1 cells are equipped not only with germline encoded and adaptable receptors but also with cytoplasmic and nuclear factors able to detect site-specificmetabolic profiles. Thus, B1 cells express high levels of
retinoic acid receptors ( RAR) and sense the retinoic acid
( RA ) produced by peritoneum associated adipose tissues,
such as omentum and mesentery. Signaling through RAR isessential for regulation of the nuclear factor of activated T
cells ( NFAT ) c1 ( through activation of the constitutive promoter ) , which in turn, is critical for the survival, proliferation
and antibody production by B1 cells. In mice, a vitamin-A
deficient diet results in reduction of NFATc1 expression in B1
cells, and the almost complete loss of innate-like memory
B1 cells. Hence, vitamin A-deficient mice are unable tomount appropriate immune responses to bacterial antigens.
Such environmental regulation of B1 cell biology may be
a part of a complex and fine-tuned regulatory pathway
involved in immune homeostasis.

5. Tsuji M, Suzuki K, Kitamura H, Maruya
M, Kinoshita K, Ivaylo II, Itoh K, Littman
DR, Fagarasan S. Requirement for
Lymphoid tissue inducer cells in isolated
follicle formation and T cell-independent
immunoglobulin A generation in thegut. Immunity, 2008, 29, 261-71

75

Laboratory for

Host Defense
Team Leader

Tsuneyasu Kaisho

Research Scientists : Katsuaki Hoshino, Hiroaki Hemmi
Technical Staff: Yuri Fukuda, Ikuko OgaharaStudent Trainees: Izumi Sasaki (JRA), Chihiro Yamazaki (JRA),		 Masanaka Sugiyama (JRA), Takeshi 				 Hirashima, Akihiko Iizuka, Eri HamadaBoth innate and adaptive immunity contribute to hostdefense in higher organisms. Innate immune receptors
function as relatively invariant pathogen sensors and are
involved in first-line eradication of pathogens. Furthermore,
innate immunity also contributes to the establishment ofadaptive immunity, in which lymphocytes use highly diverse
receptors to deal with pathogens that evade the innate
immune response. Dendritic cells (DCs) are antigen presenting cells critically involved in the sequence of these immune
responses. DCs sense various pathogen-derived molecularcomponents and exert their immunostimulatory functions byproducing inflammatory cytokines or upregulating expression of costimulatory molecules. Those components are
termed immune adjuvants because of their ability to activate
DCs. Immune adjuvants are recognized by various patternrecognition receptors including Toll-like receptors (TLRs).
Identification of new types of immune adjuvants and characterization of the mechanisms by which they activate DCs
should contribute to the development of novel immunoregulatory strategies. We are attempting to clarify how DCs areactivated through pattern recognition receptors in order todevise strategies for effective manipulation of the immune
response. Various immune adjuvants, including TLR ligands,
and gene targeted mice are important tools for this purpose.

DC subset-specific mechanisms in TLR7/9-induced
type I IFN gene induction
DCs sense nucleic acid adjuvants and produce type I interferon ( IFN) in a subset-dependent manner. Among nucleic
acid sensors, TLR7 and TLR9 are peculiar in that they recognize not only pathogen- but also host-derived nucleic
acids. Accumulating evidence suggests that TLR7/9-induced
type I IFN production plays important roles in the pathogenesis of autoimmune disorders such as systemic lupus erythematosus ( SLE) .
The plasmacytoid DCs ( PDC) form a unique DC subsetthat expresses the nucleic acid sensing TLRs, TLR7 andTLR9, and can produce type I IFN, especially IFN-α, in
response to signaling through these receptors. TLR7 andTLR9 are quite similar in their amino acid sequence and canactivate similar signaling pathways, which depend on the

Recent publications
1. K. Hoshino, I. Sasaki, T. Sugiyama,
T. Yano, C. Yamazaki, T. Yasui,
H. Kikutani, T. Kaisho. Cutting edge:
Critical role of IκB Kinase α in TLR7/9Induced type I interferon production by
conventional dendritic cells.
J. Immunol. 184, 3341-3345 (2010).

76

2. K. Gotoh, Y. Tanaka, A. Nishikimi,
R. Nakamura, H. Yamada, N. Maeda,
T. Ishikawa, K. Hoshino, T. Uruno,
Q. Cao, S. Higashi, Y. Kawaguchi,
M. Enjoji, R. Takayanagi, T. Kaisho,
Y. Yoshikai and Y. Fukui. Selective
control of type I IFN induction by
the Rac activator DOCK2 during

TLR-mediated plasmacytoid
dendritic cell activation. J. Exp.
Med. 207, 721-730 (2010).

3. T. Sugiyama, K. Hoshino, M. Saito,
T. Yano, I. Sasaki, C. Yamazaki , S.
Akira, T. Kaisho. Immunoadjubant
effects of polyadenylic-polyuridylic
acids through TLR3 and TLR7.
Int. Immunol. 20, 1-9 (2008).

Figure :cytoplasmic adapter molecule, MyD88. Downstream of
MyD88, the signaling pathways become bifurcated into
NF-κB and IRF-7 activation pathways, leading to the induction of inflammatory cytokines and type I IFNs, respectively.
We have demonstrated that the IκB kinase-α ( IKKα) , serine
threonine kinase, is critically involved in this type I IFN induction pathway in PDC ( Hoshino et al. Nature 440:949, 2006) .
IKKα associates with and phosphorylates IRF-7, which is
critical for type I IFN gene expression in response to TLR7/9
signaling. A critical role of IKKα was also elucidated throughthe analysis on DOCK2-deficient mice ( Gotoh et al. JEM
207:721,2010) . DOCK2 is a Rac activator and involved inchemokine receptor signaling. DOCK2-deficient mice
showed defects in TLR7/9-induced type I IFN production.
Furthermore, IRF-7 activation by TLR7/9 was impaired in
DOCK2-deficient pDC. Activation of IKKα, but not of any
other signaling molecules was defective, indicating that
DOCK2 is critical for IKKα activation, which is required fortype I IFN induction by TLR7/9 signaling.
We have further tested how IKK is involved in humanpDC function. The IKK inhibitor impaired type I IFN, but not
TNF-α, production by human pDC activated by TLR7/9 signaling. TLR7/9-induced IRF-7 activation was also impaired
by IKK inhibitor. These findings indicate that the IKK-IRF-7
axis should also function in human pDC ( Arth. Res. Therap.,
12:R87, 2010) .

Regulatory mechanisms for a TLR3-expressing DC
subsetConservation of the XCR1
chemokine receptor and its ligand
XCL1 in humans and mice.
In mouse splenic DC subsets, XCR1 isexpressed specifically in the CD8α+ cells.
An XCR1 ligand, XCL1, is constitutively
expressed by NK cells, which increase XCL1
production upon IL-2 stimulation. Activated
CD8+, but not CD4+, T cells produce large
amounts of XCL1. In humans, XCR1 isexpressed dominantly on BDCA3+ DCs,
which are the equivalent of murine CD8α+
DCs. The human XCL1 expression profile isalso similar to the murine profile. Thicknessof the green arrows represents relative
amounts of XCL1 chemokine production.

CD8α+ DC among murine splenic DC subsets. The CD8α+
DCs are uniquely specialized for crosspresentation of exogenous antigens to CD8+ T cells, thereby playing key roles inanti-viral and anti-tumor immunity. TLR3 signaling can augment the crosspresenting activity of CD8α+ DC. In order toclarify the molecular mechanisms for regulating CD8α+ DC
functions, we have first analyzed gene expression profiles ofDC subsets. A chemokine receptor, XC chemokine receptor
1 ( XCR1) , was highly and exclusively expressed in CD8α+
DCs ( Yamazaki et al. BBRC 397:756,2010) . TheXcr1 gene
was not expressed in the other splenic cells such as T cells,
B cells or NK cells. An XCR1 ligand, XCL1, was constitutively expressed only by NK cells. XCL1 was detected at
0.5-1.0 ng/ml in the serum and NK cell depletion decreased
the serum XCL1 level. NK cells in vitro increased XCL1 production in response to IL-2, but not to IL-12, IL-15, IL-18 or
IFN-α. CD8+ T cells did not constitutively express XCL1, but
produced high amounts upon activation with anti-CD3/28,
whereas CD4+ T cells failed to express XCL1 in either
unstimulated and activated conditions.
We have then analyzed XCR1 and XCL1 expression inhuman cells. Human XCR1 was functionally expressed by
BDCA3+ DCs, which are an equivalent of murine CD8α+
DCs. Furthermore, XCL1 expression was high in NK cells
and activated CD8+ T cells. Thus, the expression pattern of
XCR1 and its ligand is conserved both in mouse and human.
This system should thus play a critical role in innate andadaptive cytotoxic immunity in many species ( Figure) .

TLR3 is a sensor for dsRNA that is specifically expressed in

4. K. Hoshino, T. Kaisho. 2008. Nucleic
acid sensing Toll-like receptors
in dendritic cells. Curr. Opin.
Immunol. 20, 408-413 (2008).

5. T. Kaisho, T. Tanaka. Turning NF-κB
and IRFs on and off in DC. Trends
Immunol. 29, 329-336 (2008).

77

Laboratory for

Infectious
Immunity
Team Leader

Satoshi Ishido

Research Scientists : Mari Ohmura-Hoshino
		 Eiji Goto
		 Yohei Matsuki
		 Mizuho Kajikawa
Student Trainee: Eiji Uota

Technical Staff: Masami Kawasumi-Aoki
		 Mari Yoshida-MitoWe have demonstrated that the expression level of
MHC class II ( MHC II) is regulated through MARCH-Imediated ubiquitination of the MHC II β chain and have been
intensively examining the physiological role of this process.
In this regard, last year, we have found that MHC II ubiquitination is required for the maintenance of dendritic cells( DCs) . Therefore, one important project of my lab is to reveal
the molecular basis of DC maintenance by MHC II ubiquitination. Once we know that molecular mechanism, we canformulate specific strategies for our final goal. In addition, we
have started collaborative projects with several groups tounderstand how antigen-presentation is regulated by MHC II
ubiquitination. This information should also provide insight
into the physiological role of MARCH-1.
MARCH-I belongs to a family of membrane-bound E3
ubiquitin ligases, named MIR. MIR family members ( MIRs)
recognize different membrane molecules ( e.g. MHC class I,
ICAM-1) . However, it is still unclear how MIRs recognize theirsubstrates and how MIR-mediated ubiquitination induces
lysosomal degradation. To examine these issues, we havegenerated a Tet-on expression system for MIR E3 ligases.

This experimental system was generated by using B cells
and HeLa cells, and enables us to analyze the detailed relationship between the status of ubiquitination and the trafficking of substrate molecules. Ubiquitination-mediatedregulation of transmembrane proteins ( e.g. cytokine receptors) contributes to critical cell signaling events, thus, using
this experimental model we should be able to provide important insight into fields beyond immunology.
MHC II is constitutively ubiquitinated in the steady state.
However, MHC II ubiquitination is quickly down-regulated
upon DC activation/maturation. Interestingly, MARCH-I
expression is also down-regulated by DC activation/maturation. Given that MARCH-I ubiquitinates only the peptidebound form of MHC II, which is able to stimulate cognate
CD4 T cells, down-regulation of MARCH-I is thought to be
an important initiation signal for immune responses. To test
this hypothesis, we examined whether MARCH-I-deficientDCs efficiently present model antigens through MHC II. As
we expected, MARCH-I-deficient DCs efficiently presented
HEL and I-E alpha through MHC II, as judged by specificantibody responses. In addition, the half life of peptide-pre-Recent publications
1. Goto, E., Yamanaka, Y., Ishikawa, A.,
Aoki-Kawasumi, M., Mito-Yoshida,M., Ohmura-Hoshino, M., Matsuki, Y.,
Kajikawa, M., Hirano, H., and Ishido, S*.
Contribution of K11-linked ubiquitination to
MIR2-mediated MHC class I internalization.
J Biol Chem. 2010, 285(46):35311- 9.

78

2. Walseng, E., Furuta, K., Bosch, B.,
Weih, KA., Matsuki, Y., Bakke, O.,
Ishido, S., and Roche, PA. Ubiquitination
Regulates MHC Class II-Peptide
Complex Retention and Degradation in
Dendritic Cells. Proc. Natl. Acad. Sci.
U S A. 2010, 107(47):20465-70.

3. Ohmura-Hoshino, M., Matsuki, Y.,
Mito -Yoshida, M., Goto, E., Aoki-Kawasumi,
M., Nakayama, M., Ohara, O., and Ishido,
S. Cutting edge: Requirement of MARCHI-mediated MHC II ubiquitination for the
maintenance of conventional dendritic cell.
J. Immunol., 2009, 183 (11):6893-7.

4. Goto, E., M. Mito-Yoshida, M. Uematsu,
Y. M. Aoki, Matsuki, M. Ohmura-Hoshino,
M. Miyagishi, H. Hotta, and S. Ishido.
An excellent monitoring system
for surface ubiquitination-induced
internalization in mammals. PLoS
ONE, 2008, 30;3 (1):e1490.

Figure :senting MHC II was extended in MARCH-I-deficient DCs.
Thus, loss of MHC II ubiquitination by down-regulation of
MARCH-I enhances antigen-presentation. Currently, we are
examining how enhanced antigen-presentation alters
immune responses ( e.g. Th1, Th2, Th17 development) .
As second project, we are examining how MIR E3s recognize their substrates and regulate the trafficking of substrates. During this analysis, we found that a novel type ofpolyubiquitin chain can function as an internalization signal.
Polyubiquitination is the sequential reaction between lysine
residues on ubiquitin moieties, catalyzed by E3 ubiquitin
ligase ( E3) , and generates the polyubiquitin chain on substrate proteins. All seven lysine residues ( K6, K11, K27, K29,
K33, K48 and K63) on ubiquitin can be utilized for the formation of polyubiquitin chains. Among them, K48 and K63
were classically reported as the acceptor lysine residues forthe generation of polyubiquitin chains. The K48-linked polyubiquitin chain and the K63-linked chain are well-establishedinducers of proteasome-dependent degradation and signaltransduction, respectively. However, the other linkages are
not clearly established.
We previously generated a tetracycline-inducible ( Teton ) system for a member of the MIR family. MIR family mem-

Hypothetical model of downregulation by MIR2
Based on our current understanding,the most logical model at this point
may be that Epsin binds to K63-linked
di-ubiquitin and longer chains, andthat the presence of K11 (either asa mixed chain, as a cap or as a side
branch) facilitates some events related
to Ub-dependent endocytosis. The K11
linkages might support the extensionof the polyubiquitin chain required
for efficient internalization, or theselinkages might generate a suitable chain
topology for the association between
K63 linkages and epsin1 in vivo. In
addition, K11 linkages may interact
with a second ubiquitin-interacting
protein that is needed for endocytosis
and/or subsequent sorting.

bers are membrane-bound E3 ubiquitin ligases and havebeen reported to induce ubiquitination-dependent internalization in vivo. This Tet-on system could be used to visualize
the detailed status of the polyubiquitin chain, which induces
the in vivo internalization. Recently, the Gygi research group
has also successfully developed a detection system forpolyubiquitin chains generated in vivo using mass spectrometry. By using the Tet-on expression system for MIR2 andquantitative mass spectrometry, we have addressed theissue of how the polyubiquitin chain contributes to receptor
internalization. We have found that the polyubiquitin chain
generated by MIR2 is a K11- and K63-mixed linkage chain
and that this mixed polyubiquitin chain is required for efficient epsin1-dependent internalization of MHC I ( Figure) .
Although further studies are required, our working hypothesis is the K11 and K63 mixed polyubiquitin chain is a common internalization signal utilized by MIR family members.
These results indicate that the structure of a functional
polyubiquitin chain is not simple, as had been thought.
Therefore, it will be important examine the detailed structureof ubiquitin chains that contribute to biological events otherthan internalization/trafficking, e.g., protein degradation andgene transcription, and to reveal the molecular basis of how
such complex chains are generated in vivo.

5. Matsuki, Y., M. Ohmura-Hoshino, E.
Goto, M. Aoki, M. Mito-Yoshida, M. Uematsu,
T. Hasegawa, H. Koseki, O. Ohara, M.
Nakayama, K. Toyooka, K. Matsuoka, H.
Hotta, A. Yamamoto, and S. Ishido. Novel
regulation of MHC class II function in B
cells. EMBO J., 2007 26:846-54.

79

Laboratory for

Innate Cellular
Immunity
Team Leader

Masato Tanaka

Research Scientists : Kenichi Asano
		 Qiu Chunhong		 Gen Nishitai
Technical Staff: Ai Kurita
		 Minako Aihara
Student TraineeEvery type of cell has its own life span and aged cells
undergo apoptosis for their turnover. The immune system eliminates harmful cells, including virus-infected cells orcancer cells, by inducing their apoptosis. Although a substantial number of cells constantly undergo apoptosis evenin physiological conditions, it is very difficult to detect suchcells, primarily because they are swiftly eliminated by phagocyte cells, such as macrophages and dendritic cells ( DCs) .
Phosphatidylserine ( PS) is exposed on the apoptotic cellsurface, and most phagocytes recognize this lipid throughspecific receptors or soluble molecules. A defect in thisclearance process has been shown to cause unremitting
inflammation in acute injury models. In addition to the resolution of inflammation, apoptotic cell clearance by phagocytes plays a crucial role in maintenance of self tolerance. In
the periphery, tissue-resident dendritic cells ( DCs) constantly
phagocytose apoptotic cells generated during normal tissue
turnover, and migrate to draining lymph nodes where theypresent antigens derived from the apoptotic cells. This presentation of self-antigens leads to deletion or anergy of any
self-reactive T cells, thereby providing another mechanism: Kazunori Karasawa

to maintain T cell tolerance. Failure of apoptotic cell clearance results in autoimmune disorders. The Laboratory for
Innate Cellular Immunity is investigating the molecularmechanisms for recognition and phagocytosis of dying cellsby phagocytes, and the pathological relevance of impaired
phagocytosis to inflammatory disorders including autoimmune diseases.

Role of CD169-positve sinus macrophages inanti-tumor Immunity
The generation of tumor antigen-specific cytotoxic T cells( CTLs) is considered crucial for the induction of anti-tumor
immunity. To generate and activate T cell immunity against
tumor antigens, antigen presenting cells ( APCs) must initiallyacquire tumor cell-associated antigens, and one of the major
sources of tumor antigens is dead tumor cells. Massive
tumor cell death is induced by anti-cancer therapy, and particulate matter from the dead tumor cells provides large
amounts of tumor-associated antigens. However, previous
studies have been unable to clarify the mechanism by which
APCs in the lymph node ( LN) internalize and crosspresent

Recent publications
1. Asano K., Nabeyama A., Miyake Y.,
Qiu C.H., Kurita A., Tomura M.,
Kanagawa O., Fujii S.I., and Tanaka
M. CD169-positive Macrophages
Dominate Antitumor Immunity by
Crosspresenting Dead Cell-Associated
Antigens. Immunity 34, 85-95, (2011)80

2. Chow A., Lucas D., Hidalgo A.,
Mendez-Ferrer S., Hashimoto D.,
Scheiermann C., Battista M., Leboeuf
M., Prophete C., Rooijen N., Tanaka M.,
Merad M., and Frenette P. Bone
Marrow CD169+ Macrophages
Promote the Retention of Hematopietic

Stem and Progenitor Cells in the
Mesenchymal Stem Cell Niche. J.
Exp. Med. 208, 261-71 (2011)

3. Nabeyama A., Kurita A., Asano K.,
Miyake Y., Yasuda T., Miura I., NishitaiG., Arakawa S., Shimizu S., WakanaS., Yoshida H., and Tanaka M. xCT
Deficiency Accelerates Chemically
Induced Tumorigenesis. Proc Natl Acad
Sci U S A 107, 6436-6441 (2010)

these types of antigens to CD8+ T cells. Antigen presentation in peripheral LNs is thought to be coordinately performed by migratory DCs that enter the LN from peripheral
tissues via the lymphatics, and LN-resident APCs. However,
little is known about the role of different APCs in the clearance of dead cells and the presentation of dead cell-associated antigens in peripheral tissue or LNs.
We recently demonstrated a critical role of CD169+
macrophages located in the LN sinus in induction of antitumor immunity by dead tumor cells. Subcutaneous immunization with irradiated tumor cells induces activation oftumor antigen-specific CD8 T cells, and consequently protects mice from a syngeneic tumor. Subcutaneously injecteddead cells reach LNs via lymphatic flow and are mainly taken
up by CD169+ macrophages. Tumor antigen-specific CD8+ T
cell activation and subsequent anti-tumor immunity areseverely impaired in mice depleted of CD169+ macrophages.
CD11c+, CD169+ macrophages phagocytose dead tumor
cells transported to the LN and directly crosspresent
cell-associated antigens to stimulate CD8+ T cell proliferation. By contrast, neither migratory dendritic cells ( DCs)
nor lymph node-resident conventional DCs are essential forthe crosspresentation of tumor antigens. Thus, we haveidentified CD169+ macrophages as lymph node-resident
APCs dominating early activation of tumor antigen-specificCD8+ T cells.

xCT is required for survival of activatedmacrophages and protection from tumorigenesis
Macrophages are key players in the initiation and regulationof inflammation. Macrophages infiltrating into an inflammatory site, e.g., an acute injury or local infection, recognize
invading microorganisms or endogenous adjuvants released
by injured cells. In response to this recognition, macrophages
produce inflammatory cytokines and chemokines. Although
the precise mechanisms of inflammation-induced tumorigenesis remain unclear, cytokines or growth factors persistently produced at the chronic inflammatory site may
stimulate the proliferation of DNA-damaged cells.
During the course of inflammation and its resolution,
macrophages are exposed to various cytotoxic substances,including reactive oxygen species. Thus, macrophages
require a mechanism for protection against oxidative stressto survive at the inflammatory site. We recently showed that
xCT, a component of the anionic amino acid transport system xc-, was significantly upregulated in activated infiltrating
cells, including macrophages and neutrophils, at the inflam-

4. Qiu C. H., Miyake Y., Kaise H., Kitamura
H., Ohara O., and Tanaka M. Novel
Subset of CD8α+ Dendritic Cells
Localized in the Marginal Zone is
Responsible for Tolerance to Cellassociated Antigens. J. Immunol.
182, 4127-4136 (2009)

Figure :CD169-positive macrophages (Green) are the gate keepersof the lymph node. They reside in the lymph node sinus
and make initial contact with particles in the lymphatic
fluid. Dead tumor cells (Red) that flow into the lymph
node are phagocytosed by CD169+ macrophages.

matory site. System xc- mediates the uptake of extracellular
L-cystine and is consequently responsible for maintenanceof intracellular glutathione levels. We established an xCT
loss-of-function mouse mutant line by N-ethyl-N-nitrosourea
mutagenesis. Macrophages from xCT mu/mu mice exhibited
cell death in association with the excessive release of High
Mobility Group Box Chromosomal Protein 1 upon stimulation with LPS, suggesting that xCT deficiency causes
unremitting inflammation due to the impaired survival of activated macrophages at the inflammatory site. Subcutaneous
injection of 3-methylcholanthrene ( 3-MCA ) induces the generation of fibrosarcomas in association with inflammation.
When 3-MCA was injected subcutaneously into mice, xCTmRNA was upregulated in situ. In xCT mu/mu mice, inflammatory cytokines such as IL-1β and TNFα were overexpressed
and the generation of 3-MCA-induced fibrosarcomas was
accelerated. These results clearly indicate that the defectof the oxidative stress protective system impaired survivalof activated macrophages and subsequently enhanced
tumorigenesis.

5. Tanaka M., Miyake Y., and Asano K.
Maintenance of Self-tolerance by
Apoptotic Cell Clearance. Front.
Biosci. 13, 6043-6049 (2008)81

Research Unit for

Inflammatory Regulation

Takashi Tanaka

Unit Leader
		Technical Staff : Emiri HagaThe inflammatory response is an important host defense
mechanism to sense and eliminate invading microbial
pathogens. Innate immune cells, such as macrophages anddendritic cells, first detect pathogens by their sensors of
pathogen associated molecular patterns ( e.g. Toll-like receptors [ TLR ]) . TLR signaling then leads to activation of thetranscription factor NF-κB, 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. Depending on theirnature, these inflammatory responses can then direct
T-helper ( Th ) lymphocyte differentiation into distinct subsets,such as Th1, Th2 and Th17 cells, to combat various pathogens. These initially helpful inflammatory responses must be
terminated at appropriate time points; otherwise an overblown response can damage normal tissue and may lead to
autoimmune or allergic diseases. Our research goal is to
identify key regulators of inflammation-related signal transduction pathways, and to clarify the molecular mechanisms
for regulating inflammatory responses. These studies should
contribute to the development of new therapeutic tools tocontrol the exaggerated inflammation seen in certain humandiseases. Our research now focuses on the role of PDLIM2( PDZ and LIM-domain protein-2) and related LIM proteins in
the negative regulation of inflammatory responses.

Molecular mechanisms to regulate
PDLIM2-mediated termination of NF-κB activation
PDLIM2, also known as SLIM ( STAT-interacting protein) , is a
nuclear protein that contains both PDZ and LIM domains
and belongs to a large family of LIM proteins. PDLIM2 wasoriginally isolated in CD4+ T cells as a nuclear ubiquitin E3
ligase for the STAT4 transcription factor, thereby suppressing Th1 cell differentiation ( Tanaka T et al, Immunity, 2005) .
We have also demonstrated that PDLIM2 negatively regulates NF-κB activity in dendritic cells and subsequent inflammatory responses, acting as a nuclear ubiquitin E3 ligase
targeting the p65 subunit of NF-κB. ( Tanaka T et al, Nat.
Immunol., 2007) . PDLIM2 binds to and promotes polyubiquitination of p65 through its LIM domain. In addition, PDLIM2
targets p65 to discrete intranuclear compartments, called
PML nuclear bodies. Polyubiquitinated p65 is ultimately
degraded by proteasomes in these compartments. However, it remains unclear how the activity of PDLIM2 is regulated. We are therefore attempting to isolate proteins thatbind to PDLIM2 and control its activity. We have recently
found that heat shock protein 70 ( HSP70 ) translocates into
the nucleus in response to TLR stimulation, binds to PDLIM2,
and augments the activity of PDLIM2 to degrade p65 andsuppress NF-κB signaling. We have further demonstrated
that HSP70 also interacts with BAG-1, a protein that can

Recent publications
1. Yan, P., Fu, J., Qu, Z., Li, S.,
Tanaka, T., Grusby, M.J., Xiao,
G. PDLIM2 suppresses HTLV-I
Tax-mediated tumorigenesis bytargeting Tax into the nuclear
matrix for proteasomal degradation.
Blood, 113, 4370-4380 (2009)82

2. Kaisho, T. and Tanaka, T. Turning
NF-κB and IRFs on and off in DCs.
Trends Immunol., 29, 329-336 (2008)

3. Tanaka, T. Grusby, M.J. and Kaisho,T. PDLIM2-mediated termination oftranscription factor NF-κB activation
by intranuclear sequestration anddegradation of the p65 subunit.
Nat. Immunol. 8, 584-591 (2007)

4. Nakahira, M., Tanaka, T., Robson,
B.E., Mizgerd, J.P. and Grusby, M.J.
Regulation of Signal Transducer andActivator of Transcription Signalingby the Tyrosine Phosphatase PTP-BL.
Immunity 26, 163-176 (2007)

Figure1: PDLIM2 binds to the p65 subunit of NF-κB andpromotes p65 polyubiquitination in the nucleus.
PDLIM2 then targets p65 into discrete intranuclear
compartments, called PML nuclear bodies. HSP70
binds to PDLIM2 and facilitates delivery of the NFκB-PDLIM2 complex to the proteasome cooperatively
with BAG1. Polyubiquitinated p65 is ultimately
degraded by the proteasome in PML nuclear bodies.

bind to both HSP70 and the proteasome, and facilitates thedelivery of the NF-κB-PDLIM2 complex to the proteasome.
Consistently, knockdown of either HSP70 or BAG-1 by smallinterfering RNA resulted in larger amounts of nuclear p65
and augmented production of proinflammatory cytokines.
These data delineate an essential role of HSP70 and BAG-1
in PDLIM2-mediated termination of NF-κB activation.

The role of LIM proteins in the in vivo inflammatory
response
The LIM protein family is classified into three groups and
PDLIM2 belongs to Group 3 LIM proteins, which containone or multiple LIM domains at their C-terminus. About 10
proteins have been identified in Group 3, but their functionsin the immune system remain unclear. We have been using
in vivo models to clarify how these LIM proteins differentiallyregulate inflammatory responses. Granuloma formation is an
important host defense mechanism against intracellularbacteria. However, uncontrolled granulomatous responses
cause tissue damage and impair normal organ function inseveral human autoimmune and inflammatory diseases.
Recent studies have suggested that exaggerated Th17 cellmediated responses may contribute to the development of

Figure 2: PDLIM2 and PDLIM4 regulate Th17 cell-mediatedinflammatory responses by different mechanisms. PDLIM2interacts with STAT3 and promotes its ubiquitin/proteasomedependent degradation, whereas PDLIM4 binds to STAT3,
but recruits PTP and enhances its dephosphorylation.

granulomatous inflammation. We showed that PDLIM2
negatively regulated Th17 development and granulomatous
responses by acting as a nuclear ubiquitin E3 ligase targeting STAT3, the transcription factor critical for commitment
to the Th17 lineage. PDLIM2 promoted polyubiquitinationand proteasome-dependent degradation of STAT3, thereby
disrupting STAT3-mediated gene activation. PDLIM2 deficiency resulted in increased amounts of nuclear STAT3,
enhanced Th17 cell differentiation, and exacerbated Propionibacterium acnes-induced liver granuloma formation.
Moreover, we found that PDLIM4, another LIM protein family
member, also negatively regulated Th17 differentiation by
suppressing STAT3 activation. Interestingly, however,
PDLIM4 did not promote STTA3 degradation but instead
inhibited tyrosine phosphorylation of STAT3. We further
demonstrated that PDLIM4 bound to and recruited a protein
tyrosine phosphatase, which facilitated dephosphorylationof STAT3. Thus, PDLIM2 and PDLIM4 suppress STAT3 signaling through distinct mechanisms. Our findings delineate
an essential role of these LIM proteins in negatively regulating Th17-mediated inflammatory responses and provide a
potential therapeutic target for inflammatory diseases.

83Research Unit for

Therapeutic
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 have unique immunoregulatory features that include the ability to rapidly
produce large quantities of cytokines. We have attempted
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. We have recently
established an immunotherapeutic strategy using artificial
adjuvant vector cells ( AAVCs) , composed of glycolipidloaded, antigen-encoding mRNA-transfected allogeneic
fibroblasts for enhancing both innate ( NKT and NK cells) as
well as adaptive immunity ( T cells) . We elucidated the mechanism of DCs in detail in the AAVC-treated mice. Also, tolead to the launch of clinical studies, we have been performing preclinical studies in collaboration with Dr. Fujii, Cellular
Immunotherapy Unit. Second, we have studied the biological interactions between NK cells and DCs following
DC therapy. The NK antitumor response in DC-vaccinatedmice depended on CD4+ T cells, but not on CD8+ T cells or
NKT cells.

Antigen mRNA-transfected allogeneic fibroblasts
loaded with NKT cell ligand confer antitumor
immunity
We previously demonstrated that the administration oftumor/Gal cells could generate innate and adaptive immunity through the maturation of DCs in a tumor-specific manner. In the current study, instead of tumor cells, we usedallogeneic fibroblast cells loaded with α-GalCer and transfected with antigen-encoding mRNA, thus combining the
adjuvant effects of iNKT cell activation with delivery of anantigen of choice to DCs in vivo. We found that these cells
produce antigen protein and activate NK and iNKT cells.
When injected into MHC mismatched mice, they elicitedantigen-specific T cell responses and provided tumor protection, suggesting that these immune responses dependon host DCs. Also, antigen-expressing fibroblasts loadedwith α-GalCer lead to a more potent T cell response thanthose expressing NK cell ligands. Thus, glycolipid-loaded,

Recent publications
1. Shimizu K, Hidaka M, Bickham K,
Moriwaki M, Fujimoto K, Fujii S. Humanleukemic cells loaded with α-GalCer
activate murine NKT cells in situ.
Int J Hematol 92, 152-60 (2010)84

2. Watarai H, Fujii S, Yamada D,
Rybouchkin A, Sakata S, Nagata Y,
Iida-Kobayashi M, Sekine-Kondo
E, Shimizu K, Shozaki Y, Sharif J,
Matsuda M, Mochiduki S, Hasegawa T,
Kitahara G, Endo TA, Toyoda T, Ohara
O, Harigaya K, Koseki H, Taniguchi M.

Murine induced pluripotent stem cells
can be derived from and differentiate
into natural killer T cells. J Clin
Invest 120, 2610-2618 (2010)

3. Fujii S, Motohashi S, Shimizu K,
Nakayama T, Yoshiga Y, Taniguchi
M. Adjuvant activity mediatedby iNKT cells. Semin Immunol
22, 97-102 (2010)

Figure :Endogenous dendritic cells in vivo play a key role for induction ofadaptive immunity in the AAVC therapy model.
OVA-specific CD8 T cells (OT-1) were transferred into WT mice, which were then injected with OVA mRNAtrasnfected AAVCs, and robust OT-1 cell proliferation was observed. However, if we used CD11c-DTR miceas recipients and depleted host CD11c+DC by DT injection, the OT-1 cells did not proliferate, indicatingthat host dendritic cells, but not AAVC, play a key role in cross-priming in the AAVC therapy system.

mRNA-transfected allogeneic fibroblasts act as adjuvant
vector cells ( AVCs ) to promote iNKT cell activation, leading
to DC maturation and T cell immunity. By harnessing the
innate immune system and generating an adaptive immune
response to a variety of antigens, this unique tool could
prove clinically beneficial in the development of immunotherapies for malignant and infectious diseases.

Anti-tumor NK cell response by dendritic cell (DC)therapy
We have examined the relationship between NK cells andendogenous DCs following administration of DCs. We vaccinated mice with DCs and evaluated long term resistanceto tumor challenge. When DC-treated mice were challenged

4. Fujii S, Goto A, Shimizu K. Antigen
mRNA-transfected, allogeneic
fibroblasts loaded with NKT-cell
ligand confer antitumor immunity.
Blood 113, 4262-72 (2009)

with tumor cells even several months later, NK cells werequickly activated to express CD69 and produce IFN-γ. The
NK cells could resist a challenge with several different tumors
in vivo. Even if the NK cells were depleted with anti-NK1.1
Ab treatment, the activated cells recovered, indicating thattumor-responsive NK cells were being generated continuously as a result of vaccination with DCs and were not true
memory cells. The NK antitumor response in DC-vaccinatedmice depended on CD4+ T cells, but not on CD8+ T cells or
NKT cells. However, both vaccine DCs and endogenous
DCs were required for the immune response. These results
indicate that DC therapy in mice induces long lasting innate
NK cell activation through a pathway that requires host DCs
and CD4+ T cells.

5. Shimizu K, Fujii S. DC therapy
induces long-term NK reactivity
to tumors via host DC. Eur J
Immunol. 39, 457-68 (2009)85

Laboratory forGroup Director

Immune Regulation

Masaru Taniguchi

Senior Research Scientist : Hiroshi Watarai
Research Scientists: Takuya Tashiro, Masao Shiozaki, Michishige Harada
Visiting Scientist: Nyambayar Dashtsoodol
Technical Staff: Sakura Sakata, Etsuko Sekine, Yuko Nagata, Tomokuni Shigeura, Yuko Sasaki,
		 Chihiro Sasaki, Diana Eissens

C

D1d-restricted invariant natural killer T ( NKT ) cells
bridge innate and acquired immunity and play animportant role in both protective and regulatory responses.
The nature of the 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 exposure to IL-12-producing cells results in proinflammatory Th1-type responses. Particularly in the antitumor response, NKT cells mediate adjuvant activity by theirproduction of IFN-γ, which in turn activates both the innate
and acquired immune systems. Thus, upon activation of
NKT cells, both MHC - and MHC + tumor cells can be efficiently eliminated. Based on our understanding of thesemechanisms, NKT cell-targeted adjuvant cell therapies havebeen developed and have shown great promise in initialclinical trials on cancer patients.

Identification of CD4-CD8- double-negative naturalkiller T cell precursors in the thymus
It is well known that CD1d-restricted Vα14 invariant naturalkiller T ( NKT ) cells are derived from cells in the CD4+CD8+
double-positive ( DP) population in the thymus. However,the developmental progression of NKT cells at earlier stages
remains unclear, and the possible existence of NKT cell precursors in stages earlier than the DP stage remains to betested. We have demonstrated that NKT cell precursors that
express invariant Vα14-Jα18 transcripts, but are devoid of
surface expression of the invariant Vα14 receptor, are present in the late CD4-CD8- double-negative ( DN) 4 stage andhave the potential to generate mature NKT cells both in vivo
and in vitro. Moreover, this DN4 population in CD1dKO miceis similar to that with NKT cell potential in wild-type C57BL /6
mice, but failed to develop into NKT cells in vitro. However,these CD1dKO precursors could develop into NKT cells
when co-cultured with normal thymocytes or in an in vivo
experimental setting, indicating that functional NKT cell precursors are present in CD1dKO mice. Together, theseresults demonstrate that the thymic DN4 fraction contains
NKT cell precursors. Our findings have provided new insights
into the early development of NKT cells prior to surface
expression of the invariant Vα14 antigen receptor and suggest a possible alternative developmental pathway of NKT
cells.

Recent publications
1. Matsuoka N, Itoh T, Watarai H,
Sekine-Kondo E, Nagata N, Okamoto
K, Mera T, Yamamoto H, Yamada S,
Maruyama I, Taniguchi M, Yasunami Y.
High-mobility group box 1 is involvedin the initial events of early loss oftransplanted islets in mice. J. Clin.
Invest. 120 :735-743 (2010).

86

2. Watarai H, Rybouchkin A, Hongo
N, Nagata Y, Sakata S, Sekine E,
Dashtsoodol N, Tashiro T, Fujii
S, Shimizu K, Mori K, Masuda K,
Kawamoto H, Koseki H, Taniguchi M.
Generation of functional NKT cells in
vitro from embryonic stem cells bearing
rearranged invariant Vα14-Jα18 TCRαgene. Blood. 115 : 230-237 (2010).

3. Iwata A, Watanabe N, Oya Y, Owada
T, Ikeda K, Suto A, Kagami S,
Hirose K, Kanari H, Kawashima S,
Nakayama T, Taniguchi M, Iwamoto
I, Nakajima H. Protective roles of B
and T lymphocyte attenuator in NKT
cell-mediated experimental hepatitis.
J. Immunol. 184 :127-133 (2010).

4. Taniguchi M, Tashiro T, Dashtsoodol N,
Hongo N, Watarai H. The specialized
iNKT cell system recognizes glycolipid
antigens and bridges the innate
and acquired immune systems withpotential applications for cancer
therapy. Int. Immunol. 22 :1- 6 (2010).

Figure :Methods for generating clonal NKT cells from
iPS cells. iPS cells derived from somatic cells
give rise to diversified lymphocytes due to thegene rearrangement during their differentiation(upper). On the other hand, (i.e. NKT cell) iPS
cells derived from a mature lymphocyte (e.g.,an NKT cell in this example) generate clonal
lymphocytes with desired functions (lower).

Generation of functional NKT cells in vitro fromembryonic stem cells ( ESCs) bearing a rearranged invariant Vα14-Jα18 TCRα gene
Establishment of a system for efficient generation of NKT
cells from ESCs would enable us to identify the cells with
NKT-cell potential and to obtain NKT cells with desired functions. For this purpose, we used cloned ES ( NKT-ES) cells
generated by the transfer of nuclei from mature NKT cells
and established a culture system that preferentially developed functional NKT cells. We also identified early NKT
progenitors, which first appeared on day 11 as a c-kit +population in cocultures on OP9 cells expressing the Notch
ligand delta-like1 ( OP9/Dll-1) , and then became c-kit lo/- on
day 14. Interestingly, in the presence of Notch signals, NKTES cells differentiated only to thymic-type CD44lo CD24hi
NKT cells producing mainly interleukin-4 ( IL-4) , whereas
NKT cells resembling CD44hi CD24lo liver NKT cells producing mainly interferon gamma ( IFN-γ ) and exhibiting strong
adjuvant activity in vivo developed in “switch” cultures, whichwere deprived of Notch signaling starting at day 14. The
cloned ES culture system offers a new opportunity for elucidation of the molecular events in NKT-cell development andfor the establishment of NKT-cell therapy.

High-mobility group box 1 ( HMGB1) is involved in
the initial events of the early loss of transplanted
islets in mice
Islet transplantation for the treatment of type 1 diabetes mellitus is limited in its clinical application mainly due to early lossof the transplanted islets, resulting in low transplantation efficiency. NKT cell–dependent IFN-γ production by Gr-1+CD11b+
cells is essential for this loss, but the upstream events in theprocess remain undetermined. We demonstrated that
HMGB1 plays a crucial role in the initial events of early loss oftransplanted islets in a mouse model of diabetes. Pancreatic
islets contain abundant HMGB1, which was released into thecirculation soon after transplantation of islets into the liver.
Treatment with an HMGB1-specific antibody prevented theearly islet graft loss and inhibited IFN-γ production by NKT

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

cells and Gr-1+CD11b+ cells. Moreover, there was no early
islet graft loss in mice lacking either of the known HMGB1
receptors, TLR2 or receptor for advanced glycation end
products (RAGE), but such loss did occur in mice lacking the
known HMGB1 receptor TLR4. Mechanistically, HMGB1
stimulated hepatic mononuclear cells (MNCs) in vivo and invitro; in particular, it upregulated CD40 expression andenhanced IL-12 production by DCs, leading to NKT cell activation and subsequent NKT cell–dependent augmented IFN-γ
production by Gr-1+CD11b+ cells. Thus, treatment with either
IL-12– or CD40L-specific antibody prevented the early islet
graft loss. These findings indicate that the HMGB1-mediatedpathway eliciting early islet loss is a potential target for intervention to improve the efficiency of islet transplantation.

Murine induced pluripotent stem cells ( iPSCs ) canbe derived from and differentiate into NKT cells
NKT cells demonstrate antitumor activity when activatedto produce Th1 cytokines by DCs loaded with
α-galactosylceramide, the prototypic NKT cell-activating
glycolipid antigen. However, most cancer patients do nothave sufficient numbers of NKT cells to induce an effective
immune response in this context, indicating the need for asource of NKT cells that could be used to supplement theendogenous cell population. iPSCs hold tremendous potential for cell-replacement therapy, but whether it is possible togenerate functionally competent NKT cells from iPSCs had
not been rigorously assessed. We successfully derived
iPSCs both from embryonic fibroblasts from mice harboring
functional NKT cell-specific rearranged T cell receptor loci inthe germline and from splenic NKT cells from WT adult mice.
These iPSCs could be differentiated into NKT cells in vitro
and secreted large amounts of the Th1 cytokine IFN-γ.
Importantly, iPSC-derived NKT cells recapitulated the known
adjuvant effects of natural NKT cells and suppressed tumor
growth in vivo. These studies demonstrate the feasibility of
expanding functionally competent NKT cells via an iPSC
phase, an approach that may be adapted for NKT cell-targeted therapy in humans.

can be derived from and differentiate
into natural killer T cells. J. Clin.
Invest. 120 :2610-2618 (2010).

87

Laboratory for

Dendritic Cell
Immunobiology
Team Leader

Katsuaki Sato

Technical Staff : Kaori Sato		 Yumiko Sato		 Kawori Eizumi
Student Trainees: Tomohiro Fukaya (JRA)		 Hideaki Takagi (JRA)		 Honami TayaD

endritic cells ( DCs) are essential antigen-presenting
cells ( APCs) that initiate primary immune responses.
DCs consist of heterogeneous subsets, mainly classified
into conventional DCs ( cDCs) and plasmacytoid DCs ( pDCs) ,which are distinguishable by surface and intracellular phenotypic markers, immunologic function, and anatomic distribution. Immature DCs ( iDCs) serve as sentinels, recognizing
invading pathogens or virus-infected cells through various
pattern-recognition receptors. Subsequently, they become
mature DCs ( mDCs ) with up-regulated expression of MHC
and costimulatory molecules under inflammatory conditions.
The mDCs then move via the afferent lymphatics into the
T-cell areas of secondary lymphoid tissues, where they prime
rare antigen-specific naive T cells for differentiation into
effector T ( Teff ) cells, including T helper type ( TH ) 1 cells, TH 2
cells, and TH17 cells, depending on environmental cues.
DCs thereby play a crucial role in the link between innate
and adaptive immunity. Conversely, iDCs are also crucial forthe induction of immunological tolerance under steady-state
conditions. The 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, afunction of likely importance in self-tolerance as well as inimmune disorders and transplant rejection. However, theprecise functional role of each DC subset in immune
responses remains unclear. Our goal is to clarify the role of
DC subsets in the immune system in vivo and to identify themolecular basis for the regulation of their function.

Crucial roles of B7-H1 and B7-DC expressed on
mesenteric lymph node dendritic cells in the generation of antigen-specific CD4+Foxp3+regulatory T
cells in the establishment of oral tolerance
Oral tolerance is a key feature of intestinal immunity, generating systemic tolerance to food antigens. However, themolecular mechanism mediating oral tolerance remains
unclear. In this study, we examined the role of the B7 family
members of costimulatory molecules in the establishment oforal tolerance. Deficiencies of B7-H1 and B7-DC abrogatedoral tolerance; instead there was an immune response
accompanied by enhanced antigen-specific CD4+T cell-

Recent publications
1. Fukaya T., Takagi H., Sato Y., SatoK., Eizumi K., Taya H., Shin T., Chen
L., Dong C., Azuma M., Yagita H.,
Malissen B., Sato K. Crucial rolesof B7-H1 and B7-DC expressed on
mesenteric lymph node dendritic cells
in the generation of antigen-specific88

CD4+Foxp3+regulatory T cells in
the establishment of oral tolerance.
Blood 116, 2266-2276 (2010)

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

3. Sato K., Eizumi K., Fukaya T., Fujita
S., Sato Y., Takagi H., Yamamoto M.,
Yamashita N., Hijikata A., Kitamura H.,
Ohara O., Yamasaki S., Saito T., Sato K.
Naturally occurring regulatory dendritic
cells regulate murine cutaneous
chronic graft-versus-host disease.
Blood 113, 4780-4789 (2009)

CD19/CD11c/CD3εFigure :Splenic localization of CD11chighcDCs and BST2+pDCs. Immunofluorescent
microscopic analysis was performed on frozen sections that were stained for CD19
(green), CD3ε (blue) and CD11c (red; A) or BST2 (red; B). Original magnification; 4x.

responses and IgG1 production. Mesenteric lymph node
( MLN) dendritic cells ( DCs) displayed higher levels of
B7-H1 and B7-DC than systemic DCs, whereas they showedsimilar levels of CD80, CD86, and B7-H2. Relative to systemic DCs, the MLN DCs enhanced the antigen-specificgeneration of CD4+Foxp3+inducible regulatory T ( iTreg ) cells
from CD4+Foxp3-T cells rather than CD4+effector T ( Teff )cells, owing to their dominant expression of B7-H1 and
B7-DC. Furthermore, the antigen-specific conversion of
CD4+Foxp3-T cells into CD4+Foxp3+iTreg cells occurred in
MLNs to a greater extent than in peripheral organs during
oral tolerance under steady-state conditions. Moreover, thisconversion required B7-H1 and B7-DC more than other B7
family members, whereas it was severely impaired under
inflammatory conditions. In conclusion, our findings suggestthat B7-H1 and B7-DC expressed on MLN DCs are essential
for establishing oral tolerance through the de novo generation of antigen-specific CD4+Foxp3+ iTreg cells.

Naturally occurring CD49b+CD200R3+regulatory
DCs induce regulatory T cell-mediated peripheral
tolerance
Despite their key functions in multiple aspects of the immune

4. Candolfi M., Yagiz K., Foulad D.,
Alzadeh G.E., Tesarfreund M.,
Muhammad A.K., Puntel M., Kroeger
K.M., Liu C., Lee S., Curtin J.F., King
G.D., Lerner J., Sato K., Mineharu
Y., Xiong W., Lowenstein P.R., Castro
M.G. Release of HMGB1 in response toproapoptotic glioma killing strategies:

CD19/BST2 /CD3εefficacy and neurotoxicity. Clin.
Cancer Res. 15, 4401-4414 (2009)

system, there is much to be learned about the DCs, including which subset is specialized for the induction of immunological tolerance. We have identified a naturally occurring DC
subset with a regulatory function, termed regulatory DCs( DC regs ). DCregs generated from bone marrow in vitro ( BMDC regs ) exclusively expressed CD200 receptor 3 ( CD200R3) ,which exerted a suppressive function in the antigen-specific
CD4+T-cell response. CD49+CD200R3+cells showed similarities in phenotype and function to BM-DC regs, which formally distinguishes them from other leukocytes, suggestingthat they are the natural counterpart of BM-DC regs. In vivo,
CD49+CD200R3+cells not only generated antigen-specificanergic CD4+T cells but also induced the peripheral conversion of CD4+CD25+Foxp3+regulatory T ( Treg ) cells from
CD4+CD25-Foxp3- T cells. In addition, CD49+CD200R3+cells
as well as BM-DC regs protected against experimental autoimmune encephalomyelitis ( EAE) , an effect mediated throughthe suppression of the development of pathogenic T helper
type ( TH )1 cells and TH17 cells, and the depletion of these
subsets enhanced EAE pathogenesis. In conclusion,
CD49+CD200R3+cells act as naturally occurring DC regs tomaintain immunological tolerance and to regulate immune
pathogenesis.

5. Fujita S., Yamashita N., Ishii Y., SatoY., Sato K., Eizumi K., Fukaya T.,
Nozawa R., Takamoto Y., YamashitaN., Taniguchi M., Sato K. Regulatory
dendritic cells protect against allergic
airway inflammation in a murine
asthmatic model. J. Allergy Clin.
Immunol. 121, 95 -104 (2008)89

Laboratory forTeam Leader

Immunochaperones

Hei-ichirô Udono

Research Scientist
Technical Staff
Student Trainee: Shusaku Mizukami: Chiaki Kajiwara, Takashi Matsuura, Yu Kato
: Shizuha Uda

C

D8+ T cells recognize naturally processed peptides in
the context of MHC class I molecules. During viral
infection or malignant transformation, intracellular changes
in non-APC ( antigen presenting cells) must be reported to
CD8+ T cells, which are indispensable in fighting virusinfected cells and cancer cells. However, these abnormal
cells cannot act by themselves as APC to prime T cells,
although they express tumor or viral antigens on their cellsurface. In addition, many viruses cannot infect professional
APC, such as DCs ( dendritic cells) , and thus the APC cannot present such viral antigens directly to CD8+ T cells.
Therefore, DCs must internalize neighboring tumor orinfected cells, digest them, and then present antigen peptides to CD8+ T cells in the context of MHC class I molecules.
This pathway is called cross-presentation and is believed to
be specific to DCs and not other APC type such as macrophages or B cells. We have evidence that molecular chaperones such as hsp90 play important roles in this antigen
cross-presentation. One of our research goals is to identify
the mechanisms of cross-presentation, especially focusing
on the role of the molecular chaperone hsp90. During thestudy of cross-presentation, we developed a mAb that canefficiently detect cell surface hsp90 on DCs and macrophages. Since hsp90 has no transmembrane domain, it
must be associated with unknown molecules for anchorage
to the cell surface. We are currently examining the physiological significance of cell surface hsp90 in the context of theimmune response.

The role of hsp90 in antigen cross-presentation
In general, the main functions of HSPs are ( i ) facilitate protein folding/refolding and prevent aggregation of newly synthesized proteins on the ribosome; ( ii ) maintain the normal
function of mature proteins; ( iii ) target misfolded and/or
damaged mature proteins for degradation by the proteasome; ( iv ) transport proteins between distinct intracellularcompartments such as cytosol and mitochondria. Proteinstargeted to the proteasome undergo degradation in order torecycle amino acids, and the byproducts of this process( degradation products, peptides) are utilized as antigen
peptides, mainly presented by MHC class I molecules. Thus,
proteasomal degradation is important at the end of the life
cycle of all proteins to prevent waste of raw materials and isused by the immune system to alert T cells of any alterations

Recent publications
1. Ichiyanagi T, Imai T, Kajiwara C,
Mizukami S, Nakai A, Nakayama
T, Udono H. Essential role ofendogenous HSP90 of dendritic cells
in antigen cross-presentation. J.
Immunol. 185, 2693-2700 (2010)90

2. Ohkusu-Tsukada K, Toda M, Udono
H, Kawakami Y, Takahashi K. Targeted
inhibition of the p38 MAP kinase of
IL-10-secreting CD25 - T regulatory
cells in cancer immunotherapy. Eur.
J. Immunol. 40,1011-1021 (2010)

3. Udono H, Ichiyanagi T, Mizukami
S, Imai T. Heat Shock Proteins in
Antigen Trafficking -Implications on
Antigen Presentation to T cells- Int. J.
Hyperthermia 25, 617-625 (2009)

4. Yamano T, Mizukami S, Murata S,
Chiba T, Tanaka K, Udono H. Hsp90
mediated assembly of the 26S
proteasome is involved in MHC
class I antigen processing. J. Biol.
Chem. 283, 28060-28065 (2008)

Figure :HSPs mediate degradationof a few percent of newly
synthesized proteins aswell as mature proteins. In
dendritic cells, HSP90 seems
to facilitate the degradation ofeven internalized exogenous
proteins following theirtranslocation to the cytoplasm.
The proteolysis step depends
on the ubiquitin-proteasome
system to produce peptides
presentable by MHC class I
molecules and amino acids thatare reused for protein synthesis.

in the intracellular antigenic milieu. Each step towards proteolysis by the proteasome is regulated, at least in part, by
HSPs. In considering how HSPs are involved in cross-presentation ( in terms of translocation of endosomal antigen to
the cytosol ) , there might be two distinct mechanisms. First,
HSPs direct carboxyl terminus of hsc70-interacting protein( CHIP) -mediated polyubiquitinylation of retro-translocatedunfolded proteins for proteasomal degradation. Second,
HSPs direct retro-translocation of proteins from the endosome to cytosol, a mechanism that is supported by evidence
that Bip and cytosolic hsp70/hsp90 drive polypeptides into
the ER and the mitochondria, respectively. Our recent observations clearly indicate that HSPs play a pivotal role in antigen trafficking, which links to antigen cross-presentation as
well as endogenous antigen processing.

The role of cell surface hsp90 on dendritic cells
HSP90 is an abundant cytosolic molecular chaperone associated with protein folding, transport and degradation. Witha novel mAb, 6H8, we found that HSP90 is also expressedon the surface of bone marrow derived dendritic cells( BMDCs ) and macrophages. The HSP90 expression level isenhanced by type I and II interferons but not by other cytokines. The 6H8 mAb bound to DCs was rapidly internalized,
and the immobilized form stimulates BMDCs to produce
IL-12. Incubation of 6H8 coupled with OVA ( 6H8-OVA ) with

DCs resulted in efficient cross-presentation of the dominant
epitope, OVA 257-264, a process which was blocked by free
6H8 mAb, indicating that the cross presentation truly
depends on cell surface HSP90. Alexa-labeled 6H8 mAb
accumulated on CD8+ but not on CD8- DCs in vivo one hour
after intravenous injection. Administration of less than amicrogram of a 6H8-OVA conjugate induced vigorous
proliferation of adoptively transferred OT-I CD8+ T cells, a
response that did not occur in DC-deficient mice or TAP(transporter associated antigen processing) 1-deficient mice.
Furthermore, 6H8-OVA induced CTL in naïve mice and protected them from lung metastasis of melanoma MO5 cells
expressing OVA.
These results indicate that targeting cell surface HSP90
by 6H8 bearing antigens of interest enables cross-priming
but not cross-tolerance of antigen-specific CD8+ T cells,raising the possibility of a novel antibody-based vaccine
strategy for induction of cytotoxic T cell immunity.
In addition to this application strategy using cell surface
hsp90, we are currently investigating more fundamental
aspects – the physiological significance of cell surface hsp90
and the mechanism by which hsp90 is localized on the cellsurface of DCs and macrophages. A full answer to thesequestions may open an avenue to a novel role of the molecular chaperone hsp90 in immunity.

5, Yamano T, Sugahara H, Mizukami S,
Murata S, Chiba T, Tanaka K, Yui K and
Udono H. Allele-selective effect of PA28
in MHC class I antigen processing.
J.Immunol. 181, 1655-1664 (2008)91

Research Unit for
Unit leader

Immune Homeostasis

Shohei Hori

Research Scientists : Masashi Tachibana, Maria Encarnita Mariotti-Ferrandiz (JSPS Foreign Post-Doctoral Fellow)
Technical Staff: Takahisa Miyao, Guanying Wang, Kuniko KamiyaAsmall subpopulation of T lymphocytes known as regulatory T ( Treg ) cells plays a central role in preventing
pathological immune responses including autoimmunity,
inflammation and allergy, thereby ensuring self-tolerance
and immune homeostasis. This has been well illustrated by
the findings that the development and function of Treg
cells is controlled by the transcription factor Foxp3, and that
defective generation of functional Treg cells underlies thecatastrophic autoimmune pathology that develops in
Foxp3-mutant scurfy mice and in human patients with the
IPEX syndrome.
The identification of Foxp3 as a central regulator ofTreg cell differentiation and function has provided a key to anumber of outstanding unresolved questions concerning
their role in tolerance and immune regulation, and thephysiology of these cells including their origins, the mechanisms controlling their development and function, and theirantigen specificity. Resolving these issues is the goal of thislaboratory.

Resolving the controversies over Treg cell plasticity
Early studies of organ-specific autoimmunity provoked byneonatal thymectomy, and of transplantation tolerance
induced by thymic epithelium grafts have suggested that

Foxp3+ Treg cells represent a thymus-committed cell lineage, which is distinct from CD4+ helper and CD8+ cytotoxic
T cell lineages. The discovery of Foxp3 as the “master regulator” of Treg cells has further strengthened this notion of acommitted lineage by suggesting that it is Foxp3 that programs and maintains their regulatory functions. The irreversibility of the Treg cell fate has been believed to be necessary
to ensure the robustness of self-tolerance and immune
homeostasis in a changing environment.
Recent findings have challenged this notion of a committed Treg cell lineage by suggesting that these cells retain
developmental plasticity- the capacity to be “reprogrammed”
to various Foxp3- helper T ( Th ) cell subsets in response toenvironmental perturbations such as lymphopenia andinflammation. However, this issue of Treg cell lineage plasticity remains controversial because unequivocal evidence forlineage reprogramming is lacking and because there areother recent studies that argue against the plasticity phenomena. We have previously proposed that Foxp3+ T cells
are heterogeneous and consist of the majority populationcommitted to the Treg lineage and a minority uncommitted( or un-programmed ) population that retains plasticity. We
have addressed the nature of the “uncommitted” Foxp3+ T
cells converting to Foxp3- Th cells under physiological, lym-

Recent publications
1. 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, I.I., Umesaki, Y., Itoh, K., Honda,
K. Induction of colonic regulatory T cells
by indigenous Clostridium species.
Science 331: 337-341 (2011)92

2. Hori, S. Developmental plasticity of
Foxp3+ regulatory T cells. Curr. Opin.
Immunol. 22, 575-582 (2010)

3. Tsuji M., Komatsu N., Kawamoto
S., Kanagawa O., Honjo T., Hori S.*,
Fagarasan S.* (*corresponding/senior authors) Preferential generationof follicular B helper T cells from
Foxp3+ T cells in gut Peyer’s patches.
Science 323, 1488-1492 (2009)

4. Komatsu N., Mariotti-Ferrandiz M.E.,
Wang Y., Malissen B., Waldmann
H., Hori S. Heterogeneity of natural
Foxp3+ T cells: a committed regulatory
T cell lineage and an uncommitted
minor population retaining plasticity.
Proc. Natl. Acad. Sci. U.S.A.
106, 1903-1908 (2009)

Figure :A model of Treg lineage commitment and plasticity. During thymic or peripheral Treg cell differentiation, TCR and other signals specify
precursor cells to the Treg cell differentiation pathway and induce Foxp3 expression. At this stage, the Foxp3 locus remains methylated
and the induced Foxp3 expression can be easily extinguished by external perturbations such as inflammatory signals. The locus gradually
becomes demethylated but such partially demethylated states may still be susceptible to external perturbations. Once the locus
becomes fully demethylated, Foxp3 binds to it in cooperation with other transcription factors and self-enforces its own transcription.

phopenic, and inflammatory conditions. By using serial
adoptive transfer as well as fate mapping strategies, we
showed that there is certain time window in thymic andperipheral Foxp3+ T cell differentiation during which Foxp3
expression is transient. After this time window, Foxp3
expression becomes fixed and Foxp3+ T cells become irreversibly committed to the Treg cell fate. These findings
resolve the current controversies over Treg cell plasticity andindicate that Treg lineage commitment is a multilayered process, determined not by Foxp3 expression per se but by
higher-order regulation, which involves “epigenetic” modifications of the Foxp3 locus and Foxp3-dependent selfenforcement of Foxp3 transcription ( Figure) .

Impact of naturally occurring Foxp3 genemutations on Treg cell differentiation and function
In order to understand the molecular mechanisms by which
Foxp3 controls Treg cell differentiation and function, we havebeen addressing whether and how Foxp3 mutations that
had been identified in human IPEX impair Treg cell development and function. We previously showed that all the missense mutations examined were amorphic or hypomorphic
in that, upon retroviral transduction, these mutants fail toconvert conventional T cells into fully functional Treg cells. To
determine the impact of these mutations in vivo, we haveestablished three independent knock-in mouse strains harboring foxp3 mutations that mirror those in human IPEX. We
found that hemizygous mutant males developed a scurfylike lymphoproliferative autoimmune disease, although miceharboring hypomorphic mutations developed a milder disease than mice with scurfy or amorphic mutations. Thymic
differentiation, peripheral maintenance, and suppressivefunctions of Foxp3+ Treg cells were impaired in two of thethree mutant mice. By contrast, Foxp3+ Treg cells from theother mutant strain showed compromised peripheral
homeostasis of Treg cells, while displaying normal thymic
differentiation, in vitro suppressor function, and overall gene
expression profiles. Strikingly, this homeostasis defect was
accounted for by the absence of a particular Treg cell subset. These results suggest that the defective differentiation
and/or maintenance of this Treg cell subset underlie thedevelopment of autoimmune pathology in this mutant strain.
We are currently testing this hypothesis and addressing how
this particular Foxp3 gene mutation results in this remarkable phenotype.

5. Komatsu, N. and Hori, S. Fullrestoration of peripheral Foxp3+
regulatory T cell pool by radioresistanthost cells in scurfy bone marrow
chimeras. Proc. Natl. Acad. Sci.
U.S.A. 104, 8959-8964 (2007)

93

Laboratory forGroup Director

Cytokine Signaling

Toshio Hirano

Senior Research Scientists : Toshiyuki Fukada, Keigo Nishida
Research Scientists: Satoru Yamasaki, Wakana Ohashi (Research Associate), Shintaro Hojyo (Research Associate)
Technical Staff: Ayumi Ito, Masami Kawamura, Mayumi Hara
Student Trainees: Byn Beum-Ho (JRA), Aiko Hasegawa

Despite their increasing prevalence in developed countries, the molecular mechanisms leading to autoimmune
diseases and allergies remain poorly understood. The ultimate
goal of the Cytokine Signaling Research Group is to help elucidate the underlying molecular and immunological mechanisms of these diseases from the viewpoint of signaltransduction within the immune system. Recently, we havereported that heavy metal cations such as Zinc ( Zn) might act
as intracellular signaling molecules, i.e., molecules whose
intracellular status is altered in response to 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 have not been resolved. Therefore, we arefocusing our attention on the largely unknown universe of
signaling through Zn transporter proteins, to understand therole of Zn in immune and other physiological systems.

Mast cell biology and molecular mechanisms of its
functions
We are investigating cellular and molecular mechanisms of
the involvement of mast cells in allergy, inflammation, andautoimmune diseases. Many researchers have believed thatmast cells are major players in allergic responses, and IgEdependent activation of mast cells leads to degranulation andcytokine production. However, how these distinct mast cell

responses are differentially controlled is still unclear. We dissected Fc epsilon RI-mediated signal transduction in mastcell degranulation and cytokine production. First, we foundthat the Zinc transporter, ZnT5/Slc30a5 is required for contact
hypersensitivity, a mast cell-mediated delayed-type allergic
response, but not for passive cutaneous anaphylaxis, animmediate response. In mast cells from Znt5-KO mice, Fc
epsilon RI-induced cytokine production was diminished, but
degranulation was intact. ZnT5 was essential for Fc epsilon
RI-induced translocation of PKC to the plasma membrane
and the nuclear translocation of NF-kappa B. In addition, we
found that the zinc finger– like motif of PKC was required forits plasma membrane translocation and binding to diacylglycerol. Thus, ZnT5 is selectively required for the mast cell-mediated delayed-type allergic response and is a novel player in
PKC signaling (Nishida et al., J Exp. Med, 2009). Second,
recently we found that Gab2 knock-in mice that express
Gab2 mutated in either the PI-3K or SHP-2 binding sites havea defect in mast cell-mediated immediate-type allergic
responses, but not in delayed-type responses. We also
showed that the PI-3K-binding site, but not the SHP-2-binding site, was involved in Fc epsilon RI-dependent ARF1 activation and granule translocation to the plasma membrane
during the degranulation process, further dissecting the signals required for degranulation. Finally, we established themolecular framework of the Fyn/Gab2/PI-3K-dependent

Recent publications
1, Nishida, K., S. Yamasaki, A. Hasegawa,A. Iwamatsu, H. Koseki, and T. Hirano.
Gab2, via PI-3K, regulates ARF1 in
FcεRI-mediated granule translocationand mast cell degranulation. J.
Immunol. 187: 932-941, (2011)94

2, Hojyo, S*., T. Fukada*, S. Shimoda,
W. Ohashi, B-H. Bin, H. Koseki, T.
Hirano. (*equal contribution) The zinctransporter SLC39A14/ZIP14 controls
G-protein coupled receptor-mediatedsignaling required for systemic growth.
PLoS ONE 6(3): e18059. (2011)

3, Murakami, M*., Y. Okuyama*, H. Ogura*,
S. Asano, Y. Arima, M. Tsuruoka, M.
Harada, M. Kanamoto, Y. Sawa, Y.
Iwakura, K. Takatsu, D. Kamimura,T. Hirano. (*equal contribution)
Local microbleeding facilitates
IL-6– and IL-17–dependent arthritis
in the absence of tissue antigen

recognition by activated T cells. J.
Exp. Med. 208, 103-114, (2011)

Figure1: A: Model of the Fc epsilon RI-mediated Fyn/Gab2/PI-3K/ARF1 signaling
pathway involved in granule translocation and mast cell degranulation andthe anaphylaxis response. B: Inhibition of ARF1 inhibits the IgE-mediatedanaphylaxis response. Both ears were injected with antigen-specific IgE
antibodies. After 16-18 h, the antigen was administered intravenously
together with a blue dye and the amount of dye extravasating into the ear
tissue was measured. One ear was pretreated with saline (Upper panel) andthe other with an ARF-1 inhibitor (Lower panel) before antigen injection.

ARF1-mediated granule translocation and mast cell degranulation (Fig.1) (Nishida et al., J. Immunol., 2011). Thus, this is
the first demonstration of a molecular mechanism to differentiate the immediate and delayed allergic responses.

Role of Zn and its transporters in immune andnon-immune tissues
We found that the Zn transporter Slc39a6/Zip6/Liv1 is a
STAT3 target gene and have shown that it has a role in cell
migration during early zebrafish development. (Yamashita
et al., Nature. 2004). We also found that LPS-induced
maturation of DCs is partly mediated through lowering theintracellular concentration of free Zn by down regulating Zntransporters, including Slc39a6 (Kitamura et al., Nature
Immunology., 2006), suggesting involvement of Zn in MHC
class II cell surface expression through regulating endocytosis
and membrane trafficking. In order to understand the role andfunction of Zn and Zn transporters in vivo, we havegenerated 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), in
which there are defects in the maturation of osteoblasts,chondrocytes, odontoblasts, and fibroblasts. Impairment ofbone morphogenic protein (BMP) and TGF-β signaling wasobserved in the corresponding tissues and cells of the knockout mice. Homozygosity for an SLC39A13 loss of function
mutation was identified in human sibs affected with a uniquevariant of EDS that recapitulates the phenotype observed in
Slc39a13-KO mice. Hence, our results have revealed a crucial role of SLC39A13 in connective tissue development at

4, Kitabayashi C., T. Fukada, M.
Kanamoto1, W. Ohashi, S. Hojyo, T.
Atsumi, N. Ueda, I. Azuma, H. Hirota,
M. Murakami, and T. Hirano. Zinc
suppresses Th17 development via
inhibition of STAT3 activation. Int.
Immunol. 22, 375-386, (2010)

Figure2 : Schematic model of the regulation of GPCR-mediatedsignaling by Slc39a14. Slc39a14 regulates basal cAMPlevels by suppressing PDE activity, which enhances the
GPCR-cAMP-CREB pathway to control systemic growth.

least in part due to its involvement in BMP/TGF-β signaling
pathways (Fukada et al, PLoS ONE, 2008). In addition, we
have investigated the role of Slc39a14 by generating deficientmice. The Slc39a14-KO mice showed progressive systemic
growth defects, and impaired signaling of G-protein -coupled
receptors (GPCR) was observed in the growth plate, pituitary
gland, and liver. We found that Slc39a14 -mediated Zn transport plays a role in control of GPCR-cAMP response elementbinding protein (CREB) signaling via regulating cAMP levels
by suppressing phosphodiesterase (PDE ) activity (Figure 2,
Hojyo et al, PLoS ONE, 2011), suggesting a crucial role for
Slc39a14 in control of mammalian systemic growth.
Recently, we showed that Zn suppresses Th17-mediatedautoimmune diseases by inhibiting the development of Th17
cells via attenuating STAT3 activation. In mice injected with
type II collagen to induce arthritis, Zn treatment inhibited Th17
cell development. IL-6-mediated activation of STAT3 and invitro Th17 cell development were all suppressed by Zn. Mechanistically, we found that Zn binding changed the α-helicalsecondary structure of STAT3, disrupting its association with
JAK2 kinase and with a phospho-peptide that included a
STAT3-binding motif from the IL-6 signal transducer gp130.
Thus, we conclude that Zn suppresses STAT3 activation,
which is a critical step for Th17 development (Kitabayashi etal, Int. Immunol., 2010).
Together these results support the idea that Zn has roles
in mediating and controlling intracellular signaling events. We
propose the term “Late Zn signaling” for this type of Zn signaling, in contrast to “Early Zn signaling”. The former is dependent on changes in the transcription of Zn transporter genes,
whereas the latter is not (Hirano et al, Adv. Immunol., 2008).

5, Nishida K*., A. Hasegawa*, S. Nakae,
K. Oboki, H. Saito, S. Yamasaki,
and T. Hirano. (*equal contribution)
Zinc transporter Znt5/Slc30a5 isrequired for the mast cell-mediateddelayed-type allergic reaction but not
the immediate-type reaction. J. Exp.
Med. 206, 1351-1364, (2009)95

Laboratory for

Signal Network
Team Leader

Masato Kubo

Research Scientist: Yuriko Nakatani

Visiting Scientist: Yasutaka Motormura

Technical Staff: Yoshie Suzuki

Student Trainee: Minoru Sawaguchi

Student Trainee / Part Timer : Keisuke SuzukiTcells play a central role in the effector and regulatory
functions in immunological surveillance, and aberrationsin these functions can lead to various immunological disorders. T helper 1 ( TH1) cells secrete interleukin-2 ( IL-2) , IFN-γ
and TNF-α during the cellular immune response to intracellular pathogens and viruses. TH 2 cells, which are mainly
protective against extracellular pathogens, but also accountfor allergic immune responses, produce IL-4, IL-5, IL-6,
IL-10 and IL-13. TH17 cells, which are important for defense
against certain bacteria and fungal pathogens, especially at
mucosal and epithelial surfaces, express IL-17 and IL-22
and are important effector cells in autoimmune tissue inflammation. These cytokines secreted by the different effector
helper T cells play a critical role in controlling the outcome of
immunological surveillance. The helper T cell subsets aredifferentiated from common precursor cells, naïve T cells,
and these cells accomplish their differentiation by acquiring
the capacity to produce certain types of cytokines. The
overriding goal of our laboratory is to understand the molecular mechanisms of cytokine gene regulation during helper
T cell differentiation.

Regulation of Il4 gene expression in type 2 helper T
cells by an Il4 intronic enhancer HS2
Transcription of the TH 2-associated cytokine genes, IL-4,
IL-5 and IL-13 is controlled by the TH 2 cell master regulator
GATA3. However, the molecular basis of GATA3-mediatedgene regulation during TH2 cell development is an inconclusive and controversial area of research. We recently showedthat binding of GATA3 to the DNase-I hypersensitivity site 2( HS2) in the second intron of the Il4 locus is specificallyrequired for chromosomal modifications at the Il4 locus thatallow transcription of Il4.
Several regulatory elements in the TH 2 locus have been
identified; however whether TH 2-associated cytokine expression is controlled by a single element or by the coordinatedactivity of multiple elements is not known. To address thisissue, we generated a series of mutant mice that lack each
HS element in the Il4–Il13 locus and assessed the affect ofthese mutations on cytokine production. TH 2 cells from micethat lack HS2 produced only low levels of IL-4 whereas theexpression of IL-13 and IL-5 remained normal. By contrast,
naive T cells that lack the conserved GATA3-response

Recent publications
1. Tanaka, S., Motomura, Y., Suzuki,
Y., Yagi, R., Inoue, H., Miyatake,S., Kubo, M.: The enhancer HS2
critically regulates GATA-3-mediatedIl4 transcription in TH 2 cells. Nat.
Immunol. 12, 1, 77-85, 201196

2. Park, JH., Adoro, S., Guinter, T.,
Erman, B., Alag, A., Catalfamo, M.,
Kimura, M.Y., Cui Y., Lucas, P.J., Gress,
R.E., Kubo, M., Hennighausen, L.,
Feigenbaum, L., Singer, A.: Signaling
by intrathymic cytokines, not T cell
receptors, specifies CD8-lineage

choice and promotes differentiationof cytotoxic-lineage T cells Nat.
Immunol. 11, 3, 257-265, 2010

3. Tanaka, S., Yoshimoto, T., Naka, T.,
Nakae S., Iwakura Y., Cua, D. and Kubo,
M.; Natural Occurring Il-17 producing
T cells regulate the initial phase ofneutrophil mediated airway responses.
J. Immunol. 183, 7523-7530, 2009

Figure :Modification of chromatin structureat Th2 cytokine loci during helper T
cell differentiation. The chromatin
structure at the Th2 cytokine lociis compacted in naïve T cells,
and antigen stimulation via the Tcell receptor randomly promotesa permissive situation though
acetylation (Ac) of core histones(H3). GATA-3 binds to tits cognate
binding site localized in each Th2
cytokine gene, and this binding
gives rise to further acetylationand methylation (Me) of particularlysine residues on histones. By
contrast, GATA-3 is not expressedin Th1 and Th17 cells, therefore,
the chromatin structure in the Th2
cytokine loci remains condensed.

element ( GCRE) in the Il13 locus gave rise to TH 2 cells producing normal levels of IL-4 but not IL-13, indicating that thiselement regulates Il13 transcription.
These gene expression defects were reflected by abnormalities in transcription-permissive epigenetic changes normally observed at the Il4 and Il13 loci. Indeed, the permissive
histone code; acetylation of histone H3 at Lys9 and Lys14
and trimethylation of histone H3 at Lys4 was impaired in
HS2-deficient TH 2 cells, but only at the Il4 locus. By contrast,
deletion of GCRE resulted in impaired methylation of histone
H3 at Lys4 at the Il13 locus but not the Il4 locus. In the TH 2
differentiation process, GATA3 functions as a master regulator, so it is possible that binding of GATA3 to HS2 and CGRE
is required for TH 2 differentiation. HS2 and CGRE are crucial
GATA3-binding sites in the Il4 and Il13 locus, respectively,
and GATA3-binding to these regions independently regulates IL-4 and IL-13 expression.

Role of basophils and mast cells in inflammatory
responses
We established a diphtheria toxin ( DT ) -based conditionaldeletion system using Il4 enhancers specific for either mastcell ( MC ) s or basophils to drive the expression of the DT
receptor ( DTR) ( Mas-DTR and Bas-DTR mice) . While diphtheria toxin treatment of Bas-DTR mice resulted in specificdeletion of basophils, treatment of Mas-DTR mice resulted
in deletion of both MCs and basophils. Using these mice,
we sought to tease out the role of MCs and basophils in

4. Okamoto, M., Van Stry, M., Chung,L., Koyanagi, M., Sun, X., Suzuki,
Y., Ohara, O., Kitamura, H., Hijikata,A., Kubo, M.*and M., Bix,*; MINA53,
an Il4 co-repressor controllingTh2-bias Nat. Immunol. 10, 872-879,
2009. *equally correspondent.

5. Kano, S., Sato, K., Morishita, Y.,
Vollstedt, S., Kim, S., Taki, S., Honda, K.,
Kubo, M., & Taniguchi, T.; Regulation ofTh1 vs. Th17 differentiation: Selective
contribution of the transcription
factor IRF1 to the IFN-γ-IL-12 axis of
signaling networks in CD4+ T cells.

several IgE-mediated disease models. We found that MCs
and basophils played distinctive roles in the early and chronic
phases of the IgE-mediated allergic response. In an asthma
model, MCs appeared to play a critical role in the effector
phase but not in the induction phase. Although basophil
deletion partially impaired cysteine protease-mediated IL-4
production by antigen-specific T cells, basophils were dispensable for asthmatic responses, and for IgE
production induced by systemic antigen immunization andby Trichinella spiralis infection. By contrast, MC deletion
resulted in increased serum IgE levels, which were stably
sustained for more than 15 days. Thus, these newly established Mas-DTR and Bas-DTR mice have allowed us todemonstrate distinct roles of MCs and basophils in IgEmediated allergic responses and in regulation of serum
IgE levels.
Using the Mas-DTR mouse model, we also examined
the role of mast cells in contact hypersensitivity ( CHS) . CHS
was attenuated when MCs were depleted during the sensitization phase. In addition, both maturation and migration ofskin dendritic cells ( DCs) were abrogated by MC depletion.
Consistent with these findings, co-culture with bone marrow-derived MCs ( BMMCs) enhanced maturation andchemotaxis of BMDCs in vitro. These results suggest that
MCs interact with DCs in the skin and enhance DC functions. This interaction might be essential for establishing thesensitization phase of CHS.

Nat. Immunol. 9:34-41, 2008

97

Laboratory for

Immunogenetics
Team Leader

Hisahiro Yoshida, M. D., Ph. D.

Research Scientist : Takuwa Yasuda
Technical Staff: Hitomi Fukiage
		 Akimi Banno
		 Ayako Kobayashi
		 Mikiko Sato
Student TraineeT

he main activity of our team is the screening of a large
ethylnitrosourea ( ENU ) mutant mouse panel and is acollaborative effort with the RIKEN Genomic Sciences Center. An important goal of this project for RCAI is the development of novel mouse models for various immunologicaldisorders, notably, allergic and autoimmune disorders. Since
ENU mutagenesis introduces approximately 3,000 point
mutations on 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 with allergens and adjuvants to identify allergic condition 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 singlebase 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.

: Machiko Sugiyama

During the first 4 years of this project we have screened
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 fromanimals at 16 weeks of age. We have thus analyzed themutant phenotypes by pathological, cytological and molecular biological evaluations of affected tissues, lymphoidorgans and blood cells.
A phenotype identified in a mutant candidate line isreexamined at a later developmental stage and, if the same
phenotype is confirmed, we begin mating the mice for phenotype inheritance tests. The responsible mutant loci aremapped by backcrossing mutant individuals with the C3H/
HeJ strain for gene detection by single-nucleotide polymorphism analysis. After approximate mapping, candidate genesequences are compared with wild type sequences 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

Recent publications

98

1. Matsushima Y, Kikkawa Y., TakadaT., Matsuoka K., Seki Y., Yoshida H.,
Minegishi Y., Karasuyama H., andYonekawa H. An Atopic DermatitisLike Skin Disease with HyperIgE–emia Develops in Mice Carrying a
Spontaneous Recessive Point Mutationin the Traf3ip2 (Act1/CIKS) Gene.
J Immunol. 185(4),2340-9 (2010)

2. Wada H., Yasuda T., Miura I., Watabe
K., Sawa C., Kamijuku H., Kojo S.,
Taniguchi M., Nishino I., Wakana S.,
Yoshida H., Seino K. Establishmentof an improved mouse model for
infantile neuroaxonal dystrophy thatshows early disease onset and bears
a point mutation in Pla2g6. Am J
Pathol. 175(6), 2257-63 (2009)

3. Kakugawa K., Yasuda T., Miura I.,
Kobayashi A., Fukiage H., Satoh R.,
Matsuda M., Koseki H., Wakana S.,
Kawamoto H., Yoshida H. A novel
gene essential for the developmentof single positive thymocytes. Mol
Cell Biol. 29(18), 5128-35. (2009)

4. Aoki H. ,Yoshida H. ,Hara A. ,Suzuki
T. ,and Kunisada T.: Transplantationof melanocytes into iris: method foriris repigmentation.. Transplantation
85, 492--494. (2008)

Stepwise AD-like disease development in the AD mutant
Initiation

Chronic

Progression

Skin Barrier Permeability Destruction
Onset of Dermatitis
Elevation of Serum IgE
Elevation of Serum
IgG2b & IgG2c
2w

8w

10w

12w

16w

20w

Skin permeability defect was conﬁrmed at 2 weeks after birth
Itchness started and persisted
Mechanical stress by scratching & wound healingTh2 condition started and persisted

Chronological accumulation of clinicalevents in this mutant animal

Th1 and Th17 condition
started and persisted

Figure 1: An atopic dermatitis-like mutant mouse was established from the
ENU screen. The above scheme illustrates the stepwise progressionof 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).

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 typical human allergic disease. The ear skinbecame thicker and red and, as the mice aged, they started
to scratch the ear skin or face. The serum IgG1 and IgElevels of these mice gradually increased at 3 weeks after thedermatitis onset. Pathologically, the epidermal layer was
hypertrophic and many inflammatory cells were found in thedermis of the lesion. Finally, they developed chronic facialand ear skin inflammation and not only Th2 serum immunoglobulin but also Th1 serum immunoglobulin levels increased.
These stepwise symptoms and findings are compatible withthe criteria for human infantile atopic dermatitis ( AD ) . We
have mapped the phenotype-causing point mutation to a
distinct gene and directly confirmed this causality by genetic
manipulation to introduce the same mutation into the geneof the wild type animal. A bone marrow transplantation study
demonstrated that the disease is induced by mutant skintissue and not by mutant bone marrow cells. We have clarified the disease onset mechanism and succeeded in disease
onset prevention based on the results of our studies ( manuscript under submission) . We are now further analyzing the
stepwise progression of this AD-like disease in the mutant

ENU mutant phenotype screen; 80 pedigrees, 4 years

Phenotype2nd screen byAllergen
Immunization

145 lines

Immunoglobulins
Leukocyte number or proﬁles
Lymphoid organ development
Autoimmune related
Leukocyte function

80 lines
45 lines
9 lines
12 lines
10 lines

Allergy related phenotypes(Th2-biased phenotype)①IgG1 high②IgG1, IgE high③IgE high④Mast cell related⑤Eosinophil related⑥Allergic disease⑦IgE production to allergen

experimental
allergic conjunctivitisorallergic asthma

60 lines
6 lines
15 lines
12 lines
10 lines
5 lines
3 lines
9 lines

Wild type

ENU mutant
with asthmatic
reaction

Figure 2: Summary of four years of ENU mutant screening. In total,
approximately 8,000 mutant mice from 80 genomes have been screened
and 145 phenotypes related to immunity or allergy have been identified.
The images in the bottom right show the 3 dimensional renderings of thelungs of WT and mutant mice after OVA immunization and challenge.

mouse to better understand the allergic disease progressionin skin and the immune system.
By ENU recessive mutant screening, we have identified
and established more than 145 mutant lines of immune orblood disease models. In keeping with our expectation,
more than 50 lines had phenotypes related to allergic diseases. From these mutant lines, we are now analyzing amutant 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 2010, we have mapped thirty two independent mutantcausing loci to distinct regions, and nine of them have been
identified to be point mutations in independent genes. Alongwith 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 for the human neurodegenerative disease
infantile neuroaxonal dystrophy ( INAD ) ( Wada et al., 2009 ) ,and 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 model for a primary
immunodeficiency disease ( Kakugawa et al., 2009 ) .

5. Hamanaka S, Nabekura T, Otsu M,
Yoshida H, Nagata M, Usui J, Takahashi
S, Nagasawa T, Nakauchi H, Onodera
M. Stable transgene expression inmice generated from retrovirallytransduced embryonic stem cells.
Mol. Ther. 15(3),560-5(2007)99

Laboratory forTeam Leader

Vaccine Design

Yasuyuki Ishii

Research Scientists
Visiting Scientists
Technical Staff: Hidetoshi Akimoto, Satoshi Komaniwa, Emi Fukuda: Kenichi Masuda, Omar Duramad: Haruka Katagiri, Risa Nozawa

Allergen-specific immunotherapy ( ASIT ) is used globallyfor treatment of various forms of pollinosis. However, inJapan, the only ASIT is subcutaneous immunotherapy ( SCIT )for Japanese cedar pollinosis and this therapy is not popular
due to the long treatment time and unclear mechanism ofaction. We have been studying two types of vaccine technologies whose aim is the clinical application of ASIT astherapy for Japanese cedar pollinosis. The first vaccine candidate is a PEGylated recombinant fusion protein of Cry j 1
with Cry j 2, which has much less risk of anaphylaxis thannatural allergen extracts, and can be utilized for not only for
SCIT but also for sublingual immunotherapy ( SLIT ) . The
fusion protein project was licensed to Torii Pharmaceutical
Co. The second candidate vaccine is liposomal α-GalCer, aligand for invariant natural killer T ( iNKT ) cells, encapsulating
the Cry j 1/2 fusion protein. The liposomes not only reduced
ongoing IgE antibody formation but also suppressed tertiary
antibody responses. Experiments are ongoing to elucidate
the detailed mechanisms of the liposome vaccine. Preclinical and phase I/IIa studies to confirm the proof of concept
are being done in collaboration with a biotech company,
REGiMMUNE Co, and supported by the Japan Science andTechnology Agency ( JST ) .

Mode of action of liposomal α-GalCer-antigen
To elucidate the detailed mechanism of liposome vaccinemediated suppression of IgE antibody responses, target
cells of the liposome vaccine after intravenous injection wereidentified by using rhodamine-labeled liposomal α-GalCer-OVA protein. Splenic B220+CD1d high cells, but not
B220+CD1d low cells, preferentially incorporated liposomes.
Immunofluorescence analysis of spleen sections showedthat the B220+CD1d high cells were localized in the marginalzone area and interacted there with iNKT cells. We foundthat CXCR6, which is a major chemokine receptor for iNKT
cells, and its ligand, CXCL16 were expressed on iNKT cells
and marginal zone B220+CD1d high cells, respectively. To test
whether CXCR6/CXCL16 was involved in the interaction of
the iNKT cells with the B220+CD1d high cells, co-administration of neutralizing antibody against CXCL16 and rhodaminelabeled liposomes into NKT-Venus cloned mice was carriedout. As shown in Figure, iNKT cells failed to traffic to themarginal zone area. The significance of interaction of iNKT
cells with marginal zone B220+CD1dhigh cells was supportedby the result that co-administration of neutralizing antibody
against CXCL16 with liposomal α-GalCer-OVA in OVAprimed mice diminished the immunosuppressive effects of
the liposome vaccine. Taken together, these results suggest

Recent publications
1. Okayama T, Matsuno Y, Yasuda
N, Tsukui T, Suzuta Y, Koyanagi
M, Sakaguchi M, Ishii Y, Olivry
T, Masuda K. Establishmentof a quantitative ELISA for themeasurement of allergen-specific100

IgE in dogs using anti-IgE antibody
cross-reactive to mouse and dog
IgE. Vet. Immunol. Immunopathol.
2011, 139 (2-4):99 -106.

2. Duramad O, Laysang A, Li J, Ishii Y andNamikawa R, Pharmacologic Expansionof 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 2011, 17, 1154-68

Figure :Venus-NKT cells (green) failed to traffic to the MZ B220+ cells, which havetaken up the liposomal antigen (red) after anti-CXCL16 antibody treatmentthat the interaction of iNKT cells with marginal zone
B220+CD1d high cells is essential for the immunosuppressive
activity of the liposomal α-GalCer -Cry j 1/2 fusion protein.
The next question was why IgE antibody response are
preferentially suppressed by the liposome treatment. One
possibility is the production of IL-21 which could induce
apoptosis of Bε cells. To test this possibility, in vitro cultureof liposome-treated B220+CD1d high cells, OVA-primed CD4+T
cells with or without iNKT cells was performed. The result of
quantitative PCR analysis indicated that IL-21 mRNA expression was only enhanced in the presence of iNKT cells.

Development of liposomal α-GalCer -Cry j 1/2
fusion protein (Code: RGI-1001)
Supported by a JST grant, development of RGI-1001 wasstarted at the end of 2008. Preclinical studies including
manufacturing, pharmacology, toxicology, pharmacokinetics
and pharmacodynamics are ongoing.
In the manufacturing process, each component of
RGI-1001 was independently produced. The first GMP lot ofα-GalCer, KRN7000 was released three years ago. Feasibility studies of liposome formulations had also been done.
Optimized liposome compositions for protein encapsulation
were determined. High level of production of recombinant
Cry j 1/2 fusion protein was successful in a fed-batch culture

system. A scalable purification process was developed,
which made it possible to provide GLP grade material. GMP
lot release is in preparation.
In pharmacology studies using non-GLP liposomal
α-GalCer -Cry j 1/2 fusion protein, the Cry j-sensitizedmouse asthma model showed that treatment with liposomes
could suppress not only the Cry j-specific IgE antibody
response after Cry j antigen challenge but also airway hyperreactivity. Cry j-sensitized monkey studies were just started
to confirm the efficacy of RGI-1001 in a primate model.
In toxicology studies, several safety concerns havealready been alleviated. One is that RGI-1001 might interferewith host defense against viral and bacterial infection. Studies of murine CMV and Listeria monocytogenes showed thatthe treatment with RGI-1001 did not suppress host defense.
Anaphylaxis risk of recombinant Cry j 1/2 fusion protein was
examined by using Cry j-sensitized mice. The results indicated that the anaphylaxis score and body temperature
were not affected, even when a large amount of the recombinant protein was intravenously injected into the sensitizedmice with high IgE antibody titers.
Next year, GLP toxicology and other studies required for
IND filing should be started.

3. Ishii Y, Motohashi S, Shimizu K,
Nakayama T, Taniguchi M, andFujii S, Application of NKT cells in
immunotherapy, Curr. Immunol.
Rev, 2010, 6, 109-115.

101

Research Unit for
Unit Leader

Human Disease Model

Fumihiko Ishikawa

Senior Research Scientist : Yoriko Saito
Technical Staff: Mariko Tomizawa-Murasawa, Akiko Sone, Nahoko Suzuki, Hiroshi Kajita
Graduate Students: Yuki Aoki, Shinsuke Takagi, Yuho NajimaT

he ultimate goal of our lab is to understand normal
human hematopoiesis and immunity and to conquer
human diseases that impact these systems. To do so, we
have created a humanized mouse model through intravenous injection of human stem cells into newborn NOD/SCID/
IL2rgKO mice. Transplantation of normal or diseased humanstem cells results in reconstitution of human immunity orrecapitulation of human diseases in the recipient mice. This
year, in the area of normal human immunity, we have analyzed the development of human innate immunity and innate
immune response in vivo. Among various human diseases,
we have continued to focus our investigations on human
myeloid and lymphoid leukemias for better understanding ofthese disease entities. In addition to these projects, we havebegun to study human iPS cells along with somatic stem
cells. Specifically, we are currently seeking a strategy for
inducing hematopoietic stem/progenitor cells from human
iPS cells and to understand the role of transcription factors
in hematopoietic development.
Reconstitution of human adaptive and innate immunity
in mice
Lack of an HLA-restricted human immune response has long

been one of the major obstacles in humanized mouse research.
To overcome this limitation, in collaboration with Dr. Leonard
D. Shultz at The Jackson Laboratory, we have created HLAclass A02 transgenic NOD/SCID/IL2rgKO mice. Human CD8+
T cells developing in the HLA class I positive NOD/SCID/
IL2rgKO recipients expressed functional molecules such as
granzyme, in accordance with their phenotypic maturationfrom naïve, central memory, to effector memory cells. Furthermore, we demonstrated that these human CD8+ T cells arefunctional in cytokine production and cytotoxicity, recognizing
EBV-infected cells and virus-associated peptides. These
functions are inhibited by the addition of anti-HLA class I antibody. Since we have detected a human CTL response against
a species-specific virus, we are currently analyzing the HLArestricted human T cell response to peptides.
Regarding human innate immunity, human myeloid cell
development in humanized mice has yet to be determined. We
therefore set out to analyze differentiation and function ofhuman myeloid cells in the recipients transplanted with purified
human cord blood CD34+CD38- HSCs. In the recipient bonemarrow and spleen, we detected HLA-DR- granulocytes and
HLA-DR+ antigen presenting cells within the human CD45+ cell
fraction. CD15+CD33low neutrophils, c-Kit+CD203c+ mast

Recent publications
1. Saito Y, Kitamura H, Hijikata A, TomizawaMurasaza M, Tanaka S, Takagi S, UchidaN, Suzuki N, Sone A, Najima Y, Ozawa H,
Wake A, Taniguchi S, Shultz LD, Ohara O,
Ishikawa F. Identification of therapeutictargets for quiescent chemotherapyresistant human AML stem cells. Sci.
Transl. Med., 2,17ra9, 2010102

2. 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. Cell cycle entry
potentiates elimination of quiescentchemotherapy-resistanthuman
AML stem cells. Nat. Biotechnol.
28-3,275-280, 2010

3. Shultz LD, Saito Y, Najima Y, Tanaka
S, Ochi T, Tomizawqa M, Doi T, Sone
A, Suzuki N, Fujiwara H, Yasukawa M,
Ishikawa F. Generation of functional
human T-cell subsets with HLArestricted immune responses in HLAclass I expressing NOD/SCID/IL2rγnull
humanized mice. Proc. Nat. Acad.

Sci. U. S. A.107,13022-13027, 2010

Figure :Chemotherapy can efficiently eliminate non-stem leukemia cells in the bone marrow cavity, but not AML stem cells,
which reside in the endosteal region of the bone marrow. Cytokines or antibodies disrupt the interaction between AMLstem cells and their niche, thereby inducing the stem cells to enter cell cycle and become susceptible to chemotherapy.

cells, and c-Kit-CD203c+ basophils were observed among
human granulocytes developing in vivo. Among HLA-DR+
human APCs, CD14+ monocytes, BDCA1/3+ conventional
DCs and plasmacytoid DCs were present in the bone marrowand spleen of the recipient mice. These human myeloid cells
express cytokine receptors and TLRs in a similar manner as
seen in primary human myeloid cells. We are continuing toevaluate the function of these human myeloid cells in terms ofcytokine production and inflammatory responses.

Targeting human acute myeloid leukemia (AML)
stem cells
AML is one of the most intractable hematologic malignancies in adults. This year, we have continued our efforts tobetter understand the mechanism for AML relapse and tocreate effective therapies enabling AML patients to survive
long-term through collaboration with Dr. Shuichi Taniguchi at
Toranomon Hospital and Dr. Osamu Ohara at RIKEN.
To date, we have determined that CD34+CD38- AML
cells satisfy the functional criteria for leukemia stem cells(LSCs) and that LSCs exhibit significant chemotherapyresistance. Through in vivo analysis, we found that human
LSCs preferentially reside in the endosteal region of the bonemarrow and are cell cycle quiescent. The relative chemotherapy-resistance of LSCs may be accounted for by theircell cycle status and specific location.
Therefore, to achieve long-term relapse-free survival of
AML patients, we need to eliminate the chemotherapyresistant human LSCs residing within the endosteal regionof the bone marrow. As a potential novel therapeutic strategy, first, we modified the cell cycle status of LSCs by

4. Ishikawa F, Yoshida S, Saito Y, Hijikata
A, Kitamura H, Tanaka S, Nakamura R,
Tanaka T, Tomiyama H, Saito N, Fukata
M, Miyamoto T, Lyons B, Ohshima
K, Uchida N, Taniguchi S, Ohara O,
Akashi K, Harada M, Shultz L D.
Chemotherapy-resistant human AMLstem cells home to and engraft within

the bone marrow endosteal region.
Nat. Biotechnol. 25,1315-1321, 2007.

inhibiting the stem-niche interaction at the endosteal
region. To accomplish this, we have treated AML engrafted
mice with G-CSF for five days. Though we observed significant case-dependent variability among patients, G-CSF
induced LSCs to enter cell cycle in all the cases examined.
Consistent with the successful cell cycle induction, conventional chemotherapy following G-CSF treatment
resulted in significantly enhanced eradication of AML stem
cells at the niche. By analyzing other cytokines and antibodies for their effect on cell cycle status in AML stem
cells, we aim to construct cytokine-enhanced chemotherapy as a post-remission, curative anti-LSC therapy.
Global gene expression profiling has given us muchinformation on potential therapeutic targets expressed byhuman AML stem cells. In 2010, we identified FCGR2A
and IL2RA as genes encoding plasma membrane molecules that could be promising targets for antibody drug
development. We are continuing to pursue the identification of anti-LSC markers through global expression profiling followed by functional validation. To cope with thesignificant biological heterogeneity in AML, we are performing analyses of more homogeneous patient groups basedon clinically-verified AML classifications including thoseusing morphology (FAB classification) and cytogenetic
abnormalities. In addition to plasma membrane molecules,
we are focusing on WT1, a zinc-finger transcription factor
and well known tumor-associated antigen, as a potential
target for effective immune therapy. By using HLA class I
Tg NOD/SCID/IL2rgKO mice, we are currently developing
methods that allow efficient induction of WT1-targeting
human CD8+ T cells in vivo.

5. Shultz LD, Ishikawa F, Greiner DL.
Humanized mice in translational
biomedical research. Nat. Rev.
Immunol. 7,118-130, 2007103

Research Unit for

Cellular
Immunotherapy
Unit Leader

Shin-ichiro Fujii

Technical staff: Hidetoshi Sugahara
		 Jun Shinga
Student TraineeHuman Vα24+ NKT cells bearing an invariant Vα24Jα18
antigen receptor are activated by a specific ligand,α-GalCer, in a CD1d-dependent manner. We previously
showed that circulating Vα24+ NKT cells present in lungcancer patients were functional. We have been developing a
joint clinical study with Chiba University using
α-galactosylceramide ( α-GalCer) -pulsed autologous dendritic cell ( DC) therapy to evaluate the immunological andclinical responses to NKT cell therapy in advanced non-small
cell lung cancer patients. Based on our initial analyses, wehave encouraging immunological and clinical results inthe phase I/IIa trials ( A collaboration with RCAI Director
Dr. Taniguchi and Drs. Nakayama and Motohashi in Chiba
University) .
We have also studied the role of DCs in situ for tumor
immunity by focusing on the link between innate and adaptive immunity. Recently, we successfully developed a strategy for the induction of antigen-specific T cell responses
using tumor-associated antigen-expressing, α-GalCerloaded allogeneic fibroblasts. Based on these findings in
mice, we will attempt to launch clinical studies, in collabora-: Yusuke Satotion with Dr. Shimizu, Therapeutic Model Research Unit.

Translational research: Increased number of NKT
cells in the lung after α-GalCer-loaded dendritic
cell ( DC/Gal ) therapy (A collaboration with Chiba
University and RCAI Director Dr. Taniguchi )We have been developing a Phase I/IIa clinical study of theapplication of NKT cell therapy for advanced non-small celllung cancer ( NSCLC) patients refractory to standard treatment as a post second line therapy ( stage IIIB, IV or recurrence) . α-GalCer-pulsed APCs ( 1x109/m2 PBMC-derived
DCs) were intravenously administered four times. In thisstudy, when we compared the responder group, which had
an increased number of IFN-γ producing NKT cells, and thenon-responder group, the MST ( median survival time) was
longer in the responder group ( 31.9 months versus 9.7
months) . Thus, the increased IFN-γ production by NKT cells
upon α-GalCer stimulation was significantly associated withclinical outcome. Also, we detected a larger number of NKT
cells capable of producing IFN-γ in the lung tumor. These
phase I/IIa trial results are encouraging and warrant further

Recent publications
1. Shimizu K, Hidaka M, Bickham K,
Moriwaki M, Fujimoto K, Fujii S. Humanleukemic cells loaded with α-GalCer
activate murine NKT cells in situ.
Int J Hematol 92, 152-60 (2010)

104

2. Watarai H, Fujii S, Yamada D,
Rybouchkin A, Sakata S, Nagata Y,
Iida-Kobayashi M, Sekine-Kondo
E, Shimizu K, Shozaki Y, Sharif J,
Matsuda M, Mochiduki S, Hasegawa T,
Kitahara G, Endo TA, Toyoda T, Ohara
O, Harigaya K, Koseki H, Taniguchi M.

Murine induced pluripotent stem cells
can be derived from and differentiate
into natural killer T cells. J Clin
Invest 120, 2610-2618 (2010)

3. Fujii S, Motohashi S, Shimizu
K, Nakayama T, Yoshiga Y,
Taniguchi M. Adjuvant activity
mediated by iNKT cells. Semin
Immunol 22, 97-102 (2010)

Figure :Efficacy of AAVCs for induction of Innate and Adaptive Immunity
In the murine models, we evaluated the efficacy of artificial adjuvant vector cells (AAVCs) that had been
loaded with α-GalCer, and were transfected with ova Ag mRNA. When we used the AAVCs in the B16 lungmetastasis model, we observed the eradication of tumor cells. Also, when we treated EG7 tumor (ova-expressing
tumor) bearing mice with AAVC, we observed resistance to tumor cells, but not in CD8-deficient mice.

evaluation of the survival benefit of this immunotherapy.
To understand the mechanism of the DC therapy response
and identify predictive biological markers, we have been
analyzing NKT cells in the lung tumors in murine models
and cancer patients.

Antigen mRNA-transfected allogeneic fibroblasts
loaded with NKT cell ligand confer antitumor
immunity (A collaboration with the University ofTokyo, Yamaguchi University and RCAI Drs. Ishii
and Shimizu)
We previously demonstrated that administration of tumor/
Gal cells can generate both innate and adaptive immunity
through the maturation of DCs in a tumor specific manner. In
the current study, instead of tumor cells, we used allogeneic
fibroblast cells loaded with α-GalCer and transfected with
antigen-encoding mRNA (CD1dNIH/Gal, Fig.), thus combining the adjuvant effects of iNKT cell activation with delivery

4. Fujii S, Goto A, Shimizu K. Antigen
mRNA-transfected, allogeneic
fibroblasts loaded with NKT-cell
ligand confer antitumor immunity.
Blood 113, 4262-72 (2009)

5. Shimizu K, Fujii S. DC therapy
induces long-term NK reactivity
to tumors via host DC. Eur J
Immunol. 39, 457-68 (2009)of antigen to DCs in vivo. We found that these cells produce
antigen protein and activate NK and iNKT cells. Wheninjected into MHC mismatched mice, they elicited antigenspecific T cell responses and provided tumor protection,suggesting that these immune responses depend on hostDCs. Also, antigen-expressing fibroblasts loaded with
α-GalCer generated a more potent T cell response thanthose expressing NK cell ligands. Thus, glycolipid-loaded,
mRNA-transfected allogeneic fibroblasts act as artificial
adjuvant vector cells ( AAVCs) to promote iNKT cell activation, leading to DC maturation and T cell immunity. By harnessing the innate immune system and generating an
adaptive immune response, this unique tool could prove
clinically beneficial in the development of immunotherapies
for malignant and infectious diseases. Presently, we havebeen setting up preclinical studies to establish AAVCs inhumans.

6. Fujii S. Exploiting dendritic cells andnaturatl killer T cells in immunotherapy
against malignancies. Trends
Immunol. 29, 242-9 (2008)

105

Laboratory for

Cellular Systems
Modeling
Team Leader

Mariko Okada-Hatakeyama
Research Scientists : Takeshi Nagashima
		 Yuichi Shiraishi
		 Yuko Saeki
Technical Staff: Noriko Yumoto
		 Kaoru TakahashiThe Laboratory for Cellular Systems Modeling has been
carrying out research to identify and reconstruct biological networks and basic regulatory principles from quantitative experimental data using computational, theoreticaland experimental biology approaches. The main topic of ourcurrent research is mechanistic understanding of membrane
receptor signaling and its transcriptional control in the cellfate decision process.
For this purpose, we take both bottom-up and topdown approaches. For bottom-up studies, kinetic mathematical modeling of a pathway using computational andexperimental approaches is carried out. This tactic is usefulwhen the components and their interconnections in thereaction network are already known. Based on our in-house
data and the literature, we constructed ordinary differential
equation (ODE ) -based kinetic models that allow us to understand intracellular signaling dynamics. For the past few
years, we have worked on the model to explain digital (all-ornone) outputs of c-Fos transcription factor activation (Cell,
2010). The model can explain that a structure where there isStudent Trainees: Felix Jonas (IPA), Hanna Iribe

Trainee: Karin Mitosch

co-existence of ERK signal and c-Fos transcription (an ANDgate structure) can generate the digital output of c-Fos. The
model predicted and our experimental results verified that
DUSP, an ERK phosphatase, together with as yet unknown
negative regulators determine c-Fos dynamics. This modelis thought to be valid for production of transcription factors
during B-cell development and neurite elongation of PC12
cells; therefore it is most likely that the model captures general network principles of the signal network during a varietyof cellular differentiation process.
In addition, for the modeling of signaling pathways, we
try to obtain information on molecular binding interactions
and kinetics in the pathway. For this purpose, we closelycollaborate with Dr. Yasushi Sako in RIKEN ASI to determine
association and diffusion constants of protein-protein interactions in living cells. Particularly we are focusing on the
dynamics of SH2 domain proteins and ligand-receptor interactions because critical intracellular signaling dynamics
often originate based on the binding behavior of membrane
receptors. Our analysis so far indicates that interactions

Recent publications

106

1. 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, OkadaHatakeyama M. Integrated Quantitative
Analysis of the Phosphoproteome
and Transcriptome in TamoxifenResistant Breast Cancer. J. Biol.
Chem., 286, 818-896 (2011).

2. Shiraishi Y, Kimura S, Okada M.
Inferring Cluster-based Networks
from Differently Stimulated
Multiple Time-course Gene
Expression Data. Bioinformatics
26, 1073-1081 (2010).

3. Nakakuki T, Birtwistle MR, Saeki Y,
Yumoto N, Ide K, Nagashima T, Brusch
L, Ogunnaike BA, Okada-Hatakeyama
M*, Kholodenko BN.* Ligand-Specificc-Fos Expression Emerges fromthe Spatiotemporal Control of ErbB
Network Dynamics. Cell 141, 884-896(2010). * corresponding authors.

4. Suenaga A, Hatakeyama M, Kiyatkin
AB, Radhakrishnan R, Taiji M,
Kholodenko BN. Molecular Dynamics
Simulations Reveal that Tyr-317
Phosphorylation Reduces Shc Binding
Affinity for Phosphotyrosyl Residuesof Epidermal Growth Factor Receptor.
Biophys. J. 96, 2278-2288 (2009).

Figure:Interaction network ofphosphorylated proteinsin the HRG-stimulated
wild type (WT) and drug
resistant (TamR) cells.
The reconstructed network
comprises one large subnetworkand small subnetworks withfewer than four nodes. Nodesize reflects the number ofinteraction partners. Nodecolor indicates phosphorylation
changes: red, up-regulation;
blue, down-regulation;
purple, up- and downregulation; gray, no change.

between receptors and Grb2 or Shc, p85 and p110 of PI3K
in living cells might be different from the schemes that had
been suggested based on in vitro studies. Molecular dynamics (MD) simulation done in collaboration with Dr. Makoto
Taiji in RIKEN also indicated an unexpected role of Shc as apotential molecular switch to turn on/off the signal comingfrom the receptor, in that binding affinity to the receptor
might be reduced after the phosphorylation at Tyr317(Biophys.
J, 2009). Thus, by combining various experimental andcomputational approaches, we are able to construct more
realistic mathematical models of signaling networks andidentify key regulators that determine cell fate.
As for the top-down approach, we perform transcriptome and proteome analyses to determine overall systems
properties. We performed phospho-proteome analysis using
LC mass spectrometry in collaboration with Dr. Oyama( Tokyo Univ.) et al. to understand drug-resistance mechanisms in breast cancer cells (J. Biol. Chem., 2010).The
analysis indicated an overall down-regulation of protein
phosphorylation in drug-resistant cells whereas specificsignaling pathways were up-regulated. Integrative statisticalanalysis with gene expression data indicated that the activation of AP-1 and CREB transcription factors was dysregu-

lated in the drug-resistant cells. We thus proposed and thenexperimentally verified that GSK3beta phosphorylation status might be considered as a marker for drug-resistance.
These analyses showed that the approach of using
combinatorial analysis of signaling and transcription allows
us to understand the drug-resistance properties at the
network level, which should narrow down the potential
therapeutic targets.
To realize our goal of understanding signal-transcription
networks, we developed several computer algorithms andmethods to analyze time-course data, infer networks and to
estimate model parameters. The methods are useful to estimate positive and negative regulatory relationship among
the molecules when their functions are experimentally
unknown. Particularly, we developed a method to simultaneously cluster and infer regulatory relationship among genes
from the time-course gene expression data (Bioinformatics,
2010). The method allowed us to predict functional regulatory relationship of genes in breast cancer cells under several conditions. The method is also useful for analysis of any
time-course data as well to estimate molecular functions.

5. Seo J-H, Suenaga A, Hatakeyama
M, Taiji M, Imamoto A. Structuraland functional basis of an essential
role for CRKL in FGF8-induced
feedforward pathway. Mol. Cell.
Biol. 29, 3076-3087 (2009).

107

Laboratory for

Immunogenomics
Group Director

Osamu Ohara

Research Scientists : Takashi Watanabe , Yoko Kuroki,
		 Yoshitaka Shirasaki (SPDR)
Research Associatess : Atsushi Hijikata, Mai Yamagishi
Technical Staff: Masatoshi Ito, Nobutake Suzuki,
		 Atsuo Kobayashi, Tomoko Hasegawa,		 Keiko Takahashi, Naomi Inagaki,
		 Ryosuke Yashi (temporary employee),
		 Fumie Yokoyama (temporary employee),
		 Noriko Utsumi (temporary employee),
		 Emi Abe (temporary employee)
Student Traineess: Nanako Shimura (Junior Research Associate),
		 Asahi Nakahara, Masayuki Ishii

It has been our important and basic mission to function asa “Gateway” to genomics for immunologists. Thus, ourresearch activities have been three-pronged since our group
was launched: ( 1) central support activities; ( 2) strategic andcollaborative research activities; and ( 3) exploratory research
activities aimed at new technology development. However,these research activities are never mutually exclusive, butshould be synergistically stimulated. In 2010, we haveplaced a significant emphasis on development of new technology to eventually enhance strategic and collaborative
research activities as well as the central support activities.
Our current emphasis is on the development of technology
for single-cell analyses at the mRNA and the protein levels.
We believe that single-cell genomic data together with bioimaging data will serve as the foundation of systems immunology, where cell-to-cell interactions play a crucial role indetermining the behavior of the whole immune system. Our
recent proteomic method development, an assay system forsecretion of cytokines from single cells, also enables us toretrieve the single cells on demand, as described below.

Characterization of multiple alternative forms ofheterogeneous nuclear ribonucleoprotein K byphosphate-affinity electrophoresis
Post-translational modifications of proteins often play animportant biological role and, among these modifications,
phosphorylation is one of the most common and dynamic.
For example, the phosphorylation of heterogeneous nuclear
ribonucleoprotein K ( hnRNP K) is thought to play an important role in cell cycle regulation and signal transduction.
However, the relationship between hnRNP K phosphorylation and cellular events has only been indirectly examined,
and the phosphorylated forms of endogenous hnRNP K
have not been biochemically characterized in detail. Taking
hnRNP K as a model protein for development of analysis
method for protein phosphorylation, we extensively examined the phosphorylated isoforms of endogenous hnRNP K
by direct protein-chemical characterization. The proteins
were isolated by phosphate-affinity electrophoresis, whichuses polyacrylamide-bound Mn2+-Phos-TagTM ligand as acopolymer in the separating gel matrix, followed by immunoblotting and mass spectrometry. Phosphate-affinity
electrophoresis enabled us to detect and separate the

Recent publications
1. Kimura Y, Nagata K, Suzuki N,
Yokoyama R, Yamanaka Y,
Kitamura H, Hirano H, Ohara
O. Characterization of multiple
alternative forms of heterogeneous
nuclear ribonucleoprotein K byphosphate-affinity electrophoresis.
Proteomics 10, 3884-3895 (2010)108

2. Suyama M, Harrington ED, Vinokourova
S, von Knebel Doeberitz M, Ohara
O, Bork P. A network of conserved
co-occurring motifs for the regulationof alternative splicing. Nucleic Acids
Res. 38, 7916-7926 (2010)

3. 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 analysing mutations fromhuman genetic diseases. DNA
Res. 17, 197-208 (2010)

4. Keerthikumar S, Raju R, Kandasamy K,
Hijikata A, Ramabadran S, Balakrishnan
L, Ahmed M, Rani S, Selvan LD,
Somanathan DS, Ray S, Bhattacharjee
M, Gollapudi S, Ramachandra YL,
Bhadra S, Bhattacharyya C, Imai K,
Nonoyama S, Kanegane H, Miyawaki
T, Pandey A, Ohara O, Mohan S.

Figure1: hnRNP K phosphorylation profiles revealed byphosphate-affinity two-dimensional electrophoresis.
Nuclear extracts from J774.1 cells treated with and without
lipopolysaccharide (LPS- and LPS+) were separated
by phosphate-affinity two-dimensional electrophoresis.
The white and the black arrows indicate the positions ofphosphorylated and unphosphorylated protein spots,
respectively, on phosphate-affinity SDS-PAGE gel images.

phosphorylated forms of hnRNP K with high sensitivity.
When we used two-dimensional gel electrophoresis withphosphate-affinity SDS-polyacrylamide gel electrophoresis
in the second dimension, the nuclear fraction contained
more than 20 spots of endogenous hnRNP K on the twodimensional map. We found that the multiple forms of
hnRNP K were produced by combinations of alternative
splicing of the single HNRNPK gene and phosphorylation of
Ser116 and/or Ser284. Furthermore, the subcellular localization of these proteins revealed by the two-dimensional
map correlated with their phosphorylation states and alternative splicing patterns. The results also indicated that themultiple forms of hnRNP K were differentially modulated in
response to external stimulation with bacterial lipopolysaccharide or serum. This example demonstrates very well thehigh resolving power of phosphate-affinity two-dimensionalgel electrophoresis, which will enable us to analyze complex
phosphorylation states of proteins in the immune system.

Real-time monitoring of protein secretion fromsingle cells
While mRNA profiling at the single-cell level is now
feasible by various established methods, we still lack theability to obtain protein profiles of single cells, providing asnapshot for comprehensive characterization of their functional state. This is a significant challenge and thus, one of
the focal points of our research is to develop a protein
analysis method at the single cell level ( Sasuga et al., Anal
Chem. ( 2008) 80, 9141-9149) . As an extension of the previous study, we have now succeeded in monitoring secretionof proteins from each single cell in real-time over 12 hours.
This measurement could be performed in parallel for about
150 single cells with the current analysis platform. A combination of fully automated total internal reflection fluorescence

RAPID : Resource of Asian Primary
Immunodeficiency Diseases. Nucleic
Acids Res 37, D863-7 (2009)

Figure2: Real-time monitoring of protein secretion from single cells.
(A) A schematic of real-time observation of cytokine
secretion in micro fabricated wells by total internal reflection
fluorescence imaging. Cytokines secreted from an immune cellare detected by a real-time sandwich immunosorbent assay.
(B) Secretion of IL-1β from a single MC/9 cell. Red spots at the bottom of
the microwells correspond to single molecules of the IL-1β/sandwich
antibody complex. The number of red fluorescent spots increases
with time only in the case of the secreting cell (upper panel).

microscopic technology ( TIRFM ) and microfluidics technology modified to be compatible with TIRFM has made thisfeasible. Taking advantage of these technologies, it becomes
possible to assess the single cell response to stimulation bymonitoring secretion activity with time. An important aspectof this technology is that monitoring of protein secretion is
non-invasive as it sensitively reflects the state of the cell.
This has interesting implications. For example, we could
analyze mRNA profiles and/or epigenetic states of cells at aparticular moment of interest, which could be identified by
the protein secretion profile. In fact, our preliminary results
showed that individual cells undergoing protein secretion
analysis could be separately retrieved and then characterized in terms of mRNA profiles and/or genomic sequences.
If we can successfully develop this system to make it moreuser-friendly and high-throughput, various applications of
the technology will become possible. Most interestingly,
because the immune system is composed of various typesof immune cells, this technology may enable us to uncoverunexpected complexity, which would be missed in the averaged bulk biochemical data. This concept is well-illustrated
in the case of somatic mosaicism of chronic infantile neurologic, cutaneous, articular syndrome caused by NLRP3
mutation. Together with bioimaging data, single-cell genomic
data of heterogeneous cell populations would greatly assist
us in tackling multiscale problems in the immune system.

5. Ohara O. From transcriptome
analysis to immunogenomics: current
status and future direction. FEBS
Lett 583, 1662-1667 (2009)109

Research Unit for
Unit Leader

Immunoinformatics

S. Sujatha Mohan

Research Associate : Suresh Kumar RamadossThe main mission and focus of our research unit is todevelop and maintain an open access bioinformatics platform and data resource in order to gain insights into primary
immunodeficiency diseases (PID) through genomic, transcriptomic and proteomic data. It has been our basic aim to provide
relevant, up-to-date and validated information on PID in aneasily decipherable and usable format as per global community standards. This would assist physicians in the early diagnosis and effective treatment of PID in order to minimize clinicaldeterioration, provide researchers with vital clues to analyze
candidate PID genes, and create awareness among PID
patients to lead a near normal quality life and also support
affected family members with relevant PID information.

Seq genomic, cDNA and protein sequences as per the recommendations of the Human Genome Variations Society(HGVS). The frequent updates of RAPID have resulted inexponential data growth since its launch, as depicted in Fig.
1. Each mutation entry in the database is also linked to“Mutation@A glance”, a web-based graphical user interface(GUI)-enabled tool that allows users to visualize the mutation
position both at the level of DNA and protein sequences toobtain homology based three-dimensional structures withvarious types of information such as SNP, protein domains
and functional sites, as well as to predict deleterious andnovel mutations using the SIFT program.

RAPID : Resource of Asian Primary Immunodeficiency
Diseases – “An integrated informational platform”
We have recently developed an open access PID database
designated “Resource of Asian Primary Immunodeficiency
Diseases (RAPID)”, a web-based compendium of molecularalterations and gene expression at the mRNA and protein levels of all PID genes reported from PID patients in the public
literature. The database also includes other pertinent information about protein-protein interactions, mouse studies andmicroarray gene expression profiles in various organs and cellsof the immune system. RAPID (published in Nucleic Acids
Research, 2009) can be accessed at http://rapid.rcai.riken.jp.
PID gene mutations in RAPID were mapped to NCBI Ref-

Figure1: RAPID data growth as of December 2010. No. of PubMed
refers to number of annotated reference papers with
mutation data and No. of mutations indicates non-redundant
entries of mutation data annotated in RAPID.

Recent publications
1. Hiroshi Masuya, Yuko Makita, Norio
Kobayashi, et al. The RIKEN integrated
database of mammals. Nucleic
Acids Research, 39 (Databaseissue) : D861-70 (2011)110

2. Atsushi Hijikata, Rajesh Raju,
Shivakumar Keerthikumar et al.
Mutation@A Glance: an integrative
web application for analyzing mutations
from human genetic diseases. DNA
Research, 17 (3) :197-208 (2010).

3. Kandasamy, K.*, Mohan, S.S*, Raju,
R., et al. NetPath: A Public Resource ofCurated Signal Transduction Pathways.
Genome Biology, 11 (1):R3 (2010).
*These authors contributed equally.

4. Kandasamy, K., Keerthikumar, S.,
Rajesh, R., et al. PathBuilder – Open
Source Software for Annotating andDeveloping Pathway Resources.
Bioinformatics, 25 (21):2860-2 (2009).

PID candidate gene prediction
RAPID also hosts the candidate PID gene list obtained from
Support Vector Machine ( SVM) based parameter classification approach. We have predicted 1442 candidate PID
genes using 69 binary features of 148 known PID genes and3162 non-PID genes as a training data set ( published in
DNA Research, 2009) . Currently, 19 SVM candidate genes
have been confirmed as PID-causing genes, while others
remain attractive candidates that are to be analyzed inpatients where the disease etiology cannot be ascribed toany of the known PID genes.
RAPID mutation data analysis
With our enormous collection of PID mutation data, we have
implemented an in silico approach to deduce interacting
domains of PID genes which lose function due to observed
mutations. The workflow of this approach is shown in Fig. 2.
A submitted manuscript describing this approach has been
accepted for oral presentation at the International Conference on Bioscience, Biochemistry and Bioinformatics( ICBBB 2011) , Singapore which is to be published by IEEEand will be archived in the IEEE Xplore.
Development of a PID pathway resource
We have developed an internal pathway annotation tool ( Fig.
3) to construct immune cell / tissue-specific molecular pathways of PID processes thereby highlighting various signaling
and regulatory events that are curated from the publishedliterature. These pathway reactions are depicted using 3-D
visualization and high-throughput expression data analysis
software tools such as Protein Lounge, ePath (www.proteinlounge.com ) and PathVisio ( www.pathvisio.org) . This tool
should aid users in evaluating gene expression patterns by

Figure2: Overview of
our in silico approach toprioritize interacting domains
associated with occurrence ofhigh frequency PID genespecific mutations. In thisprocess, domain spanning
regions for a PID gene andits interactors are retrieved
from BioMart. Proteinprotein interaction (PPI) anddomain-domain interaction(DDI) data sets are collectedfrom HitPredict & HPRD
and DIMA web resources
respectively. Functional
domain annotation andprioritization are performed
using UniProt and RAPID,
respectively, to gather PID
gene-specific mutations
reported with equal to or
more than 80% of observed
mutation frequency forfurther integrated analysis.

5. Shivakumar Keerthikumar, Sahely
Bhadra, Kumaran Kandasamy,
et al. Prediction of candidate
primary immunodeficiency disease
genes using a support vector
machine learning approach - DNA
Research,16 (6) : 345-51 (2009).

Figure3: Internal pathway annotation tool showing various features tocurate PID pathway-specific data from the published literature.

overlaying expression data on the pre-constructed pathways
as well as in analyzing defined pathophysiology of PID alongwith specific identification of potential candidate genes
involved in PID for further experimental validation.

PID phenotype standardization
The main challenge for in silico genotype-phenotype correlation for any genetic diseases is to standardize phenotype
ontology terms. We are in the process of collecting PID
mutation specific phenotypes from the literature and Immunodeficiency resources ( IDR) . The generated phenotype list
is then mapped to standard phenotype ontologies obtained
from Human Proteome Ontology ( HPO) , human diseases( DOID) and Symptom ontology browser ( SYMP) as provided
by the ontology lookup service ( http://www.ebi.ac.uk/ontology-lookup/ ) . We also recommend the list of unmapped PID
phenotypes to the HPO team to build a standard ontology.
This kind of analysis should bridge a gap between genotype
and phenotype correlation, thereby improving phenotypebased genetic analysis of PID genes. Also, it should aid clinicians in confirming early PID diagnosis and in implementing
proper therapeutic interventions.
Collaboration and funding
The PID project has been initiated in collaboration with theInstitute of Bioinformatics ( IOB, Bangalore, India), the Immunogenomics research group at RIKEN RCAI, Japan and theKazusa DNA Research Institute (KDRI), Japan. This laboratory
was supported by The Asia S&T Strategic Cooperation Promotion Program, Special Coordination Funds for Promoting
Science and Technology by the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) (Project Title: Generation of primary immunodeficiency disease database from Asia)
between July, 2007 and March 2010. The project is now being
solely funded by a RCAI, RIKEN internal funding source.

6. Shivakumar Keerthikumar, Rajesh Raju,
Kumaran Kandaswamy, et al. RAPID:
Primary Immunodeficiency disease
database. Nucleic Acids Research,
37 (Database issue) : D863-7 (2009).

111

Research Unit for
Unit Leader

Immunodynamics

Takaharu Okada

Research Scientists : Masahiro Kitano (SPDR), Yoshikazau Ando
Technical Staff: Noriko TakahashiThe goal of our research is to understand the mechanisms regulating cell migration and interactions in thetissues that shape immune responses. For this purpose weuse real time imaging, in particular multi-photon microscopy,
to analyze in vivo cellular migration and interactions. This
microscopy method, which was introduced only recently tothe field of immunology, has been revealing striking dynamics of immune cells in the lymphoid organs, underlining theimportance of this approach to resolve the complexity of theimmune system. This year, we have been focusing onunderstanding how cellular dynamics are regulated by lymphocyte differentiation during the formation of germinal centers ( GCs ) , the primary site for long-term, high-affinity
antibody responses. GC B cells and follicular helper T ( Tfh )cells play pivotal roles in GC reactions, and the transcription
factor BCL6 is attracting a lot of attention in the field because
it is now recognized as the master regulator for both of thesecell types. However, in vivo dynamics of BCL6 protein
expression in B cells and T cells have not been examined,
despite the knowledge that Bcl6 mRNA levels are some-times a poor indicator of BCL6 protein expression. In particular, it is currently uncertain where in the tissue B cells
begin to upregulate BCL6 protein, and how BCL6 influences
B cell dynamics, leading to their clustering in GCs. Moreover,little is known about the stability of BCL6 expression inTfh cells despite its potential importance in determining
their fate.

Roles for BCL6 upregulation in controlling pre-GC
B cell dynamics
In order to understand BCL6 protein expression dynamics
during the antibody response, we generated a novel fluorescent reporter mouse strain by gene targeting. Utilizing thereporter animals we found that antigen-engaged B cells
upregulated BCL6 while they were in the outer part of B cellfollicles before clustering in GCs. Two-photon microscopic
analysis indicated that this early upregulation of BCL6 in
pre-GC B cells contributed to sustaining their interactions
with Tfh cells in the outer follicle. The BCL6-dependent sustainment of cellular conjugate formation may play an impor-

Recent publications
1. Tanizaki H, Egawa G, Inaba K, Honda
T, Nakajima S, Moniaga CS, Otsuka
A, Ishizaki T, Tomura M, Watanabe
T, Miyachi Y, Narumiya S, Okada T,
Kabashima K. Rho-mDia1 pathway
is required for adhesion, migration,
and T-cell stimulation in dedritic cells.
Blood 116, 5875-5884 (2010)112

2. Okada T. Two-photon microscopy
analysis of leukocyte trafficking andmotility. Semin. Immunopathol.
32, 215-225 (2010)

3. Katagiri K., Katakai T., Ebisuno Y., Ueda
Y., Okada T., Kinashi T. Mst1 controls
lymphocyte trafficking and interstitial
motility within lymph nodes.
EMBO J. 28, 1319-1331 (2009)

4. Grigorova I.L., Schwab S.R., Phan T.G.,
Pham T.H., Okada T., Cyster J.G.
Cortical sinus probing, S1P1dependent entry and flow-based
capture of egressing T cells.
Nat. Immunol. 10, 58-65 (2009)

Figure :BCL6 expression and migration of antigen-engaged B cells and helper T cells in the lymph node.
After initial interaction with helper T cells at the B-T boundary region, antigen-engaged B cells are localized
in the outer follicle region. In the outer follicle, some of these cells are committed into the plasma cell (PC)pathway, whereas others upregulate BCL6, which enables them to enter the GC region in the inner part of thefollicle (left). Helper T cells partially upregulate BCL6 without interacting with B cells, presumably throughinteractions with dendritic cells. Helper T cells further upregulate BCL6 through cognate interactions with B
cells to become Tfh cells. In the outer follicle, Tfh cells preferentially form conjugates with BCL6-upregulating
pre-GC B cells. After their development, some of Tfh cells downmodulate BCL6. These BCL6-low Tfh
cells, which are found even in GCs, terminate proliferation and upregulate the IL-7 receptor (right).

tant role in the GC formation, as our preliminary results
suggest that blockade of integrin molecules at the time whenantigen-engaged B cells and Tfh cells are in the outer follicle
partially impaired their conjugation and almost completely
abrogated subsequent GC formation. Moreover, the dynamic
imaging analysis revealed that if antigen-engaged B cells
were impaired in upregulation and function of BCL6 theyfailed to enter the GC region in the inner part of the B cellfollicles, indicating that BCL6 upregulation in the outer follicle
directs pre-GC B cell migration into the GC. These results
clarify the role of BCL6 upregulation in dynamics of B cells
committed to the GC pathway.

Heterogeneity of BCL6 expression in Tfh cells
BCL6 expression dynamics in helper T cells during the antibody response was also investigated. We observed that asmall fraction of antigen-engaged helper T cells in the T cellzone expressed BCL6. Furthermore, antigen-engaged CD4+
T cells partially upregulated BCL6 protein even in B cell-

5. Yamamoto S., Shimizu S., Kiyonaka
S., Takahashi N., Wajima T., Hara Y.,
Negoro T., Hiroi T., Kiuchi Y., Okada T.,
Kaneko S., Lange I., Fleig A., Penner
R., Nishi M., Takeshima H. and Mori
Y. TRPM2-mediated Ca2+ influx
induces chemokine production

deficient animals, suggesting that helper T cells begin to
express BCL6 protein in the T cell zone and further increase
its expression as they became Tfh cells. When BCL6 function was genetically compromised, helper T cells were
defective in the formation of the cells with the highest
expression levels of CXCR5 and PD-1. Two-photon microscopy of LNs and subsequent flow cytometric analysis of
the same LNs showed that the Bcl6 mutant helper T cells
were most severely reduced in the GCs. These results
support a model in which Tfh cells with the highest expression of CXCR5 and PD-1 are the Tfh cell population inhabiting GCs. Interestingly, after their development, Tfh cells
gradually downmodulated BCL6 protein. BCL6-low Tfh
cells were less proliferative than BCL6-high cells, and upregulated the IL-7 receptor. These results highlight the heterogeneity of BCL6 protein expression in Tfh cells, and suggest
functional consequences. We propose that BCL6-low Tfh
cells are precursors of memory helper T cells derived fromTfh cells.

in monocytes that aggravatesinflammatory neutrophil infiltration.
Nat. Med. 14, 738-747. 2008.

113

Research Unit for

Molecular Systems Immunology
Unit Leader

Makio Tokunaga

Student Trainees : Akira Okada, Yuma Itou, Jun Takimoto, Katsuo Ichinomiya, Masahiro ShimozawaOur laboratory has developed technologies that allow
immune responses and signaling processes to be visualized at the single-molecule level. Single molecule imaging
coupled with the ability to simultaneously visualize several
different proteins in cells has enabled the quantification ofmolecular dynamics, interactions, and kinetics. Based onthese three-dimensional and temporal parameters, we examine numerical modeling and computer simulations of cellfunctions. Using the combination of single molecule quantification and “in silico” modeling, we aim to open 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 andHILO microscopy. The main technical challenge of singlemolecule fluorescence imaging is increasing the signal/
background ratio. We have been involved in the development of total internal reflection fluorescence ( TIRF ) microscopy, a light-microscopic technique. TIRF is now widelyused 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
approach, 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 beamand by minimizing the illumination area. The incident laser
beam is highly inclined by a large refraction, and is laminatedas a thin optical sheet at the specimen side. In HILO microscopy, this thin optical sheet is used for illumination.
To evaluate the HILO microscopy technique, we reconstructed three-dimensional images of the nuclear pore complex ( NPC) in cells from z-scanned serial images ( Fig. 1b) .
Clear point-like images of the Sp1 transcriptional factor wereobtained without the need for deconvolution to remove outof-focus haze. The fluorescence intensity of the NPCSp1
point images corresponded well to the theory of image
formation. Further, there was much less photobleaching
than in conventional confocal microscopy because of thelower intensity and non-focused nature of the illumination.
We examined the thickness of the illumination beam bymeasuring illumination intensity profiles in the z-direction.
We decreased the illumination thickness with the reductionof the illumination diameter. Notably, the full width at half
maximum of the profile, i.e., the illumination thickness, wasRecent publications
1. Shiina N., Tokunaga M.: RNA GranuleProtein 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).

114

2. Yokosuka T., Kobayashi W.,
Takamatsu M., Sakata-Sogawa
K., Zeng H., Yagita H., Tokunaga
M., Saito T.: Spatiotemporal basisof CTLA-4-mediated negative
regulation of T-cell activation.
Immunity 33, 326-339 (2010).

3. Fukagawa, A., Hiroshima, M., Sakane,
I., Tokunaga M.: Stochastic emergenceof multiple intermediates detectedby single-molecule quasi-staticmechanical unfolding of protein.
BIOPHYSICS, 5, 25-35 (2009).

4. Tokunaga, M., Imamoto, N.,
Sakata-Sogawa, K.: Highly inclined
thin illumination enables clear
single-molecule imaging in cells. Nat.
Methods 5 (2), 159 -161 (2008).

a
a

b

b

Figure 1: Molecular imaging enables one to visualize and quantify
molecular dynamics, interactions, and kinetics in cells formolecular systems biology. (a) HILO microscopy for molecularimaging in cells. Illumination by a highly-inclined and thin beamincreases image intensity and decreases background intensity,
yielding a signal/background ratio up to about eightfold
greater than that of epi-illumination. A high ratio yielded clear
single-molecule images and three-dimensional images. (b)To evaluate the HILO microscopy technique, we visualized
single molecules of the Sp1 transcription factor in a living cell.
Molecular dynamics and interactions can be quantified byimage analysis using these single molecule images. Bar, 5 µm.

less than 7 µm at the diameter below 20 µm.
Further, we evaluated the signal/background ratio ofimages in HILO. Inclination of the illumination beam increases
intensities of the fluorescence images up to 2.8-fold compared with epi-illumination. The 2.8-fold increase is in excellent agreement with the theory. By contrast, the background
intensity is substantially decreased by illumination inclination. As the background is composed of out-of-focus
images, the decrease is explained by the reduction of theilluminated range. As a result, illumination inclination
increased the ratio of image to background ( signal/background ) up to 3.1-3.5-fold. Reduction of the illumination
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 thesignal/background ratio up to 7.6-fold.
To explore potential new uses of this technology, we
performed quantitative analysis of nuclear import to demonstrate its application to kinetic studies. We could visualize
single molecules of GFP-importin β mediating the import ofcargo through nuclear pores in cells as bright spots on the
nuclear envelope. Molecular interactions with the assembled
NPC were quantified by single molecule analysis. Retention

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 Sp1transcription factor visualized in a living cell. Molecular dynamics
and interactions can be quantified by image analysis using thesesingle molecule images. Bar, 5 µm. (b) Aiming at understanding
immune cells as molecular systems, we plan to construct “insilico” cell models based on single-molecule quantification.
Bidirectional research is essential to reconstruct cell functions
in silico; research from molecules to systems by single molecule
analysis, and feedback research from systems to molecules.

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 tounderstand the molecular mechanism of nuclear import, anumerical model of import was constructed using thesekinetic parameters. Computer simulation was carried out
based on the model with two types of binding sites. The
simulation fit very well with both the results of single-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 ) . Direct comparison with molecular imaging is indispensable for thesimulation, since the values of the parameters have a huge
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 fordeveloping molecular systems biology in immunology andother fields.

5. Yokosuka, T., Kobayashi, W.,
Sakata-Sogawa, K., Takamatsu, M.,
Hashimoto-Tane, A., Dustin, M. L.,
Tokunaga, M., Saito, T.: Spatiotemporal
regulation of T cell co-stimulation by
TCR-CD28 microclusters through
PKCq translocation. Immunity
29 (4), 589-601 (2008).

115

Research Unit for

Thymic Environments
Unit Leader

Willem van Ewijk

Research Scientist: Katrin Ishii-SchradeIn the thymic environment, lympho-stromal interaction
forms the basis for both lymphoid and stromal cell development. On the one hand, thymic epithelial cells ( TECs)
promote maturation of T lymphocytes. On the other hand,developing thymocytes induce maturation and functional
stability of the thymic stroma, a phenomenon earlier designated as “thymic crosstalk” ( Ewijk W van, Shores EW, Singer
A. Immunol Today. 1994, 15: 214-217 ) .

The artificial thymus in vitro
Notch signaling in developing hematopoietic cells induces
restriction of these cells along the T-cell lineage ( Radtke F.,
et al. Immunity 1999, 10 : 547-558 ) .
Conversely, Notch signaling in TECs promotes theestablishment of appropriate thymic microenvironments( Masuda et al. Mol. Immunol., 2009) . Based on these findings, we have set up an in vitro culture system to generate T
lymphocytes. We have infected the mesenchymal cell line
TSt4 with a retrovirus containing the Notch ligand DLL4.
Previously, we found that on TSt4/DLL4 monolayers,
hematopoietic progenitor cells become committed to the Tcell lineage and develop into CD4+CD8+ ( DP ) thymocytes,
but do not differentiate further. Apparently, these pre-selection DP cells are awaiting signals to be positively selected.

Recent publications
1. Vroegindeweij, E, Itoi M, Satoh R,
Zuklys S, Crobach S, Germeraad WTV.
Cornelissen JJ, Cupedo T, Holländer G,
Kawamoto H, van Ewijk W. Thymic cysts
originate from Foxn1 positive thymic
medullary epithelium.
Mol. Immunol. 2010 47(5):1106 -13116

2. Masuda, K, Germeraad WTV, Satoh
R, Itoi M, Katsura Y, van Ewijk W,
Kawamoto H. Activation of Notch
signaling in thymic epithelial cells
induces development of thymic
microenvironments.
Mol. Immunol. 2009, 46:1756 -1767

TSt4/DLL4 cells cannot provide such key signals, although
they express MHC class I molecules. Thus, although the
TCR-MHC interaction is the cornerstone of positive selection, there must be other candidate molecules that contribute to the process by as yet undiscovered mechanisms. In
2010, we began focusing on one such candidate molecule,the recently discovered proteasome subunit β5t. β5t, which
is only expressed in thymic cortical epithelial cells ( cTECs) , iscrucial for the development of CD8SP cells ( Murata S,
Sasaki K, Kishimoto T, Niwa S, Hayashi H, Takahama Y,
Tanaka K. Science 2007, 316:1291-1292 ) .
Is β5t the missing molecule that works together with
TCR and MHC to initiate positive selection of CD8SP cells
from pre-selection DP cells?
To answer this question, we have examined whether
retrovirally transduced β5t expressed in TSt4/DLL4 cells,bone marrow derived dendritic cells ( BMDC) , DP cells, or B
cells can confer on these cells the ability to positively select
CD8SP cells from pre-selection DP cells in vitro ( Fig.1A,C) .
None of the β5t expressing cells induced CD8SP cell
development using the monolayer culture system. Because
a 2-D organized monolayer culture itself may not provide an
optimal microenvironment for positive selection, we have redesigned our initial system into a ( 3-D) re-aggregate cultureFigure 1:
A) Developing T cells (blue) and β5t expressing antigen presenting cells(green) growing on mono-layered TSt4/DLL4 stromal cells (yellow).
B) The same cells as in A) growing as a tight reaggregate on a filter that floats on medium.
C) We have labeled BMDCs (green) to study how well they grow in monolayer
culture together with DP cells and Tst4/DLL4 cells (both not visible)

system ( RTOC) where pre-selection DP cells can be mixed
with β5t over-expressing cells in known numbers to form
discrete, small volume aggregates, ensuring maximal cellto-cell interaction ( Fig.1B) . We found that, again, none of the
β5t expressing antigen presenting cells promoted CD8SP
development. However, cTECs isolated from deoxyguanosine ( dGuo) treated fetal thymic lobes were able to generate
CD8 SP cells.
We conclude that simple overexpression of β5t in theseantigen presenting cells is not sufficient to confer the capacity for positive selection. Thus, additional unknown molecules, unique to cTECs associated in a network configuration,must act in concert with β5t, TCR, and MHC in the processof CD8SP development.

In vivo microenvironments
The normal thymic environment is largely composed of epithelial cells. Thymic fibroblasts form a minority population
and are only found in the thymic capsule, in septae, aroundblood vessels, and as free fibroblasts in the medulla.
In following up our crosstalk experiments, we noticed
that an early block in T cell development induces majorchanges in the thymic environment, leading both to thegeneration of thymic cysts, as well as to changes in thedevelopment of mesenchymal cells. Regarding the development of thymic cysts, we have now provided formal proof

Figure2: Thymic lobes of normal and hCD3Tg fetal micethat cysts differentiate from the 3-D organized thymic
epithelium ( 2) .
Regarding the development of mesenchymal cells in thethymus, we noticed that loss of thymic crosstalk signaling
leads to expansion of these cells in the thymic environment.
Thus, in the thymus of fetal hCD3tg mice, which have a
block in T cell development at the TN-1 stage, the organization of TEC’s is disturbed, going hand in hand with a marked
increase in mesenchymal cells, detected by ERTR7 expression ( Fig 2 ) . Similarly, fetal thymi cultured with dGuo show
increased numbers of fibroblasts.
BrdU labeling experiments showed that fibroblasts proliferate under these experimental conditions. We are presently focusing on the role of epithelial derived cytokines instimulating the growth of thymic mesenchymal cells.
In sum, our data indicate:( 1) Further research is required to enable the production of
fully differentiated T cells in vitro.
( 2) In the in vivo thymus, TECs dominate the thymic environment in the presence of crosstalk signaling. Absenceof crosstalk signaling impairs TECs, but stimulates
growth of fibroblasts. Such increased activity of mesenchymal cells might prepare a scaffold for repair of theaffected thymic epithelial environment.

117

Research Unit for

Immunoepigenetics
Unit Leader

Miguel Vidal

Research Scientist : Kaori Hisada
Technical staff

P

olycomb group ( PcG) complexes function as transcriptional repressors in cells undergoing cell fate transitions.
Thus, maintenance of stem cells and utilization of their fulldevelopmental potential are some of the essential functions
regulated by PcG activity. PcG complexes are chromatin
modifiers endowed with histone modifying activities ( trimethylation and monoubiquitylation of histones H3 and H2A) .
How these modifications relate to transcriptional repression
and how PcG complexes are recruited to their targets are a
subject of active research.
We identified RYBP ( Ring1 and YY1 binding protein) asa PcG histone H2A monoubiquitin ligase interactor that alsointeracts with YY1, features that made it appear as a candidate for PcG recruiting. RYBP function, however, is poorly
characterized, in part due to the embryonic lethal phenotype
of constitutive RYBP mutant alleles.

RYBP functional studies in an embryonic stem (ES)cell model
RYBP it thought to be part of the pluripotency network oftranscriptional regulators of ES cells. To study RYBP func-: Asako Shibano

tion we have generated ES cells that carry both a floxed
RYBP allele and a ubiquitously expressed allele encoding a
Cre-ER recombinase that can be activated by hydroxytamoxifen. RYBP-deficient ES cells maintain their self-renewal
properties and show a limited alteration in the mRNA expression pattern of protein-coding genes. We have addressed
the possibility of redundancy by knocking down the onlyRYBP paralog, Yaf2, using an shRNA approach, but preliminary results do not support such a function. Interestingly, a
subset of upregulated mRNAs in RYBP-deficient ES cells
encode germline specific products and are also derepressedin ES cells lacking components of the DNA methylation
machinery. To gain insight in RYBP mechanisms we generated ( in collaboration with Drs. M. Endoh and T. Endo at
RCAI ) maps of chromatin association for RYBP in wild type,Dnmt1-deficient and eed-deficient ES cells. Preliminary
analysis of the results showed RYBP association to characteristic Polycomb targets whose repression is independentof RYBP. By contrast, RYBP silenced targets, including
genes marked by partial DNA methylation, show poor association of PcG subunits. In both cases, depletion of

Recent publications
1. Román-Trufero, M., Méndez-Gómez,H., Pérez, C., Hijikata, A., Fujimura, Y.,
Endo, T., Koseki, H., Vicario-Abejón, C.
and Vidal, M. (2009) Maintenance of
undifferentiated state and self-renewalof embryonic neural stem cells by
Polycomb protein Ring1B.
Stem Cells 27: 1559-1570

118

2. Vidal, M. (2009) Role of Polycomb
proteins Ring1A and Ring1B
in the epigenetic regulationof gene expression Int. J.
Dev. Biol. 53:355-370

3. Hirabayashi and, Y., Suzki, N., Tsuboi,
M., Endo, T.A., Toyoda, T., Shinga, J.,
Koseki, H., Vidal, M. Gotoh, Y. (2009)
Polycomb limits the neurogenic
competence of neural precursor cells to
promote astrogenic fate transition.
Neuron 63: 600-613

4. Calés, C., Román-Trufero, M.,
Pavón, L., Serrano, I., Melgar, T.,
Endoh, M., Pérez, C., Koseki, H. andVidal, M. (2008) Inactivation of the
Polycomb group protein Ring1B
unveils an antiproliferative role inhematopoietic cell expansion andcooperation with tumorigenesis

Figure 1: RYBP protein expression in preimplantation embryos.

Figure 2: RYBP association to chromatin of ES cells of the indicated genotypes.
Representative loci of the groups of genes whose transcriptional output was upregulated or unchanged in RYBP-deficient
ES cells. Arrows indicated transcription start sites and direction of elongation. CpG islands are indicated in red.

H3K27me3 marks or of CpG methylation ( eed and Dnmt1deficient ES cells) has little effect on RYBP chromatin association. Future work will involve validation of the hypothesis
raised by these results suggesting that RYBP, which isexpressed from the earliest developmental stages, may contribute to DNA methylation marking and the recruitment of
Ring1A/Ring1B Polycomb complexes.

associated to Ink4a deletion. Mol.
Cell. Biol. 28:1018-1028

119

Central Facilities
C

entral Facilities in RCAI provides all researchers in the Center with access to the most advanced
equipment and technologies. The Central Facilities consist of five sections: the FACS, Confocal, and Monoclonal Antibody Laboratories managed by Takashi Saito, the Genomics Laboratory
managed by Osamu Ohara, and the Animal Facility managed by 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 2010, FACS Lab. added new FACS machines, FACS Aria II and
Canto II. In addition, the lab installed ImageStream100 ( Fig. 1) that combines the speed, sensitivity, andquantitation of flow cytometry with the visual detail of microscopy in a single platform. Ikuo Ishige, Engineer
from BM Equipment Co. Ltd., provides training courses, consultations for experiments and operation andanalysis services for using ImageStream.

Table 1: FACS instruments in the Central Facilities
Machine types

Machines

FACS cell analyzer

Calibur

5

Canto

1

FACS cell sorter

Imaging flow cytometer

Number of machines

Aria

2

Aria

1

Vantage

2

Diva (digital vantage)

1

ImageStream100

1

Figure 1 : Ikuo Ishige with ImageStream100

For the users of FACS machines ( cell analyzers and cell sorters) , Hanae Fujimoto and Yukiko Hachiman provide
various services, mainly in the following three areas.

1. Technical support and training
In 2010, the facility offered 9 technical courses ( 4 for cell sorting and 5 for cell analysis) both in English andJapanese. Courses were held at 3 different levels, Calibur basic, Canto II and Aria basic. A total of 41
researchers in RCAI took the courses in 2010.

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 2010, the number of operation service provided by the lab
was 266.

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, engineersfrom Becton Dickinson visit once a week to provide maintenance and technical support.

Monoclonal Antibody Lab.
Technical Staff : Tomomi Aoyama, Mayuko Matsuda

The Monoclonal Antibody ( mAb) Lab. aims to produce mAbs that meet the needs of RCAI researchers, and120

also focus on more strategic development of mAb, such as those for allergy-related molecules, and several
GPCRs that are difficult targets. The activity is partially supported by Becton Dickinson, and two technical staff
members, Tomomi Aoyama and Mayuko Matsuda, are engaged to produce mAbs. In 2010, the Lab. established mAbs against 15 different antigens, which were requested from 7 laboratories. The lab also succeededin the establishment of an antibody against the human mast cell IgE receptor that inhibits IgE-binding andwould have potential therapeutic applications.

Confocal Lab.
Visiting Scientist : Yasutaka Wakabayashi (Leica Microsystems)

The Confocal Lab. provides imaging equipment and technical support. The Confocal Lab is managed incollaboration with Leica Microsystems. There are 6 Confocal microscope systems:
1. Inverted system with visual and multi-photon MP laser that is suitable for time-lapse imaging of living cells
and organs.
2. Inverted system with a 405 nm laser that is suitable for a time-lapse imaging of living cells in a controlledenvironment ( CO2, temperature, and humidity) .
3. Leica SP5 was newly introduced in February, 2011. SP5 is the successor of SP2. Brighter and more beautiful image will be possible because of the improved optical system.
4. Upright system with visual and UV lasers that is suitable for standard fixed specimen observation.
5. Newly installed inverted intravital system with visual laser, MP, OPO laserand high speed scanner that can be used for in vivo imaging of various
tissues. Especially OPO laser can excite red fluorophore in deeper area
because its tunable wave length is longer than MP.
6. Intravital upright system with single visual laser, double MP lasers and highspeed scanner that can be used for in vivo imaging and for some applications such as light stimulation ( Fig 2) .
During 2010, Yasutaka Wakabayashi from Leica provided training course for18 people. The total running time of the microscopes was over 1860 hours.
Figure 2 : Yasutaka Wakabayashi withintravital upright system

Genomics Lab.
Research Scientist: Takashi Watanabe
Research Associates : Mai Yamagishi,
		 Atsushi Hijikata
Technical Staff: Tomoko Hasegawa,
		 Nobutake Suzuki,
		 Akio Kobayashi,		 Ryosuke Yashi,
		 Fumie Yokoyama

The Genomics Lab. provides various services to themembers of the Center: proteomics analysis, multiplex suspension array, DNA microarray, DNA
sequencing, cDNA/Genomic clone distribution, andPrimer/labeled probe distribution for qRT-PCR
analysis of immune cells ( Table 2) .

Table 2 : Services provided by the Genomics Lab. in 2010
Proteomics
Two-dimentional electrophoresis
Mass Spectrometry Analysis
Multiplex suspension array
Affymetrix Genechip (Exon array, Gene array, miRNA array)
Human
Mouse
Total
DNA sequencing
36cm capillary
50cm capillary
Total
cDNA clone delivery
Primer/labeled probe delivery# of samples
14367# of samples
3414# of samples
148
441
589
# of samples
18,473
13,42531,898# of clones
121# of sets551# of teams
1
2# of teams
13# of teams
6
1723# of teams
18
16
34# of teams
6# of teams
4121Because progress in genome technology is very rapid, the Genomics Lab. is keen to provide the Center withthe most updated technology on demand. As an example, a Fluidigm BioMark system is newly introduced to
expedite single-cell quantitative PCR processes. The BioMark system enables us to obtain a high throughput
qPCR data based on single cell using a 48.48 Dynamic Array chip and/or more high-throughput array chips( Fig. 3) .

Figure 3 : BioMark™ system and BioMark™ 48.48 Dynamic Arrays

Animal Facility
Senior Research Scientist: Takanori Hasegawa
Technical Scientist: Shinobu Mochizuki
Technical Staff: Tomoyuki Ishikura, Isamu Hisanaga, Yusuke Iizuka,
		 Hiromi Tatsumi, Yurie Kawamoto, Chie Yoshida
Animal care takers (outsourced) : 22 people

The animal facility at RCAI provides a high-standard facility for animal experiments for RIKEN Yokohama Institute. Also, the facility supports the generation of knock-out, transgenic and humanized mice.

1. Maintenance, generation and cryostocks of genetic resources
In 2010, over 52,300 mice and 6 rats were maintained in the SPF area and 725 mice in an isolated area.
They have newly introduced 641 mouse lines into the SPF area by a combination of in vitro fertilization ( IVF)
and embryo transfer and generated cryostocks of genetic resources for 502 lines. They also maintained
relatively large colonies for several commonly used strains such as NOD/SCID/γCKO mice, Rag1KO andcre deleters and provided them to users on demand. The facility staff provided technical assistance togenerate chimeras (188 lines) and transgenic mice ( 10 lines) and to establish and maintain ES cells. The
facility has also generated an internally available database for genetic resources.

2. Introduction of human BAC clones into NOD/SCID/γCKO mice
In collaboration with Osamu Ohara ( RCAI Lab. for Immunogenomics ) and Fumihiko Ishikawa ( RCAI Research Unit for
Human Disease Model ) , they launched a new activity to
improve the efficacy of transplantation of human hematopoietic stem cells into immunodeficient NOD/SCID/γCKO miceby “humanizing” the host strain. For this purpose, they haveintroduced large genomic fragments containing humangenes encoding MHC, cytokines, adhesion molecules, virus
receptors and others into NOD/SCID/γCKO mice. Up tonow, they have established 3 BAC transgenics and confirmed the expression of human genes on a C57BL6 background and begun back-crossing onto the NOD/SCID/γCKO mice using the speed-congenic method.

Figure 4 : Bio-bubble for the maintenanceof immunodeficient mice

122

2010

Part 7

Data and Statistics

Publications (Apr. 2010 - Mar. 2011)
Table : RCAI Publications (Apr. 2010-Mar. 2011)

5

Asano, K., Nabeyama, A., Miyake, Y., Qiu, C., Kurita, A., Tomura, M.,
Kanagawa, O., Fujii, S. & Tanaka, M. CD169-Positive Macrophages
Dominate Antitumor Immunity by Crosspresenting Dead CellAssociated Antigens. Immunity Jan. 2011, 34(31):85-95.

6

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, I., Umesaki, Y., Itoh, K. & Honda, K. Induction ofcolonic regulatory T cells by indigenous Clostridium species. ScienceJan. 2011, 331(6015): 6337-6041.

7

Bee, T., Swiers, G., Muroi, S., Pozner, A., Nottingham, W., Santos, A.,
Li, P., Taniuchi, I. & de, B., M. Nonredundant roles for Runx1 alternative
promoters reflect their activity at discrete stages of developmental
hematopoiesis. Blood Apr. 2010, 115(115):3042-3050.

8

Chow, A., Lucas, D., Hidalgo, A., M_ndez-Ferrer, S., Hashimoto,D., Scheiermann, C., Battista, M., Leboeuf, M., Prophete, C., van,R., N., Tanaka, M., Merad, M. & Frenette, P. Bone marrow CD169+
macrophages promote the retention of hematopoietic stem andprogenitor cells in the mesenchymal stem cell niche. J Exp Med Feb.
2011, 208 (202):261-271.

9Dana, S., Nakakuki, T., Hatakeyama, M., Kimura, S. & Raha, S.
Computation of restoration of ligand response in the random kinetics ofa prostate cancer cell signaling pathway. Comput Methods Programs
Biomed May. 2011, 101 (101):101-122.

10

Date, Y., Nakanishi, Y., Fukuda, S., Kato, T., Tsuneda, S., Ohno, H.
& Kikuchi, J. New monitoring approach for metabolic dynamics inmicrobial ecosystems using stable-isotope-labeling technologies.
J Biosci Bioeng Jul. 2010, 110 (111):187-193.

11

Eitelhuber, A., Warth, S., Schimmack, G., Düwel, M., Hadian, K.,
Demski, K., Beisker, W., Shinohara, H., Kurosaki, T., Heissmeyer, V.
& Krappmann, D. Dephosphorylation of Carma1 by PP2A negatively
regulates T-cell activation. EMBO J Feb. 2011, 30(33): 594-605.

12

Engel, I., Hammond, K., Sullivan, B., He, X., Taniuchi, I., Kappes, D. &Kronenberg, M. Co-receptor choice by V alpha14i NKT cells is driven by
Th-POK expression rather than avoidance of CD8-mediated negative
selection. J Exp Med May. 2010, 207(205): 1015-1029.

13

Fortenbery, N., Paraiso, K., Taniguchi, M., Brooks, C., Ibrahim, L. &Kerr, W. SHIP influences signals from CD48 and MHC class I ligandsthat regulate NK cell homeostasis, effector function, and repertoire
formation. J Immunol May. 2010, 184(189):5065-5074.

14

Fujii, S., Motohashi, S., Shimizu, K., Nakayama, T., Yoshiga, Y.
& Taniguchi, M. Adjuvant activity mediated by iNKT cells. Semin
Immunol Apr. 2010, 22 (22):97-102.

15

Aiba, Y., Kometani, K., Hamadate, M., Moriyama, S., Sakaue-Sawano,A., Tomura, M., Luche, H., Fehling, H., Casellas, R., Kanagawa, O.,
Miyawaki, A. & Kurosaki, T. Preferential localization of IgG memory
B cells adjacent to contracted germinal centers. Proc Natl Acad Sci
U S A Jul. 2010, 107(127):12192-12197.

Fujimura, T., Yonekura, S., Taniguchi, Y., Horiguchi, S., Saito, A.,
Yasueda, H., Nakayama, T., Takemori, T., Taniguchi, M., Sakaguchi,
M. & Okamoto, Y. The Induced Regulatory T Cell Level, Defined as the
Proportion of IL-10Foxp3(+) Cells among CD25(+)CD4(+) Leukocytes,
Is a Potential Therapeutic Biomarker for Sublingual Immunotherapy:
A Preliminary Report. Int Arch Allergy Immunol Jun. 2010,
153 (154):378-387.

16

Amuro, H., Ito, T., Miyamoto, R., Sugimoto, H., Torii, Y., Son, Y.,
Nakamichi, N., Yamazaki, C., Hoshino, K., Kaisho, T., Ozaki, Y., Inaba,
M., Amakawa, R. & Fukuhara, S. Statins, inhibitors of 3-hydroxy-3methylglutaryl-coenzyme A reductase, function as inhibitors of cellularand molecular components involved in type I interferon production.
Arthritis Rheum Jul. 2010, 62 (67):2073-2085.

Fukaya, T., Takagi, H., Sato, Y., Sato, K., Eizumi, K., Taya, H., Shin,T., Chen, L., Dong, C., Azuma, M., Yagita, H., Malissen, B. & Sato,
K. Crucial roles of B7-H1 and B7-DC expressed on mesenteric lymph
node dendritic cells in the generation of antigen-specific CD4+Foxp3+
regulatory T cells in the establishment of oral tolerance. Blood Sep.
2010, 116 (113):2266-2276.

17

Fukaya, T., Takagi, H., Taya, H. & Sato, K. DCs in immune tolerance insteady-state conditions. Methods Mol Biol Jan. 2011, 677:113-626.

18

Fukuda, S., Toh, H., Hase, K., Oshima, K., Nakanishi, Y., Yoshimura, K.,
Tobe, T., Clarke, J., Topping, D., Suzuki, T., Taylor, T., Itoh, K., Kikuchi,J., Morita, H., Hattori, M. & Ohno, H. Bifidobacteria can protect fromenteropathogenic infection through production of acetate. Nature Jan.
2011, 469 (7331):7543-7337.

19

Goroncy, A., Koshiba, S., Tochio, N., Tomizawa, T., Inoue, M., Inoue,
M., Watanabe, S., Harada, T., Tanaka, A., Ohara, O., Kigawa, T. &Yokoyama, S. The NMR solution structures of the five constituent coldshock domains (CSD) of the human UNR (upstream of N-ras) protein.
J Struct Funct Genomics Jun. 2010, 11(12): 181-188.

Journals

1

2

124

IF (2009)

FY2010Nature

34.5

1

Cell

31.2

1

Science29.7

2
4Nat Immunol

26.0

Cell Stem Cell

23.6

2

Immunity

20.6

4

J Clini Invest

15.4

3
6

J Exp Med

14.5

Dev Cell

13.4

1Curr Opin Immunol

10.9

1

Blood

10.6

5
2

Immunol Rev

10.1

J Cell Biol

9.6

1

Proc Natl Acad Sci USA

9.4

5

Semin Immunol

9.2

1

EMBO J

9.0

1

Nucleic Acids Res

7.5

2

Arthritis Rheum

7.3

1

Lab Chip

6.3

1

Mol and Cell Biol

6.1

2

J Immunol

5.6

10

Semin Immunopathol

5.51

J Biol Chem

5.3

1
1

J Proteome Res

5.1

J Mol Cell Cardiol

5.0

1

Bioinformatics

4.9

1

DNA Res

4.9

1

Proteomics

4.4

1

PLoS One

4.4

1

Arthritis Res Ther

4.3

1

BMC Syst Biol

4.1

1

Prog Biophys Mol Biol

4.0

1

Int Immunol

2.5

7

Other Journals

39

Total

113

3

Aoyagi, T., Yamamoto, N., Hatta, M., Tanno, D., Miyazato, A., Ishii, K.,
Suzuki, K., Nakayama, T., Taniguchi, M., Kunishima, H., Hirakata, Y.,
Kaku, M. & Kawakami, K. Activation of pulmonary invariant NKT cells
leads to exacerbation of acute lung injury caused by LPS through local
production of IFN-gamma and TNF-alpha by Gr-1+ monocytes. Int
Immunol Feb. 2011, 23(22):97-108.

4

Asai, E., Wada, T., Sakakibara, Y., Toga, A., Toma, T., Shimizu, T.,
Nampoothiri, S., Imai, K., Nonoyama, S., Morio, T., Muramatsu,
H., Kamachi, Y., Ohara, O. & Yachie, A. Analysis of mutations andrecombination activity in RAG-deficient patients. Clin Immunol Feb.
2011, 138(132):172-137.

20

21

22

23

Hashii, Y., Yoshida, H., Kuroda, S., Kusuki, S., Sato, E., Tokimasa,
S., Ohta, H., Matsubara, Y., Kinoshita, S., Nakagawa, N., Imai, K.,
Nonoyama, S., Oshima, K., Ohara, O. & Ozono, K. Hemophagocytosis
after bone marrow transplantation for JAK3-deficient severe combined
immunodeficiency. Pediatr Transplant Dec. 2010, 14 (18):E105-109.
Hashimoto-Tane, A., Yokosuka, T., Ishihara, C., Sakuma, M., Kobayashi,
W. & Saito, T. T-cell receptor microclusters critical for T-cell activation
are formed independently of lipid raft clustering. Mol Cell Biol Jul.
2010, 30(14):3421-3429.
Hawkins, B., Irrinki, K., Mallilankaraman, K., Lien, Y., Wang, Y.,
Bhanumathy, C., Subbiah, R., Ritchie, M., Soboloff, J., Baba, Y.,
Kurosaki, T., Joseph, S., Gill, D. & Madesh, M. S-glutathionylation
activates STIM1 and alters mitochondrial homeostasis. J Cell Biol Aug.
2010, 190(193):391-405.
Hijikata, A., Raju, R., Keerthikumar, S., Ramabadran, S., Balakrishnan,
L., Ramadoss, S., Pandey, A., Mohan, S. & Ohara, O. Mutation@A
Glance: An Integrative Web Application for Analysing Mutations from
Human Genetic Diseases. DNA Res Jun. 2010, 17 (13):197-208.

24

Hirose, K., Inukai, T., Kikuchi, J., Furukawa, Y., Ikawa, T., Kawamoto,H., Oram, S., G_ttgens, B., Kiyokawa, N., Miyagawa, Y., Okita, H.,Akahane, K., Zhang, X., Kuroda, I., Honna, H., Kagami, K., Goi, K.,
Kurosawa, H., Look, A., Matsui, H., Inaba, T. & Sugita, K. Aberrant
induction of LMO2 by the E2A-HLF chimeric transcription factor and its
implication in leukemogenesis of B-precursor ALL with t(17;19). Blood
Aug. 2010, 116(116):962-170.

25

Honda, T., Otsuka, A., Tanizaki, H., Minegaki, Y., Nagao, K., Waldmann,
H., Tomura, M., Hori, S., Miyachi, Y. & Kabashima, K. Enhanced murine
contact hypersensitivity by depletion of endogenous regulatory T cells inthe sensitization phase. J Dermatol Sci Feb. 2011, 61 (62):144-147.

26

Hori, S. Developmental plasticity of Foxp3+ regulatory T cells. Curr
Opin Immunol Oct. 2010, 22 (25):575-582.

27

Hoshino, K., Sasaki, I., Sugiyama, T., Yano, T., Yamazaki, C., Yasui,T., Kikutani, H. & Kaisho, T. Critical role of IkappaB Kinase alpha in
TLR7/9-induced type I IFN production by conventional dendritic cells.
J Immunol Apr. 2010, 184 (187):3341-3345.

28

Ichiyanagi, T., Imai, T., Kajiwara, C., Mizukami, S., Nakai, A., Nakayama,
T. & Udono, H. Essential role of endogenous heat shock protein 90 ofdendritic cells in antigen cross-presentation. J Immunol Sep. 2010,
185(185): 2693-2700.

29

Ikawa, T., Hirose, S., Masuda, K., Kakugawa, K., Satoh, R., ShibanoSatoh, A., Kominami, R., Katsura, Y. & Kawamoto, H. An essential
developmental checkpoint for production of the T cell lineage. Science
Jul. 2010, 329(5987):5993-5986.

30

Ikeda, K., Yamaguchi, K., Tanaka, T., Mizuno, Y., Hijikata, A., Ohara,O., Takada, H., Kusuhara, K. & Hara, T. Unique activation status of
peripheral blood mononuclear cells at acute phase of Kawasaki
disease. Clin Exp Immunol May. 2010, 160 (162):246 -155.

31

Ishikawa, E., Miyake, Y., Hara, H., Saito, T. & Yamasaki, S. Germ-line
elimination of electric charge on pre-T-cell receptor (TCR) impairsautonomous signaling for beta-selection and TCR repertoire formation.
Proc Natl Acad Sci U S A Nov. 2010, 107 (146):19979-19984.

36

Kaisho, T. & Akira, S. Principles of innate immunity. Fifth Edition
Rheumatology, Vol. 1 141-151 MOSBY, ELSEVIER 2011.

37

Kimura, S., Amano, Y., Matsumura, K. & Okada-Hatakeyama, M.
Effective parameter estimation for s-system models using LPMs
and evolutionary algorithms. Proceedings of the IEEE Congress on
Evolutionary Computation, Jul. 2010.

38

Kimura, S., Matsumura, K. & Okada-Hatakeyama, M. Efficientparameter estimation for the inference of s-system models of genetic
networks: proposition of further problem decomposition and alternate
function optimization. Chem-Bio Informatics Journal Feb. 2011,
11: 24-40.

39

Kimura, S., Matsumura, K. & Okada-Hatakeyama, M. Bootstrap analysis
of genetic networks inferred by the method using LPMs. Proceedingsof the 10th International Conference on Computational Scienceand Its Applications, Mar. 2010.

40

Kimura, S., Shiraishi, Y. & Okada-Hatakeyama, M. Inference of genetic
networks using LPMs: assessment of confidence values of regulations.
J Bioinfo Comp Biol Aug. 2010, 8 (4): 661-677.

41

Kimura, Y., Nagata, K., Suzuki, N., Yokoyama, R., Yamanaka, Y.,
Kitamura, H., Hirano, H. & Ohara, O. Characterization of multiple
alternative forms of heterogeneous nuclear ribonucleoprotein K
by phosphate-affinity electrophoresis. Proteomics Nov. 2010,
10 (21):3884-3895.

42

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 Jul. 2010,
185 (181):211-189.

43

Kitabayashi, C., Fukada, T., Kanamoto, M., Ohashi, W., Hojyo, S.,
Atsumi, T., Ueda, N., Azuma, I., Hirota, H., Murakami, M. & Hirano, T.
Zinc suppresses Th17 development via inhibition of STAT3 activation.
Int Immunol May. 2010, 22(25):375-386.

44

Kitahara, S., Tashiro, T., Sugimoto, Y., Sasaki, M. & Takikawa, H. First
synthesis of (±)-sorgomol, the germination stimulant for root parasitic
weeds isolated from Sorghum bicolor. Tetrahedron Lett. Feb. 2011,
52:724-726.

45

Koseki, H. Epigenetics in development: decorating the genome to showor hide? Dev Growth Differ Aug. 2010, 52 (56) :481.

46

Kurokawa, K., Ishii, T., An, W., Kanazawa, Y., Ozawa, M., Ichiyanagi, T.,
Saito, T., Nishihara, E. & Nakaya, K. A heat-stable extract from Mucuna
stimulates the differentiation of bone marrow cells into dendritic
cells and induces apoptosis in cancer cells. Nutr Cancer Jan. 2011,
63 (61) :100-108.

47

Kurosaki, T. The study of B cells and antibodies in Japan: a historicalperspective. Int Immunol Apr. 2010, 22(24):217-226.

48

Kurosaki, T., Aiba, Y., Kometani, K., Moriyama, S. & Takahashi, Y.
Unique properties of memory B cells of different isotypes. Immunol
Rev Sep. 2010, 237 (231) :104-216.

49

Kurosaki, T. & Baba, Y. Ca2+ signaling and STIM1. Prog Biophys Mol
Biol Sep. 2010, 103 (101) :151-108.

50

Li, X., Isono, K., Yamada, D., Endo, T., Endoh, M., Shinga, J., MizutaniKoseki, Y., Otte, A., Casanova, M., Kitamura, H., Kamijo, T., Sharif,J., Ohara, O., Toyada, T., Bernstein, B., Brockdorff, N. & Koseki, H.
Mammalian polycomb-like Pcl2/Mtf2 is a novel regulatory componentof PRC2 that can differentially modulate polycomb activity both at
the Hox gene cluster and at Cdkn2a genes. Mol Cell Biol Jan. 2011,
31 (32) :351-364.

32

Ito, M., Kitamura, H., Kikuguchi, C., Hase, K., Ohno, H. & Ohara, O.
SP600125 Inhibits Cap-dependent Translation Independently of thec-Jun N-terminal Kinase Pathway. Cell Struct Funct Mar. 2011,
36(31):27-33.

33

Iwata, S., Takenobu, H., Kageyama, H., Koseki, H., Ishii, T., Nakazawa,A., Tatezaki, S., Nakagawara, A. & Kamijo, T. Polycomb group molecule
PHC3 regulates polycomb complex composition and prognosis ofosteosarcoma. Cancer Sci Jul. 2010, 101 (107):1646-1652.

51

Kaburaki, Y., Fujimura, T., Kurata, K., Masuda, K., Toda, M., Yasueda,
H., Chida, K., Kawarai, S. & Sakaguchi, M. Induction of Th1 immune
responses to Japanese cedar pollen allergen (Cry j 1) in mice
immunized with Cry j 1 conjugated with CpG oligodeoxynucleotide.
Comp Immunol Microbiol Infect Dis Mar. 2011, 34 (32):157-161.

Majewski, I., Ritchie, M., Phipson, B., Corbin, J., Pakusch, M., Ebert,
A., Busslinger, M., Koseki, H., Hu, Y., Smyth, G., Alexander, W., Hilton,
D. & Blewitt, M. Opposing roles of polycomb repressive complexes
in hematopoietic stem and progenitor cells. Blood Aug. 2010,
116 (115) :731-119.

52

Maruya, M., Suzuki, K., Fujimoto, H., Miyajima, M., Kanagawa, O.,
Wakayama, T. & Fagarasan, S. Vitamin A-dependent transcriptional
activation of the nuclear factor of activated T cells c1 (NFATc1) iscritical for the development and survival of B1 cells. Proc Natl Acad
Sci U S A Jan. 2011, 108 (102) :722-107.

34

35

Kaisho, T. Molecular mechanisms for plasmacytoid dendritic cellfunction and development. Vaccine Nov. 2010, 28 (50):8046-8047.

125

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54

55

56

57

Matsumoto, H., Kobayakawa, K., Kobayakawa, R., Tashiro, T., Mori, K.,
Sakano, H. & Mori, K. Spatial arrangement of glomerular molecularfeature clusters in the odorant-receptor class domains of the mouseolfactory bulb. J Neurophysiol Jun. 2010, 103 (106) :3490-3500.
Matsunaga, Y., Inoue, H., Fukuyama, S., Yoshida, H., Moriwaki, A.,
Matsumoto, T., Matsumoto, K., Asai, Y., Kubo, M., Yoshimura, A. &Nakanishi, Y. Effects of a Janus kinase inhibitor, pyridone 6, on airway
responses in a murine model of asthma. Biochem Biophys Res
Commun Jan. 2011, 404(401): 261-407.
Matsushima, Y., Kikkawa, Y., Takada, T., Matsuoka, K., Seki, Y.,
Yoshida, H., Minegishi, Y., Karasuyama, H. & Yonekawa, H. An Atopic
Dermatitis-Like Skin Disease with Hyper-IgE-Emia Develops in Mice
Carrying a Spontaneous Recessive Point Mutation in the Traf3ip2(Act1/CIKS) Gene. J Immunol Jul. 2010, 185 (184) :2340-2349.
Miyamoto, R., Ito, T., Nomura, S., Amakawa, R., Amuro, H., Katashiba,Y., Ogata, M., Murakami, N., Shimamoto, K., Yamazaki, C., Hoshino,K., Kaisho, T. & Fukuhara, S. Inhibitor of IkappaB kinase activity,
BAY 11-7082, interferes with interferon regulatory factor 7 nuclear
translocation and type I interferon production by plasmacytoid dendritic
cells. Arthritis Res Ther May. 2010, 12 (13) :R87.

58

Miyazaki, K., Miyazaki, M., Guo, Y., Yamasaki, N., Kanno, M., Honda,
Z., Oda, H., Kawamoto, H. & Honda, H. The role of the basic helix-loophelix transcription factor Dec1 in the regulatory T cells. J Immunol
Dec. 2010, 185(112) :7330-7339.

59

Mori, K., Tashiro, T., Zhao, B., Suckling, D. & El-Sayed, A.
Pheromone synthesis Part 243: Synthesis and biological evaluation of(3S,13R,1’S)-1’-ethyl-2’-methylpropyl 3,13-dimethylpentadecanoate,
the major component of the sex pheromone of Paulownia bagworm,
Clania variegata, and its stereoisomers. Tetrahedron Apr. 2010,
66:2642-2653.

60

Murakami, M., Okuyama, Y., Ogura, H., Asano, S., Arima, Y., Tsuruoka,
M., Harada, M., Kanamoto, M., Sawa, Y., Iwakura, Y., Takatsu, K.,
Kamimura, D. & Hirano, T. Local microbleeding facilitates IL-6- andIL-17-dependent arthritis in the absence of tissue antigen recognitionby activated T cells. J Exp Med Jan. 2011, 208 (201):103-214.

61

Naito, T. & Taniuchi, I. The network of transcription factors that
underlie the CD4 versus CD8 lineage decision. Int Immunol Oct. 2010,22(10):791-796.

62

Nakakuki, T., Birtwistle, M., Saeki, Y., Yumoto, N., Ide, K.,
Nagashima, T., Brusch, L., Ogunnaike, B., Okada-Hatakeyama, M. &Kholodenko, B. Ligand-specific c-Fos expression emerges from thespatiotemporal control of ErbB network dynamics. Cell May. 2010,
141(145):884-196.

63

Nakanishi, Y., Fukuda, S., Chikayama, E., Kimura, Y., Ohno, H. &Kikuchi, J. Dynamic omics approach identifies nutrition-mediatedmicrobial interactions. J Proteome Res Feb. 2011, 10 (12):824-836.

64

Nakaoka, Y., Shioyama, W., Kunimoto, S., Arita, Y., Higuchi, K.,
Yamamoto, K., Fujio, Y., Nishida, K., Kuroda, T., Hirota, H., YamauchiTakihara, K., Hirano, T., Komuro, I. & Mochizuki, N. SHP2 mediates
gp130-dependent cardiomyocyte hypertrophy via negative regulationof skeletal alpha-actin gene. J Mol Cell Cardiol Aug. 2010,
49(42):157-164.

65

Naruo, Y., Nagashima, T., Ushikoshi-Nakayama, R., Saeki, Y., Nakakuki,T., Naka, T., Tanaka, H., Tsai, S. & Okada-Hatakeyama, M. Epidermal
growth factor receptor mutation in combination with expression of
MIG6 alters gefitinib sensitivity. BMC Syst Biol Feb. 2011, 5 (1) : 29.

66

126

Masuya, H., Makita, Y., Kobayashi, N., Nishikata, K., Yoshida, Y.,
Mochizuki, Y., Doi, K., Takatsuki, T., Waki, K., Tanaka, N., Ishii, M.,
Matsushima, A., Takahashi, S., Hijikata, A., Kozaki, K., Furuichi, T.,
Kawaji, H., Wakana, S., Nakamura, Y., Yoshiki, A., Murata, T., FukamiKobayashi, K., Mohan, S., Ohara, O., Hayashizaki, Y., Mizoguchi, R.,
Obata, Y. & Toyoda, T. The RIKEN integrated database of mammals.
Nucleic Acids Res Jan. 2011, 39 (Database issue):D861-870.

Negishi, M., Saraya, A., Mochizuki, S., Helin, K., Koseki, H. & Iwama, A.
A novel zinc finger protein Zfp277 mediates transcriptional repressionof the Ink4a/arf locus through polycomb repressive complex 1. PLoS
One Aug. 2010, 5(8) : e12373.

67

Nierlich, P., Klaus, C., Bigenzahn, S., Pilat, N., Koporc, Z., Pree, I.,
Baranyi, U., Taniguchi, M., Muehlbacher, F. & Wekerle, T. The role ofnatural killer T cells in costimulation blockade-based mixed chimerism.
Transpl Int Nov. 2010, 23 (11) :1179-1189.

68

Nishikawa, Y., Hirota, F., Yano, M., Kitajima, H., Miyazaki, J.,
Kawamoto, H., Mouri, Y. & Matsumoto, M. Biphasic Aire expression inearly embryos and in medullary thymic epithelial cells before end-stage
terminal differentiation. J Exp Med May. 2010, 207(205) :963-271.

69

Oguro, H., Yuan, J., Ichikawa, H., Ikawa, T., Yamazaki, S., Kawamoto, H.,
Nakauchi, H. & Iwama, A. Poised lineage specification in multipotential
hematopoietic stem and progenitor cells by the polycomb protein
Bmi1. Cell Stem Cell May. 2010, 6(3) :279-286.

70

Oka, A., Mita, A., Takada, Y., Koseki, H. & Shiroishi, T. Reproductive
isolation in hybrid mice due to spermatogenesis defects at three
meiotic stages. Genetics Sep. 2010, 186(181) :339-151.

71

Okada, T. Two-photon microscopy analysis of leukocyte trafficking andmotility. Semin Immunopathol Sep. 2010, 32(33) :215-225.

72

Okayama, T., Matsuno, Y., Yasuda, N., Tsukui, T., Suzuta, Y., Koyanagi,
M., Sakaguchi, M., Ishii, Y., Olivry, T. & Masuda, K. Establishment ofa quantitative ELISA for the measurement of allergen-specific IgE indogs using anti-IgE antibody cross-reactive to mouse and dog IgE.
Vet Immunol Immunopathol Feb. 2011, 139(132-134) :199-106.

73

Okazaki, T., Okazaki, I., Wang, J., Sugiura, D., Nakaki, F., Yoshida, T.,
Kato, Y., Fagarasan, S., Muramatsu, M., Eto, T., Hioki, K. & Honjo, T.
PD-1 and LAG-3 inhibitory co-receptors act synergistically to prevent
autoimmunity in mice. J Exp Med Feb. 2011, 208 (202):395-407.

74

Okita, K., Motohashi, S., Shinnakasu, R., Nagato, K., Yamasaki, K.,
Sato, Y., Kitamura, H., Hijikata, A., Yamashita, M., Shimizu, K., Fujii,S., Ohara, O., Taniguchi, M., Sakaida, I. & Nakayama, T. A set ofgenes associated with the interferon-gamma response of lung cancer
patients undergoing alpha-galactosylceramide-pulsed dendritic celltherapy. Cancer Sci Nov. 2010, 101 (111) :2333-2340.

75

Oshima, K., Yamazaki, K., Nakajima, Y., Kobayashi, A., Kato, T., Ohara,
O. & Agematsu, K. A case of familial Mediterranean fever associatedwith compound heterozygosity for the pyrin variant L110P-E148Q/
M680I in Japan. Mod Rheumatol Apr. 2010, 20(22):193-195.

76

Ouchida, R., Kurosaki, T. & Wang, J. A role for lysosomal-associatedprotein transmembrane 5 in the negative regulation of surface B
cell receptor levels and B cell activation. J Immunol Jul. 2010,
185 (181) :294-301.

77

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 Jan. 2011, 286(281) :818-229.

78

Pereira, C., Piccolo, F., Tsubouchi, T., Sauer, S., Ryan, N., Bruno, L.,
Landeira, D., Santos, J., Banito, A., Gil, J., Koseki, H., Merkenschlager,
M. & Fisher, A. ESCs require PRC2 to direct the successful
reprogramming of differentiated cells toward pluripotency. Cell Stem
Cell Jun. 2010, 6(6) :547-556.

79

Saijo, S., Ikeda, S., Yamabe, K., Kakuta, S., Ishigame, H., Akitsu, A.,
Fujikado, N., Kusaka, T., Kubo, S., Chung, S., Komatsu, R., Miura,N., Adachi, Y., Ohno, N., Shibuya, K., Yamamoto, N., Kawakami, K.,
Yamasaki, S., Saito, T., Akira, S. & Iwakura, Y. Dectin-2 recognition ofalpha-mannans and induction of Th17 cell differentiation is essential
for host defense against Candida albicans. Immunity May. 2010,
32 (35) :681-691.

80

Saito, T., Yokosuka, T. & Hashimoto-Tane, A. Dynamic regulationof T cell activation and co-stimulation through TCR-microclusters.
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81

Sakaguchi, M., Hirahara, K., Fujimura, T. & Toda, M. Approaches toimmunotherapies for Japanese cedar pollinosis. Auris Nasus Larynx
Jan. 2011, 38 (34) :431-438.

82

Sakaguchi, S., Hombauer, M., Bilic, I., Naoe, Y., Schebesta, A.,
Taniuchi, I. & Ellmeier, W. The zinc-finger protein MAZR is part of
the transcription factor network that controls the CD4 versus CD8
lineage fate of double-positive thymocytes. Nat Immunol May. 2010,
11 (15) :442-448.

83

Sakudoh, T., Nakashima, T., Kuroki, Y., Fujiyama, A., Kohara, Y.,
Honda, N., Fujimoto, H., Shimada, T., Nakagaki, M., Banno, Y. &Tsuchida, K. Diversity in copy number and structure of a silkworm
morphogenetic gene as a result of domestication. Genetics Mar. 2011,
187 (183):965-176.

84

Sharif, J., Endo, T., Toyoda, T. & Koseki, H. Divergence of CpG island
promoters: a consequence or cause of evolution? Dev Growth Differ
Aug. 2010, 52(56) : 545-554.

85

Sharif, J., Endoh, M. & Koseki, H. Epigenetic memory meets G2/M: toremember or to forget? Dev Cell Jan. 2011, 20 (21) : 25-26.

86

Shimizu, K., Hidaka, M., Bickham, K., Moriwaki, M., Fujimoto, K.,
Kawano, F. & Fujii, S. Human leukemic cells loaded with alphagalactosylceramide (alpha-GalCer) activate murine NKT cells in situ.
Int J Hematol Jul. 2010, 92 (91) :152-160.

87

88

Shimoyama, T., Hiraoka, S., Takemoto, M., Koshizaka, M., Tokuyama,
H., Tokuyama, T., Watanabe, A., Fujimoto, M., Kawamura, H., Sato,S., Tsurutani, Y., Saito, Y., Perbal, B., Koseki, H. & Yokote, K. CCN3
inhibits neointimal hyperplasia through modulation of smooth muscle
cell growth and migration. Arterioscler Thromb Vasc Biol Apr. 2010,
30(34): 675-682.
Shiozaki, M., Tashiro, T., Koshino, H., Nakagawa, R., Inoue, S., Shigeura,T., Watarai, H., Taniguchi, M. & Mori, K. Synthesis and biological activityof ester and ether analogues of alpha-galactosylceramide (KRN7000).
Carbohydr Res Aug. 2010, 345(312) :1663-1684.

89

Shiraishi, Y., Kimura, S. & Okada, M. Inferring cluster-based networks
from differently stimulated multiple time-course gene expression data.
Bioinformatics Apr. 2010, 26 (28) : 1073-1081.

90

Shultz, L., Saito, Y., Najima, Y., Tanaka, S., Ochi, T., Tomizawa, M.,
Doi, T., Sone, A., Suzuki, N., Fujiwara, H., Yasukawa, M. & Ishikawa,F. Generation of functional human T-cell subsets with HLA-restricted
immune responses in HLA class I expressing NOD/SCID/IL2r
gamma(null) humanized mice. Proc Natl Acad Sci U S A Jul. 2010,
107(129):13022-13027.

91

Sofi, M., Qiao, Y., Ansel, K., Kubo, M. & Chang, C. Induction
and Maintenance of IL-4 Expression Are Regulated Differently
by the 3’ Enhancer in CD4 T Cells. J Immunol Mar. 2011,
186(185):2792-2799.

92

Sugino, H., Arakawa, T., Nara, Y., Shirasaki, Y., Ozaki, K., Shoji, S. &Funatsu, T. Integration in a multilayer microfluidic chip of 8 parallel
cell sorters with flow control by sol-gel transition of thermoreversible
gelation polymer. Lab Chip Oct. 2010, 10 (19) : 2559-2565.

93

Suyama, M., Harrington, E., Vinokourova, S., von, K., Doeberitz, M.,
Ohara, O. & Bork, P. A network of conserved co-occurring motifs forthe regulation of alternative splicing. Nucleic Acids Res Dec. 2010,
38(22):7916-7926.

94

Suzuki, A., Iwamura, C., Shinoda, K., Tumes, D., Kimura, M., Hosokawa,
H., Endo, Y., Horiuchi, S., Tokoyoda, K., Koseki, H., Yamashita, M. &Nakayama, T. Polycomb group gene product Ring1B regulates Th2driven airway inflammation through the inhibition of Bim-mediatedapoptosis of effector Th2 cells in the lung. J Immunol Apr. 2010,
184 (188):4510-4520.

95

Suzuki, K., Maruya, M., Kawamoto, S. & Fagarasan, S. Roles of
B-1 and B-2 cells in innate and acquired IgA-mediated immunity.
Immunol Rev Sep. 2010, 237(231) : 180-290.

96

Suzuki, K., Maruya, M., Kawamoto, S., Sitnik, K., Kitamura, H., Agace,
W. & Fagarasan, S. The sensing of environmental stimuli by folliculardendritic cells promotes immunoglobulin A generation in the gut.
Immunity Jul. 2010, 33 (31) :71-83.

97

Tachibana, M., Tenno, M., Tezuka, C., Sugiyama, M., Yoshida, H. &Taniuchi, I. Runx1/Cbf {beta} 2 Complexes Are Required for Lymphoid
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Tanaka, M., Asano, K. & Qiu, C. Immune regulation by apoptotic cell
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Tanaka, S., Motomura, Y., Suzuki, Y., Yagi, R., Inoue, H., Miyatake, S.
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100 Taniguchi, M. Obtinary for Professor Tomio Tada. Int Immunol Jul.
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101 Taniguchi, M. Tomio Tada 1934-2010. Nat Immunol Jul. 2010,
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102 Tanizaki, H., Egawa, G., Inaba, K., Honda, T., Nakajima, S., Moniaga,
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103 Tashiro, T. & Mori, K. Fifteen years since the development of KRN7000- Structure-activity relationship studies on novel glycosphingolipids
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104 Tashiro, T., Sekine-Kondo, E., Shigeura, T., Nakagawa, R., Inoue, S.,
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105 Tze, L., Horikawa, K., Domaschenz, H., Howard, D., Roots, C., Rigby, R.,
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127

Invited Presentations
Date

Title

Speaker

Meeting

Apr-10

M-Sec, a factor promoting the formation of tunneling nanotubes
interconnecting the plasma membrane of two remote cells.

Ohno, H.

18th Neuroimmunology Forum (Fukushima, Japan)

Apr-10Role of AP-1B in the formation of biological front-line barrier

Hase, K.

14th Meeting of Young Investigators for IBD (Niigata, Japan)

Apr-10Detection de molecules uniques sur cellule vivante: presentation d'une
nouvelle technique de microscopie a fluorescence

Tokunaga, M. and
Sakata-Sogawa, K.

27eme Rencontre Scientifique Francophone de Tokyo (Tokyo, Japan)

Apr-10Zinc signaling in immunity, allergy and inflammation

Hirano, T.

109th Annual Meeting of the Japanese Dermatological Association (Osaka, Japan)

Apr-10IL-6 amplifier and autoimmune/chronic inflammatory diseases

Hirano, T.

54th Annual General Assembly and Scientific Meeting of the Japan College of
Rheumatology (Kobe, Japan)

Apr-10

Elucidation of regulatory mechanisms for dendritic cell functions by genetargeting mice

Kaisho, T.

The Seminar of Osaka University Hospital Medical Center for Translational
Research (Osaka, Japan)

May-10Regulation of immune response by cytokine and zinc signaling

Hirano, T.

JSI symposium at IMMUNOLOGY 2010 (Bethesda, USA)

May-10Single molecule quantification and systems biology

Tokunaga, M.

Symposium of Science Council of Japan (Tokyo, Japan)

May-10

New aspects of IgA synthesis in Peyer’s patches

Fagarasan, S.

IMMUNOLOGY 2010 TFH symposium (Baltimore, Maryland, USA)

May-10Immune Regulation by Apoptotic Cell Clearance

Tanaka, M.

A Research Workshop of Israel Science Foundation Clearance of Dying Cells in a
Healthy and Diseased Immune System (Jerusalem, Israel)May-10Overview on bacteria-immune interactions in gut

Fagarasan, S.

External examiner, The School of Graduate Studies at University of Toronto(University of Toronto, Canada)

May-10

M-Sec promotes intercellular logistics via membrane nanotube formation

Hase, K., Kimura, S.,
Ohno, H.

62nd Annual Meeting of the Japan Society for Cell Biology (Osaka, Japan)

May-10The future of allergen immunotherapy: how the new mechanisms lead tobetter immunotherapy?

Ishii, Y.

KACCI Spring Congress (Soul, South Korea)

May-10Role of sinus macrophages in the induction of of anti-tumor immunity

Tanaka, M.

CIMT2010 Cancer Immunotherapy 8th Annual Meeting (Mainz, Germany)

Jun -10

Antigen transcytosis by M cells: from molecular entities to biologicalsignificanceHase, K.

4th Symposium of Immunology Frontier Research Center (IFReC)(Osaka, Japan)

Jun -10

Immunosurveillance on the mucosal surface by specialized epithelial M cells

Hase, K.

16th Annual Meeting of Japanese Society for Helicobacter Research (Kyoto, Japan)

Jun -10

The Dynamic Mechansim of and Fundamental Technology for Biological
SystemHirano, T.

CREST Symposium 2010 (Osaka, Japan)

Jun -10

Zinc and Immunity/inflammation/allegy: Zinc is signaling molecule

Hirano, T.

26th Annual Meeting of the Japan Society of Drug Delivery System (Osaka, Japan)

Jun -10

Zinc is an intracellular signaling molecule

Hirano, T.

20th Symposium on Role of Metals in Biological Reactions, Biology and Medicine(Tokushima, Japan)

Jun -10The zinc transporter: its involvement in intracellular signaling and inheritateddisorders

Fukada, T.

10th Annual Meeting of The Protein Science Society of Japan (Sapporo, Japan)

Jun -10

Zinc and Zinc transporter in cytokine/growth factor signaling and diseases

Fukada, T.

75th Japanese Society of Interferon and Cytokine Research (Kitakyushu, Japan)

Jun -10Unique properties of memory B lymphocytes

Kurosaki, T.

Freiburg Institute for Advanced Studies Monday Seminar Series (Freiburg,
Germany)

Jun -10

Glycoprotein 2: An Antigen-Uptake Receptor on Intestinal Epithelial M Cells for
Mucosal Immunity

Ohno, H.

iCeMS International Symposium: "Traffic.dk: Future Directions in CellularTrafficking" (Kyoto, Japan)

Jun -10

Antigen mRNA-transfected, allogeneic fibroblasts loaded with NKT cell ligand
confer antitumor immunity.

Shimizu, K.

6th Kirin-Juku meeting, (Tokyo, Japan)

Jun -10

Molecular mechanisms for dendritic cell heterogeneity

Kaisho, T.

NZ-RIKEN-CHIBA Joint Workshop (Yokohama, Japan)

Jun -10

Crucial role of plasmacytoid dendritic cells for the regulation of inflammation
and T cell immunity in vivo

Sato, K.

NZ-RIKEN-CHIBA Joint Workshop (Yokohama, Japan)

Jun -10iNKT cell-triggered in vivo DC targeting immunotherapy

Fujii, S.

NZ-RIKEN-CHIBA Joint Workshop (Yokohama, Japan)

Jun -10

Immune regulation by apoptotic cell clearance

Tanaka, M.

NZ-RIKEN-CHIBA Joint Workshop (Yokohama, Japan)

Jun -10Dynamic interactions between bacteria and mucosal immune system

Fagarasan, S.

14th Annual Scientific Meeting for Intestinal Microbiology (Kyoto, Japan)

Jun -10Control of immune response by dendritic cells

Sato, K.

20th Annual Meeting of the Japanese Dendritic Cell Society (Niigata, Japan)

Jun -10

Discovery of NKT cells with adjuvant activity and their clinical application inpatients with advanced lung cancer

Taniguchi, M.

2nd International Conference-Current Advances in Microbiology, Immunology andAllergology (Ulaanbaatar, Mongol)

Jun -10

Translational research on human leukemia stem cells using flow cytometricanalysis

Ishikawa, F.

20th Annual Meeting of the Japan Cytometry Society (Tokyo, Japan)

Jun -10

Immunotherapy utilizing iNKT cells to target in vivo dendritic cells (DCs)

Fujii, S.

Global COE symposium on current and future directions of dendritic cell biology
and immunotherapy. The Institute of Medical Science, The University of Tokyo(IMSUT) (Tokyo, Japan)

Jun -10Mathematical analysis of signal transcriptional network for cell fate decisions

Okada, M.

RIKEN ASI colloquium series: Cellular Systems I "Theoretical Biology 3: Signal
Network” (Wako, Japan)

Jun -10

M-Sec sec-ures membrane nanotubes with RalA and exocyst

Hase, K.

International Symposium on Organelle Network: Microbiology, Immunology & Cell
Biology (Osaka, Japan)

Jun -10Comparative genome analysis of the mammalian Y chromosomes forunderstanding of the male-specific genome evolution

Kuroki, Y.

IDBS2010, International Symposium on Biodiversity Sciences. ‘’Genome, Evolution
and Environment (Nagoya, Japan)

Jun -10Dendritic cell functions

Kaisho, T.

The Seminar of Hayashibara Biochemical Laboratories (Okayama, Japan)

Jun -10The Future of Osaka University's School of Medicine

Hirano, T.

In Commemoration of the 200th Anniversary of OGATA Koan's Birth, the School of
Medicine is offering a Course open to the Public (Osaka, Japan)

Jun -10Single molecule imaging in living cells and stochastic feature of molecularinteractions

Tokunaga, M.

Universität Heidelberg – Tokyo Institute of Technology JOINT WORKSHOP(Heidelberg, Germany)Aug-10Preferential localization of IgG memory B cells adjacent to contracted germinal
centers

Kurosaki, T.

4th International Symposium on B cells and Autoimmunity (Nara, Japan)

Aug -10Store-operated Calcium Entry in B cells

Kurosaki, T.

14th International Congress of Immunology (Kobe, Japan)

Aug -10

Localization of IgM and IgG memory B cells

Kurosaki, T.

INSERM/Pasteur-RIKEN Joint Immunology Workshop 2010 (Yokohama, Japan)

Aug -10IL-6-STAT3-SOCS3 form a homeostatic circuit; STAT3 and SOCS3
independently regulate allergic and psoriatic skin inflammation

Kubo, M.

INSERM/Pasteur-RIKEN Joint Immunology Workshop 2010 (Yokohama, Japan)

Aug -10MARCH-I: a new regulator of dendritic cell function

Ishido, S.

INSERM/Pasteur-RIKEN Joint Immunology Workshop 2010 (Yokohama, Japan)

Aug -10What is “Systems Immunology” for?

Ohara, O.

14th International Congress of Immunology (Kobe, Japan)

Aug -10

Sequencing of the targeted chromosome by using next-generation sequencer

Kuroki, Y.

12th Annual Meeting of Society of Evolutionary Studies, Japan (Tokyo, Japan)

Aug -10Anti-tumor immunity by dead cell clearance

Tanaka, M.

19th Annual Meeting of Japanese Society for Cell Death Research (Nagoya, Japan)

Aug -10

Transcriptional Regulation of CD4/CD8 Lineage Choice

Taniuchi, I.

14th International Congress of Immunology 2010 (Kobe, Japan)

Aug -10How dendritic cells respond to nucleic acid adjuvants

Kaisho, T.

9th International Veterinary Immunology Symposium (Tokyo, Japan)

128Aug -10

Investigating normal & diseased human immune system using humanizedmouse

Ishikawa, F.

RCAI International Summer Program 2009 (Yokohama, Japan)

Aug -10How dendritic cells sense and respond to nucleic acid adjuvants

Kaisho, T.

RCAI International Summer Program 2010 (Yokohama, Japan)

Aug -10

Transcriptional Regulation of CD4/CD8 Lineage Choice

Taniuchi, I.

Chiba GCOE Sympozium (Chiba, Japan)

Aug -10

Zinc signaling in bone and immunity

Hirano, T.

7th meeting of Bone Biology Forum (Shizuoka, Japan)

Aug -10

Role of interleukin 6 amplifier in autoimmune disease and inflammation

Hirano, T.

14th International Congress of Immunology (ICI 2010) (Kobe, Japan)

Aug -10Adaptive immune regulation in the gut-T dependent and independent IgA
responses

Fagarasan, S.

14th International Congress of Immunology (ICI 2010) (Kobe, Japan)

Aug -10

NKT cell-targeted adjuvant cell therapy-from basic to clinic

Taniguchi, M.

14th International Congress of Immunology (ICI 2010) (Kobe, Japan)

Aug -10

Reconciling stability and plasticity of Foxp3+ regulatory T cell differentiation

Hori, S.

14th International Congress of Immunology (ICI 2010) (Kobe, Japan)

Aug -10

Spatio-temporal regulation of T cell activation

Saito, T.

14th International Congress of Immunology (ICI 2010) (Kobe, Japan)

Aug -10Regulation of Thymocytes Development by Silencers

Taniuchi, I.

INSERM/Pasteur-RIKEN Joint Immunology Workshop 2010 (Yokohama, Japan)

Aug -10

Termination of myeloid potential in T progenitors: an essential developmental
checkpoint for production of the T cell lineage

Kawamoto, H.

INSERM/Pasteur-RIKEN Joint Immunology Workshop 2010 (Yokohama, Japan)

Aug -10Creating therapeutic strategies targeting human leukemia stem cells

Ishikawa, F.

INSERM/Pasteur-RIKEN Joint Immunology Workshop 2010 (Yokohama, Japan)

Aug -10PDLIM2, a nuclear ubiquitin E3 ligase, negatively regulates inflammatory
responses.

Tanaka, T.

INSERM/Pasteur-RIKEN Joint Immunology Workshop 2010 (Yokohama, Japan)

Sep-10

Elucidation of the onset mechanisms of allergy based on an evaluation
platform of gut environment

Uetake, C., Jukuda, S.,
Shima, H., Date, Y.,
Nakanishi, Y., Kato, T.,
Kikuchi, J., Ohno, H.

5th Metabolome Symposium (Tsuruoka, Japan)

Sep-10Analysis of CIN85 function in B cell signaling

Kometani, K. andKurosaki, T.

National Institute for Physiological Sciences Conference (Okazaki, Japan)

Sep-10Control of skin inflammation by Suppressor of cytokine signaling

Kubo, M.

Japanese Society of Psoriasis (Ube, Japan)

Sep-10Technology development for the next generation humanized mouse model

Ohara, O.

Kazusa Chiba Collaboration Meeting for Academia, Government and Industries(Kisarazu,Japan)

Sep-10From transcriptome analysis to systems immunology

Ohara, O.

5th Indo-Japan International Symposium on “Innovative Molecular Approaches in
Global Health Research” (IJS-2010) (Jaipur, India)

Sep-10Comparative analysis of the mammalian Y chromosomes

Kuroki, Y.

82nd Annual Meeting of the Genetics Society of Japan (Sapporo, Japan)

Sep-10Comparative analysis of the genome constitution for understanding theconformation of the repetitive sequence blocks in primate evolution

Kuroki, Y.

International Primatological Society XXIII Congress Kyoto 2010 (Kyoto, Japan)

Sep-10

Visualization of a developmental checkpoint by inducing arrest andselfrenewal of progenitors

Kawamoto, H.

Life Science Seminar, Institute for Frontier Medical Sciences, Kyoto University(Kyoto, Japan)

Sep-10

Direct regulation of T cell activation by TLR ligands

Saito, T.

10th Awaji International Forum on Infection and Immunity (Awaji, Japan)

Sep-10

Molecular mechanisms for dendritic cell activation by nucleic acid adjuvants

Kaisho, T.

Vaccine Forum 2010 (Tokyo, Japan)

Sep-10Immune regulation mechanism and clinical application of alphagalactosylceramide

Ishii, Y.

Vaccine Forum 2011 (Tokyo, Japan)

Sep-10Now, what are the elements expected to cancer research? – Specificproposals for cancer research –Hirano, T.

69th Annual Meeting of the Japanese Cancer Association (Osaka, Japan)

Sep-10

iPS-derived NKT cells

Taniguchi, M.

40th Annual Meeting of the German Society for Immunology (Leipzig, Germany)

Sep-10Chemotherapy resistance of quiescent AML stem cells

Ishikawa, F.

72nd Annual Meeting of the Japan Society of Hematology (Yokohama, Japan)

Sep-10Control of immune response by regulatory dendritic cells

Sato, K.

72nd Annual Meeting of Japanese Society of Hematology (Yokohama, Japan)

Sep-10Lineage stability and plasticity of Foxp3+ regulatory T cells

Hori, S.

72nd Annual Meeting of Japanese Society of Hematology (Yokohama, Japan)

Sep-10

Revision of the relationship of blood cell lineages: insight into the generationof LGL upon dasatinib administration

Kawamoto, H.

Corporate Seminar, Annul Meeting of Japanese Society of Hematology(Yokohama, Japan)

Oct -10Modeling cellular signaling functions in breast cancer cells

Okada, M.

11th International Conference on Systems Biology (ICSB 2010) (Edinburgh, UK)

Oct -10The myeloid-based model: revised scheme for hematopoiesis

Kawamoto, H.

Network Medicine Seminar, Tohoku University (Sendai, Japan)

Oct -10Regulation CD4/CD8 lineage choice by transcriptional silencer in the thymus
and the gut

Taniuchi, I.

Seminar at CSI (CSI Research Meeting) (Singapore)

Oct -10Locus specific and non-specific regulation of silencers function

Taniuchi, I.

Aegean Sympo (5th International Conference on Gene Regulation in Lymphocyte
Development ) (Creta, Greece)

Oct -10RAPID: Resource of Asian Primary Immunodeficiency Diseases

Mohan, S.

Center for the Study of Immunodeficiencies at the Hospital Necker-Enfants
Malades (Paris, France)

Oct -10

Negative regulation of lymphocyte activation by LAPTM5-mediated TCR andBCR downmodulation

Wang, J. Y.

China Agricultural University (Beijing, China)

Oct -10

Zinc signaling in immunity, allergy and inflammation

Hirano, T.

52nd Annual Meeting of the Japanese Society of Gastroenterology (Yokohama,Japan)

Oct -10

From tolerance to symbiosis and back

Fagarasan, S.

Resistance and Tolerance to Infection (Mosteiro do Convento da Arrábida, Portugal)

Oct -10

Zinc is an intracellular signaling molecule: early and late signal

Hirano, T.

60th Fujihara Seminar Zinc Signaling and Cellular Functions (Osaka, Japan)

Oct -10The zinc transporter Zip13: its involvement in connective tissue development
and pathogenesis of mouse and human

Fukada, T.

61st Fujihara Seminar Zinc Signaling and Cellular Functions (Osaka, Japan)

Oct -10

Role of Zinc transporter, Znt5/Slc30a5 in mast cell mediated-allergic response

Nishida, K.

62nd Fujihara Seminar Zinc Signaling and Cellular Functions (Osaka, Japan)

Oct -10T cell lineage commitment regulated by the transcription factor Bcl11b

Ikawa, T.

114th Pediatric Hematologist-oncologist Workshop (Tokyo, Japan)

Nov -10

BCL6 Protein Expression Shapes Pre-Germinal Center B Cell Dynamics andFollicular Helper T Cell Heterogeneity

Okada T.

LIAI -RCAI Joint Workshop 2010 (Yokohama, Japan)

Nov -10

The adaptor molecule Gab2, via PI-3K, regulates ARF1 in Fc epsilon RImediated granule translocation and mast cell degranulation

Nishida, K.

LIAI -RCAI Joint Workshop 2010 (Yokohama, Japan)

Nov -10

CIN85 regulates canonical NF-kB pathway and subsequent B cell responses

Kurosaki, T.

The CSI(Chinese Society of Immunology)-iFReC Joint Symposium on Immunology(Hangzhou, China)

Nov -10Dynamic regulation of positive/negative T cell co-stimulation

Saito, T.

The CSI (Chinese Society of Immunology)-iFReC Joint Symposium on Immunology(Hangzhou, China)

Nov -10On IgA, vitamin A and gut homeostasis

Fagarasan, S.

Frontiers of Immunology in Health and Diseases (Cold Spring Harbor Asia, Suzhou,
China)

Nov -10

Molecular mechanisms for dendritic cell subset activation by nucleic acids-From mice to human-

Kaisho, T.

The Seminar of Hokkaido University Graduate School of Pharmaceutical Sciences(Sapporo. Japan)

Nov -10

Transcriptional Control of CD4/CD8 Lineage Choice

Taniuchi, I.

LIAI-RCAI Joint Workshop (Yokohama, Japan)

129

Nov -10

Retention and termination of myeloid potential in T progenitors: an essential
developmental checkpoint for production of the T cell lineage

Kawamoto, H.

LIAI-RCAI Joint Workshop (Yokohama, Japan)

Nov -10

Molecular mechanisms for dendritic cell responses against nucleic acidadjuvants

Kaisho, T.

LIAI-RCAI Joint Workshop (Yokohama, Japan)

Nov -10Involvement of zinc transporters in physiology and pathogenesis

Fukada, T.

2nd Metallomics Research Forum (Kyoto, Japan)

Nov -10Regulation of T cell activation: a study using molecular imaging

Yokosuka, T.

2nd Bio-frontier Seminar "Imaging analysis for the regulation of the immune
systems and diseases" (Tokyo, Japan)

Nov -10Creating novel and curative therapies for adult leukemia

Ishikawa, F.

Science Forum by AAAS (The American Association for the Advancement of
Science) (Yokohama, Japan)

Nov -10

Molecular mechanisms for dendritic cell subset functions

Kaisho, T.

60th Annual Meeting of Japanese Society of Allergology (Tokyo, Japan)

Nov -10Development of allergy vaccines for IgE antibody suppression

Ishii, Y.

13th Workshop of Japan Health Sciences Foundation (Tokyo, Japan)

Nov -10Regulation of immune responses by Foxp3+ regulatory T cells

Hori, S.

60th Annual Meeting of Japanese Society of Allergology (Tokyo, Japan)

Nov -10

Crucial roles of B7-H1 and B7-DC expressed on mesenteric lymph nodedendritic cells in the generation of antigen-specific CD4+Foxp3+regulatory T
cells in the establishment of oral tolerance

Sato, K.

60th Annual Meeting of Japanese Society of Allergology (Tokyo, Japan)

Nov -10

Food Allergy, Intestinal Immunity and Oral Tolerance

Ohno, H.

60th Annual Meeting of Japanese Society of Allergology (Tokyo, Japan)

Dec -10Specialized epithelial M cells contribute to immunosurveillance on themucosal surface

Hase, K.

2010 Symposium by Japan Soceity of Immunology (Tokyo, Japan)

Dec -10Regulation of lymphocyte dynamics during the immune response by thetranscription factor BCL6

Okada, T.

Biochemistry and Molecular Biology 2010 (Kobe, Japan)

Dec -10

IL-6 amplifier and four step model

Hirano, T.

3rd Saitama Research Meeting of Therapeutic Approch of Anti-interleukin 6(Saitama, Japan)

Dec -10

Zinc signaling in immunity, allergy and inflammation

Hirano, T.

26th Annual Meeting of the Japanese Society of Pediatric Oncology (Osaka, Japan)

Dec -10

Plasma cells differentiation from memory B cells

Kometani, K.

5th Chiba University Global COE Symposium (Tokyo, Japan)

Dec -10

Collaborative activity by academia, government and industries based onhuman DNA clones

Ohara, O.

Biochemistry and Molecular Biology 2010 Biotechnology Seminar (Kobe, Japan)

Dec -10

Molecular dynamics in living cells using single molecule imaging andquantification

Tokunaga, M. and
Sakata-Sogawa, K.

Biochemistry and Molecular Biology 2010 Biotechnology Seminar (Kobe, Japan)

Dec -10

Understanding normal and diseased human immunity using humanized mouse

Ishikawa, F.

2010 Symposium by Japan Soceity of Immunology (Tokyo, Japan)

Dec -10

Elucidation of the effectirvness of probiotics against lethal infection ofenterohemorrhagic E. coli O157:H7

Ohno, H.

2nd Danon Asian Probiotics Confention (Tokyo, Japan)

Dec -10How vitamin A impact on innate and adaptive immunity

Fagarasan, S.

Annual Meeting of the Japanese Society for Immunology (Tokyo, Japan)

Dec -10

Lineage restriction process from hematopoietic stem cells to T cell progenitors

Kawamoto, H.

Award Lecture for 13th JSI Award (Tokyo, Japan)

Dec -10

Biological roles of essential trace elements and the prospect for their clinicalapplications

Fukada, T.

2nd SPring-8 Next Plan 2019 Symposium (Tokyo, Japan)

Dec -10

Memory B cell generation and functional maturation; distinct cellular pathways
establish B cell memory

Takemori, T.

Development and Maintenance of Immune Memory (Tokyo, Japan)

Dec -10Essential Role of Membrane Trafficking Factor AP-1B in Gut Immune
Homeostasis

Ohno, H.

Biochemistry and Molecular Biology 2010 (Kobe, Japan)

Dec -10

M-Sec: a promoter of tunneling nanotube formation

Ohno, H.

Biochemistry and Molecular Biology 2010 (Kobe, Japan)

Dec -10

Molecular dynamics in living cells using single molecule imaging andquantification

Tokunaga, M. and
Sakata-Sogawa, K.

Biochemistry and Molecular Biology 2010 (Kobe, Japan)

Dec -10

Construction of an Informational Platform for Primary Immunodeficiency
Diseases in Asia

Mohan, S.

1st Asia-Pacific Symposium on Primary Immunodeficiency Diseases (Yokohama,Japan)

Dec -10

Immune regulation by dead cell clearance

Tanaka, M.

Biochemistry and Molecular Biology 2010 (Kobe, Japan)

Jan -11

iPS-Derived NKT Cells and their Adjuvant Effects

Taniguchi, M.

2011 Keystone Symposia on NK and NKT Cell Biology (Breckenridge, USA)Jan -11

Lineage restriction process from hematopoietic stem cells to T cellprogenitors: developmental checkpoints visualized by inducing arrest and self
renewal of progenitors

Kawamoto, H.

Stem Cell Therapy Forum, The Institute of Medical Science, Tokyo University(Tokyo, Japan)

Jan -11

Visualization of a developmental checkpoint by inducing arrest andselfrenewal of progenitors

Kawamoto, H.

Nihon University Stem Cell Biology Forum (Tokyo, Japan)

Jan -11

Dynamics of proteins in the nucleus by single molecule analysis

Tokunaga, M. and
Sakata-Sogawa, K.

International Symposium on the Physicochemical Field for Genetic Activities(Awaji, Japan)

Jan -11

Dual specificity receptor for damaged cells and pathogens

Saito, T.

US-Japan Cooperative Medical Science Program (USJCMSP), Adaptive and Innate
Immune Responses to Neglected Tropical Diseases (San Diego, USA)Jan -11

Molecular mechanisms for dendritic cell responses against nucleic acids

Kaisho, T.

1st symposium of Scientific Research on Innovative Areas, "Homeostatic
inflammation" (Tokyo, Japan)

Jan -11

Dynamic regulation of T-cell costimulation by TCR microclusters and thec-SMAC

Yokosuka, T.

13th International Membrane Research Forum/ The 6th iCeMS International
Symposium, Featuring Membrane Meso Mechanisms (Kyoto, Japan)

Feb -11

Dendritic cell subset responses against nucleic acid adjuvants

Kaisho, T.

29th Annual Meeting of Japan Society of Immunology and Allergology in
Otolaryngology (Oita, Japan)

Feb -11

RAPID: Resource of Asian Primary Immunodeficiency Diseases A road ahead

Mohan, S.

2nd Asia-Pacific symposium on Primary Immunodeficiency Diseases (Chiba,
Japan)

Feb -11

Visualization of a developmental checkpoint by inducing arrest andselfrenewal of progenitors

Kawamoto, H.

Special Seminar, Yamaguchi University (Yamaguchi, Japan)

Feb -11

Genetic factor determining allergic trait

Kubo, M.

20 th Molecular Immunology Forum Tokyo (Tokyo, Japan)

Feb -11

Molecular Mechanisms that regulate early T cell lineage commitment

Ikawa, T.

20th Molecular Immunology Forum Tokyo (Tokyo, Japan)

Mar -11

Transcriptional control of thymocytes development

Taniuchi, I.

TCUID (Towards Comprehensive Understanding of Immune Dynamism) 2011
(Osaka, Japan)

Mar -11Integrated omics approach identified acetate produced by probiotic
bifidobacteria to protect host from enteropathogenic infection

Fukuda, S and Ohno, H.

4th Stage Surface Barrier Immunology Study Group (SBARIS) 3rd Meeting (Tokyo,
Japan)Mar -11

Analysis on the influence of natural medicine on mucosal immune system

Sato, K.

Seminar on Collaborative Reserch in Institute of Natural Medicine, University of
Toyama (Toyama, Japan)

Mar -11

NKT cell-mediated adjuvant activity

Taniguchi, M.

World Immune regulation Meeting -V (Davos, Switzerland)

Mar -11

Interaction dynamics of B cells and T cells in the lymph node

Okada T.

115 th Annual Meeting of Japanese Association of Anatomists(Symposium 1S1-PM1) (Morioka, Japan)

Mar -11

Molecular dynamics for T cell activation and costimulation

Saito, T.

Keystone Symposia, Lymphocyte Activation and Gene Expression (Breckenridge,
CO, USA)Mar -11

Control of immune response by dendritic cells

Sato, K.

Seminar on Institute of Natural Medicine, University of Toyama (Toyama, Japan)

130

RCAI Seminars
Date

Speaker

Title

05-Apr-10

Ken Shirasu(RIKEN Plant Science Center, JAPAN)

Common immunity systems in plants and animals

11-Apr-10

Koichi Kobayashi(Dana-Farber Cancer Institute, Harvard Medical School, USA)

14-Apr-10

Shingo Iwami(PRESTO, JST, JAPAN)

Computational Virology and Immunology Based on in vivo / invitro Experiments

08-Jun-10

Kenji Nakanishi(Hyogo College of Medicine, JAPAN)

Induction of innate and acquired type allergy by basophils

30-Jun-10

Hajime Karasuyama(Tokyo Medical and Dental University, JAPAN)

Novel roles for basophils in allergy and protective immunity

15-Jul-10

Yosuke Takahama(Institute for Genome Research, The University of Tokushima,JAPAN)

Role of thymic cortex-specific self-peptides in positive
selection of T cells

28-Jul-10

Kazuhiro Aoki
(Complex Carbohydrate Research Center, University of Georgia, USA)

Regulation of protein N- and O-glycosylation in the Drosophilamelanogaster embryo by a Toll-like receptor

15-Aug-10

Jeffrey Browning(Biogen Idec, USA)

Exploration of the BAFF and Lymphotox in Pathways in Miceand Man

31-Aug-10

Dorina Avram(Center for Cell Biology and Cancer Research, Albany Medical
College, USA)

BCL11B controls glycolipid presentation by DP thymocytes
independent of the control of early iNKT precursors

05-Sep-10

Hidehiro Yamane(National Institute of Allergy & Infectious Diseases, NIH, USA)

Early Signaling Events Underlying the Fate Determination ofNaïve CD4+ T Cells towards Effector T Helper Subsets

05-Sep-10

Klaus Fruh
(Vaccine and Gene Therapy Institute, Oregon Health and Science
University, USA)

Viral Immune evasion

27-Sep-10

James Douglas Engel(University of Michigan Medical School, USA)GATA3 reguktaion of early T cell and hematopoietic progenitor
development

Recognition of microbial components by Nod2:The role in the
innate immunity and Crohn’s disease

131

132

27-Oct-10

Richard Kroczek(Robert Koch Institute, GERMANY)

Understanding the CD8+ T cells – DC communication axis: astarting point for a new type of vaccine?

31-Oct-10

Boris N. Kholodenko(University College Dublin, IRELAND)

Spatio-temporal coding of signal specificity and network
reconstruction

03-Nov-10

Ichiro Hirao(RIKEN Systems and Structural Biology Center, JAPAN)

Expansion of the genetic alphabet by unnatural base pairsystems

15-Nov-10

Reiko Onishi(University of Pittsburgh, USA)A unique role for IL-17RA in IL-17 signal transduction

05-Dec-10

Jennifer Stow
(Institute for Molecular Bioscience, The University of Queensland,
AUSTRALIA)

Recycling endosomes-compartments for secretion and cell
polarty

09-Dec-10

Randy L. Johnson(University of Texas, MD Anderson Cancer Center, USA)

Genetic analysis of the mammalian Hippo signaling pathway
in organ size control and tumor suppression

12-Dec-10

Esteban C.Dell’Angelica(University of California,Los Angeles, USA)

Protein Complexes Involved in the Biogenesis of
Lysosome-related Organelles

20-Dec-10

Florent Ginhoux(Singapore Immunology Network (SIgN), Agency for Science,
Technology and Research, SINGAPORE)

Revisiting the ontogeny of the mononuclear phagocyte
system:Embryonic versus adult hematopoiesis contribution

23-Dec-10

Michael Reth
(University of Freiburg, Max-Planck Institute of Immunobiology,
BIOSS Excellence Cluster, GERMANY )The silence of the B cells

26-Dec-10

Michael Reth
(University of Freiburg, Max-Planck Institute of Immunobiology,
BIOSS Excellence Cluster, Germany)

Synthetic biology programme in Freiburg

30-Jan-11

David Grunwald(Kavli Institute of Nano Science, Technical University Delft, The
NETHERLANDS)

Crossing the Gate: Export of Single mRNAs in vivo

27-Feb-11

Frederic Geissmann(Center for Molecular and Cellular Biology of inflammation (CMCBI),King’s College, UK )In vivo functions of monocytes

02-Mar-11

Lawrence E. Samelson(Center for Cancer Research, National Cancer Institute, NIH, USA)

Signaling at the T cell antigen receptor

Budget, Personnel and Patents

RCAI Budget FY2001-2010(JPY 100 Million)
RCAI Budget (JPY 100 Million)

Note: Budgets for FY2001-2003 include
construction expenses for RCAI facility

100 million yen

70

RCAI Budget (JPY 100 Million )

60

2001

41.74

50

2002

54.23

2003

60.48

2004

40.10

2005

39.02

2006

35.90

2007

34.56

10

2008

32.61

0

2009

31.86

2010

30.83

40
30
20

2002

2000

2004

2006

2008

2010

Fiscal Year

RCAI staff composition(as of Mar. 2011)

RCAI Personnel

RCAI Personnel

400

Category

Number350

Director

1

300

Senior Advisor

1

250Group Director

7Team Leader

14

Unit Leader

10

Coordinator

1

100

Senior Scientist

1250

Research Scientist

340Special Postdoctoral Researcher

8

Foreign Postdoctoral Researcher

1

Research Associate

7

Junior Research Associate

17International Program Associate

5Student Trainee

58
2

200
150

2000

2002

2004

2006
Fiscal Year

RCAI Personnel
2001

84

2002

156

2008

2010

2003

191

Research Fellow

2004

328

Research Consultant

2

2005

363

Visiting Scientist

79

2006

366

Senior Technical Scientist

1

2007

339

2008

326

2009

380

2010

400

RCAI Patents FY 2010 (as of Mar. 2011)

Technical Scientist

5Technical Staff I

35

Technical Staff II

65

Assistant21

Temporary Employment

8others

6

Total

400

There were 28 patents filed from
April 2010-March 2011.

133

Original Photos of the Cover and Front pages
Cover
An internal RCAI collaboration among the Laboratories for Immune
Regulation and Developmental Genetics and the Research Unit for
Cellular Immunotherapy demonstrated the feasibility of expanding
functionally competent NKT cells via an induced pluripotent stem
cell ( iPSC ) phase, an approach that may be adapted for NKT celltargeted therapy in humans ( Watarai et al., J Clin Invest, 2010 ).
( See Part 1 Creation of New Paradigms, p. 10).
A cluster of undifferentiated iPSCs derived from reprogrammed NKTs
highly express pluripotency markers Oct3/4 (left, green) and SSEA13 (right,
green). DAPI (red) labels the nuclei. (Image courtesy of the collaborative
groups.)Front page of Part 1 Creation of New Paradigms
The follicular dendritic cell (FDC) is a specialized stromal cell in the germinal
center. The Laboratory for Mucosal Immunity discovered that FDCs in the gut
sense bacterial and food components and secrete the B-cell chemoattractant
CXCL13 (Suzuki et al. Immunity, 2010 ). (See Part 1 Creation of New Paradigms, p. 3)
A cross-section of Peyer's patches showing B cell follicles (B220 stained in magenta)
built upon FDCs (CXCL13 stained in blue). Image courtesy of the Laboratory for Mucosal
Immunity

Front page of Part 2 Technology and Innovation
The transition from an early innate response to the adaptive immune response can beorchestrated by a special subset of B cells named B1 cells. The Laboratory for Mucosal
Immunity showed that B1 cells sense the retinoic acid (RA ) produced by peritoneum associated adipose tissues such as omentum, and activate the transcription factor NFATc1,
which is critical for the survival, proliferation and antibody production of B1 cells ( Maruya
et al. Proc. Natl. Acad. Sci. USA, 2011). ( See Part 6 Laboratory Activities, p. 74-75)
Aggregates of peritoneal B1 cells (stained for IgM (green)) in the omentum. Image courtesy of theLaboratory for Mucosal Immunity

Front page of Part 3 Nurturing Young Scientists
To elucidate the mechanism by which a candidate allergy vaccine mediates suppression of IgE antibody
responses, the Laboratory for Vaccine Design identified the target cells of the liposome vaccine. Splenic
B220+CD1d high cells in the marginal zone preferentially incorporate liposomes and express CXCL16,
which then recruits iNKT cells that express CXCR6, the receptor for CXCL16. ( See Part 6 Laboratory
Activities, p. 100-101)

iNKT cells (green) failed to traffic to liposome antigen-capturing B220+ cells (red) in the marginal zone when antiCXCL16 antibody was co-administrated with the liposomes. No Ab treatment (left), treated with anti-CXCL16 Ab (right).
Image courtesy of the Laboratory for Vaccine Design

134

DAPI

Sera

Merge

Front page of Part 4 Collaborative Networks
WT

Laptm5 -/-LAPTM5 is a lysosomal protein highly expressed in resting B
cells but down regulated in germinal center B cells. The Laboratory for Immune Diversity found that LAPTM5 negatively
regulates cell surface expression of the B cell antigen receptor
by promoting its lysosomal degradation. (See Part 6 Laboratory Activities, p. 68-69)
Increased levels of anti-nuclear autoantibodies (green) in the sera ofLaptm5-/- mice. Hep2 cells were stained with sera from WT (upper
panel) or Laptm5-/- mice (lower panel). Image courtesy of the Laboratory for Immune Diversity

Front page of Part 5 Outreach Activities
The Research Unit for Thymic Environment found that loss of thymic
crosstalk signaling leads to expansion of mesenchymal cells in the thymus (See Part 6 Laboratory Activities, p. 116-117).
In the thymus of fetal hCD3e transgenic mice, in which T cell development isblocked, the organization of thymic epithelial cells is disturbed, increasing mesenchymal cells detected by ERTR7 expression (green). Image courtesy of the
Research Unit for Thymic Environment

Front page of Part 6 Laboratory Activities
Using single molecule fluorescence microscopy, the Research
Unit for Single Molecule Imaging, in collaboration with the
Research Units for Molecular Systems Immunology, visualized the
dynamics of NF-κB and its suppressor IκBa. In resting cells, bothproteins were localized in the cytoplasm. Upon stimulation, degradation of IκBa caused translocation of NF-κB to the nucleus(See Part 6 Laboratory Activities, p. 64-65).
HeLa cells expressing both NF-κB-RFP (red) and IκBα-GFP (green) were
stimulated with human TNF-α. (A) Before stimulation. (B) 30min after stimulation. Scale bar: 20 μm. Image courtesy of the Research Unit for Single Molecule Imaging.

Front page of Part 7 Data and Statistics
The laboratory for Developmental Genetics identified afraction of tubulins that persistently associated with thenuclear lamina and exhibited a filamentous structure (SeePart 6 Laboratory Activities, p. 56-57)
Subnuclear colocalization of α (red) and β tubulin (green) with
Lamin-B (red) in mouse ES cells. Image courtesy of the Laboratory
for Developmental Genetics.