RIKEN Center for Integrative Medical Sciences
Organization Chart

RIKEN President
Ryoji Noyori

Director (Acting)
Shigeo Koyasu

Deputy Director
Michiaki Kubo
Takashi Saito

Integrative Medical Sciences
Planning OfficeCore for Homeostatic
Regulation

Haruhiko Koseki
Toshihiro Tanaka

Core for Precise Measuringand ModelingLab. for Cell Signaling: Takashi Saito

Lab. for Developmental Genetics: Haruhiko KosekiLab. for Lymphocyte Differentiation: Tomohiro Kurosaki

Lab. for Integrative Genomics: Osamu Ohara

Lab. for Transcriptional Regulation: Ichiro Taniuchi

Lab. for Disease Systems Modeling: Hiroaki KitanoLab. for Immune Cell System: Shigeo Koyasu

Lab. for Cell Functional Imaging: Shigeo Koyasu

Lab. for Human Disease Models: Fumihiko Ishikawa

Lab. for Immunogenetics: Hisahiro YoshidaLab. for Intestinal Ecosystem: Hiroshi Ohno

Lab. for Integrated Bioinformatics: Todd Duane Taylor

Lab. for Mucosal Immunity: Sidonia FagarasanLab. for Tissue Dynamics: Takaharu Okada

Lab. for Gut Homeostasis: Kenya Honda

Lab. for Integrated Cellular Systems: Mariko Okada

Lab. for Immune Homeostasis: Shohei HoriLab. for Molecular Live-Cell Quantification: Makio Tokunaga

Lab. for Skin Homeostasis: Masayuki Amagai

Lab. for Metabolomics: Makoto Arita

Lab. for Metabolic Homeostasis: Naoto Kubota
Lab. for Immune Crosstalk: Hilde Cheroutre
Lab. for Inflammatory Regulation: Takashi Tanaka
Lab. for Cytokine Regulation: Masato Kubo
Lab. for Homeostatic Network: Shigeo Koyasu

RIKEN Center for Integrative
Medical Sciences Advisory Council
Max Cooper (chair)Peter Sorger

Mark Lathrop (vice chair)

Rudi Balling

Ronald N. Germain

Kiyoshi Takatsu

Paul W. Kincade

Hajime Karasuyama

Bernard Malissen

Yutaka Kawakami

William E. Paul

Michel Georges

Dale Umetsu

Edison Tak-Bun Liu

Arthur Weiss

Katsushi Tokunaga

Masaru Taniguchi

John O’Shea

Hiroyuki Aburatani

Shizuo Akira

Fiona Powrie

Senior Advisor

Core for Genomic Medicine

Program for Medical
Innovations

Lab. for Genotyping Development: Michiaki Kubo

Lab. for Immune Regulation: Masaru Taniguchi

Lab. for Genome Sequencing Analysis: Hidewaki Nakagawa

Lab. for Immunotherapy: Shin-ichiro Fujii

Lab. for Medical Science Mathematics: Tatsuhiko Tsunoda

Lab. for Vaccine Design: Yasuyuki Ishii

Lab. for Statistical Analysis: Atsushi Takahashi

Lab. for Allergic Disease: Toshiaki KawakamiLab. for Pharmacogenomics: Taisei Mushiroda

RIKEN-TORII Joint Research Team: Masaru Taniguchi

Lab. for International Alliance on Genomic Research:

Drug Discovery Antibody Platform Unit: Toshitada Takemori

Ming Ta Michael Lee
Lab. for Cardiovascular Diseases: Toshihiro Tanaka
Lab. for Autoimmune Diseases: Kazuhiko Yamamoto
Lab. for Digestive Diseases: Kazuaki Chayama
Lab. for Bone and Joint Diseases: Shiro Ikegawa
Lab. for Endocrinology, Metabolism and Kidney Diseases:

Shiro Maeda
Lab. for Respiratory and Allergic Diseases: Mayumi TamariiContents

Organization ........................................................................... iContents .................................................................................. ii

Director’s Report .................................................................... iv

Part 1Lab Activities
Sections
Core for Homeostatic Regulation .................................. 2
Core for Precise Measuring and Modeling ................... 2
Core for Genomic Medicine ........................................... 3
Program for Medical Innovations ................................. 3
Lab Activities
Laboratory for Cell Signaling ......................................... 4
Laboratory for Lymphocyte Differentiation ................. 5
Laboratory for Transcriptional Regulation ................... 6
Laboratory for Immune Cell System ............................. 7
Laboratory for Human Disease Models ........................ 8
Laboratory for Intestinal Ecosystem ............................. 9Laboratory for Mucosal Immunity ................................ 10
Laboratory for Gut Homeostasis ................................... 11
Laboratory for Immune Homeostasis ........................... 12
Laboratory for Skin Homeostasis .................................. 13
Laboratory for Metabolic Homeostasis ......................... 14
Laboratory for Immune Crosstalk ................................. 15
Laboratory for Inflammatory Regulation ..................... 16
Laboratory for Cytokine Regulation ............................. 17
Laboratory for Homeostatic Network ........................... 18
Laboratory for Developmental Genetics ....................... 19
Laboratory for Integrative Genomics ............................ 20
Laboratory for Disease Systems Modeling ................... 21
Laboratory for Immunogenetics .................................... 22
Laboratory for Integrated Bioinformatics ..................... 23
Laboratory for Tissue Dynamics ................................... 24
Laboratory for Integrated Cellular Systems .................. 25
Laboratory for Molecular Live-Cell Quantification .... 26
Laboratory for Genotyping Development .................... 27
Laboratory for Genome Sequencing Analysis .............. 28
Laboratory for Medical Science Mathematics .............. 29
Laboratory for Statistical Analysis ................................. 30
Laboratory for Pharmacogenomics ............................... 31
Laboratory for International Alliance onii

Genomic Research ..................................................... 32
Laboratory for Cardiovascular Diseases ....................... 33
Laboratory for Autoimmune Diseases .......................... 34
Laboratory for Digestive Diseases ................................. 35
Laboratory for Bone and Joint Diseases ....................... 36
Laboratory for Endocrinology, Metabolism andKidney Diseases ......................................................... 37
Laboratory for Respiratory and Allergic Diseases ....... 38
Laboratory for Immune Regulation .............................. 39
Laboratory for Immunotherapy ..................................... 40
Laboratory for Vaccine Design ...................................... 41
Laboratory for Allergic Disease ..................................... 42
RIKEN-TORII Joint Research Team ............................. 43
Drug Discovery Antibody Platform Unit ..................... 44
Central Facilities
Facs Lab ............................................................................ 45
Confocal Lab .................................................................... 45
Genomics Lab .................................................................. 46
Animal Facility ................................................................. 46
Young Chief Investigator Program.......................... 47
YCI Laboratory for Mathematical Modeling of
Immune System ......................................................... 47
YCI Laboratory for Stem Cell Competency ................. 48
YCI Laboratory for Immune Regeneration .................. 48
YCI Laboratory for Integrative Genomics .................... 49
YCI Laboratory for Cellular Bioenergetic Network .... 49
Other Programs
RIKEN International Program Associate (IPA)............ 50
RIKEN Foreign Postdoctoral Researcher (FPR)
Program....................................................................... 50
RIKEN Junior Research Associate (JRA) Program...... 50
RIKEN Special Postdoctoral Researcher (SPDR)
Program....................................................................... 50
Award Winners ................................................................ 51

Part 2
Research Projects
Research highlights
Intestinal Bacteria Show ‘Community Spirit’ ............... 54
Friends in Low Places Preserve Gut Health ................. 55
Rheumatoid Arthritis Research Shows the Potentialof Large-scale Genetic Studies for Drug
Discovery .................................................................... 56
Connecting the Genetic Dots in Connective Tissue
Disorders .................................................................... 57
Getting Ahead of the Curve ........................................... 58
Compound that Could Prevent Acute Blood Cancer
Relapse Identified ...................................................... 59
Genetic Variations May Help Identify Best
Candidates for Preventive Breast Cancer Drugs .... 60
Drug Treatment Improves Survival of InsulinProducingcells ............................................................ 61
Fighting Asthma Drug Resistance ................................. 62
Rapid Recall to Fight Familiar Foes .............................. 63
Helping Cells Make a Commitment .............................. 64
Building Blocks Help Silence Genes .............................. 65

Projects
iPS Project ........................................................................ 66
Identification of Novel Causative Genes for Common
Variable Immunodeficiency .................................... 66
Modeling Skin Diseases .................................................. 67
Collaboration with the SardiNIA Project ..................... 67
Linkage to RIKEN Program for Drug Discovery andMedical Technology Platforms (DMP).................... 68
Humanized Mouse Research .......................................... 68
NKT Project ..................................................................... 69
Allergy Project ................................................................. 69
Influenza Project .............................................................. 70
PGRN-CGM International Collaborative Studies ....... 70
Collaboration with Asian Institutes and SEAPharm ... 71
International Cancer Genome Consortium (ICGC).... 71
Commissioned Research
The BioBank Japan Project ............................................. 72
Genome-guided drug Therapy Optimization Project(G-TOP) ..................................................................... 72

Part 3
EventsRIKEN IMS Summer Program (RISP) 2013 ...................... 74
The 8th IMS-JSI International Symposium on
Immunology 2013 ........................................................... 74
The 8th PGRN-RIKEN Strategic Alliance Meeting ........... 75

LJI & IMS Workshop ............................................................. 75
Harvard Summer School 2013 ............................................. 76
Adjunct Professorship Programs ......................................... 76

Part 4
Data and Statistics
Publications ............................................................................ 78
Guest seminars ....................................................................... 86

Budget, Personnel and Patents ............................................. 87iii

Director’s ReportIn 2012, RIKEN drafted a complete reorganization plan for the
RIKEN’s third 5-year term (2013-2018). The aim of the reorganization was to promote science and technology that leads to innovation, and leads to the discovery of solutions for critical scientific,
technical and social issues, while still pursuing basic research.
The RIKEN Center for Integrative Medical Sciences (IMS) was
newly established in April, 2013 by combining two existing research centers, RIKEN Research Center for Allergy and Immunology (RCAI 2001-2013) and RIKEN Center for Genomic Medicine(CGM 2008-2013). RCAI was established in 2001 as the first large
research institute dedicated to allergy and immunology in Japan thatis fully supported by the Japanese Government. CGM was established in 2008 by reorganization of RIKEN SNPs Research Center(SRC 2000-2008). SRC was a part of the Millennium Genome Projects initiated by the former Prime Minister, the late Keizo Obuchi. SRC successfully developed an accurate and high-throughput
system for SNP analysis (genome-wide association study; GWAS).
To accomplish the mission of discovering genetic elements that mediate human diseases and therapeutic effects and adverse reactionsof various drugs, CGM conducted GWAS studies using more than
200,000 patients’ samples for 47 diseases that were stocked in the
BioBank Japan (BBJ).
Both CGM and RCAI have been recognized worldwide as distinguished institutes with a large number of influential results ineach field. CGM’s approaches have uncovered the corelations between many human genetic polymorphisms and diseases. RCAI’s
approaches were extremely effective in elucidating molecular mechanisms by which certain genes regulate immune reactions using
various gene-manipulated animal models. To understand the molecular mechanisms of human disease onset that are caused by genealterations, we believed that amalgamation of these two approaches
would be quite effective. Based on the history and achievementsof two research centers, IMS integrates both mechanistic researchiv

developed in immunology and human genome research and transcends the existing scientific boundaries. IMS will also employ systems approaches and mathematical modeling expertise to create a
new research area “integrative medical sciences.”
The goal of IMS is to understand the robust system that maintains the homeostatic regulation of our body and to elucidate themechanisms of disease onset as a consequence of malfunction of
homeostasis. Homeostasis is regulated by robust gene networks thatorchestrate the functions of cells, tissues and organs and the interplay between organs. The regulation of gene networks is modified
by environmental factors affecting epigenetic modifications. Alteration of gene networks and interplay between cells and organs is
induced by a combination of genetic factors and environmental factors and causes diseases if the degree of alteration proceeds beyonda certain threshold. We thus focus on the interplay of disease-related genes and environment factors, such as commensal microbiota
affecting epigenetic alterations.
Elucidation of molecular mechanisms of disease onset by focusing on disease-related genes will uncover the target genes or target
gene networks important for the prevention of diseases. Personal
genomic information is also pivotal for the prediction of disease onset and avoidance of the adverse effect of certain drugs. Thus, IMS
aims to elucidate how homeostasis is maintained and how its disruption initiates disease based on our knowledge of human genome
research, which defines human genetic and epigenetic diversity andconsequent disease susceptibility. IMS will contribute to the development of personalized preventive medicine and therapeutics.
IMS builds three cores and one program, Core for Homeostatic Regulation, Core for Precise Measuring and Modeling, Core for
Genomic Medicine and Program for Medical Innovations. These
divisions collaborate with each other and create platforms for 1) human genome analysis, 2) big data analysis and mathematical modeling, 3) disease models (e.g. humanized mice, human iPS cells), and
4) functional analysis using immunology and molecular and cellular biology approaches. These platforms will enable IMS to elucidate
how the genetic and environmental alterations affect homeostasis.
By understanding the disease onset mechanisms, IMS will apply
our discoveries to predictive/preventive medicine and personalized
medicine, and thus contribute to innovative medical science for the
next generation.

Shigeo Koyasu
Director (Acting)
RIKEN Center for Integrative Medical Sciences (IMS)Part 1Lab Activities

Core for Homeostatic RegulationT

he ultimate goal of the Core for Homeostatic Regulation is toelucidate the mechanisms of onset of human diseases and tocreate new scientific paradigms. This Core clarifies the regulation of
homeostasis in individuals, focusing on the immune, metabolism
and environmental response systems. In addition, the Core for Homeostatic Regulation will validate the disease models established by
the Core for Precise Measuring and Modeling in a multi-tier timeframe from before to after the onset of diseases.
The Core for Homeostatic Regulation is composed of 15 laboratories divided into four areas;[1] Immune homeostasis: Cell signaling, Lymphocyte differentiation, Immune homeostasis and Metabolic homeostasis[2] Lymphocyte development, Transcriptional regulation and
Human disease model[3] Mucosal immunity: Intestinal ecosystem, Mucosal immunity,
Immune cell systems, Gut homeostasis and Immune crosstalk[4] Allergy and inflammation: Skin homeostasis, Inflammatory
regulation and Cytokine regulation

Sections

These areas elucidate the basic mechanism of immune regulation
at cellular, tissue and systemic levels. We eventually aim to analyze
the onset of autoimmune diseases, metabolic disorders [1], primary
immunodeficiency [2], inflammatory bowel disease and colitis [3],and atopic dermatitis and allergic diseases [4].

Figure: Induction of colonic regulatory T cells by fatty acids derived from gut
microbiota.
Intestinal homeostasis is regulated by the interaction between gut microbiota and intestinal cells. Systemic analysis of the interaction elucidates the intestinal homeostasis
and onset of mucosal diseases.

Core for Precise Measuring and ModelingT

he ultimate goal of the Core for Precise Measuring and Modeling is to elucidate the mechanisms that govern human disease onset. This will be accomplished through collection of a wide
variety of quantitative data to build a computational and predictive
network of the process. Toward this end, this core must intensively
collaborate with the Cores of Homeostatic Regulation and Genome
Medicine as well as the program for Medical Innovations at IMS.
Precise quantitative measurements are done for profiling of mRNA,
protein (Laboratory for Integrative Genomics), and metabolites(Laboratory for Metabolomics) in a genome-wide manner and also
for more dynamic properties of the biological systems by in vivo/in vitro bio-imaging (Laboratories for Tissue Dynamics, Molecular
Live-Cell Quantification, and Cell Functional Imaging). The production of genetically engineered mice is another indispensable research activity in this core (Laboratories for Developmental Genetics and Immunogenetics). After being processed by bioinformatics(Laboratory for Integrated Bioinformatics), the datasets are used
for modeling (Laboratories for Disease Systems Modeling and In-

Figure: “Data-Model-Simulation-Validation (DMSV)” Cycle
A practical goal of the Core for Precise Measuring and Modeling is to implement the“DMSV” cycle in the IMS project in close collaboration with other cores at IMS.

2

tegrated Cellular Systems). In particular, the Laboratory for Disease
Systems Modeling plays a main role in systems biological researchand software platform development at IMS. Because we have to derive a “Data-Model-Simulation-Validation” cycle to reach the ultimate goal, intra-core collaboration is essential. As the first leading
project, the core is fully involved in the atopic dermatitis project at
IMS. Needless to say, each laboratory in the core is also doing theirown cutting-edge research to be prepared to address intriguing but
unexplored questions in medical sciences.

Core for Genomic Medicine

T

he Core for Genomic Medicine is performing genomic research on human diseases, especially the common diseases.
The aims of the Core for Genomic Medicine are 1) to identify genetic variations related to disease susceptibility, outcome and drug
responses (efficacy/adverse reaction), 2) to provide useful information about possible molecular targets for drug discovery, 3) to examine the interactions between genetic and environmental factors
to understand the pathogenesis and the progression of diseases, 4)
finally to construct the evidence base for the implementation of personalized medicine.
To identify genetic variations related to disease susceptibility
and drug responses, the Core for Genomic Medicine first showed
the proof of concept of the genome-wide association study (GWAS)
in 2002. To advance this strategy, the Core for Genomic Medicine
has organized laboratories to facilitate comprehensive genomic research on common diseases. To produce comprehensive genomic
information, the Laboratory for Genotyping Development is mainly
working on large-scale SNP genotyping for GWAS and the Laboratory for Genome Sequencing Analysis is mainly working on wholegenome sequencing of cancer genomes. To analyze large genomic
variation data from many samples, the Laboratory for Statistical
Analysis is mainly working on the analysis of GWAS data and theLaboratory for Medical Science Mathematics is mainly working on

Sections

the analysis of cancer genome sequencing data. These laboratories
are in close communication with each other to provide high quality
genomic information analysis results to the research group of pharmacogenomics (Laboratory for Pharmacogenomics and Laboratory
for International Alliance on Genomic Research), laboratories fordisease-causing mechanisms (Laboratory for Cardiovascular Diseases, Autoimmune Diseases, Digestive Diseases, Bone and Joint
Diseases, Endocrinology, Metabolism and Kidney Diseases, andRespiratory and Allergic Diseases) and many other collaborators
worldwide for further analysis.

Figure: Twelve laboratories tackle the most advanced research
The Core for Genomic Medicine consists of four research groups and eight researchteams. Each team is linked systematically with the others and works toward theimplementation of personalized medicine.

Program for Medical Innovations

Seven original projects for clinical applications have been conducted in 2013: 1) A biochemical drug using a PEGylated Cryj1/2 fusion recombinant protein for Cedar Pollinosis has been underdevelopment by the Torii pharmaceutical company and IMS. 2) A
chemical compound recently developed by using the α-galactosylceramide (α-GalCer) analog, RCAI-X, selectively induces apoptosis of IgE but not IgG B cells and preferentially suppresses IgE
production. It therefore potentially could be applied to any type ofallergic disorder, such as pollenosis, food allergy, as well as allergic asthma. 3) NKT cell-targeted therapy for head and neck tumors
has been conducted in collaboration with Professor Okamoto in
Chiba University, and was authorized by the Japanese governmentas Advanced Medical Treatment B in 2013. 4) NKT cell-targeted
therapy for stage IIA/IIB lung cancers after surgical resection has
been conducted in collaboration with National Hospital Organizations. 5) The artificial adjuvant vector cell as an anti-tumor vaccine
has been developed. This vaccine can be dosed with tumor antigen

mRNA together with α-GalCer, activates both innate and acquired
protective immunity, and induces long-term memory of more than
one year in mice and dogs following a single administration. 6) The
human iPS project for clinical use of in vitro generated NKT cells
was accepted as the Center for Clinical Application Research (TypeB) in the Research Center Network for Realization of Regenerative
Medicine, Japan in 2013. 7) By using humanized mice, leukemia
stem cells have been identified, and a drug candidate for effective
treatment of leukemia has been developed.

Figure: Human iPS project on cancer
The project is accepted as the Center for Clinical Application Research (Type B) in
the Research Center Network for Realization of Regenerative Medicine, Japan.

3

Laboratory for

Lab activities

Cell Signaling
Group Director : Takashi Saito

Figure: Regulation of generation of inflammatory T cells by Bach2.
Bach2 suppresses the expression of genes required
to differentiate into Th2 and memory T cells, andmaintains the naïve status of T cells. Bach2-deficient T cells express genes related to inflammatory/
memory T cells, and re-expression of Bach2 resulted in a return to the phenotype of naïve T cells.

Recent Major Publications
Yokosuka T, Takamatsu M, Kobayashi-Imanishi W,
Hashimoto-Tane A, Azuma M, Saito T. Programmed cell
death-1 forms negative costimulatory microclusters
that directly inhibit T cell receptor signals by recruitment of phosphatase SHP2. J Exp Med 209, 1201-17(2012)
Tsukumo S, Unno M, Muto A, Takeuchi A, Kometani K,
Kurosaki T, Igarashi K, Saito T. Bach2 maintains T cells
in naïve state by suppressing effector memory-related
genes. Proc Natl Acad Sci 110, 10735-40 (2013)
Liang Y, Cucchetti M, Roncagalli R, Yokosuka T, Malzac
A, Bertosio E, Imbert J, Nijman IJ, Suchanek M, Saito T,
Wulfing C, Malissen B, Malissen M. Rltpr, a lymphoidlineage-specific actin-uncapping protein is essential for
CD28 co-stimulation and regulatory T cell development.
Nat Immunol 14, 858-66 (2013)

Invited Presentations
Saito T. Dynamic regulation and modulation of T cell
activation. Immunology 2013: AAI Annual Meeting,
Honolulu, USA. May 4th, 2013.
Saito T. Molecular dynamics for T cell activation andco-stimulation. Frontiers in Immunology Conference,
Tokyo, Japan. August 2nd, 2013.
Saito T. Dynamic regulation of T cell activation andco-stimulation. 4th International Symposium: Regulators of Adaptive Immunity, Erlangen, Germany. September 28th, 2013.
Saito T. Dynamic regulation of T cell activation andco-stimulation. 43rd Annual Scientific Meeting of theAustraliasian Society for Immunology (ASI ASM 2013),
Wellington, New Zealand. December 2nd, 2013.
Saito T. Nucleic acids sensing by T cells initiates Th2 celldifferentiation. Germany-Japan Immunology Seminar
2013, Shizuoka, Japan. December 7th, 2013.

4W

e have analyzed the mechanism and regulation of T cell activation anddifferentiation by analyzing the function of genes and signaling molecules
whose expression is up-regulated during, or which regulate T cell activation anddifferentiation. Analyzing the genes whose expression is altered during T cell development, we identified Bach2. Bach-2 is transcriptional repressor and has beenthought to be specific for B cells, but we found it is expressed equally well in T cells
and to increase its expression during T cell maturation. Bach-2 deletion in T cells
reduces naïve T cell numbers and increases memory-type T cells. The Bach2-/naïve T cells highly express genes related to effector memory T cells, and rapidly
produce Th2 cytokines upon T cell stimulation. These findings indicate that Bach2
suppresses effector memory-related genes to maintain the naive T-cell state andregulates generation of effector-memory T cells (Fig.).
We have analyzed the functional contribution of innate-related signals for T
cell activation and function, particularly the function of TLRs expressed on T
cells. We found that effector Th1 but not Th2 cells were directly activated through
TLR2 for IFNγ production. Nucleic acids also induce T cell co-stimulation for cytokine production and proliferation, a process that is independent of TLR/RLRs.
Unlike innate cells, not only CpG but also other forms of DNA could induce T cell
co-stimulation, particularly when complexed with LL37 or histones. Nucleic acid-mediated T cell co-stimulation is induced by an as yet-unknown unique sensor
and, physiologically, DNA released from dead cells at inflammation/infection sites
induces Th2 differentiation by inducing IL-4 in naïve T cells, triggering allergic
responses.

Laboratory for

Lab activities

Lymphocyte
Differentiation
Group Director : Tomohiro Kurosaki

Figure: Bcl6 activation mechanisms in naive versus memory T cells
During primary immune responses, naïve T cells areprimed by antigen-presenting dendritic cells anddifferentiate into effector cells. Some of the activatedT cells up-regulate Bcl6 expression. The Bcl6+ cells
express the CXCR5 chemokine receptor, which leads
them to the T-B border where cognate interactions
between Bcl6+ T cells and antigen-specific B cells occur.
In contrast to primary responses, memory TFH cells areactivated directly by antigen-presenting memory B cells
and quickly gain high levels of Bcl6 expression and redifferentiate into effector TFH cells, which contribute toactivation of memory B cells.

Recent Major Publications
Kometani K, Nakagawa R, Shinnakasu R, Kaji T,
Rybouchkin A, Moriyama S, Furukawa K, Koseki H,
Takemori T, Kurosaki T. Repression of the Transcription
Factor Bach2 Contributes to Predisposition of IgG1
Memory B Cells toward Plasma Cell Differentiation.
Immunity 39, 136-47 (2013)
Sasaki Y, Sano S, Nakahara M, Murata S, Kometani K,
Aiba Y, Sakamoto S, Watanabe Y, Tanaka K, Kurosaki
T, Iwai K. Defective immune responses in mice lacking
LUBAC-mediated linear ubiquitination in B cells. EMBO
J 32, 2463-76 (2013)
Capietto AH, Kim S, Sanford DE, Liehan DC, Hikida M,
Kurosaki T, Novack DV, Faccio R. Downregulation of
PLCγ2/β-catenin pathway promotes activation and expansion of myeloid-derived suppressor cells in cancer. J
Exp Med (in press)

Invited Presentations
Kurosaki T. Calcium Signaling in B Lymphocytes. 5thInternational Conference on B cell and Autoimmunity,
Como, Italy. August 20th, 2013.
Kurosaki T. Calcium Signaling in B Lymphocytes. Immune-related Pathologies: Understanding Leukocyte
Signaling and Emerging therapies (IMPULSE 2013),
Matrahaza, Hungary. September 2nd, 2013.
Kurosaki T. B cell intrinsic and extrinsic mechanisms
for rapid responsiveness of IgG1 type memory B cells.
Oversea Scholar Seminar, Tsinghua University, Beijing,
China. November 14th, 2013.
Kurosaki T, Kometani K, Ise W, Shinnakasu R. B cell
intrinsic and extrinsic mechanisms for rapid responsiveness of IgG1 type memory B cells. The 36th Annual
Meeting of the Molecular Biology Society of Japan,
Kobe, Japan. December 5th, 2013.
Kurosaki T. Calcium Signaling in B Lymphocytes. Keystone Symposia: Biology of B Cell Responses, Keystone,
USA. February 11th, 2014.

M

emory humoral responses are typically more rapid, have a greater magnitude, andconsist of antibodies of higher affinity than in the primary response. Our laboratory has now focused on clarifying the functional characterization of memory B cells andmemory T cells and on revealing the mechanisms underlying the robustness of memory
antibody responses.
Characterization of memory TFH cells
CD4 T cells are critical for memory B cell generation and their subsequent activation. Previously, we demonstrated that CXCR5+ CD4 T cells reside nearby the IgG1 memory B cells
in the follicles (Aiba et al, PNAS, 2010) at the memory phase. Based on these results, we hypothesized that a fraction of antigen-specific effector T cells, especially TFH cells, survives
over the contraction phase and becomes memory CXCR5+ TFH cells, which participate inrecall antibody responses. Thus, by using the transgenic TCR model, we first verified ouridea. In addition, our data suggest that memory B cells function as APCs to rapidly induce
Bcl6 in TFH memory T cells, thereby contributing to robust humoral memory responses.
Regulation of Bach2 during immune responses
We previously proposed that reorganization of transcription factors (for instance, repression of Bach2) takes place during generation of IgG1 type memory B cells after primary antigen exposure, and is critical for rapid responsiveness of IgG1 memory B cells(BCR-extrinsic model) (Kometani et al, Immunity, 2013). To directly test this model, we
have established mice in which Bach2 could be overexpressed or knocked out in an inducible manner. When Bach2 was overexpressed just before secondary responses, IgG1 type
memory B cells became skewed toward generation of GC B cells rather than differentiating
into plasma cells. Conversely, deletion of Bach2 promoted preferential differentiation into
plasma cells. These results clearly suggest that the expression level of Bach2 is involved in
the functions of memory B cells.

5Laboratory for

Lab activities

Transcriptional
Regulation
Group Director : Ichiro Taniuchi

Figure: Toward understanding of mechanismsthat couple TCR signals with cell fate decision
T lymphocytes sense environmental cues via the
TCR. This information needs to be appropriately
integrated into developmental programs that
control cell fate. Studies in my laboratory focus on
regulatory mechanisms that link environmental
cues with cell fate determination.

Recent Major Publications
Seo W, Ikawa T, Kawamoto H, Taniuchi I. Runx1/Cbfβ isessential for early B lymphocyte development throughregulation of Ebf1 expression. J Exp Med 209, 1255-62(2012)
Tanaka H, Naito T, Muroi S, Seo W, Chihara R, Miyamoto
C, Kominami R and Taniuchi I. Epigenetic Thpok silencing limits the time window to choose CD4+ helper-lineage fate in the thymus. EMBO J 32, 1183-94 (2013)
Mucida D, Husain M.M, Muroi S, van Wijk F, Shinnakasu
R, Naoe Y, Reis B, Huang Y, Lambolez F, Docherty M, Attinger A, Shui J.W, Kim G, Lena C, Sakaguchi S, Miyamoto C, Wang P, Atarashi K, Park Y, Nakayama T, Honda K,
Ellmeier W, Kronenberg M, Taniuchi I* and Cheroutre
H*. Transcriptional Reprogramming of Mature CD4 T
helper Cells generates distinct MHC class II restricted Cytotoxic T Lymphocytes. Nat Immunol 14, 281-9 (2013)*corresponding authors

Invited Presentations
Taniuchi I. Local Chromatin Loop in CD4/CD8 lineage
Choice. 6th International Workshop of Kyoto T Cell Conference (KTCC2013), Kyoto, Japan. June 5th, 2013.
Taniuchi I. Reconstitution approach to understandregulatory mechanisms of the Zbtb7b and Cd8 gene
expression. 4th Synthetic Immunology Workshop, Kyoto,
Japan. November 16th, 2013.
Taniuchi I. Runx transcription factors and inflammation.
The 63rd Annual Meeting of Japanese Society of Allergology, Tokyo, Japan. November 28th, 2013.

6O

ne of major questions in developmental biology is how extracellular information is sensed by interface receptors and is integrated into a developmental program encoded by the genome. Besides such a profound question, current
advances in our understanding of functional modulation of effector T cell subsets
in different environments raised the challenging question of how retention andloss of developmental plasticity is counterbalanced during the initial commitment
process. Research in my laboratory has been addressing these important issues
by studying mechanisms that control helper versus cytotoxic T cell differentiation in the thymus as well as in the periphery. In particularly, we have focused on
antagonistic interplay between two transcription factors, ThPOK and Runx. Our
research goal is to advance our understanding of how external information, recognized by TCR in this case, is integrated into transcriptional and epigenetic controlof these factors’ expression in the initial lineage selection process in the thymus,
and how peripheral environmental cues modulate their expression.
Our second objective is to unravel functions of Runx transcription factor complexes that act as heterodimers of Runx proteins and the non-DNA binding Cbfb
protein. Runx complexes are evolutionally conserved and play multiple and central roles to control differentiation and function of many types of hematopoietic cells. Our goal is to reveal regulatory mechanisms that modulate function ofRunx complexes as well as to provide insights into how Runx complexes regulate
immune system development and immune responses. We are addressing thesequestions mainly by analyzing a series of mutant mouse strains harboring specificmutations in the Runx family genes and by identification and characterization ofRunx interacting molecules including functional RNAs.

Laboratory for

Lab activities

Immune Cell System
Group Director : Shigeo Koyasu

Figure: Pimozide attenuates corticosteroid resistance in a severe asthma model.
Mice were treated with IL-33 and TSLP for 3 days toinduce asthmatic airway inflammation (left). Dexamethasone, a typical steroid used for treating asthma(middle), or a combination of dexamethasone andpimozide (right) were administered at 24 and 1 hr
before IL-33+TSLP treatments. Shown are sections of
the airways stained periodic acid–Schiff (PAS)-alcian
blue to examine mucin secretion. It can be seen thatdexamethasone alone is insufficient to suppress the
airway inflammation characterized by thickening of airway epithelia and mucin secretion shown in violet (leftand middle) but a combination of dexamethasone andpimozide effectively suppressed such asthmatic airway
inflammation (right).

Recent Major Publications
Koyasu S, Moro K. Th2-type innate immune responses
mediated by natural helper cells. Ann N Y Acad Sci
1283, 43-9 (2013)
Furusawa J, Moro K, Motomura Y, Okamoto K, Zhu J,
Takayanagi H, Kubo M, Koyasu S. Critical role of p38 and
GATA3 in natural helper cell function. J Immunol 191,
1818-26 (2013)
Kabata H, Moro K, Fukunaga K, Suzuki Y, Miyata J,
Masaki K, Betsuyaku T, Koyasu S, Asano K. Thymic
stromal lymphopoietin induces corticosteroid resistancein natural helper cells during airway inflammation. Nat
Commun 4, 2675 (2013)

Invited Presentations
Koyasu S. Role of natural helper cell, a member for thegroup 2 ILC (ILC2), in steroid resistance of allergic inflammation. Keystone Symposia: Advances in the knowledge
and treatment of autoimmunity, British Columbia, Canada. April 8th, 2013.
Koyasu S. Natural helper cells and type-2 innate immune
responses. 5th Symposium & Master Classes on Mucosal
Immunology: “Cytokines and border patrol”, Rotterdam,
Kingdom of the Netherlands. May 13th, 2013.
Koyasu S. Thymic stromal lymphopoietin induces
corticosteroid resistance in natural helper cells in the inflamed airways. Aegean Conferences, 6th International
Conference on Autoimmunity: Mechanism and Novel
Treatments, Corfu, Greece. October 4th, 2013.
Moro K. Role of natural helper cells, a member of group
2 innate lymphoid cells. 12th FIMSA Advanced Training
Course, Chiang Mai, Thailand. October 22nd, 2013.

W

e focus on a new lymphoid tissue in mouse, rat and human mesentery,fat-associated lymphoid cluster (FALC), and an innate lymphocyte population, the Natural Helper (NH) cell, a type of group 2 innate lymphoid cell (ILC2),both of which we discovered. NH cells constitutively produce low levels of IL-5,
IL-6 and IL-13 and support the proliferation of B1 cells in the peritoneal cavity
and IgA production by B cells expressing surface IgA. NH cells respond to IL-33
and a combination of IL-2 and IL-25 during helminth infection and produce large
amounts of IL-5 and IL-13. IL-5 induces eosinophilia and IL-13 induces goblet
cell hyperplasia during the innate phase of helminth infection (Moro et al., (2010)
Nature 463: 540-544). In 2013, we demonstrated a critical role of Gata3 in the
differentiation of NH cells and their expression of IL-5 and IL-13. IL-33 and a
combination of IL-2 and IL-25 induced IL-5 and IL-13 production through the
p38-Gata3 pathway. RORα is also expressed at high levels in NH cells. We showedthat RORα is involved in the differentiation of NH cells but it is dispensable forcytokine production by mature NH cells. We also discovered in 2013 that NH
cells are involved in steroid resistance of asthma. NH cells are as sensitive to corticosteroids as T cells and eosinophils but thymic stromal lymphopoietin (TSLP)
synthesized during airway inflammation induced corticosteroid resistance of NH
cells in vitro and in vivo by controlling phosphorylation of STAT5 and expressionof Bcl-xL. Blockade of TSLP with a neutralizing antibody or blocking the TSLP/STAT5 signaling pathway with low molecular weight STAT5 inhibitors such as
pimozide restored corticosteroid sensitivity of NH cells. Based on these results,
the TSLP-STAT5 pathway could be a new therapeutic target in severe, corticosteroid-resistant asthma.

Moro K. Natural Helper Cell “a new player in innate
immune system”. Annual Meeting of the JapaneseSociety for Immunology 2013, Chiba, Japan. December
12th, 2013.

7

Laboratory for

Lab activities

Human Disease Models
Group Director : Fumihiko Ishikawa

Figure: A pyrrolo-pyrimidine compound eliminates human AML stem cells.
Histopathological examination with HE staining andimmunohistochemical staining demonstrated that invivo administration of RK-20449 resulted in eliminationof human AML cells in NSG bone marrow over time.
Normal mouse erythrocytes and myeloid cells recoveredat day 6 and at day 13 while the number of human AML
cells in the bone marrow was decreased. Human AML
cells were stained with an anti-hCD45 antibody (brown).

Recent Major Publications
Takagi S, Saito Y, Hijikata A, Tanaka S, Watanabe T,
Hasegawa T, Mochizuki S, Kunisawa J, Kiyono H, Koseki
H, Ohara O, Saito T, Taniguchi S, Shultz LD, Ishikawa
F. Membrane-bound human SCF/KL promotes in vivo
human hematopoietic engraftment and myeloid differentiation. Blood 119, 2768-2777 (2012)
Valent P, Bonnet D, De Maria R, Lapidot T, Copland M,
Melo JV, Chomienne C, Ishikawa F, Schuringa JJ, Stassi
G, Huntly B, Herrmann H, Soulier J, Roesch A, Schuurhuis GJ, Wohrer S, Arock M, Zuber J, Cerny-Reiterer S,
Johnsen HE, Andreeff M, Eaves C. Cancer stem cell definitions and terminology: the devil is in the details. Nat
Rev Cancer 12, 767-775 (2012)
Saito Y, Yuki H, Kuratani M, Hashizume Y, Takagi S, Honma T, Tanaka A, Shirouzu M, Mikuni J, Handa N, Ogahara I, Sone A, Najima Y, Tomabechi Y, Wakiyama M,
Uchida N, Tomizawa-Murasawa M, Kaneko A, Tanaka S,
Suzuki N, Kajita H, Aoki Y, Ohara O, Shultz LD, Fukami T,
Gogo T, Taniguchi S, Yokoyama S, Ishikawa F. A pyrrolo-pyrimidine derivative targets human primary AML
stem cells in vivo. Sci Transl Med 5, 181ra52 (2013)

Invited Presentations
Ishikawa F. Chemotherapy-Resistance of Human AML
Stem Cells. 2013 USA-Japan Science Conference, Hawaii,USA. March 26th, 2013.
Ishikawa F. Targeting Chemotherapy-resistant AML
Stem Cells. 1st Shanghai International Workshop on
Stem Cells in Cancer, Shanghai, China. April 18th, 2013.
Ishikawa F. Niche for human leukemia stem cells. 54th
meeting for Japan Society of Histochemistry & Cytochemistry, Tokyo, Japan. September 27th, 2013.
Ishikawa F. Developing Therapeutic Strategies Targeting
AML Stem Cells. 4th International Workshop on Humanized Mice, Seoul, Korea. October 2nd, 2013.
Ishikawa F. Review talk on humanized mouse research.
Annual Meeting of the Japanese Society for Immunology 2013, Chiba, Japan. December 11th, 2013.

8O

ur laboratory has been creating humanized mice, by injecting human cord
blood HSCs into immune-deficient NOD/SCID/Il2rgKO (NSG) newborns.
Though the NSG humanized mouse model has enabled us to achieve high levelsof human hematopoietic chimerism in multiple organs, limitations remain due tothe species barrier between human immune cells and the mouse microenvironment. To overcome such limitations, in the past few years we have been developing mice with a more humanized microenvironment. We previously reported thathuman CD8+ T cells developed in HLA class I expressing NSG mice exhibited
HLA-restricted functions such as cytokine production and cytotoxicity throughrecognition of EBV-infected B cells (Shultz, Saito, et al., PNAS 2010). Currently,
we have been evaluating whether the HLA-expressing humanized mice could be amodel to evaluate immune-therapy for malignancies.
In human leukemia research, to date, we reported that CD34+CD38- AML
cells are largely cell cycle quiescent and are resistant to chemotherapy (Nature Biotechnology, 2007 & 2010). Furthermore, we found that HCK, a Src family kinase,
was over-represented in human AML CD34+CD38- cells compared with normal
CD34+CD38- HSCs (Science Translational Medicine 2010). Through large-scale library screening and in silico simulation for binding between HCK and small molecules, we identified a pyrrolo-pyrimidine compound, RK-20449, that can bind
the ATP binding site of HCK and efficiently inhibit kinase activity of HCK. Fromin vitro experiments using 25 AML patient samples, we found that RK-20449 waseffective especially against FLT3-ITD mutated AML cells. Finally, by using FLT3ITD mutated AML engrafted humanized mice, we confirmed that in vivo administration of RK-20449 could almost completely eliminate human AML cells in therecipient circulation and in the recipient bone marrow (Fig.). We concluded that
RK-20449 is a promising agent for targeting FLT3-ITD mutated AML stem cells(Saito et al. Science Translational Medicine 2013).

Laboratory for

Lab activities

Intestinal Ecosystem
Group Director : Hiroshi Ohno

Figure: Schematic representation of the induction of colonic regulatory T-cell differentiation
by gut microbial-derived butyrate
Butyrate, a short chain fatty acid produced as a resultof fermentation by gut commensal microbiota offood-derived indigestible dietary carbohydrate fiber,
induces differentiation of extrathymic regulatory T cells
from naïve T cells in the colonic lamina propria. This issuggested to result mainly from epigenetic changes via
the histone deacetylase-inhibiting capacity of butyrate
in both naïve T cells and antigen-presenting dendritic
cells. The combination of microbial short-chain fatty
acids is also suggested to promote production of TGFβ,which is required for regulatory T-cell differentiation, byintestinal epithelial cells.

Recent Major Publications
Kanaya T, Hase K, Takahashi D, Fukuda S, Hoshino K, Sasaki
I, Hemmi H, Knoop KA, Kumar N, Sato M, Katsuno T, Yokosuka O, Toyooka K, Nakai K, Sakamoto A, Kitahara Y, Jinnohara T, McSorley SJ, Kaisho T, Williams IR, Ohno H. The Ets
transcription factor Spi-B is essential for the differentiationof intestinal M cells. Nat Immunol 13, 729-36 (2012)
Hase K, Nakatsu F, Ohmae M, Sugihara K, Shioda N,
Takahashi D, Obata Y, Furusawa Y, Fujimura Y, Yamashita
T, Fukuda S, Okamoto H, Asano M, Yonemura S, Ohno H.
AP-1B-mediated protein sorting regulates polarity andproliferation of intestinal epithelial cells in mice. Gastroenterology 145, 625-35 (2013)
Furusawa Y, Obata Y, Fukuda S, Endo TA, Nakato G, Takahashi D, Nakanishi Y, Uetake C, Kato K, Kato T, Takahashi
M, Fukuda NN, Murakami S, Miyauchi E, Hino S, Atarashi
K, Onawa S, Fujimura Y, Lockett T, Clarke JM, Topping DL,
Tomita M, Hori S, Ohara O, Morita T, Koseki H, Kikuchi J,
Honda K, Hase K, Ohno H. Commensal microbe-derived
butyrate induces colonic regulatory T cells. Nature 504,
446-50 (2013)

Invited Presentations
Ohno H. Differentiation and function of M cells, a uniquesubset of intestinal epithelial cells specialized for mucosal
antigen-uptake. Immunology 2013: AAI Annual Meeting,
Honolulu, USA. May 6th, 2013.Ohno H. Differentiation andfunction of M cells, a unique subset of intestinal epithelial
cells specialized for mucosal antigen-uptake. The 16th
International Congress for Mucosal Immunology: Plenary
session, Vancouver, Canada. July 19th, 2013.Ohno H. Commensal microbe-derived butyrate epigenetically inducescolonic regulatory T cell differentiation. 7th International
Leukocyte Signal Transduction Conference, Kos, Greece.
September 12th, 2013.

G

ut microbiota play important roles in the normal physiology as well as pathology of the host. However, the gut does not unconditionally accept commensal microbiota. Our intestinal immune system somehow senses the kind andamount of bacteria existing in the gut lumen and tries to contain the total number
and composition of the gut microbiota. The aim of this laboratory is to understandthe mechanisms by which the host and its gut commensal microbiota interact,
especially focusing on how gut microbes are delivered across the intestinal epithelial barrier to be recognized by the intestinal immune system, how gut microbiota
shape host defense and immune systems, and how host-gut microbiota interactions affect host health and disease status.
The delivery of particulate antigens such as bacteria is thought to be mainly
achieved by a unique epithelial cell subset, M cells, residing in a limited region ofepithelial layer covering the lymphoid follicles of gut-associated lymphoid tissue
such as Peyer’s patches. We are studying the function and differentiation of M cells
at the molecular mechanistic level. We have identified bacterial uptake receptors
on M cells, such as glycoprotein 2 and cellular prion protein. We also have recently
discovered that Spi-B, an Ets family transcription factor expressed in immatureepithelial progenitor cells in the crypt, is essential for these cells to differentiate
into M cells.
Regarding host-gut microbiota interactions, we employ a comprehensive multiple omics approach, combining exhaustive metagenomic, (meta)transcriptomic,
and metabolomic analyses. By applying this approach, we have shown that genes
encoding ATP-binding-cassette (ABC)-type carbohydrate transporters present incertain bifidobacteria contribute to protecting mice against death induced by enterohaemorrhagic Escherichia coli O157:H7. We have also found that a short chain
fatty acid, butyrate, produced by gut microbiota is important for the induction ofregulatory T cells from naïve T cells in the colonic lamina propria.

9

Laboratory for

Lab activities

Mucosal Immunity
Team Leader : Sidonia Fagarasan

Figure: The coating of commensal bacteria withIgA facilitates maintenance of diversified bacterial species in the gut
A section of Peyer’s patch Gram stained (left panel)
showing mostly Gram positive segmented bacteria
trapped in the mucus layer (yellow). Under normal
conditions, a large fraction of the commensal bacteria
are coated with IgAs produced by the plasma cells in
the lamina propria (shown in the right panel).

Recent Major Publications
Kawamoto S, Tran TH, Maruya M, Suzuki K, Doi Y,
Tsutsui Y, Kato LM, Fagarasan S. The inhibitory receptor
PD-1 regulates IgA selection and bacterial compositionin the gut. Science 336, 485-9 (2012)
Kato LM, Kawamoto S, Maruya M, Fagarasan S. Gut
TFH and IgA: key players for regulation of bacterial
communities and immune homeostasis. Immunol Cell
Biol 92, 49-56 (2014)
Magri G, Miyajima M, Bascones S, Mortha A, Puga I,
Cassis L, Barra CM, Comerma L, Chudnovskiy A, Gentile M, Llige D, Cols M, Serrano S, Arostegui JI, Juan
M, Yague J, Merad M, Fagarasan S, Cerutti A. Innate
immune cells integrate stromal and immune signals toenhance antibody production by splenic marginal zone
B cells. Nat Immunol (in press)

Invited Presentations
Fagarasan S. IgA synthesis: a form of immune adaptation extending beyond gut. Keystone Symposia: B Cell
Development and Function (X1), Keystone, Colorado,USA. February 12th, 2013.
Fagarasan S. IgA synthesis: a form of immune adaptation extending beyond gut. 6th International Singapore
Symposium of Immunology: From basic Immunology toeffective immunotherapies, Singapore. June 6th, 2013.
Fagarasan S. TFR/TFH cells in mucosal immunity.
Gordon Research Conferences: T follicular Helper Cells,
Hong-Kong, China. July 23rd, 2013.
Fagarasan S. Symbiotic regulatory loop between Foxp3+
T cells, IgA and bacteria in the gut. 15th International
Congress of Immunology, Milan, Italy. August 27th,
2013.
Fagarasan S. Symbiotic regulatory loop between
acquired immune system and gut microbiota. The 3rd
lymphoid tissue meeting: Plenary Lecture, Rotterdam,
The Netherlands. September 15th, 2013.

10

O

ur previous studies demonstrated that the absence of immunoglobulin A(IgA), the major effector molecule of the adaptive immunity in the gut, or
the impaired IgA selection in germinal centers (GC) due to deregulated T cell control, severely affects the balance of gut bacterial communities, resulting in massive
activation of the immune system in the entire body. The absence of a subset of
Foxp3+ T cells induced by bacterial antigens also modifies the composition of thegut microbiota by evoking mucosal TH2 inflammation. Interestingly, the Foxp3+ T
cells induce GC and IgA responses by generating GC T cells (TFH and TFR cells),
and their depletion causes a rapid loss of specific IgA responses in the intestine.
Together, all these observations pointed to the existence of a Foxp3-IgA axis inmaintaining the gut microbiota balance. It remained unclear however, how these
specific arms of the adaptive immune system mediate host-microbial interactionsin the gut. We found that Foxp3+ T cells contribute to diversification of the gut microbiota, particularly of species belonging to Firmicutes. The control of indigenous
bacteria by Foxp3+ T cells involved their regulatory functions outside and insideof the GCs, suppression of inflammation and regulation of IgA selection in Peyer’s
patches, respectively. The diversified and selected IgA repertoires generated in thepresence of Foxp3+ T cells in the GCs (TFR cells) associate with specific coating ofa large diversity of bacterial species and their maintenance rather than exclusionfrom the gut. By contrast, the IgAs generated in the absence of TFR cells associatewith abundant coating of bacteria with largely non-specific IgAs, reduced diversity
and skewed gut microbiota. Thus, the adaptive immune system, through its cellular and molecular components required for immune tolerance, and diversification
as well as selection of the antibody repertoire, mediates host-microbial symbiosis
by controlling the richness and balance of bacterial communities required for homeostasis.

Laboratory for

Lab activities

Gut Homeostasis
Team Leader : Kenya Honda

Figure: Treg-inducing 17 strains of Clostridia
The image is a scanning electron micrograph showing
the proximal colon of germ-free mice colonized with theregulatory T cell-inducing human Clostridia strains that
we have isolated (magnification ~5,000x). The community of Clostridia strains induces regulatory T cells andcontributes to suppression of unfavorable inflammation
and allergic responses.

Recent Major Publications
Yoshimoto S, Loo TM, Atarashi K, Kanda H, Sato S,
Oyadomari S, Iwakura Y, Oshima K, Morita H, Hattori M,
Honda K, Ishikawa Y, Hara E, Ohtani N. Obesity-induced
gut microbial metabolite promotes liver cancer throughsenescence secretome. Nature 499, 97-101 (2013)
Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y,
Nishikawa H, Fukuda S, Saito T, Narushima S, Hase K,
Kim S, Fritz JV, Wilmes P, Ueha S, Matsushima K, Ohno
H, Olle B, Sakaguchi S, Taniguchi T, Morita H, Hattori M,
Honda K*. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota.
Nature 500, 232-6 (2013)*corresponding author
Furusawa Y, Obata Y, Fukuda S, Endo TA, Nakato G,
Takahashi D, Nakanishi Y, Uetake C, Kato K, Kato T,
Takahashi M, Fukuda NN, Murakami S, Miyauchi E,
Hino S, Atarashi K, Onawa S, Fujimura Y, Lockett T,
Clarke JM, Topping DL, Tomita M, Hori S, Ohara O,
Morita T, Koseki H, Kikuchi J, Honda K, Hase K, Ohno H.
Commensal microbe-derived butyrate induces colonic
regulatory T cells. Nature 504, 446-50 (2013)

Invited Presentations
Honda K. Clostridia strains from human microbiota for
Treg induction. Cell Symposia Microbiome and Host
Health, Lisbon, Portugal. May 14th, 2013.
Honda K. Immunomodulation by the gut microbiota.
Fondation Mérieux Conference: Targeting Commensal
Flora to Better Shape Protective Immune Responses for
Better Disease Prevention and Therapy, Veyrier-du-Lac
France. June 12th, 2013.
Honda K. Regulation of T cells by the gut microbiota.
FASEB Autoimmunity, Saxtons River, Vermont, USA. July
10th, 2013.
Honda K. Microbiota Regulation of T Cells in the Intestine. The 16th International Congress for Mucosal
Immunology, Vancouver, Canada. July 19th, 2013.
Honda K. Regulation of Th17 and Treg cells by the gut
microbiota. 15th International Congress of Immunology, Milan, Italy. August 27th, 2013.

T

he gut microbiota intimately interacts with the host immune system and influences pathogenesis of several immune disorders. CD4+Foxp3+ Treg cells
are the most prominent regulatory cells. They are indispensable for maintaining
immune tolerance and are also an emerging therapeutic target for inflammatory
bowel diseases (IBD). Treg cells are most abundant in the intestinal mucosa atsteady state. In germ-free (GF) or antibiotic-treated mice, the frequency of colonic
Treg cells is significantly reduced and interleukin (IL)-10, an immune suppressive
cytokine, expression by Treg cells is markedly decreased. We aimed to identify
Treg-inducing bacterial strains derived from the human microbiota and test theirpotential to attenuate diseases such as colitis and allergy, thus enabling clinicaltranslation of our previous findings. Starting from a complete human fecal sample
from a healthy Japanese volunteer, which can typically include several hundred
strains, a sequence of selection steps was applied to isolate Treg inducing strains.
These steps included chloroform treatment, 20,000-fold dilution, and serial transplantation to GF mice using gnotobiotic techniques. These steps allowed us to narrow the number of bacterial strains down to 17 without sacrificing Treg-inducing
potency. All 17 strains were successfully isolated, cultured, and stored in frozen
stocks. All strains are members of Clostridia clusters IV, XIVa, and XVIII. Whenthese 17 strains were mixed and inoculated into GF mice, they robustly induced
Tregs in the colonic LP and displayed full Treg-inducing capacity, comparable tothat of a complete fecal microbiome. The 17 strains act as a community to bothprovide bacterial antigens to T cells and to help polarize the T cells to become
Tregs by inducing the generation of a TGF-β-rich environment. Furthermore, oral
administration of the cocktail of 17 strains to adult mice attenuated disease inmodels of colitis and allergic diarrhea.We published these results in Nature.

11

Laboratory for

Lab activities

Immune Homeostasis
Team Leader : Shohei Hori

Figure: A model of regulatory T cell fate determination and maintenanceDuring Treg cell differentiation, uncommitted precursor
cells adopt either Treg cell or conventional T (Tconv)
cell fates upon activation, independently of Foxp3 expression. The commitment to the Treg cell fate is madebefore Foxp3 induction and executed by a transcription
factor network elicited by extrinsic signals from the extracellular environment. The same signals also induce
epigenetic modifications, including DNA demethylationof the Foxp3 locus. Foxp3 is incorporated into thepre-existing transcription factor network and the resulting “Foxp3 interactome” establishes the characteristic
Treg cell phenotype in cooperation with the epigenetic
modifications. Although Treg cells may down-regulate
Foxp3 expression under certain circumstances, these“latent” Treg cells retain the epigenetic memory of, andthus remain committed to, the Treg cell fate. On theother hand, when activated T cells express Foxp3 without engagement of the transcription factor network,
Foxp3 by itself cannot establish the characteristic Treg
cell phenotype. As a result, the activated Foxp3+ T cells
readily lose Foxp3 expression, adopt the alternative
Tconv cell fate and differentiate into effector Th cells.

Recent Major Publications
Miyao T, Floess S, Setoguchi R, Luche H, Fehling HJ,
Waldmann H, Huehn J, Hori S. Plasticity of Foxp3+ T
cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T
cells. Immunity 36, 262-75 (2012)
Hori S. The Foxp3 interactome: a network perspectiveof Treg cells. Nat Immunol 13, 943-5 (2012)
Toker A, Engelbert D, Garg G, Polansky JK, Floess S,
Miyao T, Baron U, Düber S, Geffers R, Giehr P, Schallenberg S, Kretschmer K, Olek S, Walter J, Weiss S,
Hori S, Hamann A, Huehn J. Active demethylationof the Foxp3 locus leads to the generation of stable
regulatory T cells within the thymus. J Immunol 190,
3180-8 (2013)

Invited Presentations
Hori S. Genetic control of regulatory T cell fitness in tissues. The 7th International Leukocyte Signal Transduction Conference, Kos, Greece. September 10th, 2013.
Hori S. Genetic control of regulatory T cell homeostasis
in non-lymphoid tissues. The 3rd Bizan Immunology
Symposium at the University of Tokushima “Immune
System Development, Deviation, and Regulation”, Tokushima, Japan. February 13th, 2014.

12Regulatory T (Treg) cells expressing the transcription factor Foxp3 play an indispensable role in the establishment and maintenance of immunological self-tolerance
and tissue homeostasis, a concept firmly established by the finding that defective generation of functional Treg cells underlies a fatal autoimmune disease that develops in
Foxp3-mutant mice and in humans suffering from the IPEX syndrome. Recent findingsthat Foxp3+ Treg cells exert tissue-protective or immune-suppressive functions under diverse circumstances have raised the question of what mechanisms ensure the robustnessof Treg cell functions in the face of various unpredictable perturbations in the extracellular environment. To answer this question, we have focused on the mechanisms that
control lineage stability and adaptability of Treg cells in changing environments.
We have performed cell fate mapping studies of Foxp3+ T cells to address the lineage
stability of Treg cells and found that Foxp3 expression per se does not specify the Treg celllineage. Thus, activated conventional T cells can promiscuously and transiently express
Foxp3 and committed Treg cells can transiently and reversibly down-regulate Foxp3. Despite this phenotypic plasticity, Treg cells retain epigenetic memory of, and thus remaincommitted to, Foxp3 expression and suppressive functions. Our findings have establishedthat Treg cells constitute a stable cell lineage committed to Foxp3-dependent suppressivefunctions.
We have also analyzed how Foxp3 gene mutations found in human IPEX impinge on
Treg cells in vivo using knock-in mutagenesis in mice, and found that one mutation preferentially impairs Treg cell homeostasis in non-lymphoid tissues without affecting theirdifferentiation and in vitro suppressive functions. Our analyses revealed that this defect isdue to altered DNA binding specificity of the mutant Foxp3. By taking advantage of thisunique animal model, we are currently investigating how Treg cells adapt to diverse andfluctuating tissue environments.

Laboratory for

Lab activities

Skin Homeostasis
Team Leader : Masayuki Amagai

Figure: Three epidermal barrier components inthe skin

Recent Major Publications
Nagao K, Kobayashi T, Moro K, Ohyama M, Adachi T,
Kitashima DY, Ueha S, Horiuchi K, Tanizaki H, Kabashima K, Kubo A, Cho YH, Clausen BE, Matsushima K,
Suematsu M, Furtado GC, Lira SA, Farber JM, Udey MC,
Amagai M. Stress-induced production of chemokines byhair follicles regulates the trafficking of dendritic cells inskin. Nat Immunol 13, 744-52 (2012)
Kubo A, Shiohama A, Sasaki T, Nakabayashi K, Kawasaki H, Atsugi T, Sato S, Shimizu A, Mikami S, Tanizaki H,
Uchiyama M, Maeda T, Ito T, Sakabe J, Heike T, Okuyama T, Kosaki R, Kosaki K, Kudoh J, Hata K, Umezawa
A, Tokura Y, Ishiko A, Niizeki H, Kabashima K, Mitsuhashi Y, Amagai M. Mutations in SERPINB7, encoding
a member of the serine protease inhibitor superfamily,
cause Nagashima-type palmoplantar keratosis. Am J
Hum Genet 93, 945-56 (2013)
Sasaki T, Shiohama A, Kubo A, Kawasaki H, Ishida-Yamamoto A, Yamada T, Hachiya T, Shimizu A,
Okano H, Kudoh J, Amagai M. A homozygous nonsense
mutation in the gene for Tmem79, a component for
the lamellar granule secretory system, produces spontaneous eczema in an experimental model of atopic
dermatitis. J Allergy Clin Immunol 132, 1111-1120.
e4 (2013)

Invited Presentations
Matsui T. Skin-specific retroviral-like aspartic protease
SASPase and evolution of mammalian skin. The Genetics Society of Japan, Tokyo, Japan. September 19th,
2013.
Amagai M. Skin barrier dysfunction and cutaneous sensitization in atopic disease. The 50th Annual Meetingof the Japanese Society of Pediatric Allergy and Clinical
Immunology, Yokohama, Japan. October 19th, 2013.
Amagai M. Stratum corneum barrier dysfunction andatopic diseases. Annual Meeting of the JapaneseSociety for Immunology 2013, Chiba, Japan. December
11th, 2013.

W

hen the immune system meets external antigens in the skin, it tends toreact to them. By contrast, when the immune system meets antigens in thegut, it tends to tolerate them. However, the exact mechanisms for these opposite
immune reactions are still largely unknown. Our laboratory attempts to dissect
and understand the skin as an immune organ. Especially we are studying skin barrier formation, function, and its dysfunction in atopic diseases.
Skin is composed of three components: epidermis, dermis and subcutaneous fat tissue. The epidermis is a keratinized stratified squamous epithelium andforms an effective barrier essential for preventing the invasion of microorganisms,
chemical compounds and allergens. The epidermis is composed of four distinct
layers; stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG) andstratum corneum (SC) from bottom to top (Fig.).
Among many elements of the skin barrier, we are focusing on the SC as an
air-liquid barrier, tight junctions as a liquid-liquid barrier, and the Langerhans
cell network as an immunological barrier (Fig.). Tight junctions are formed in thesecond layer of SG or SG2 cells and Langerhans cells extend their dendrites above
these tight junctions to capture external antigens. Filaggrin deficiency, which is apredisposing factor for atopic disease, enhances SC penetration of external antigens.
The SC is 12 to 15 accumulated layers of corneocytes, which are terminallydifferentiated dead keratinocytes. Therefor, all the essential components for SC are
produced in SG1 cells. We have established a method to isolate SG1 and SG2 cells
and are characterizing SG1, 2-specific genes and proteins. It will be interesting todetermine which proteases and/or protease inhibitors are key players in SC homeostasis and how their balance is regulated.

13

Laboratory for

Lab activities

Metabolic Homeostasis
Team Leader : Naoto Kubota

Figure: TCF7L2 expressed in pancreatic beta
cells plays a crucial role in glucose metabolism
through regulation of the beta cell mass.
Blood glucose levels (ratio baseline) during an insulin
tolerance test (ITT).
(a) in 10-week-old wild-type (white diamonds) and B
cell-specific dominant negative TCF7L2 transgenic (DN)
mice (black squares) from each independent DN mouseline (n=12). Blood glucose (b) and serum insulin levels(c) during the oral glucose tolerance test (OGTT) in
11-week-old wild-type (white diamonds) and DN mice
(black squares) from each line (n=8–12).Whole-pancreas insulin content (d–f) of 12-week-old wild-type (Wild)
and DN mice from each line (n=12). Results for line 390(d), 440 (e) and 469 (f). Immunohistochemical analysis of the pancreas for insulin (red) and BrdU (green)
incorporation in 12-week-old mice (g). Scale bar, 100
μm. Beta cell area ratio per whole pancreas (h), area ofthe beta cells in each islet (i), number of BrdU-positive
cells (j) and number of apoptotic cells (k) per beta cellarea of 10,000 μm2 in 12-week-old mice (n=8–10).
Data are shown as mean ± SEM. *p<0.05, DN mice vswild-type mice

Recent Major Publications
Kadowaki T, Ueki K, Yamauchi T, Kubota N. SnapShot:
Insulin signaling pathways. Cell 148, 624- 624.e1(2012)
Kadowaki T, Kubota N, Ueki K, Yamauchi T. SnapShot:physiology of insulin signaling. Cell 148, 834-834.e1(2012)
Takamoto I, Kubota N, Nakaya K, Kumagai K, Hashimoto S, Kubota T, Inoue M, Kajiwara E, Katsuyama H,
Obata A, Sakurai Y, Iwamoto M, Kitamura T, Ueki K,
Kadowaki T. TCF7L2 in mouse pancreatic beta cells
plays a crucial role in glucose homeostasis by regulating beta cell mass. Diabetologia (in press)

Invited Presentations
Kubota N. Molecular mechanisms of type 2 diabetes
and insulin resistance. The 36th Naito Conference on
Molecular Aspects of Energy Balance and Feeding Behavior, Sapporo, Japan. September 12th, 2013.

14

In recent years there has been a rapid increase in the incidence of type 2 diabetes inboth Western and Asian countries. This high prevalence is most likely the result of acomplex interplay between genetic, such as reduced insulin secretion, and environmental factors, such as high-fat diet and decreased physical activity. However, the precise
molecular mechanisms underlying the development and progression of type 2 diabetes
remained unclear. The goal of our team is to identify the molecular mechanism of insulin
secretion and insulin resistance.
A) Molecular mechanism of insulin secretion
Most of the common variant single-nucleotide polymorphisms (SNPs) identified by genome-wide association study (GWAS) are associated with defective pancreatic islet function. However, the functional role of genes identified by GWAS remained unclear. We
discovered that transcription factor 7-like 2 (TCF7L2), which confers the largest effect on
the risk of type 2 diabetes in Western populations, plays a crucial role in glucose metabolism through regulation of the beta cell mass (Diabetologia in press).
B) Molecular mechanism of insulin resistance
Insulin resistance is defined as a condition in which physiological insulin signals are
impaired or dysfunctional for some reason, such as ER stress, adipokine abnormality
or chronic inflammation. Once insulin binds to insulin receptors, insulin receptor substrate (IRS)-1 and IRS-2 are activated and begin transmitting intracellular insulin signals.
These result in various insulin-mediated activities, including glucose and lipid metabolism, protein synthesis and cell proliferation. Until now, we have been studying the role of
IRS-1 and IRS-2, which show ubiquitous expression patterns (Diabetes 2000, Circulation
2003, J Clin Invest 2004, Cell Metab 2008, Cell Metab 2011, Cell 2012). We are currently
working to elucidate the molecular mechanism of insulin resistance via IRS-1 and IRS-2
in cells and tissues such as liver, skeletal muscle, macrophages, and the central nervous
system.

Laboratory for

Lab activities

Immune Crosstalk
Team Leader : Hilde Cheroutre

Figure: Intraepithelial T cells protect the mucosal
barrier of the intestine from pathogen- and inflammation-induced pathology.
T cells that reside within the epithelium of the intestine
are phenotypically heterogeneous but they are all
specialized to protect the mucosal barrier against pathogen- and immune cell-induced pathology. In contrast
to the T cells in the periphery, epithelial T lymphocytes
are all antigen-experienced T cells that encountered
their antigen initially during selection in the thymus(agonist selected CD8αα TCRαβ and TCRγδ precursor
cells) or as mature cells in the periphery (CD8αβ CTL and
CD4 CTL). Although the various epithelial T cell subsets
display different antigen specificity and MHC restriction
and although they follow different paths of effector
differentiation, they all acquire cytolytic and regulatory
capacity. The functional specialization of intraepithelial
T lymphocytes adapts them to provide optimal protection in the face of preserving the integrity of the delicate
mucosal barrier.

Recent Major Publications
Fu G, Casas J, Rigaud S, Rybakin V, Lambolez F, Brzostek
J, Hoerter JA, Paster W, Acuto O, Cheroutre H, Sauer
K, Gascoigne NR. Themis sets the signal threshold for
positive and negative selection in T-cell development.
Nature 504, 441-5 (2013)
Mucida D, Husain MM, Muroi S, van Wijk F, Shinnakasu
R, Naoe Y, Reis BS, Huang Y, Lambolez F, Docherty M,
Attinger A, Shui JW, Kim G, Lena CJ, Sakaguchi S, Miyamoto C, Wang P, Atarashi K, Park Y, Nakayama T, Honda K, Ellmeier W, Kronenberg M, Taniuchi I, Cheroutre H.
Transcriptional reprogramming of mature CD4+ helper T
cells generates distinct MHC class II-restricted cytotoxic
T lymphocytes. Nat Immunol 14, 281-9 (2013)

Invited Presentations
Cheroutre H. CD4 CTL: a major new player in health anddisease. Immunology 2013: AAI Annual Meeting, Major
Symposia A: Tissue-Resident Lymphocytes, Honolulu,
USA. May 4th, 2013.
Cheroutre H. Mucosal T Cells in Inflammation and Homeostasis. The 16th International Congress for Mucosal
Immunology: Pioneering Frontiers in Mucosal Regulation, Vancouver, Canada. July 20th, 2013.
Cheroutre H. Unique Adaptive Immune Defense at the
Mucosal Frontline of the Intestine. 15th International Congress of Immunology, Symposium: Innate lymphocytes andmucosal Immunity, Milan, Italy. August 22nd-27th, 2013.
Cheroutre H. 2013 Grand Challenges Meeting: Vaccine
Discovery and Translational scientific track session, Riode Janeiro, Brazil. October 28th-30th, 2013.

O

ur research continues to elucidate mechanisms of mucosal immune protection and regulation. In a recent study, we uncovered an unexpected degreeof plasticity for CD4 T helper (Th) cells, which upon antigenic stimulation, areable to terminate the expression of the Th transcription factor, ThPOK, and differentiate into cytotoxic T lymphocytes (CTL). At steady state, CD4 CTL remainquiescent and express a self-regulated phenotype. However under challenging
conditions, these cells have the potential to transform into potent inflammatory
killer effector cells (Mucida et al., Nature Immunology 2013).
Overall, based on the insights we are gaining from our research, a clear picture
has begun to emerge showing that the immune defense of the intestine adapts tothe local environment and specializes to provide the most efficient and immediate protection in the face of preserving the integrity of the most critical mucosal
barrier of the body.
In another study, we are aiming to understand the various mechanisms andprocesses that lead to central tolerance. Our previous research showed that in addition to conventional selection, a process of so called “agonist” selection operates
in the thymus, which preserves self-specific thymocytes and functionally differentiates these precursor cells to become beneficial pre-programmed protective or
regulatory T cells. In an effort to understand what factors control the decisivecheckpoint during thymic selection, we identified “Themis” as a critical switch(Guo et al., Nature 2013). We are now trying to elucidate the molecular and cellular factors and events that connect the Pre-TCR and TCR signal strength withthymic selection and the fate decision of the developing T cells.

Cheroutre H. 85th Anniversary Research Foundation
Flanders (FWO): Session on Inflammation and Immunity,
Ghent, Belgium. December 17th, 2013.

15

Laboratory for

Lab activities

Inflammatory Regulation
Team Leader : Takashi Tanaka

Figure: LIM is a new family of negative regulators of NF-κB signaling.
PDLIM2 and PDLIM7 are ubiquitin E3 ligases forthe p65 subunit of NF-κB, form heterodimers andcooperatively promote p65 degradation. By contrast,
PDLIM1 sequestrates p65 in the cytoplasm and inhibits
its nuclear translocation, thereby suppressing NF-κB
signaling.

Recent Major Publications
Yan P, Fu J, Qu Z, Li S, Tanaka T, Grusby MJ, Xiao G. PDLIM2 suppresses HTLV-I Tax-mediated tumorigenesis bytargeting Tax into the nuclear matrix for proteasomal
degradation. Blood 113, 4370-80 (2009)
Tanaka T, Yamamoto Y, Muromoto R, Ikeda O, Sekine
Y, Grusby MJ, Kaisho T, Matsuda T. PDLIM2 inhibits T
Helper 17 cell development and granulomatous inflammation through degradation of STAT3. Sci Signal 4,
ra85 (2011)
Sasaki I, Hoshino K, Sugiyama T, Yamazaki C, Yano T,
Iizuka A, Hemmi H, Tanaka T, Saito M, Sugiyama M,
Fukuda Y, Ohta T, Sato K, Ainai A, Suzuki T, Hasegawa
H, Toyama-Sorimachi N, Kohara H, Nagasawa T, Kaisho
T. Spi-B is critical for plasmacytoid dendritic cell function and development. Blood 120, 4733-43 (2012)

Invited Presentations
Tanaka T. Negative regulation of T-helper cell differentiation by LIM proteins. The 132th Annual Meeting of
the Pharmaceutical Society of Japan, Hokkaido, Japan.
March 30th, 2012.
Tanaka T. Clarifying the molecular mechanisms that
regulate inflammatory responses. RIKEN - Novo Nordisk
A/S Scientific Forum, Yokohama, Japan. April 19th,
2012.
Tanaka T. Negative regulation of inflammatory responses by LIM proteins. The 12th Biennial International
Endotoxin & Innate Immunity Society Meeting, Tokyo,
Japan. October 24th, 2012.
Tanaka T. Clarifying the molecular mechanisms that
regulate inflammatory responses. The Osaka Minami
Medical Center: Talk with the expert seminar, Osaka,
Japan. October 11th, 2013.
Tanaka T. Regulation of inflammatory responses by LIM
proteins. The 5th LJI & IMS-RCAI Workshop, Yokohama,Japan. October 31st, 2013.

16

T

he inflammatory response is an important host defense mechanism to sense
and eliminate invading microbial pathogens. Dendritic cells first detect pathogens and activate the transcription factor NF-κB, which enters the nucleus andinduces the expression of a series of inflammation-related genes. These initiallyhelpful inflammatory responses must be terminated at the appropriate time point,
otherwise excessive responses can damage normal tissue and may cause autoimmune or allergic diseases. Our research goal is to identify key regulators of inflammation-related signal transduction pathways and to clarify the complete pictureof the molecular mechanisms for regulating inflammatory responses. We previously identified PDLIM2 (PDZ and LIM domain protein-2), a nuclear proteinthat belongs to a large family of LIM proteins, as one of the key factors negatively
regulating inflammatory responses. We found that PDLIM2 is an E3 ubiquitin
ligase for the STAT4 and STAT3 transcription factors, thereby suppressing Th1
and Th17 cell differentiation (Tanaka T, Immunity, 2005, Tanaka T, Sci. Signal.,
2011). We have also demonstrated that PDLIM2 negatively regulates NF-κB activity and subsequent inflammatory responses, acting as a nuclear ubiquitin E3 ligase
targeting the p65 subunit of NF-κB. (Tanaka T, Nat. Immunol., 2007). We now
focus on PDLIM2 and other members of the LIM family. Recently we found thatPDLIM7 and PDLIM1 are also negative regulators of NF-κB-mediated signaling.
PDLIM7 was found to be an E3 ubiquitin ligase for p65. PDLIM7 and PDLIM2
form heterodimers and synergistically promote p65 degradation by the proteasome. On the other hand, PDLIM1 sequestrated p65 in the cytoplasm, possibly byinteraction with actin stress fibers, through binding to α-actinin, an actin binding
protein, and suppressed nuclear translocation of p65 protein. These studies should
contribute to our understanding of the pathogenesis of human autoimmune andinflammatory diseases and the development of new therapeutic tools.

Laboratory for

Lab activities

Cytokine Regulation
Team Leader : Masato Kubo

Figure: CNS2 is an essential enhancer element in
TFH cells but not inTH2 cells.
The CNS active T cells in CNS2-regulated transgenic
mice expressed several markers of TFH cells, CXCR5, PD1, and ICOS, which favored their localization in B cellfollicles and germinal centers of Peyer’s patches. The
upper part of the figure depicts the genome structureof the Il4 locus. The lower part shows the cell surface
phenotype and localization of GFP+ TFH cells.

Recent Major Publications
Harada Y, Tanaka S, Motomura Y, Harada Y, Ohno S,
Ohno S, Yanagi Y, Inoue H, Kubo M. The 3' enhancer
CNS2 is a critical regulator of interleukin-4-mediatedhumoral immunity in follicular helper T cells. Immunity
36, 188-200 (2012)
Otsuka A, Nakajima S, Kubo M, Egawa G, Honda T,
Kitoh A, Nomura T, Hanakawa S, Sagita Moniaga C, Kim
B, Matsuoka S, Watanabe T, Miyachi Y, Kabashima K.
Basophils are required for the induction of Th2 immunity to haptens and peptide antigens. Nat Commun 4,1739 (2013)
Noti M, Wojno ED, Kim BS, Siracusa MC, Giacomin
PR, Nair MG, Benitez AJ, Ruymann KR, Muir AB, Hill
DA, Chikwava KR, Moghaddam AE, Sattentau QJ,
Alex A, Zhou C, Yearley JH, Menard-Katcher P, Kubo
M, Obata-Ninomiya K, Karasuyama H, Comeau MR,
Brown-Whitehorn T, de Waal Malefyt R, Sleiman PM,
Hakonarson H, Cianferoni A, Falk GW, Wang ML,
Spergel JM, Artis D. Thymic stromal lymphopoietin-elicited basophil responses promote eosinophilicesophagitis. Nat Med 19, 1005-13 (2013)

Invited Presentations
Kubo M. Role of Notch signal in the generation offollicular helper T cells (TFH) and memory T cell. The
1st International Immunological Memory and Vaccine
Forum, Tokyo, Japan. January 29th, 2013.
Kubo M. Roles of follicular helper T (TFH) cells in antibody based protective immunity against influenza
virus. Joint Symposium of the 21st International Symposium of Macrophage Molecular and Cellular Biology& the 78th Japanese Society for Interferon and Cytokine
Research, Tokyo, Japan. May 21st, 2013.
Kubo M. Cytokine regulation in T follicular helper (TFH)
cells. Gordon Research Conferences: T Follicular Helper
Cells, Hong Kong, China. July 22nd, 2013.
Kubo M. Molecular Mechanism of Immune Responses.
The 86th Annual Meeting of the Japanese Biochemical
Society: International session, Yokohama, Japan. September 13th, 2013.

T

he expression profile of a group of genes related to T cell development anddifferentiation is ingeniously controlled by spatiotemporal processes. Epigenetic changes in cis-acting regions of the Il13/Il4 locus are strongly associated withcytokine expression profiles during TH2 differentiation. We generated a series ofgenetically targeted mice disrupting the cis-acting activity of conserved non-coding sequences (CNS) in the Il4 locus to understand lineage-specific regulation of
IL-4. The main role for TH2-derived IL-4 has been thought to be in humoral immunity. However, we provided evidence that a recently-discovered subset of follicular
T (TFH) cells generates the IL-4 that promotes B cell differentiation into antibody
producing cells. CNS2 is an essential enhancer element for IL-4 expression in TFH
cells but not in Th2 cells. Mice with a CNS2 deletion had a reduction in IgG1 andIgE production and in IL-4 expression by TFH cells. We concluded that TH2 cells are
dispensable for antibody responses and that CNS2 is a specific enhancer element
required for IL-4 expression by TFH cells controlling humoral immunity.
We established a diphtheria toxin-based conditional deletion system using an
Il4 enhancer that we previously found was specific for IL-4 production by mast
cells or basophils (Mas-TRECK and Bas-TRECK mice). Using the Bas-TRECK
system, we examined the role of thymic stromal lymphopoietin (TSLP) in basophil development. We further focused on Eosinophilic esophagitis (EoE), a foodallergy–associated inflammatory disease characterized by esophageal eosinophilia. We established a new mouse model of EoE-like disease that developed independently of IgE, but was dependent on TSLP and basophils, as targeting TSLP orbasophils during the sensitization phase limited disease. Our data suggest that the
TSLP-basophil axis contributes to the pathogenesis of EoE and could be therapeutically targeted to treat this disease.

17

Laboratory for

Lab activities

Homeostatic Network
Team Leader : Shigeo Koyasu

Figure: Zn is a newly identified inflammatory
mediator.
Zinc is a mediator of the inflammatory response.
After injury, endogenous inflammatory mediators or“danger signals” are released and activate mast cells,
which then release Zn into the extracellular space. The
released Zn directly binds to the GPR39 Zn receptor on
immune-related cells and induces the expression ofcytokines such as IL-6 and TNFα. These cytokines thencontribute to inflammatory responses such as woundhealing.

Recent Major Publications
Yamasaki S, Hasegawa A, Hojyo S, Ohashi W, Fukada T,
Nishida K, Hirano T. A Novel Role of the L-Type Calcium
Channel α(1D) Subunit as a Gatekeeper for Intracellular
Zinc Signaling: Zinc Wave. PLoS One 7, e39654 (2012)
Fukada T, Hojyo S, Furuichi T. Zinc Signal: Zinc signal: a
new player in osteobiology. J Bone Miner Metab 31,
129-35 (2013)
Tamaki M, Fujitani Y, Hara A, Uchida T, Tamura Y, Takeno
K, Kawaguchi M, Watanabe T, Ogihara T, Fukunaka A,
Shimizu T, Mita T, Kanazawa A, Imaizumi MO, Abe T,
Kiyonari H, Hojyo S, Fukada T, Kawauchi T, Nagamatsu
S, Hirano T, Kawamori R, Watada H. The diabetes susceptible gene SLC30A8/ZnT8 regulates hepatic insulin
clearance. J Clin Invest 123, 4513-24 (2013)

Invited Presentations
Fukada T. Physio-pathological roles of zinc signaling.
The 86th Annual Meeting of the Japanese Pharmacological Society, Fukuoka, Japan. March 22nd, 2013.
Fukada T. New Horizon of Zinc Biology in Signaling,
Homeostasis and Diseases. The 86th Annual Meetingof the Japanese Biochemical Society: International session, Yokohama, Japan. September 11th, 2013.
Nishida K. Role of zinc transporter Slc30a2/ZnT2 in normal wound healing. The 86th Annual Meeting of theJapanese Biochemical Society: International session,
Yokohama, Japan. September 11th, 2013.
Nishida K. Metallothioneins control FcεRI-mediated IL-4
production in basophils. The meeting of the Japanese
Research Group for Studies of Metalbioscience 2013,
Shizuoka, Japan. September 27th, 2013.
Fukada T. Basic and Clinical aspects in Zinc and Degenerative diseases of Aging. 10th International Society
for Trace Element Research in Humans (ISTERH) 2013,
Tokyo, Japan. November 19th, 2013.

18

Zinc signaling is essential for B cell maintenance.
The immune system is influenced by vital zinc (Zn) status and Zn deficiency triggers
lymphopenia and immunodeficiency, however the mechanisms of Zn-mediatedlymphocyte maintenance remain elusive. We investigated the role of ZIP10, a Zntransporter expressed in B cells, on their development. Genetic ablation of Zip10
in early B cell stages resulted in reduction of B cells, which showed increased
caspase activity in parallel with decreases in intracellular Zn levels, indicating that
ZIP10-mediated Zn signaling is essential for early B cell survival. Moreover, ZIP10
expression was regulated by JAK-STAT pathways, which was also correlated with
STAT activation in human B cell lymphoma, implicating ZIP10 as a mediator of
malignancy. Our results establish a role for ZIP10 in B cell survival during early
development and underscore the importance of Zn signaling in immune system
maintenance.
Zinc controls skin wound healing through the GPR39 / IL-6 axis.
Bedsores are painful skin lesions that affect many patients, including the increasing
populations of elderly individuals and those afflicted with metabolic syndromes.
The essential trace element Zn has an important role in accelerating skin-woundhealing, but how it does so is unclear. We reported that Zn released from mast cells
is required for wound healing. The released Zn binds to its receptor (called GPR39)
on inflammatory cells, which release the inflammation-promoting molecule, IL6. IL-6 in turn controls wound healing (Fig.). These findings explain why Zn isbeneficial for treating skin disorders including wounds and burns.

Laboratory for

Lab activities

Developmental Genetics
Group Director : Haruhiko Koseki

Figure: The Meis2 promoter/enhancer association is preceded by promoter/enhancer/RBS
tripartite interaction in midbrain.
(Top) The Meis2 gene is schematically depicted andpositions of FISH probes are shown. (Middle) Tri-color
FISH images showing topological transition of promoter(PRT) (blue), MBE (red) and RBS (green) in 6.5 dpc epiblasts and midbrain of 16-20 and 22-26 somite stageembryos. Fluorescent signals from each channel in theregions indicated by dotted boxes are shown in insets.
(Bottom) Graphic summary of promoter/enhancer/
RBS DNA topology. Co-localization of RING1B foci withpromoter (PRT), enhancer (MBE) or RBS at each stage
is shown. Note that RBS constitutively associates with
the RING1B foci.

The Developmental Genetics Research Group is pursuing a research program to elucidate the epigenetic regulation of organ development and stem cell functions by
Polycomb group (PcG) proteins and DNA methylation mechanisms.

Recent Major Publications
Endoh M, Endo TA, Endoh T, Isono K, Sharif J, Ohara
O, Toyoda T, Ito T, Eskeland R, Bickmore WA, Vidal M,
Bernstein BE, Koseki H. Histone H2A Mono-Ubiquitination Is a Crucial Step to Mediate PRC1-Dependent
Repression of Developmental Genes to Maintain ES Cell
Identity. PLoS Genet 8, e1002774 (2012)
Isono K, Endo TA, Ku M, Yamada D, Suzuki R, Sharif
J, Ishikura T, Toyoda T, Bernstein BE, Koseki H. SAM
Domain Polymerization Links Subnuclear Clustering of
PRC1 to Gene Silencing. Dev Cell 26, 565-77 (2013)
Kondo T, Isono K, Kondo K, Endo TA, Itohara S, Vidal
M, Koseki H. Polycomb Potentiates Meis2 Activation in
Midbrain by Mediating Interaction of the Promoter witha Tissue-Specific Enhancer. Dev Cell 28, 94-101 (2014)

Invited Presentations
Koseki H. DNA methylation in T cells and gut immune
homeostasis. 3rd McGill-RIKEN Workshop, Montreal,
Canada. June 21st, 2013.
Koseki H. Epigetic regulation of spermiogenesis. The
10th Annual Meeting of Asian Reproductive Biotechnology Society, Phan Thiet, Vietnam. August 21st, 2013.
Koseki H. Activation of polycomb-repressed genes.
Mouse Molecular Genetics Conference , Cambridge, UK.
September 20th, 2013.
Koseki H. Activation of polycomb-repressed genes. Annual Meeting of the Japanese Society for Immunology
2013, Chiba, Japan. December 11th, 2013.

Regulation of large scale chromatin structures by Polycomb
The PcG Repressive Complex-1 (PRC1) forms microscopically visible clusters in nuclei;
however, the impact of this cluster formation on transcriptional regulation and the underlying mechanisms that regulate this process remain obscure. We found that the sterile
Alpha Motif (SAM) domain of a PRC1 core component, Phc2, plays an essential role for
PRC1 clustering through head-to-tail macromolecular polymerization, which is essential for PRC1 clustering, recruitment of PRC1 to target genes and robust gene silencing
activity. We propose a novel role for SAM domain polymerization in this repression by
two distinct mechanisms, firstly, through capture and/or retaining of PRC1 at the PcG
targets, and, secondly, by strengthening the interactions between PRC1 and PRC2 to stabilize transcriptional repression. Our findings reveal a previously unknown regulatory
mechanism mediated by SAM domain polymerization for PcG-mediated repression ofdevelopmental loci that enables a robust yet reversible gene repression program during
development.
Chromatin dynamics associated with activation of Polycomb-repressedgenes
PcG proteins mediate repression of developmental regulators in a reversible manner.
However, it is poorly understood how PcG-repressed genes are activated by developmental cues. We used the Meis2 gene as a model to identify the role of a tissue-specificenhancer in removing PcG from its promoter. Meis2 repression in early development depends on binding of RING1B, an essential E3 component of PcG, to its promoter, coupledwith its association with another RING1B-binding site (RBS) at the 3’ end of the Meis2
gene. During early midbrain development, a midbrain-specific enhancer (MBE) transiently associates with the promoter/RBS, forming a promoter/MBE/RBS tripartite interaction in a RING1-dependent manner. Subsequently, RING1B-bound RBS dissociatesfrom the tripartite, leaving promoter/MBE engagement to activate Meis2 expression. This
study therefore demonstrates the role of PcG and/or related factors in Meis2 activation by
regulating the topological transition of cis-regulatory elements.

19

Laboratory for

Lab activities

Integrative Genomics
Group Director : Osamu Ohara

Figure: Real-time imaging of IL-1β secretion
A conceptual diagram of the platform for real-time
imaging of protein secretion at single-cell resolution.
Secreted protein was captured on site by antibody
immobilized on the bottom of a microwell. Captured
protein was immediately recognized by fluorescently-labelled detection antibody and visualized by totalinternal reflection illumination.

Recent Major Publications
Shirasaki Y, Yamagishi M, Shimura N, Hijikata A, Ohara
O. Toward an understanding of immune cell sociology:
real-time monitoring of cytokine secretion at the single-cell level. IUBMB Life 65, 28-34 (2013)
Saito Y, Yuki H, Kuratani M, Hashizume Y, Takagi S,
Honma T, Tanaka A, Shirouzu M, Mikuni J, Handa N,
Ogahara I, Sone A, Najima Y, Tomabechi Y, Wakiyama
M, Uchida N, Tomizawa-Murasawa M, Kaneko A,
Tanaka S, Suzuki N, Kajita H, Aoki Y, Ohara O, Shultz LD,
Fukami T, Goto T, Taniguchi S, Yokoyama S, Ishikawa F.
A pyrrolo-pyrimidine derivative targets human primary
AML stem cells in vivo. Sci Transl Med 5, 181ra52(2013)
Kamae C, Nakagawa N, Sato H, Honma K, Mitsuiki N,
Ohara O, Kanegane H, Pasic S, Pan-Hammarström Q,
van Zelm MC, Morio T, Imai K, Nonoyama S. Common
variable immunodeficiency classification by quantifying
T-cell receptor and immunoglobulinκ-deleting recombination excision circles. J Allergy Clin Immunol 131,
1437-40.e5 (2013)

Invited Presentations
Ohara O. Systems immunology: Tackling the complexityof the immune system. 2012 Annual Meeting of theJapanese Society for Immunology, Kobe, Japan. December 7th, 2012.
Ohara O. Exploration of disease causative mutations on
the basis of genomics. Annual Meeting of Japan Pediatric Society, Hiroshima, Japan. April 21st, 2013.
Ohara O. Perspectives of integrative genomics forunderstanding of dynamics of the biological system.
The 40th Annual Meeting of the Japanese Society of
Toxicology, Chiba, Japan. June 18th, 2013.
Ohara O. Post-GWAS animal models. 3rd Sardinian
Summer School, Pula, Italy. September 9th, 2013.
Ohara O. Next-generation DNA sequencing technology
and genomic analysis. 58th Annual Meeting of theJapan Society of Human Genetics, Sendai, Japan. November 23rd, 2013.

20

S

ince the preceding research center, Research Center for Allergy and Immunology, was launched more than 10 years ago, the very basic mission of the Laboratory for Integrative Genomics has consistently been to function as a “Gateway”
to genomics for biologists coming from other fields. To achieve this mission, we
have organized our research activities into three parts as follows: (1) central support activities; (2) strategic and collaborative research activities; and (3) exploratory research activities aimed at new technology development. Because this systemhas worked very well so far, we have continued to maintain it in the new IMS. As
for the central support activities (1), we have made every effort to introduce new
genomic technologies to the center according to the needs and the budget size ofthe center. The most prominent change in these three years is to incorporate massively parallel DNA sequencing (MPS) technology using a Roche 454 GS Junior,
Illumina MiSeq and HiSeq1500. As for the strategic research activities (2), ourgroup plays the role of “workhorse”, which drives the measurement pipeline ofcenter projects such as the Atopic Dermatitis project and the Primary Immunodeficiency (PID) project. In particular, we have developed a network of pediatriciansin Japan and functioned as a hub of the network in the PID project. As for the collaborative research activities (3), we have carried out many intramural as well as
extramural collaboration projects. As exploratory research activities (3), we havefocused our efforts on development of “single-cell” measurement technologies.
The most recent study enables us to monitor protein secretion processes from single cells in real-time (Fig.), which will be applied for analysis of autoinflammatory
diseases.

Laboratory for

Lab activities

Disease Systems ModelingGroup Director : Hiroaki Kitano

Figure: Partial map of the keratinocyte interaction network relevant to AD

L
Recent Major Publications
Hase T, Ghosh S, Yamanaka R, Kitano H. Harnessing Diversity towards the Reconstructing of Large Scale Gene
Regulatory Networks. PLoS Comput Biol 9, e1003361(2013)
Ghosh S, Matsuoka Y, Asai Y, Hsin KY, Kitano H. Toward
an integrated software platform for systems pharmacology. Biopharm Drug Dispos 34, 508-26 (2013)
Hsin KY, Ghosh S, Kitano H. Combining machine learning systems and multiple docking simulation packages
to improve docking prediction reliability for network
pharmacology. PLoS One 8, e83922 (2013)

Invited Presentations
Kitano H. Software platform for systems drug discovery.
Innovative Medicines Initiative (IMI): Workshop on
Translational Knowledge Management in Pharmaceutical R&D, Brussels, Belgium. July 12th, 2013.
Kitano H. Network-oriented drug discovery. Innovative
Medicines Initiative (IMI): Workshop on Translational
Knowledge Management in Pharmaceutical R&D, Brussels, Belgium. July 12th, 2013.
Kitano H. Systems Drug Discovery and Integrated Software Platform. Talk at ETH Zurich IMSB, Zurich, Switzerland. August 30th, 2013.
Kitano H. Mathematics for Planet Earth. Talks at RMIT
Melbourne and University of New South Wales, Melbourne and Sydney, Australia. October 7th and 10th,2013.
Kitano H. Grand Challenges of Systems Biology and Systems Biomedicine. World Health Summit 2013: Keynote
Lecture, Berlin, Germany. October 22nd, 2013.

DSM is responsible for systems modeling, computational infrastructure development
for IMS-focused projects, and exploratory research converging on the center’s goals.

· Understanding atopic dermatitis (AD) using systems biology by combining bottom-up(detailed modeling) and top-down (high-throughput data analysis) strategies is one of
our central activities.
· We are developing a detailed molecular interaction map of intra- and intercellular interactions, focused on keratinocytes and immune cells. So far, a draft map
for keratinocytes has been created; it continues to be updated based on current
literature, databases and maps previously developed. The map will serve as the
backbone for all our modeling and analysis tasks.
· We are also developing a range of large-scale analysis tools to integrate various
high-throughout data generated within IMS.
· We are creating a hierarchical dynamical model of skin representing healthy anddisease states thereby offering computational analysis of disease mechanisms andpotential therapeutic intervention. Comprehensive model development is underway to construct a detailed model incorporating existing hypotheses within thecurrent focus of experimental groups at IMS. Modeling will be extended to multi-scale settings.
· A large-scale molecular interaction map of the yeast stress response was created asa principal model of robustness inherent in eukaryotic organisms. Systemic network
analysis of the map to unravel robustness and signal separation machinery of stressresponse pathways are underway.
· A new robustness analysis combining a gTOW assay as developed for a novel high-precision computational modeling for budding yeast, with possible extension to mammalian cells.
· In collaboration with RIKEN’s FANTOM5 consortium, we are analyzing the Down
Syndrome (DS) -iPS to neuron data using systems biology approaches to understandthe time-dependent changes in the process of differentiation of a DS cell.
· Development of a series of computational infrastructures to support model development and data analysis at IMS is underway. “Project Rishi” aims to develop highly
intelligent systems to scan existing literature to extract knowledge of molecular interactions. It is based on novel large-scale text mining combined with multi-strategy
machine learning.

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Laboratory for

Lab activities

Immunogenetics
Team Leader : Hisahiro Yoshida

Figure: Pre-symptomatic disease development in
the Spade mutant
Pre-symptomatic events occurring in the Spade mutant
were detected by histological, cytological and molecular biological analysis and are summarized in the figure
as longitudinal steps.

Recent Major Publications
Watarai H, Sekine-Kondo E, Shigeura T, Motomura Y,
Yasuda T, Satoh R, Yoshida H, Kubo M, Kawamoto H,
Koseki H, Taniguchi M. Development and function ofinvariant natural killer T cells producing T(h)2- andT(h)17-cytokines. PLoS Biol 10, e1001255 (2012)
Sugiyama M, Nakato G, Jinnohara T, Akiba H, Okumura
K, Ohno H, Yoshida H. Expression pattern changes andfunction of RANKL during mouse lymph node microarchitecture development. Int Immunol 24, 369-78(2012)

Invited Presentations
Yoshida H. Skin homeostasis and atopic dermatitis
development. 8th RCAI-JSI International Symposium on
Immunology, Yokohama, Japan. June 27th, 2013.
Yoshida H. Skin homeostasis and atopic dermatitis
development. The 5th LJI & IMS-RCAI Workshop, Yokohama, Japan. October 30th, 2013.

22

U

sually disease onset is detected by symptomatic observation, however, before the onset of the disease, there must be an accumulation of many imperceptible pathogenic events in the human body as part of pre-symptomatic diseasedevelopment. If one could precisely monitor and understand the pre-symptomatic
longitudinal multiple events proceeding in a healthy individual, it should be possible to predict the timing of disease onset and to take measures to prevent it. It will
be beneficial for many of us in the coming era when everyone can know their own
genome sequence information and genetic risk factors shortly after birth.
In our laboratory, we had been working to identify the genetic factors for allergic and immune disease development by phenotype screening of chemical mutagen, N-ethyl N-nitrosourea (ENU), induced mutant mice on a C57BL/6J background. We have established an animal model of atopic dermatitis in which thedisease developed at approximately 8 weeks after birth, as detected by ear skindesquamation and scratching of the skin a few days after the first symptoms. This
phase is followed by a Th2 immune bias detected by serum IgE, IgG1 and histamine level elevation 3 weeks later, and 8 weeks after that, a Th1 immune bias
was also detected, with serum IgG2b and IgG2c elevation. At this final stage, thepathological findings identified a chronic skin inflammatory condition. Therefore
we have named this mutant Spade (Stepwise progressive atopic dermatitis).
Using this disease model, we are now trying to precisely define the pre-symptomatic events occurring not only in skin lesions but also in systemic immune
regulation, from birth to the onset of dermatitis. As shown in the figure, we have
now found that distinct pre-symptomatic events occur independently in epidermis, dermis, draining lymph node, and spleen and that the accumulation of allthese events leads to the onset of dermatitis in the late stage.

Laboratory for

Lab activities

Integrated
Bioinformatics
Team Leader : Todd D. Taylor

Figure: ORF-based approach for the taxonomic
assignment of reads of different lengths derived
from different regions of the genomic DNA
Read derived from an intergenic region (A), read containing a small 5’ region of an ORF (B), read containing
two partial ORFs at the 5’ and 3’ terminals and acomplete ORF in the middle (C), read containing only asingle complete ORF (D), read containing a long partial
ORF at one end (E), read obtained from within an ORF(F), read with sequencing error causing a single ORF
to split into two smaller ORFs (G). X, Y, Z, K, L, and M
are the genomes to which the ORFs showed matches.
The taxonomic IDs of the species of these genomes areused for making the taxonomic assignments and for
creating the taxonomic bins.

Recent Major Publications
Sharma VK, Kumar N, Prakash T, Taylor TD. Fast andaccurate taxonomic assignments of metagenomic sequences using MetaBin. PLoS One 7, e34030 (2012)
Fukuda S, Toh H, Taylor TD, Ohno H, Hattori M. Acetate-producing bifidobacteria protect the host from enteropathogenic infection via carbohydrate transporters.
Gut Microbes 3, 449-54 (2012)
Hariharan R, Simon R, Pillai MR, Taylor TD. Comparative analysis of DNA word abundances in four yeast
genomes using a novel statistical background model.
PLoS One 8, e58038 (2013)

Invited Presentations
Taylor TD. Metagenomics: an introduction and applications to health and the environment. School of
Biological Sciences, Universiti Sains Malaysia, Penang,
Malaysia. April 19th, 2013.
Taylor TD. Metagenomics and approaches to immunology. 8th RCAI-JSI International Symposium on Immunology, Yokohama, Japan. June 28th, 2013.
Taylor TD. Metagenomics and barcoding. Poznan Summer School of Bioinformatics: Molecular Evolution &Phylogenomics, Poznan, Poland. August 23rd, 2013.
Taylor TD. Online resources: databases, tools andpipelines; Introduction to the human genome projectand metagenomics. 1st Workshop in Bioinformatics,
Penang, Malaysia. December 16th, 2013.

T

he aims of the laboratory are to provide centralized bioinformatics support for theother research laboratories in this center, with a special emphasis on metagenomics
and bacterial genomics related projects. We will develop an infrastructure of pipelines
and tools for the efficient handling of both small-scale and large-scale datasets and forthe analysis of various types of data produced from expression profile analysis, transcription network analysis, biomarker discovery analysis, and so on, with a special emphasis on metagenomic and metatranscriptomic analysis. Until recently, the main area of
our research has been the analysis of various human health-and environmental- related
metagenomics systems. We are developing powerful, high-throughput open-source tools
and databases for the comprehensive analysis of metagenomic and bacterial genomic sequence data.
We have participated in various domestic and international metagenome, microbial
genome, and other genome projects, some of which are still ongoing, including analysis of 1) the human gut microbiome in 13 healthy Japanese individuals, 2) termite gut
bacteria and microbial fuel cell metagenomes, both which have the potential for waste
degradation and clean energy production, and 3) several other bacteria from a wide variety of environments. My team has developed several tools for high-throughput metagenomic and bacterial genomic sequence analysis, including 1) iMetaSys, a comprehensive
high-performance analysis pipeline and knowledgebase, 2) MetaBioME, a comprehensive platform to facilitate homology-based computational identification of novel homologous commercially useful enzymes (CUEs) from metagenomic datasets, and 3) MetaBin,
a program for fast, accurate and highly sensitive taxonomic assignments of metagenomic
sequences.
The ultimate goal of our research is to develop tools capable of efficiently processing
and analyzing high-throughput datasets leading to the modeling and prediction of behaviors and interactions of whole environments of microbes, in association with theirhosts (if applicable), metabolites, environmental conditions, and other relevant factors.

23

Laboratory for

Lab activities

Tissue Dynamics
Team Leader : Takaharu Okada

Figure: Intravital two-photon image of T cells
and dendritic cells in the lymph node
Pink and red cells are antigen-specific CD4+ and CD8+
T cells, respectively. Blue and Green cells are different
subsets of dendritic cells. Image volume: 400 μm x 400
μm x 50 μm.

Recent Major Publications
Kitano M, Okada T. Four-dimensional tracking of lymphocyte migration and interactions in lymph nodes
by two-photon microscopy. Methods Enzymol 506,
437-54 (2012)
Hirata E, Yukinaga H, Kamioka Y, Arakawa Y, Miyamoto
S, Okada T, Sahai E, Matsuda M. In vivo fluorescenceresonance energy transfer imaging reveals differential
activation of Rho-family GTPases in glioblastoma cell
invasion. J Cell Sci 125, 858-68 (2012)
Okada T, Moriyama S, Kitano M. Differentiation ofgerminal center B cells and follicular helper T cells asviewed by tracking Bcl6 expression dynamics. Immunol Rev 247, 120-32 (2012)

Invited Presentations
Okada T. Two-photon imaging of cellular dynamics during the adaptive immune response. RIKEN RCAI: Univ.
Michigan Medical Center Joint Workshop, Ann Arbor,
MI, USA. January 16th, 2013.
Okada T. 2-Photon imaging of adaptive immune responses. 2nd New Zealand-Japan Joint Immunology
Workshop, Auckland, New Zealand. February 27th,
2013.
Okada T. S1PR2-driven retention of Tfh cells in the germinal center and its contribution to antibody responses.
Gordon Research Conferences: T Follicular Helper Cells,
Hong Kong, China. July 22nd, 2013.
Okada T. Imaging of dendritic cells important forcytotoxic T cells. The 3rd International Symposium by
JSPS Core-to-Core Program: Cooperative International
Framework in TGF-β Family Signaling, Matsuyama,
Japan. October 29th, 2013.
Okada T. The role for follicular regulatory T cells in
immune homeostasis. Annual Meeting of the JapaneseSociety for Immunology 2013: International Symposium
8, Chiba, Japan. December 12th, 2013.

24

T

he goal of the laboratory is to understand the mechanisms regulating cell migration and interactions in the tissues that shape adaptive immune responses. Currently, we have limited understanding of how generation of immunologicalmemory and tolerance, two key features of adaptive immunity, are controlled bydynamic interactions among immune cells. For example, it is not understood how
dynamics of B cells and helper T cells contribute to generation of humoral immune
memory. As for cellular immunity mediated by cytotoxic T lymphocytes (CTLs),
little is known about cell-cell interactions that regulate the CTL differentiation balance between effector and memory cells. Furthermore, cell-cell interactions thatare required for peripheral tolerance of autoreactive B cells and CD8+ T cells arepoorly understood.
As a strategy for tackling the above questions, we use real time imaging, inparticular two-photon microscopy, to analyze cellular migration and interactionsin the tissues. This microscopy method has been revealing striking dynamics ofimmune cells in various organs, underlining the importance of this approach toresolve the complexity of the immune system. By applying the imaging strategy to
relevant mouse models, we aim to reveal immune cell dynamics that are critical for
generation of immunological memory and tolerance. For this aim, we have beendeveloping and studying mouse strains in which the dynamics of helper T cells specialized for B cell immune responses are perturbed, the differentiation balance of
CTLs is disrupted, cross-presenting dendritic cells (DCs) are fluorescently labeled
for in vivo imaging, or the dynamics and function of regulatory T (Treg) cell subsets
are perturbed to break peripheral tolerance of autoreactive B cells and CD8+ T cells.

Laboratory for

Lab activities

Integrated Cellular
Systems
Team Leader : Mariko Okada

Figure: Modeling of a signal-transcription network
Measurement, data integration and modeling in atheoretical framework for understanding of signal-dependent cell fate control (right). Mathematical modeland simulation of NF-κB network.

Recent Major Publications
Hiroshima M, Saeki Y, Okada-Hatakeyama M, Sako Y. Dynamically varying interactions between heregulin and ErbB
proteins detected by single-molecule analysis in living cells.
Proc Natl Acad Sci U S A 109, 13984-9 (2012)
Nomura M, Okada-Hatakeyama M. Phase responses ofoscillating components in a signaling pathway. Front
Physiol 4, 68 (2013)
Terada A, Okada-Hatakeyama M, Tsuda K, Sese J. Statistical significance of combinatorial regulations. Proc Natl
Acad Sci U S A 110, 12996-3001 (2013)

Invited Presentations
Okada M. A switch in NF-κB immune signaling. The First
Annual Winter q-bio Meeting, Honolulu, USA. February
21st, 2013.
Okada M. Regulation of cancer signaling network. The
72nd Annual Meeting of the Japanese Cancer Association,
Yokohama, Japan. October 3rd, 2013.
Shinohara H. Switch-like activity of NF-κB is generated by
CARMA1-TAK1-IKKβ positive feedback loop in BCR-signaling. Gradients and Signalling: from chemotaxis to development, Okinawa, Japan. November 14th, 2013.
Okada M. Multi-layered regulation of intracellular signaling
network. The 36th Annual Meeting of the Molecular Biology Society of Japan, Kyoto, Japan. December 3rd, 2013.

T

he aims of the laboratory are to define the general regulatory rules in signaltransduction-transcriptional networks in cell determination processes andto apply this knowledge of regulatory principles to the understanding and treatment of human diseases. For this purpose, we perform quantitative measurementsof the target biological system using various experimental methods and integratethese heterogeneous data by means of mathematical modeling. However, it is notalways the case that preexisting computational methods and theory are satisfactory to analyze the dynamic behavior of a particular biological system. Therefore,
we also develop computational algorithms that can be applied for the analysis ofactual biological data. In 2013, we developed several mathematical models of signal-transcription networks in immune cell development and cancer. In B cell signaling, based on quantitative experiments and mathematical modeling, we identified a positive feedback loop and a key molecule that induces switch activation of
the NF-κB transcription factor. We showed that a positive cooperativity in the signaling network plays an important role to determine individual cell fate (Science,
accepted). We also developed several computational methods. A newly developed
algorithm LAMP (limitless arity multiple-testing procedure) method enables us to
identify combinatorial regulation of transcription factors from time-series transcriptome data. In comparison with existing methods that can find only two-motif
combinations, our testing procedure contributed to finding larger fractions of regulatory pathways and TF complexes in human breast cancer. The method allows
us to find hidden modes of transcriptional regulation critical for determination of
specific cell stages.

Okada M. Signaling dynamics and ON/OFF transcriptional
regulation. The 3rd Open Symposium: Grant-in-Aid for Scientific Research on Innovative Areas- Protein Modificationsin Pathogenic Dysregulation of Signaling, Tokyo, Japan.
January 25th, 2014.

25

Laboratory for

Lab activities

Molecular Live-Cell
Quantification
Team Leader : Makio Tokunaga

Figure: Actin-related protein (Arp4) is an essential component of the ATP-dependent chromatin-remodeling complexes (CRC).
(A) Arp4 is thought to be involved in transcription
control and DNA damage repair. (B) (C) Fluorescence
images of Arp4-EGFP expressing HeLa cells using HILO
microscopy. Scale bar: 10 μm Arp4 is localized both in
the nucleus and cytoplasm (C), while single moleculesof Arp4 are detected in the nucleus (C).

Recent Major Publications
Hotta K, Nashimoto A, Yasumura E, Suzuki M, Azuma
M, Iizumi Y, Shima D, Nabeshima R, Hiramoto M,
Okada A, Sakata-Sogawa K, Tokunaga M, Ito T, Ando
H, Sakamoto S, Kabe Y, Aizawa S, Imai T, Yamaguchi Y,
Watanabe H, Handa H. Vesnarinone Suppresses TNFαmRNA Expression by Inhibiting Valosin-containing Protein. Mol Pharmacol 83, 930-8 (2013)
Asakawa H, Yang HJ, Yamamoto TG, Ohtsuki C,
Chikashige Y, Sakata-Sogawa K, Tokunaga M, Iwamoto
M, Hiraoka Y, Haraguchi T. Characterization of nuclear
pore complex components in fission yeast Schizosaccharomyces pombe. Nucleus 5, 1-14 (2014)

Invited Presentations
Tokunaga M, Fukagawa A, Sakata-Sogawa K. Dynamic
and entropic molecular interactions by single molecule
studies. The 69th Annual Meeting of the Japanese Society of Microscopy: Symposium Session on technologyof super-resolution microscopy and its application in
biology, Suita, Japan. May 20th, 2013.
Tokunaga M. Single molecule imaging ― TIRF andHILO microscopy ―. The 21st Cell Biology Workshop:
Training Course of Fluorescence Microscopy I, Kobe,
Japan. August 10th, 2013.
Tokunaga M. Seeing the Dynamic Face of Life by viewing a Single Molecule with Light. Symposium open to
the Public: The structure of Life Forms that manipulate
their own DNA, Suita, Japan. August 25th, 2013.

26

B

ased on emerging techniques in molecular imaging, our long-term goal is the understanding of transcriptional regulation related to cell signaling. To achieve thisgoal we quantify the interactions of signaling molecules and transcriptional regulators.
TCR microcluster formation
T cell receptor (TCR) microclusters are important to initiate and sustain T cell activation.
Although various signaling molecules were found enriched in microclusters, the mechanism of microcluster formation remains unclear. To visualize the dynamics of membraneproteins related to microcluster formation, we performed simultaneous single molecule
imaging of TCR and the tyrosine phosphatase CD45 with GFP labeled microclusters.
CD45 is known to function as an initiator of TCR activation and to be excluded from TCR
microclusters. On the activated T cell surface, TCR molecules showed slower diffusion
and longer residence time in microclusters than CD45, suggesting dynamic interaction of
TCR molecules with the microcluster. We are planning to clarify the mechanism of TCR
microcluster formation.
Transcriptional control by actin-related proteins
Actin-related proteins (Arps) are evolutionarily and structurally similar to actin. In the
nucleus, Arp4 and actin are critical components of ATP-dependent chromatin-remodeling complexes, which regulate the dynamic modification of chromatin architecture. Arp4
is reported to play a key role as a regulator of chromatin remodeling, possibly through its
direct binding to histones, however, the detailed mechanisms remain elusive. Aiming toclarify the dynamics of Arp4 in transcriptional control, we performed a quantitative imaging analysis of Arp4 in the nucleus. Both single molecule imaging by HILO microscopy
and FRAP analysis revealed dynamic movements of Arp4. After stimulation with PMA,an activator of transcription, the residence time of Arp4 became shorter. These results
suggest dynamic interactions of Arp4 with either chromatin-remodeling complexes or
chromatin.

Laboratory for

Lab activities

Genotyping
Development
Group Director : Michiaki Kubo

Figure: Illumina Human Genotyping Array system (left) and HiSeq 2500 System (right)

Recent Major Publications
Kou I, Takahashi Y, Johnson TA, Takahashi A, Guo L, Dai
J, Qiu X, Sharma S, Takimoto A, Ogura Y, Jiang H, Yan
H, Kono K, Kawakami N, Uno K, Ito M, Minami S, Yanagida H, Taneichi H, Hosono N, Tsuji T, Suzuki T, Sudo
H, Kotani T, Yonezawa I, Londono D, Gordon D, Herring
JA, Watanabe K, Chiba K, Kamatani N, Jiang Q, Hiraki
Y, Kubo M, Toyama Y, Tsunoda T, Wise CA, Qiu Y, Shukunami C, Matsumoto M, Ikegawa S. Genetic variants
in GPR126 are associated with adolescent idiopathic
scoliosis. Nat Genet 45, 676-9 (2013)
Ellinghaus D, Baurecht H, Esparza-Gordillo J, Rodríguez
E, Matanovic A, Marenholz I, Hübner N, Schaarschmidt
H, Novak N, Michel S, Maintz L, Werfel T, Meyer-Hoffert U, Hotze M, Prokisch H, Heim K, Herder C, Hirota
T, Tamari M, Kubo M, Takahashi A, Nakamura Y, Tsoi
LC, Stuart P, Elder JT, Sun L, Zuo X, Yang S, Zhang X,
Hoffmann P, Nöthen MM, Fölster-Holst R, Winkelmann
J, Illig T, Boehm BO, Duerr RH, Büning C, Brand S, Glas
J, McAleer MA, Fahy CM, Kabesch M, Brown S, McLean
WH, Irvine AD, Schreiber S, Lee YA, Franke A, Weidinger
S. High-density genotyping study identifies four new
susceptibility loci for atopic dermatitis. Nat Genet 45,
808-12 (2013)
Perera MA, Cavallari LH, Limdi NA, Gamazon ER,
Konkashbaev A, Daneshjou R, Pluzhnikov A, Crawford
DC, Wang J, Liu N, Tatonetti N, Bourgeois S, Takahashi
H, Bradford Y, Burkley BM, Desnick RJ, Halperin JL,
Khalifa SI, Langaee TY, Lubitz SA, Nutescu EA, Oetjens
M, Shahin MH, Patel SR, Sagreiya H, Tector M, Weck KE,
Rieder MJ, Scott SA, Wu AH, Burmester JK, Wadelius M,
Deloukas P, Wagner MJ, Mushiroda T, Kubo M, Roden
DM, Cox NJ, Altman RB, Klein TE, Nakamura Y, Johnson
JA. Genetic variants associated with warfarin dose in
African-American individuals: a genome-wide association study. Lancet 382, 790-6 (2013)O

ur team established a high-throughput SNP genotyping system using a combined method of multiplex-PCR and the Invader assay during the era of
the SNP Research Center (FY2000-2007). Using this system, our team contributed to the establishment of the JSNP database (http://snp.ims.u-tokyo.ac.jp/
index_ ja.html) and to the success of the International HapMap Project Phase 1(http://hapmap.ncbi.nlm.nih.gov/). From FY2003, our team has been working
as the main facility of genomic research for the BioBank Japan project and has
generated a large amount of SNP genotyping data for the association studies ofcommon diseases. In addition, since 2008 we have been performing genotypingof samples collected by NIH for pharmacogenetic study, Pharmacogenomics Research Network (PGRN), under the PGRN-RIKEN CGM Global Alliance (http://
bts.ucsf.edu/pgrn-cgm/). Moreover, we developed a new genotyping method todetect copy number variation (RETINA) that was published in Human Mutation(29:182-9, 2008). Using RETINA, we developed a new genotyping method for the
CYP2D6 gene, which has many functional variations combined with copy number variations. Since CYP2D6 is associated with the metabolism of many drugs,
CYP2D6 activity estimated by our method will help in establishing personalized
drug treatment, the right drug and the right dose. We also developed a rapid SNP
genotyping system for clinical research and are performing several clinical intervention studies based on the genotype information in the Genome-guided drug
Treatment Optimization Program (G-TOP) funded by MEXT. Our hope is to implement Genomic Medicine that will optimize medical care and health by use of
genomic information.

27

Laboratory for

Lab activities

Genome Sequencing
Analysis
Team Leader : Hidewaki Nakagawa

Recent Major Publications
Akamatsu S, Takata R, Haiman CA, Takahashi A, Inoue
T, Kubo M, Furihata M, Kamatani N, Inazawa J, Chen
GK, Le Marchand L, Kolonel LN, Katoh T, Yamano Y,
Yamakado M, Takahashi H, Yamada H, Egawa S, Fujioka T, Henderson BE, Habuchi T, Ogawa O, Nakamura Y,
Nakagawa H. Common variants at 11q12, 10q26 and3p11.2 are associated with prostate cancer susceptibility in Japanese. Nat Genet 44, 426-9 (2012)
Akamatsu S, Takahashi A, Takata R, Kubo M, Inoue T,
Morizono T, Tsunoda T, Kamatani N, Haiman CA, Wan
P, Chen GK, Le Marchand L, Kolonel LN, Henderson BE,
Fujioka T, Habuchi T, Nakamura Y, Ogawa O, Nakagawa
H. Reproducibility, performance, and clinical utility ofa genetic risk prediction model for prostate cancer inJapanese. PLoS One 7, e46454 (2012)
Fujimoto A, Totoki Y, Abe T, Boroevich KA, Hosoda F,
Nguyen HH, Aoki M, Hosono N, Kubo M, Miya F, Arai
Y, Takahashi H, Shirakihara T, Nagasaki M, Shibuya T,
Nakano K, Watanabe-Makino K, Tanaka H, Nakamura
H, Kusuda J, Ojima H, Shimada K, Okusaka T, Ueno M,
Shigekawa Y, Kawakami Y, Arihiro K, Ohdan H, Gotoh
K, Ishikawa O, Ariizumi S, Yamamoto M, Yamada T,
Chayama K, Kosuge T, Yamaue H, Kamatani N, Miyano
S, Nakagama H, Nakamura Y, Tsunoda T, Shibata T, Nakagawa H. Whole genome sequencing of liver cancers
identifies etiological influences on mutation patterns
and recurrent mutations in chromatin regulators. Nat
Genet 44, 760-4 (2012)

Invited Presentations
Nakagawa H. Comprehensive genomic analysis of livercancers with biliary phenotype. International Symposium on Cholangiocarcinoma Tokyo 2013, Tokyo, Japan.
February 9th, 2013.
Nakagawa H. Whole genome signatures of liver cancer
and personalized medicine. Cancer genomics focusedon lung cancer. Roche Korea: 12th Round Asia Oncology
Forum 2013, Seoul, Korea. August 23rd and 24th, 2013.
Nakagawa H. Whole genome sequencing analysis ofcancer and its interpretation. 72nd Annual Meeting of
the Japanese Cancer Association, Yokohama, Japan.
October 5th, 2013.
Nakagawa H. Whole genome sequencing analysis ofliver cancer, forwarding to personalized medicine. BGI:
8th International Conference on Genomics, Shenzen,
China. November 1st, 2013.
Nakagawa H. Whole genome sequencing analysis ofliver cancer, forwarding to personalized medicine. Yonsei Institute 20th Annual International Symposium: A
Roadmap to the Personalized Cancer Treatment, Seoul,
Korea. November 8th, 2013.

28

Figure: Whole genome
structure of one virus-related liver cancer
Circos plot showing intra- orinter-chromosomal rearrangements and copy number alterations in each chromosome(1~22, X, and Y).

W

e have been organizing a cancer genome project for liver cancer as a Japanese
ICGC project by collaborating with the National Cancer Research Center and the
University of Tokyo. By operating three HiSeq sequencers at full capacity, as of 2013 wecompleted 270 whole genome sequencing (WGS) sets of liver cancers (x35 for cancer
and x30 for blood). We and the Laboratory for Medical Science Mathematics performed
mathematical analysis for the WGS dataset and demonstrated a comprehensive landscapeof diverse phenotypes of liver cancers with multiple etiological backgrounds (virus-related and non-viral, hepatocellular carcinoma and biliary phenotype) for point mutations,short indels, copy-number alterations, structural variations, and virus integrations. On
average 9,700 somatic substitutions and indels were called per each tumor, and mutational signatures of liver cancer, especially multi-centric (MC) tumors, indicated an influence of etiological background on somatic mutation patterns (Fujimoto et al. Nat Genet2012) and a strong impact of chronic inflammation on the mutation process in cancer
development. We systematically performed integrated and comparative analyses of wholegenomes and transcriptomes (RNA-seq) of HBV-related liver cancers and their matched
controls, which provided strong evidence that various types of genomic mutations could
trigger diverse transcriptional changes. Some silent mutations in coding regions, deep
intronic mutations and structural changes caused splicing aberrations, and HBV integrations generated diverse patterns of virus-human fusion transcripts. These comparativeand complementary analyses provide a higher-resolution landscape of the cancer genome
and can improve the interpretation of mutational consequences of genomic alterations.
As of 2013, we also completed ~600 exome sequencing of several diseases including
arrhythmia, autoimmune diseases, and rare tumors by collaborating with other teams
within IMS. We were also involved with GWAS of prostate cancer and establishment ofa risk estimation model for prostate cancer by using 16 common SNPs (Akamatsu et al.
PLoS ONE 2012) and participated in global meta-analysis of prostate cancer GWAS.

Laboratory for

Lab activities

Medical Science
Mathematics
Group Director : Tatsuhiko Tsunoda

Figure: Development and application of our new
analysis methods

Recent Major Publications
Fujimoto A, Totoki Y, Abe T, Boroevich KA, Hosoda F,
Nguyen HH, Aoki M, Hosono N, Kubo M, Miya F, Arai
Y, Takahashi H, Shirakihara T, Nagasaki M, Shibuya T,
Nakano K, Watanabe-Makino K, Tanaka H, Nakamura
H, Kusuda J, Ojima H, Shimada K, Okusaka T, Ueno M,
Shigekawa Y, Kawakami Y, Arihiro K, Ohdan H, Gotoh
K, Ishikawa O, Ariizumi S, Yamamoto M, Yamada T,
Chayama K, Kosuge T, Yamaue H, Kamatani N, Miyano
S, Nakagama H, Nakamura Y, Tsunoda T, Shibata T, Nakagawa H. Whole Genome Sequencing of Liver Cancers
Identifies Etiological Influences on Mutation Patterns
and Recurrent Mutations in Chromatin Regulators. Nat
Genet 44, 760-4 (2012)
Shigemizu D, Fujimoto A, Akiyama S, Abe T, Nakano K,
Boroevich KA, Yamamoto Y, Furuta M, Kubo M, Nakagawa H, Tsunoda T. A practical method to detect SNVs
and indels from whole genome and exome sequencing
data. Sci Rep 3, 2161 (2013)
Kou I, Takahashi Y, Johnson TA, Takahashi A, Guo L, Dai
J, Qiu X, Sharma S, Takimoto A, Ogura Y, Jiang H, Yan
H, Kono K, Kawakami N, Uno K, Ito M, Minami S, Yanagida H, Taneichi H, Hosono N, Tsuji T, Suzuki T, Sudo
H, Kotani T, Yonezawa I, Londono D, Gordon D, Herring
JA, Watanabe K, Chiba K, Kamatani N, Jiang Q, Hiraki
Y, Kubo M, Toyama Y, Tsunoda T, Wise CA, Qiu Y, Shukunami C, Matsumoto M, Ikegawa S. Genetic variants
in GPR126 are associated with adolescent idiopathic
scoliosis. Nat Genet 45, 676-9 (2013)

Invited Presentations
Tsunoda T. Genomic medicine’s milestones and future.
International Conference on Bioinformatics 2012 (InCoB
2012): Keynote lecture, Bangkok, Thailand. October
3rd, 2012.
Tsunoda T. Supercomputing accelerates genomic medicine. 4th Biosupercomputing Symposium: International
Symposium for Next-Generation Integrated Simulationof Living Matter (ISLiM), Tokyo, Japan. December 5th,2012.
Tsunoda T. Whole genome sequencing and comprehensive mutation analysis of liver cancer. 2013 SNUCRI &SNUCH Cancer Symposium, Jeju, Korea. May 3rd, 2013.
Tsunoda T. Whole Genome Approach Is Revolutionizing
Medicine. The 10th International Workshop on Advanced Genomics, Tokyo, Japan. May 21st, 2013.
Tsunoda T. Whole genome and exome sequence analysis. The 58th Annual Meeting of the Japan Society of
Human Genetics, Sendai, Japan. November 23rd, 2013.

O

ur research centers around the development of mathematical methods to enhance
understanding of genetic variation and its impact on human diseases. To enhanceongoing GWAS analyses, we developed parallelized imputation pipelines to infer data forungenotyped SNPs using 1000 Genomes haplotypes. Further, we developed methods toextract independent loci from the high-density data and applied these methods to a number of GWAS, e.g. [3]. We recently published a type 2 diabetes (T2D) study using thesemethods to search for novel variants and to better understand existing T2D association
signals. That study discovered three novel loci associated with T2D (Human Molecular Genetics, 23: 239-246, 2014). Recently, massively parallel sequencing technology has
allowed creation of comprehensive catalogs of genetic variation. However, to overcome
relatively high sequencing error rates, sophisticated analysis methods are required. Expanding on our analysis of the first report of a Japanese individual’s whole-genome sequence (Nature Genetics 42: 931-936, 2010), we developed methods for detecting SNVs
and short indels in whole genome and exome sequencing data for single sample calling[2]. The results showed high concordance with genotyping arrays, and Sanger sequencing
revealed extremely low false positive and negative rates. As an International Cancer Genome Consortium (ICGC) member (Nature 464: 993-998, 2010), we constructed an analytical pipeline based on this method to detect somatic alterations and viral-integrationsites in cancer genomes. We reported our analysis of 27 hepatocellular carcinoma (HCC)
genomes, 25 of which were associated with hepatitis B or C virus infection, including two
sets of multicentric tumors, using this pipeline [1]. After adjusting for gene-length andbackground mutation frequency, we compiled a long-tailed list of recurrently mutatedgenes. Through gene set enrichment analysis of deleterious mutated genes, we found asignificant overrepresentation of chromatin regulators. Furthermore, principal component analysis found that the substitution patterns of the 27 HCCs correlate with the etiological backgrounds from which the tumors developed.

29

Laboratory for

Lab activities

Statistical Analysis
Team Leader : Atsushi Takahashi

Figure: Representative results of GWAS Analysis

Recent Major Publications
Hirota T, Takahashi A, Kubo M, Tsunoda T, Tomita K,
Sakashita M, Yamada T, Fujieda S, Tanaka S, Doi S, Miyatake A, Enomoto T, Nishiyama C, Nakano N, Maeda
K, Okumura K, Ogawa H, Ikeda S, Noguchi E, Sakamoto
T, Hizawa N, Ebe K, Saeki H, Sasaki T, Ebihara T, Amagai
M, Takeuchi S, Furue M, Nakamura Y, Tamari M. Genome-Wide Association Study Identifies Eight New
Susceptibility Loci for Atopic Dermatitis in the Japanese
Population. Nat Genet 44, 1222-6 (2012)Low SK, Chung S, Takahashi A, Zembutsu H, Mushiroda
T, Kubo M, Nakamura Y. Genome-Wide Association
Study of Chemotherapeutic Agent-Induced Severe
Neutropenia/Leucopenia for Patients in Biobank Japan.
Cancer Sci 104, 1074-82 (2013)
Low SK, Takahashi A, Ashikawa K, Inazawa J, Miki Y,
Kubo M, Nakamura Y, Katagiri T. Genome-Wide Association Study of Breast Cancer in the Japanese Population. PLoS One 8, e76463 (2013)

Invited Presentations
Takahashi A. Informatics in Genomic Epidemiology. The
58th Annual Meeting of the Japan Society of Human
Genetics, Sendai, Japan. November 21st, 2013.

30

T

he mission of our laboratory is to clarify the mechanisms of human diseases
and traits from the viewpoint of statistics and informatics. Recent technology
developments have enabled us to investigate human variations over the entire genome. Massive amounts of genomic data are available, and we try to identify thegenes associated with diseases/traits through performing analysis of these data.
Our center searches for genes associated with diseases and drug reactions. Ourlaboratory is in charge of performing GWAS and selecting candidate SNPs associated with diseases. We are performing many quality controls on the SNP data
based on statistical genetics and statistics to obtain interpretable results. Then ourlaboratory is performing case-control association studies or quantitative analyses.
The Biobank Japan project has collected approximately 200,000 patients with
47 diseases, along with phenotype information. IMS has genotyped a huge number of the SNPs of individuals in the Biobank Japan. By using these data, we try
to find new loci associated with diseases by GWAS. We also have constructed anddeveloped GWAS systems in IMS to perform the GWAS in a short time. Individual genotype data have been accumulated, and there are very large amounts ofgenomic data. Therefore, we have constructed a data system to manage all these
data. We are in charge of GWAS and statistical analysis and collaborate in many
projects/consortiums to find novel loci associated with diseases, drug reactions
and traits by GWAS
We are attempting to identify causal loci related to monogenic diseases by using next generation sequencing. We are in charge of development of new methods
and systems to perform such analysis from the viewpoint of statistics and informatics.

Laboratory for

Lab activities

Pharmacogenomics
Group Director : Taisei Mushiroda

Recent Major Publications
Kiyotani K, Mushiroda T, Imamura CK, Tanigawara Y,
Hosono N, Kubo M, Sasa M, Nakamura Y, Zembutsu H.
Dose-adjustment study of tamoxifen based on CYP2D6
genotypes in Japanese breast cancer patients. Breast
Cancer Res Treat 131, 137-45 (2012)

Figure: Strategy of a pharmacogenomics-guided approachto avoid adverse drug reactions: focusing on genomic biomarkers associated with risk ofskin rash induced by nevirapine(NVP), an anti-HIV therapeutic
reagent
A prospective clinical trial, GENPART Study was conducted in orderto validate a prediction method for
the NVP-induced skin rash using
genetic testing of HLA-B*35:05 andan SNP of CCHCR1, rs1576. Advancegenetic testing significantly reduced
the prevalence of NVP-induced skinrash from 18% in the control group
to 13% in the intervention group(P = 0.031), indicating the medical
usefulness of genetic testing using
the HLA-B*35:05 and CCHCR1
rs1576 markers.

Kiyotani K, Mushiroda T, Tsunoda T, Morizono T, Hosono N, Kubo M, Tanigawara Y, Imamura CK, Flockhart
DA, Aki F, Hirata K, Takatsuka Y, Okazaki M, Ohsumi
S, Yamakawa T, Sasa M, Nakamura Y, Zembutsu H.
A genome-wide association study identifies locus at
10q22 associated with clinical outcomes of adjuvant tamoxifen therapy for breast cancer patients in Japanese.
Hum Mol Genet 21, 1665-72 (2012)
Province MA, Goetz MP, Brauch H, Flockhart DA, Hebert
JM, Whaley R, Suman VJ, Schroth W, Winter S, Zembutsu H, Mushiroda T, Newman WG, Lee MT, Ambrosone
CB, Beckmann MW, Choi JY, Dieudonné AS, Fasching
PA, Ferraldeschi R, Gong L, Haschke-Becher E, Howell A,
Jordan LB, Hamann U, Kiyotani K, Krippl P, Lambrechts
D, Latif A, Langsenlehner U, Lorizio W, Neven P, Nguyen
AT, Park BW, Purdie CA, Quinlan P, Renner W, Schmidt
M, Schwab M, Shin JG, Stingl JC, Wegman P, Wingren
S, Wu AH, Ziv E, Zirpoli G, Thompson AM, Jordan VC,
Nakamura Y, Altman RB, Ames MM, Weinshilboum RM,
Eichelbaum M, Ingle JN, Klein TE. CYP2D6 Genotype
and Adjuvant Tamoxifen: Meta-analysis of Heterogeneous Study Populations. Clin Pharmacol Ther 95,
216-27 (2014)

Invited Presentations
Mushiroda T. Genetics and Genome-Guided Personalized Medicine in Japan. The 2nd Meeting of South
East Asian Pharmacogenomics Research Network (SEAPharm), Bangkok, Thailand. January 15th, 2013.
Mushiroda T. Future direction of pharmacogenomics:
identification of genes associated with drug responses
using GWAS. The 20th Annual Meeting of the JapaneseSociety for Gene Diagnosis and Therapy, Hamamatsu,
Japan. July 20th, 2013.
Mushiroda T. Avoidance of severe drug-induced skinrash based on pharmacogenomics. The 23rd Annual
Meeting of the Japanese Society of Pharmaceutical
Health Care and Sciences, Sendai, Japan. September
22nd, 2013.
Mushiroda T. Individualization of anticancer therapeutics based on genome-wide association study. The
72nd Annual Meeting of the Japanese Cancer Association: Core Symposia - Pharmacogenomics in Oncology,
Yokohama, Japan. October 4th, 2013.
Mushiroda T. Individualization of drug therapy basedon genome-wide association study. Joint Congress ofthe 23rd Annual Meeting of the Japanese Society ofClinical Neuropsychopharmacology and the 43rd Annual Meeting of the Japanese Society of Neuropsychopharmacology, Okinawa, Japan. October 25th, 2013.

A

dverse drug reactions (ADRs) are often unpredictable, owing to the fact that
responses to drugs vary among different individuals. However, it is believedthat applying knowledge of pharmacogenomics (PGx) to clinical treatment canhelp to improve predictions of efficacy and/or toxicity of drugs, leading to appropriate therapeutic regimens for individual patients and to contribute to improvement of our medical care. In fact, the U.S. Food and Drug Administration (FDA)
recommended genotyping of polymorphisms in drug-metabolizing enzymes andHLA prior to drug administration for avoidance of severe ADRs for several drugs,
such as irinotecan, atomoxetine, carbamazepine and abacavir. In attempts to identify genomic biomarkers that predict efficacy or risk of ADRs for various drugs,
such as neutropenia and leucopenia induced by cancer chemotherapeutic agentsand skin rash induced by anti-epileptics, we conduct genome-wide association
studies (GWAS) using single-nucleotide polymorphisms (SNPs), which are themost abundant polymorphisms in the human genome. To date, we have identified
several “probable valid” genomic biomarkers (HLA-B*35:05/CCHCR1 for nevirapine -induced skin rash, VKORC1/CYP2C9 for warfarin maintenance dosage,
HLA-A*31:01 for carbamazepine-induced skin rash, and CYP2D6 for tamoxifen
efficacy) that will be useful for PGx-guided drug therapy. In order to establish
PGx-based individualization of drug therapy, advantages of the genomic biomarkers should be demonstrated. Thus, we have conducted and are conducting prospective clinical trials that can evaluate the medical utility and cost-effectivenessof genetic testing. If physicians can predict in advance which patients are more
susceptible to ADRs, they could use alternative drugs or take particular care during the course of treatment to prevent the skin rash at an early stage, leading to safeand patient-friendly personalized treatment.

31

Laboratory for

Lab activities

International Alliance on
Genomic Research
Team Leader : Ming Ta Michael Lee

Figure: A New tool for joint tissue sectioning andseparation of articular cartilage and subchondralbone
Left panel: Custom tools for joint tissue sectioning thatcan cut any region of interest and a four-region model
system representing disease progression of OA. Right
Panel: sectioning and grinding for separating the overlaying cartilage and underlying subchondral bone.

Recent Major Publications
Cornejo-García JA, Liou LB, Blanca-López N, Doña
I, Chen CH, Chou YC, Chuang HP, Wu JY, Chen YT,
Plaza-Serón Mdel C, Mayorga C, Guéant-Rodríguez
RM, Lin SC, Torres MJ, Campo P, Rondón C, Laguna JJ,
Fernández J, Guéant JL, Canto G, Blanca M, Lee MT. Genome-wide association study in NSAID-induced acuteurticaria/angioedema in Spanish and Han Chinese populations. Pharmacogenomics 14, 1857-69 (2013)
Chou CH, Wu CC, Song IW, Chuang HP, Lu LS, Chang JH,
Kuo SY, Lee CH, Wu JY, Chen YT, Kraus VB, Lee MT. Genome-wide Expression Profiles of Subchondral Bone in
Osteoarthritis. Arthritis Res Ther 15, R190 (2013)
Chen CH, Lee CS, Lee MT, Ouyang WC, Chen CC, Chong
MY, Wu JY, Tan HK, Lee YC, Chuo LJ, Chiu NY, Tsang HY,
Chang TJ, Lung FW, Chiu CH, Chang CH, Chen YS, Hou
YM, Chen CC, Lai TJ, Tung CL, Chen CY, Lane HY, Su TP,
Feng J, Lin JJ, Chang CJ, Teng PR, Liu CY, Chen CK, Liu
IC, Chen JJ, Lu T, Fan CC, Wu CK, Li CF, Wang KH, Wu LS,Peng HL, Chang CP, Lu LS, Chen YT, Cheng AT. Variant
GADL1 and Response to Lithium Therapy in Bipolar I
Disorder. N Engl J Med 370, 119-28 (2014)

Invited Presentations
Lee MT. Applications of Pharmacogenetics. The 2nd
Meeting of South East Asian Pharmacogenomics
Research Network (SEAPharm), Bangkok, Thailand.
January 15th, 2013.

32

T

he main aim of our laboratory is to identify genetic associations with diseases; drug induced adverse events or drug efficacy. It is hoped that the discoveries from our research will identify useful biomarkers that could be used to predict
drug-induced adverse events, guide drug use and could also be used in disease
prediction/diagnosis. Another main focus of this laboratory is to promote collaborations both within Japan and internationally. We have set up collaborations/consortium in Asia (Southeast Asian Pharmacogenetics Consortium, SEAPharm)and Europe (Genomic Medicine Alliance) to foster collaboration with RIKEN andamong the participating research groups. We also actively recruit young scientists
from overseas to work at RIKEN and carry out research in SNP-based approaches,statistical analysis and biological analysis. Our goal is to establish an international
genetic research network that will make the Center for Integrative Medical Sciences a world leader in personalized medicine. Currently, our group’s main focus ison Pharmacogenetics (PGx) and the genetic study of complex diseases. For PGx
studies, we aim to establish a functional analysis platform that not only allows us
to study the interactions between HLA and drugs, but can also be used to confirm
the findings from genetic analysis for severe ADRs, many of which involve the
HLA molecules. In addition, we will use genome-wide association study to identify genetic variants associated with 1. Anti-infectious drug-induced liver injuries.
2. Phenytoin and co-trimoxazole induced Stevens-Johnson Syndrome (SJS) andToxic epidermal necrolysis. 3. Adverse reactions to non-steroidal anti-inflammatory drugs. For complex diseases, we aim to identify genetic variants associatedwith Hippocampal Sclerosis in Thais and are performing an epigenetic and biomarker study of osteoarthritis (OA).

Laboratory for

Lab activities

Cardiovascular Diseases
Group Director : Toshihiro Tanaka

Recent Major Publications
Onouchi Y, Ozaki K, Burns JC, Shimizu C, Terai M, Hamada H, Honda T, Suzuki H, Suenaga T, Takeuchi T, Yoshikawa N, Suzuki Y, Yasukawa K, Ebata R, Higashi K, Saji
T, Kemmotsu Y, Takatsuki S, Ouchi K, Kishi F, Yoshikawa
T, Nagai T, Hamamoto K, Sato Y, Honda A, Kobayashi
H, Sato J, Shibuta S, Miyawaki M, Oishi K, Yamaga H,
Aoyagi N, Iwahashi S, Miyashita R, Murata Y, Sasago
K, Takahashi A, Kamatani N, Kubo M, Tsunoda T, Hata
A, Nakamura Y, Tanaka T. A genome-wide association
study identifies three new risk loci for Kawasaki disease. Nat Genet 44, 517-21 (2012)
Ellinor PT, Lunetta KL, Albert CM, Glazer NL, Ritchie MD,
Smith AV, Arking DE, Müller-Nurasyid M, Krijthe BP,
Lubitz SA, Bis JC, Chung MK, Dörr M, Ozaki K, Roberts
JD, Smith JG, Pfeufer A, Sinner MF, Lohman K, Ding J,
Smith NL, Smith JD, Rienstra M, Rice KM, Van Wagoner
DR, Magnani JW, Wakili R, Clauss S, Rotter JI, Steinbeck
G, Launer LJ, Davies RW, Borkovich M, Harris TB, Lin
H, Völker U, Völzke H, Milan DJ, Hofman A, Boerwinkle
E, Chen LY, Soliman EZ, Voight BF, Li G, Chakravarti A,
Kubo M, Tedrow UB, Rose LM, Ridker PM, Conen D,
Tsunoda T, Furukawa T, Sotoodehnia N, Xu S, Kamatani
N, Levy D, Nakamura Y, Parvez B, Mahida S, Furie KL,
Rosand J, Muhammad R, Psaty BM, Meitinger T, Perz
S, Wichmann HE, Witteman JC, Kao WH, Kathiresan S,
Roden DM, Uitterlinden AG, Rivadeneira F, McKnight
B, Sjögren M, Newman AB, Liu Y, Gollob MH, Melander
O, Tanaka T, Stricker BH, Felix SB, Alonso A, Darbar
D, Barnard J, Chasman DI, Heckbert SR, Benjamin EJ,
Gudnason V, Kääb S. Meta-analysis identifies six new
susceptibility loci for atrial fibrillation. Nat Genet 44,
670-5 (2012)

Invited Presentations
Ozaki K, Tanaka T. Genetic background of cardiovascular diseases. Taiwan Human Genetics Society 2013
Spring Symposium: Taiwan-Japan Joint Symposium on
BioBank and Genomic Medicine, Taipei, Taiwan. May
25th, 2013.
Tanaka T. Genetic Epidemiology ~understanding human diversity~. Seminar at Health Sciences Universityof Mongolia, Ulaanbaatar, Mongolia. September 2nd,
2013.
Ozaki K, Tanaka T. Genetic background of myocardial
infarction. International Conference on Personalized
Medicine and Global Health 2013, Astana, Kazakhstan.
October 17th, 2013.
Onouchi Y. Genetic Factors of Kawasaki Disease. The
2nd Oriental Congress of Pediatrics, Shanghai, China.
October 19th, 2013.

Figure: Manhattan plot of meta-analysis results
for genome-wide association with atrial fibrillation

S

ince heart diseases represent more than 15% of the cause of death in the Japanese population and more than 20% of the total medical expenses in Japan, it issocially important to discover the mechanisms of these disorders. We have been thefirst to reveal genetic background effects in myocardial infarction (MI), atrial fibrillation (AF), and Kawasaki disease (KD) by comprehensive genetic analyses of theJapanese population, followed by functional in vitro analyses. We also began studies
to identify genetic factors for arteriosclerosis obliterans, or peripheral artery disease (ASO). At present, our laboratory focuses on these four cardiovascular diseases. Our ultimate goal is to provide novel diagnostic/therapeutic approaches to suchpatients. To this end, we are extending our research area from genetics to molecularbiology, including in vivo analyses of genetically engineered mice. For MI, we aresearching for novel MI susceptibility genes, followed by functional analyses of thesegenes in vitro and in vivo, including knock-out or transgenic mouse techniques. We
have just started to develop a screening system to search for novel drugs that might
prevent coronary restenosis after percutaneous coronary intervention by inhibiting
cellular BRAP- I kappa B interactions. For AF, we are performing functional analyses of AF susceptible genes in vitro and in vivo. For ASO, we have identified two
novel loci associated with the disease and are currently performing fine mapping ofthese loci and functional assessment of the associated SNPs. For KD, we are trying
to identify genetic risk factors of resistance to IVIG therapy and developing coronary artery lesions.

Onouchi Y. Genetic studies of multifactorial disease.
58th Annual Meeting of the Japan Society for Premature and Newborn Medicine, Kanazawa, Japan. December 1st, 2013.

33

Laboratory for

Lab activities

Autoimmune Diseases
Team Leader : Kazuhiko Yamamoto

Recent Major Publications
Okada Y, Shimane K, Kochi Y, Tahira T, Suzuki A, Higasa
K, Takahashi A, Horita T, Atsumi T, Ishii T, Okamoto A,
Fujio K, Hirakata M, Amano H, Kondo Y, Ito S, Takada
K, Mimori A, Saito K, Kamachi M, Kawaguchi Y, Ikari K,
Mohammed OW, Matsuda K, Terao C, Ohmura K, Myouzen K, Hosono N, Tsunoda T, Nishimoto N, Mimori T,
Matsuda F, Tanaka Y, Sumida T, Yamanaka H, Takasaki
Y, Koike T, Horiuchi T, Hayashi K, Kubo M, Kamatani N,
Yamada R, Nakamura Y, Yamamoto K. A genome-wide
association study identified AFF1 as a susceptibility
locus for systemic lupus eyrthematosus in Japanese.
PLoS Genet 8, e1002455 (2012)
Myouzen K, Kochi Y, Okada Y, Terao C, Suzuki A, Ikari
K, Tsunoda T, Takahashi A, Kubo M, Taniguchi A, Matsuda F, Ohmura K, Momohara S, Mimori T, Yamanaka
H, Kamatani N, Yamada R, Nakamura Y, Yamamoto
K. Functional variants in NFKBIE and RTKN2 involvedin activation of the NF-κB pathway are associatedwith rheumatoid arthritis in Japanese. PLoS Genet 8,e1002949 (2012)
Okada Y, Wu D, Trynka G, Raj T, Terao C, Ikari K, Kochi
Y, Ohmura K, Suzuki A, Yoshida S, Graham RR, Manoharan A, Ortmann W, Bhangale T, Denny JC, Carroll RJ,
Eyler AE, Greenberg JD, Kremer JM, Pappas DA, Jiang L,
Yin J, Ye L, Su DF, Yang J, Xie G, Keystone E, Westra HJ,
Esko T, Metspalu A, Zhou X, Gupta N, Mirel D, Stahl EA,
Diogo D, Cui J, Liao K, Guo MH, Myouzen K, Kawaguchi
T, Coenen MJ, van Riel PL, van de Laar MA, Guchelaar
HJ, Huizinga TW, Dieudé P, Mariette X, Louis Bridges
Jr S, Zhernakova A, Toes RE, Tak PP, Miceli-Richard C,
Bang SY, Lee HS, Martin J, Gonzalez-Gay MA, Rodriguez-Rodriguez L, Rantapää-Dahlqvist S, Arlestig L,
Choi HK, Kamatani Y, Galan P, Lathrop M, Eyre S, Bowes
J, Barton A, de Vries N, Moreland LW, Criswell LA, Karlson EW, Taniguchi A, Yamada R, Kubo M, Liu JS, Bae
SC, Worthington J, Padyukov L, Klareskog L, Gregersen
PK, Raychaudhuri S, Stranger BE, De Jager PL, Franke L,
Visscher PM, Brown MA, Yamanaka H, Mimori T, Takahashi A, Xu H, Behrens TW, Siminovitch KA, Momohara
S, Matsuda F, Yamamoto K, Plenge RM. Genetics ofrheumatoid arthritis contributes to biology and drug
discovery. Nature 506, 376-81 (2014)

Invited Presentations
Yamamoto K. From Genetics to Functional Insights
into the Pathogenesis of Rheumatoid Arthritis. The
Advanced in Targeted Therapies Meeting, Nice, France.
April 12th, 2013.
Yamamoto K. Genetic information and functional understanding in Rheumatoid Arthritis. Symposium of the
Asia Pacific League of Associations for Rheumatology,
Bali, Indonesia. August 31st, 2013.

34

Figure: Understanding autoimmune diseases through eQTL studies
We focus on the expression of genes as an intermediate trait to understand the mechanism of autoimmune diseases.
We perform eQTL studies for each immune cell type by using next generation sequencing technologies such as RNAseq. By combining the data from GWAS and eQTL studies, we will unravel the mechanism of disease.

M

ost autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus, are multifactorial diseases involving both genetic
and environmental factors. The aim of our laboratory is to elucidate the etiologyof these autoimmune diseases by dissecting their genetic aspect. RA is one of themost common autoimmune diseases with inflammatory arthritis. While the HLADRB1 gene polymorphism is the major determinant of RA susceptibility, several
groups worldwide including our own have performed genome-wide association
studies (GWAS) to find non-HLA risk loci. However, each individual GWAS
lacked statistical power and a substantial proportion of risk loci remained undiscovered. In world-wide collaborations, we performed meta-analyses of GWAS andfound more than 100 risk loci for RA so far.
As GWAS could only indicate the presence of disease-associated variants in theloci, we further investigated these candidate loci to seek disease-causal variants
and elucidate their biological relevance to RA. Previous expression quantitative
trait loci (eQTL) studies that examined association between genetic variants andgene expression levels have suggested that the majority of autoimmune loci are
eQTLs, where disease causing variants affect expression of the responsible genes.
Therefore, we think it is essential to focus on “gene expression” as an intermediate
trait to dissect the etiology of “disease onset” (Fig.). We are currently undertaking
genome and transcriptome analysis of each immune cell type from Japanese individuals to establish eQTL catalogues of human immune cells. Furthermore, as theprecise function of the responsible genes in vivo has been not yet clarified for mostof the candidate loci, we are investigating the function of these genes including
PADI4, CD244 and FCRL3 by using disease-model mice. Our goal is to unravel
the pathological mechanisms of disease as well as to facilitate the development of
new therapies.

Laboratory for

Lab activities

Digestive Diseases
Team Leader : Kazuaki Chayama

Figure: Case-control association plots andlinkage disequilibrium (LD) map and genomic
structure of the DEPDC5 region on chromosome
22q12.2-3
Blue diamonds represent -log10 P obtained from the
GWAS and fine mapping (upper panel). The LD map
based on D’ was drawn using the genotype of the casesand controls in the GWAS samples (lower panel). The
candidate region is indicated by two black dashedlines. The landmark SNP (rs1012068) is indicated by
the red dotted line.

Recent Major Publications
Okazaki A, Hiraga N, Imamura M, Hayes CN, Tsuge M,
Takahashi S, Aikata H, Abe H, Miki D, Ochi H, Tateno C,
Yoshizato K, Ohdan H, Chayama K. Severe necroinflammatory reaction caused by natural killer cell-mediatedFas/Fas ligand interaction and dendritic cells in humanhepatocyte chimeric mouse. Hepatology 56, 555-66(2012)
Shi N, Hiraga N, Imamura M, Hayes CN, Zhang Y, Kosaka
K, Okazaki A, Murakami E, Tsuge M, Abe H, Aikata H,
Takahashi S, Ochi H, Tateno-Mukaidani C, Yoshizato K,
Matsui H, Kanai A, Inaba T, McPhee F, Gao M, Chayama
K. Combination therapies with NS5A, NS3 and NS5B inhibitors on different genotypes of hepatitis C virus in human hepatocyte chimeric mice. Gut 62, 1055-61 (2013)
Miki D, Ochi H, Takahashi A, Hayes CN, Urabe Y, Abe H,
Kawaoka T, Tsuge M, Hiraga N, Imamura M, Kawakami
Y, Aikata H, Takahashi S, Akuta N, Suzuki F, Ikeda K,
Kumada H, Karino Y, Toyota J, Tsunoda T, Kubo M,
Kamatani N, Nakamura Y, Chayama K. HLA-DQB1*03
Confers Susceptibility to Chronic Hepatitis C in Japanese: A Genome-Wide Association Study. PLoS One 8,
e84226 (2013)

Invited Presentations
Chayama K. Treatment of Chronic Hepatitis C with First
Generation Protease Inhibitors in Asian Population:
Telaprevir-Based Triple Therapy in Japanese Population.
Taiwan Digestive Disease Week, Taipei, Taiwan. October
5th, 2013.
Chayama K. HCV Genotype, Variations Associated with
IFN and DAA Resistance. Taiwan Association for the
Study of the Liver (TASL) Conference, Taipei, Taiwan.
December 6th, 2013.

T

he objectives of our laboratory are: 1. Identification of genes related to theeffects and side effects of antiviral agents, 2. Identification of genes related to
HCV-related hepatocellular carcinoma, and 3. Identification of genes related tochronic HCV infection.
Hepatitis C virus (HCV) infection is one of the major causes of chronic liverdiseases, which may lead to liver cirrhosis and hepatocellular carcinoma (HCC).
Type-I interferons have been used as antiviral agents for HCV infection. Recent
GWAS, including ours, have shown that variants in the inosine triphosphatase(ITPA) gene are associated with ribavirin induced anemia during pegylated interferon plus ribavirin combined therapy for HCV infection, which may lead to dose
reduction or therapy discontinuation. On the other hand IL28B variants have beenfound to be associated with treatment outcome of the therapy.
We assessed efficacy and predictive factors for sustained virological response(SVR) for teraplevir-based triple therapy and published a prediction model for theoutcome of pegylated interferon plus ribavirin combination therapy (Chayama, J
Infect Dis 2011; Ochi, J Infec Dis 2012).
To identify risk factors for HCV related HCC, we conducted a genome-wide
study and identified one intronic SNP in the DEPDC5 locus associated with HCC
risk (rs1012068, combined P=1.27 ×10−13, odds ratio: 1.75) (Miki, Nat Genet 2011).
To identify genetic risk factors for chronic HCV infection, we performed
GWAS in Japanese chronic HCV patients and controls. We found one intronic SNP
in the HLA-DQ locus associated with chronic HCV infection (combined P = 3.59 ×
10−16, odds ratio: 0.79). Subsequent analysis revealed another SNP rs1130380 with astronger association (odds ratio: 0.72). This SNP causes an amino acid substitution
in the HLA-DQB1 protein specific to the DQB1*03 allele, suggesting that a common amino acid substitution in HLA-DQB1 affects susceptibility to chronic HCV
infection in the Japanese population (Miki, PLOS ONE 2013 in press).

35

Laboratory for

Lab activities

Bone and Joint Diseases
Team Leader : Shiro Ikegawa

Recent Major Publications
Kou I, Takahashi Y, Johnson TA, Takahashi A, Guo L, Dai
J, Qiu X, Sharma S, Takimoto A, Ogura Y, Jiang H, Yan
H, Kono K, Kawakami N, Uno K, Ito M, Minami S, Yanagida H, Taneichi H, Hosono N, Tsuji T, Suzuki T, Sudo
H, Kotani T, Yonezawa I, Londono D, Gordon D, Herring
JA, Watanabe K, Chiba K, Kamatani N, Jiang Q, Hiraki
Y, Kubo M, Toyama Y, Tsunoda T, Wise CA, Qiu Y, Shukunami C, Matsumoto M, Ikegawa S. Genetic variants
in GPR126 are associated with adolescent idiopathic
scoliosis. Nat Genet 45, 676-9 (2013)

Figure: Phenotypes of azebrafish morphant (MO)of gpr126(a) A lateral view of zebrafish
with measurements of bodylength and eyeball diameter.
The eyeball diameter is anaverage of the maximum(white arrow) and minimum(yellow arrow) diameters. The
body length (b) and eyeballdiameter (c) of gpr126 andcontrol MOs at 14 dpf. Alizarin
red staining for the skeleton(d) and the number of mineralized vertebrae (e) at 14
dpf. Vertebral development
is significantly delayed in
gpr126 MO. *P< 0.01; NS: notsignificant.

Nakajima M, Mizumoto S, Miyake N, Kogawa R, Iida
A, Ito H, Kitoh H, Hirayama A, Mitsubuchi H, Miyazaki
O, Kosaki R, Horikawa R, Lai A, Mendoza-Londono R,
Dupuis L, Chitayat D, Howard A, Leal GF, CavalcantiD, Tsurusaki Y, Saitsu H, Watanabe S, Lausch E, Unger
S, Bonafé L, Ohashi H, Superti-Furga A, Matsumoto
N, Sugahara K, Nishimura G, Ikegawa S. Mutations in
B3GALT6, which encodes a glycosaminoglycan linker
region enzyme, cause a spectrum of skeletal and connective tissue disorders. Am J Hum Genet 92, 927-34(2013)
Song YQ, Karasugi T, Cheung KM, Chiba K, Ho DW, Miyake A, Kao PY, Sze KL, Yee A, Takahashi A, Kawaguchi
Y, Mikami Y, Matsumoto M, Togawa D, Kanayama M,
Shi D, Dai J, Jiang Q, Wu C, Tian W, Wang N, Leong JC,
Luk KK, Yip SP, Cherny SS, Wang J, Mundlos S, Kelempisioti A, Eskola PJ, Männikkö M, Mäkelä P, Karppinen
J, Järvelin MR, O'Reilly PF, Kubo M, Kimura T, Kubo T,
Toyama Y, Mizuta H, Cheah KS, Tsunoda T, Sham PC,
Ikegawa S, Chan D. Lumbar disc degeneration is linked
to a carbohydrate sulfotransferase 3 variant. J Clin
Invest 123: 4909-17 (2013)

Invited Presentations
Ikegawa S. Genetic study of lumbar disc herniation A
Japanese study. 1st Meeting of the International Spineand Pain Consortium (ISPC), Hong Kong, China. June
28th, 2013.
Ikegawa S. Genetics of bone and joint disease: From
genome to personalized medicine. ASBMR 2013 Annual Meeting, Baltimore, USA. October 4th, 2013.
Ikegawa S. Genetic risk factors for common skeletal disorders. Croucher foundation: advanced study institutes
symposium, Hong Kong, China. December 18th, 2013.

36

W

e are now mainly working on the identification of new susceptibility genes
for adolescent idiopathic scoliosis (AIS). AIS is a complex three-dimensional spinal deformity that occurs during the pubertal growth spurt. AIS is themost common pediatric skeletal disease, affecting > 2% of school-age children.
The etiology of AIS remains largely unknown; however, many clinical and genetic
studies suggest a contribution of genetic factors in the development of AIS.
We conducted a GWAS of AIS in Japanese and identified a single locus on
chromosome 10q24.31 that surpassed a genome-wide significance level of P < 5
× 10−8 (Takahashi et al. Nat Genet 2011). The association of the locus was subsequently replicated in two independent Chinese populations and our own international meta-analysis (Sharma et al. manuscript under review). The locus has
provided the most compelling evidence of association with AIS to date.
To identify additional AIS susceptibility loci, we extended our GWAS. Througha step-wise association study which included a total of ~1,800 cases and ~26,000
controls, we identified a new AIS locus on chromosome 6q24.1 in Japanese (P =2.25 × 10-10) (Kou et al. Nat Genet 2013). The most significantly associated SNP
was in GPR126 (G protein-coupled receptor 126) and its association was replicated in Han Chinese and Caucasian populations (combined P = 1.23 × 10-14).
GPR126 is highly expressed in cartilage and its knockdown in zebrafish caused
delayed ossification of the developing spine (Fig.). Our results should provide further insights into AIS etiology and pathogenesis.

Laboratory for

Lab activities

Endocrinology, Metabolism and Kidney Diseases
Team Leader : Shiro Maeda

Figure: Manhattan plot for the discovery analysis
of directly genotyped and imputed SNPs in 5,976
T2D cases and 20,829 controls
Known loci that reached genome-wide significance (P< 5 ×10-8) in the discovery analysis (5,976 T2D casesand 20,829 controls) are indicated in black font and thethree loci that reached genome-wide significance in the
combined analysis of discovery and follow-up analyses(19,094 cases and 31,417 controls) are indicated in red.

Recent Major Publications
Cho YS, Chen CH, Hu C, Long J, Ong RT, Sim X, TakeuchiF, Wu Y, Go MJ, Yamauchi T, Chang YC, Kwak SH, Ma
RC, Yamamoto K, Adair LS, Aung T, Cai Q, Chang LC,
Chen YT, Gao Y, Hu FB, Kim HL, Kim S, Kim YJ, Lee JJ,
Lee NR, Li Y, Liu JJ, Lu W, Nakamura J, Nakashima E,
Ng DP, Tay WT, Tsai FJ, Wong TY, Yokota M, Zheng W,
Zhang R, Wang C, So WY, Ohnaka K, Ikegami H, Hara K,
Cho YM, Cho NH, Chang TJ, Bao Y, Hedman ÅK, MorrisAP, McCarthy MI, DIAGRAM Consortium, MuTHER
Consortium, Takayanagi R, Park KS, Jia W, Chuang LM,
Chan JC, Maeda S, Kadowaki T, Lee JY, Wu JY, Teo YY,
Tai ES, Shu XO, Mohlke KL, Kato N, Han BG, Seielstad
M. Meta-analysis of genome-wide association studies
identifies eight new loci for type 2 diabetes in East
Asians. Nat Genet 44, 67-72 (2011)
Imamura M, Maeda S, Yamauchi T, Hara K, Yasuda K,
Morizono T, Takahashi A, Horikoshi M, Nakamura M,
Fujita H, Tsunoda T, Kubo M, Watada H, Maegawa
H, Okada-Iwabu M, Iwabu M, Shojima N, Ohshige T,
Omori S, Iwata M, Hirose H, Kaku K, Ito C, Tanaka Y,
Tobe K, Kashiwagi A, Kawamori R, Kasuga M, Kamatani
N, Diabetes Genetics Replication and Meta-analysis(DIAGRAM) Consortium, Nakamura Y, Kadowaki T. A
single-nucleotide polymorphism in ANK1 is associatedwith susceptibility to type 2 diabetes in Japanese populations. Hum Mol Genet 21, 3042-9 (2012)
Imamura M, Shigemizu D, Tsunoda T, Iwata M, Maegawa H, Watada H, Hirose H, Tanaka Y, Tobe K, Kaku
K, Kashiwagi A, Kawamori R, Maeda S. Assessing
the clinical utility of a genetic risk score constructedusing 49 susceptibility alleles for type 2 diabetes in a
Japanese population. J Clin Endocrinol Metab 98,
E1667-73 (2013)

Invited Presentations
Maeda S, Imamura M. Genetic characteristics of type2
diabetes in the Japanese. 47th Annual Post Graduate
Course: Advance in Diabetology, Yokkaichi, Japan. February 16th, 2013.
Maeda S. Genetic Study of type 2 diabetes in Japan.
Taiwan Human Genetics Society 2013 Spring Symposium: Taiwan-Japan Joint Symposium on BioBank and
Genomic Medicine, Taipei, Taiwan. May 25th, 2013.

Research activities for type 2 diabetes (T2D):
We have performed extended analyses of our previous GWAS (Nat Genet, 2010)
by increasing the number of examined SNPs to 2,229,890 SNPs with genotype imputation, and identified the ANK1 locus as a new locus for T2D (Imamura et al.
Hum Mol Genet, 2012). In an effort of the Asian Genetic Epidemiology Network(AGEN), 8 new loci were identified by a meta-analysis of East Asian GWAS (Choet al. Nat Genet, 2012). We further performed a larger scale Japanese GWAS for
T2D using 6,209,637 SNPs in 26,805 Japanese individuals (5,976 T2D and 20,829
controls). Combined discovery and follow-up analyses (30,392 cases and 34,814
controls) identified three new loci, MIR129-LEP, GPSM1 and SLC16A13 (Fig.).
To assess the clinical utility of GWAS-derived T2D susceptibility variants in
the Japanese population, we constructed a genetic risk scores (GRS) by summing
the susceptibility alleles of 49 SNP loci for T2D and examined the association of
the GRS with the disease by receiver operating characteristic (ROC) analyses using a logistic regression model. The area under the curve (AUC) for GRS alone(model-1) and for age, sex, and BMI (model-2) was 0.624 and 0.743, respectively. Addition of the GRS to model-2 resulted in a small but significant increase in
the AUC (∆AUC = 0.03, p = 7.99 × 10−15). These results indicate that currently
available genetic information slightly improves disease prediction ability, but is
not sufficiently robust for translation into clinical practice (Imamura et al. J Clin
Endocrinol Metab, 2013).
Other research activities:
We have also participated in the identification of susceptibility loci for diabetic
nephropathy, a biomarker for visceral obesity, and causal mutations for autosomal
dominant polycystic kidney disease in Japanese populations.

37

Laboratory for

Lab activities

Respiratory andAllergic Diseases
Team Leader : Mayumi Tamari

Recent Major Publications
Hirota T, Takahashi A, Kubo M, Tsunoda T, Tomita
K, Sakashita M, Yamada T, Fujieda S, Tanaka S, Doi
S, Miyatake A, Enomoto T, Nishiyama C, Nakano N,
Maeda K, Okumura K, Ogawa H, Ikeda S, Noguchi E,
Sakamoto T, Hizawa N, Ebe K, Saeki H, Sasaki T, Ebihara
T, Amagai M, Takeuchi S, Furue M, Nakamura Y, Tamari
M. Genome-wide association study identifies eight new
susceptibility loci for atopic dermatitis in the Japanese
population. Nat Genet 44, 1222-6 (2012)

Figure: Manhattan plot of
the GWAS for Japaneseatopic dermatitis
We conducted a GWAS anda validation study. A totalof eight new atopic dermatitis susceptibility loci wereidentified with genome-wide
significance: IL1RL1-IL18R1-IL18RAP, the MHC region,
OR10A3-NLRP10, GLB1-CCR4,
CCDC80, CARD11, ZNF365-EGR2
and CYP24A1-PFDN4.

Tomita K, Sakashita M, Hirota T, Tanaka S, Masuyama
K, Yamada T, Fujieda S, Miyatake A, Hizawa N, Kubo M,
Nakamura Y, Tamari M. Variants in the 17q21 asthma
susceptibility locus are associated with allergic rhinitis
in the Japanese population. Allergy 68, 92-100 (2013)
Ellinghaus D, Baurecht H, Esparza-Gordillo J, Rodríguez
E, Matanovic A, Marenholz I, Hübner N, Schaarschmidt
H, Novak N, Michel S, Maintz L, Werfel T, Meyer-Hoffert U, Hotze M, Prokisch H, Heim K, Herder C, Hirota
T, Tamari M, Kubo M, Takahashi A, Nakamura Y, Tsoi
LC, Stuart P, Elder JT, Sun L, Zuo X, Yang S, Zhang X,
Hoffmann P, Nöthen MM, Fölster-Holst R, Winkelmann
J, Illig T, Boehm BO, Duerr RH, Büning C, Brand S, Glas
J, McAleer MA, Fahy CM, Kabesch M, Brown S, McLean
WH, Irvine AD, Schreiber S, Lee YA, Franke A, Weidinger
S. High-density genotyping study identifies four new
susceptibility loci for atopic dermatitis. Nat Genet 45,
808-12 (2013)

Invited Presentations
Tamari M. Genome-wide association study of adultasthma. The 53rd Annual Meeting of the Japanese Respiratory Society, Tokyo, Japan. April 20th, 2013.
Tamari M. Genetic Study of Allergic Diseases. Taiwan
Human Genetics Society 2013 Spring Symposium: Taiwan-Japan Joint Symposium on BioBank and Genomic
Medicine, Taipei, Taiwan. May 25th, 2013.
Tamari M. Genome-wide association study of atopic
dermatitis. The 112th Annual Meeting of the Japanese
Dermatological Association: Educational Lectures - Atopic dermatitis, Kanagawa, Japan. June 15th, 2013.
Tamari M. Genomics in Allergic Disease. European
Academy of Allergy and Clinical Immunology & World
Allergy Organization World Allergy and Asthma Congress: Symposium 24 World Allergy Forum - Omics inAllergic Disease, Milan, Italy. June 23th, 2013.
Tamari M. Genome-Wide Association Study of Allergic
Diseases. 8th RCAI-JSI International Symposium on Immunology 2013, Yokohama, Japan. June 28th, 2013.

38

T

he aim of our project is to improve our understanding of the pathophysiologyof human respiratory and allergic diseases, which are caused by a combination of genetic and environmental factors. To elucidate the genetic components, wehave conducted genome-wide association studies (GWASs) and identified several
genetic loci for bronchial asthma and atopic dermatitis (Nat. Genet. 2011 & 2012).
A recent immunochip analysis for atopic dermatitis using European populations
revealed four new susceptibility loci, 4q27 (IL2-IL21), 11p13 (PRR5L), 16p13.13(CLEC16A-DEXI) and 17q21.32 (ZNF652). We conducted a replication study using a Japanese population and confirmed the associations at the 11p13, 16p13.13
and 17q21.32 loci (Nat. Genet. 2013). Candidate genes identified by the GWASs
and immunochip analysis suggest roles for barrier functions, innate-adaptive immunity, IL-1 family signaling, regulatory T cells, and the vitamin D pathway in thepathogenesis of allergic diseases. To investigate whether polymorphisms identified by the genetic studies could affect the susceptibility to and clinical phenotypesof diseases, we will conduct functional analyses and serological studies.
The first GWAS of asthma in the European population identified a locus on
chromosome 17q21.1. Subsequent replication studies have identified genetic variants on the 17q21.1 locus associated with asthma in different ethnic populations.
We reported that genetic variants in the 17q21.1 locus were associated with allergic rhinitis and were strongly correlated in cis with transcript levels of ORMDL3
in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines in a Japanese
population (Allergy 2013).We participate in the Global Alliance for Pharmacogenomics. We have conducted GWASs and identified several loci that are involved
in individual asthma treatment response.
We will conduct further cross-disciplinary studies combining genetics, immunology and clinical epidemiology for translation of research into clinical practice.

Laboratory for

Lab activities

Immune Regulation
Group Director : Masaru Taniguchi

Figure: Anti-tumor adjuvant effects mediated bysemi-allogeneic (129xB6) F1 ES-NKT cells
Semi-allogeneic (129xB6) F1 ES-derived NKT cells weretransferred into NKT-KO mice followed by stimulation
with OVA and α-GalCer/DCs. A significant increasein the number of OVA-CD8T cells and suppressionof OVA-bearing tumor growth was observed. Thus,
semi-allogeneic NKT cells can function in vivo even if
they are eliminated in a few days by the host immune
response.

Recent Major Publications
Watarai H, Sekine-Kondo E, Shigeura T, Motomura Y,
Yasuda T, Satoh R, Yoshida H, Kubo M, Kawamoto H,
Koseki H, Taniguchi M. Development and Functionof Invariant Natural Killer T cells Producing TH2- andTH17-cytokines. PLoS Biol 10, e1001255 (2012)
Oh-Hora M, Komatsu N, Pishyareh M, Feske S, Hori S,
Taniguchi M, Rao A, Takayanagi H. Agonist-Selected
T Cell Development Requires Strong T Cell Receptor
Signaling and Store-Operated Calcium Entry. Immunity
38, 881-95 (2013)
Fujii S, Shimizu K, Okamoto Y, Kunii N, Nakayama T,
Motohashi S, Taniguchi M. NKT cells as an ideal anti-tumor immunotherapeutic. Front Immunol 4, 409(2013)

Invited Presentations
Taniguchi M. NKT Cell-mediated Adjuvant Activity on
Antitumor Responses. Federation of Clinical Immunology Societies (FOCIS2012), Vancouver, Canada. June
22nd, 2012.
Taniguchi M. NKT cells as an ideal target for anti-tumor
immunotherapy. 15th International Congress of Immunology, Milan, Italy. August 25th, 2013.
Taniguchi M. Characterization of the cell with NKT cellpotential in DN1 thymic fraction. 7th International Symposium on CD1 and NKT cells, Tours, France. September
14th, 2013.
Taniguchi M. Discovery of NKT cells and their clinicalapplication. 12th International Symposium on Sjögren's
Syndrome, Kyoto, Japan. October 9th, 2013.
Taniguchi M. RIKEN IMS-RCAI Medical Innovation Programs based on the NKT cell biology. Germany-Japan
Immunology Seminar 2013, Shizuoka, Japan. December
7th, 2013.

Identification of NKT cell precursors: Different from the present notion of NKT
cell development after CD1d-selection at the DP stage, we identified an NKT
precursor in the DN1e stage (DN1eP) in the adult and fetal thymus of CD1d-/- andwild-type mice. The DN1eP have unique gene expression profiles that differ from
NKT cells. The DN1eP already express key Th1-like signature genes prior to CD1d
selection and preferentially develop into Th1-type NKT cells in the presence ofCD1d. The development of DN1eP is dependent on IL-7R, PLZF and SAP, whilethat of DP precursors is not. Our findings reveal a novel NKT cell developmental
pathway that is different from the DP pathway.
Clinical trials of NKT-targeted therapy on advanced lung cancer (stage IIIB,
IV and recurrent tumor) after surgery/chemotherapy/radiation therapy,
and in head and neck tumors: The NKT-targeted cell therapy was approved by
the Japanese government for advanced lung cancer in 2011 and for head and necktumors in 2013, because the therapy improved their prolonged median survival
time (29.6Mo vs 4.6Mo for lung cancer and SD or PD in 100% patients treated).
From 2013, we are collaborating with Chiba University Hospital on head and necktumors and the National Hospital Organization on Stage IIA/IIB lung cancer after
surgical tumor resection.
iPS-derived NKT cells: For the future clinical application of iPS-derived NKT
cells, we made the following important observations: a) NKT cells did not induce
GvHD. b) Semi-allogeneic NKT cells induced a significant increase in the numberof CD8 T cells and suppression of tumor growth. Thus, semi-allogeneic NKT cells
can function in vivo even if they are eliminated in a few days by the host immune
system.

39

Laboratory for

Lab activities

Immunotherapy
Team Leader : Shin-ichiro Fujii

Figure: Immune response in dogs immunizedwith artificial adjuvant vector cells
Two doses of ova-expressing artificial adjuvant vector
cells (low dose; 5x106, high dose; 5x107) were administered to 3 dogs. The number of iNKT cells was evaluated by staining with murine CD1d-dimer/Gal and canine
CD3 Ab (left). The function of CD8+ T cells was assessedby IFN-γ ELISPOT after stimulation with autologous
Ag-loaded DCs (right).

Recent Major Publications
Shimizu K, Mizuno T, Shinga J, Asakura M, Kakimi
K, Ishii Y, Masuda K, Maeda T, Sugahara H, Sato Y,
Matsushita H, Nishida K, Hanada K, Dorrie J, Schaft N,
Bickham K, Koike H, Ando T, Nagai R, Fujii S. Vaccination with antigen-transfected, NKT cell ligand-loaded,human cells elicits robust in situ immune responses bydendritic cells. Cancer Res 73, 62-73 (2013)
Shimizu K, Asakura M, Shinga J, Sato Y, Kitahara S,
Hoshino K, Kaisho T, Schoenberger SP, Ezaki T, Fujii S.
Invariant NKT cells induce plasmacytoid DC cross-talkwith conventional DCs for efficient memory CD8+ T cell
induction. J Immunol 190, 5609-19 (2013)
Fujii S, Shimizu K, Okamoto Y, Kunii N, Nakayama T,
Motohashi S, Taniguchi M. NKT cells as an ideal anti-tumor immunotherapeutic. Front Immunol 4, 409,
1-7 (2013)

Invited Presentations
Fujii S. iNKT cell-triggered in vivo DC targeting immunotherapy. 2nd New Zealand-Japan Joint Immunology
Workshop, Auckland, New Zealand. February 26th,
2013.
Fujii S. Development of artificial adjuvant vector cells
targeting dendritic cells in situ. Dokkyo Medical School,
Tochigi, Japan. April 8th, 2013.
Fujii S. Development of Artificial Adjuvant Vector Cells
linking Innate and Adaptive Immunity. The 2nd SCIENCE in Shinagawa, Tokyo, Japan. April 20th, 2013.

40

T

he aims of the laboratory are to extend our basic studies for advancing immunotherapy and translational research, from basic studies back and forth to the bedsidein the field of cancer. For this, we have been conducting three NKT cell-related projects.
In this system, the synthetic glycolipid, α-galactosylceramide (α-GalCer), is presented by
CD1d molecules to invariant NKT lymphocytes. When NKT cells are activated in thismanner, they have unique immunostimulatory features that include the rapid production of IFN-γ and NK cell activation, followed by innate immunity-mediated antitumor
effects.
First, we have made attempts to establish a strategy linking innate and adaptive immunity. For this purpose, we have been studying processes of activation of adaptive immunity through full maturation of DCs soon after the activation of NKT cells in vivo. In
addition, we are investigating adequate delivery systems that have the potential to enhance antitumor immunity. We have developed artificial adjuvant vector cells as new typeof drug delivery system composed of NKT ligand and tumor associated antigen, linking
innate and adaptive immunity. Since this project was accepted by the RIKEN translational program, we are making efforts to work toward preclinical studies that will ultimately
lead to clinical trials (Fig.). As a second project, we have been working in a collaborative
study with the RIKEN iPS-group to establish iPS-NKT cells. In this project, we focus on
the preparation of primary NKT cells as the starting material for generating NKT cell-derived iPS and on the analysis of the function of iPS-derived NKT cells. Third, in our previous collaboration with Chiba University, we obtained successful immunological and clinical results using α-GalCer-loaded dendritic cell (DC/Gal) therapy in advanced lung cancer patients. To continue and extend this project, after a framework agreement, we havebeen launching a joint clinical phase I /IIa study of NKT cell therapy with the National
Hospital Organization (NHO) for early stage lung cancer patients after surgical tumor
resection. In this study, we play an important role in the analyses of immune responses.

Laboratory for

Lab activities

Vaccine Design
Team Leader : Yasuyuki Ishii

Figure: Analysis of IgE+ B cells in the germinal
centerOVA-sensitized C57BL/6 mice were injected intravenously with rhodamine-liposomal α-GalCer. Splenocytes were prepared 1 h after the injection, stainedwith anti-B220, anti-GL7 and anti-IgE mAbs and analyzed by flow cytometry.

Recent Major Publications
Shimizu K, Mizuno T, Shinga J, Asakura M, Kakimi K, Ishii
Y, Masuda K, Maeda T, Sugahara H, Sato Y, Matsushita H,
Nishida K, Hanada K, Dorrie J, Schaft N, Bickham K, Koike
H, Ando T, Nagai R, Fujii S. Vaccination with antigen-transfected, NKT cell ligand-loaded, human cells elicits robust in
situ immune responses by dendritic cells. Cancer Res 73,
62-73 (2013)
Hirai T, Ishii Y, Ikemiyagi M, Fukuda E, Omoto K, Namiki
M, Taniguchi M, Tanabe K. A Novel Approach Inducing
Transplant Tolerance by Activated Invariant Natural Killer T
Cells with Co-stimulatory Blockade. Am J Transplant 14,
554-567 (2014)

Invited Presentations
Ishii Y. Research for oral allergy vaccines inducing immune
tolerance. 2nd New Zealand-Japan Joint Immunology
Workshop, Auckland, New Zealand. February 26th, 2013.
Ishii Y. Development effective vaccine strategies to launch
promising new generation vaccine candidates. 7th Annual
World Vaccine Congress Asia 2013, Singapore. June 19th,
2013.
Ishii Y. A novel allergy therapy using IgE-suppressive drugs
generated in RIKEN. Symposium: Development of the biomedicine using a plant - the present condition and future,
Tokyo, Japan. November 8th, 2013.

Invariant natural killer T (iNKT) cells perform immunoregulatory roles suchas the suppression of IgE responses as well as having adjuvant roles in hostdefense. Although the mechanism of immune activation by iNKT cells is well understood, that of immune suppression remains unclear. Here, we show that thedelivery of α-galactosylceramide (α-GalCer), a representative iNKT cell ligand,to splenic B220-positive cell subsets including IgE-expressing B cells leads to IL21 expression by iNKT cells and results in the IgE isotype-specific suppression.
Fluorescent-labeled liposomal α-GalCer was preferentially incorporated into
B220+CD21high CD23low cell subsets expressing a high level of CD1d rather thaninto CD11b and/or CD11c-positive cells. Splenic iNKT cells migrated and co-localized with the α-GalCer-presenting B cells in a CXCL16-dependent manner.
The expression of IL-21 by iNKT cells after the co-localization was remarkably
diminished by blockade of the CXCR6/CXCL16 interaction with an anti-CXCL16
neutralizing antibody. Furthermore, the suppression of the IgE responses seen inmice administered the liposomal α-GalCer was restored by treatment with the anti-CXCL16 antibody. Unlike off-target cells of the liposomal α-GalCer, the major
target B cells, expressed IgE, IL-21 receptor and Bmf mRNAs, and were highly sensitive to IL-21-induced apoptosis in vitro. IgE-expressing cell subsets among theliposomal α-GalCer-targeted B cells included B220+GL7+ germinal center B cellsubsets (Fig.). These results collectively suggest that in vivo IgE isotype-specificsuppression by liposomal α-GalCer administration could be caused by IgE B-specific apoptosis induced by IL-21 produced by activated iNKT cells.

Ishii Y. New approaches of causal therapies for allergic
diseases. 4th Kanagawa Mirai Forum, Yokohama, Japan.
November 9th, 2013.
Ishii Y. Clinical applications of transplantation tolerance
induced by invariant natural killer T cells. 99th Ochanomizu
Cancer Academia Forum, Tokyo, Japan. November 27th,
2013.

41

Laboratory for

Lab activities

Allergic Disease
Team Leader : Toshiaki Kawakami

Figure: Hypothetical vicious cycle of allergic skininflammation
Allergen-specific TH2 cells stimulate production/secretion of periostin by fibroblasts. Periostin in turn thenstimulates keratinocytes to produce and secrete TSLP
and other inflammatory cytokines. In this scheme, TH2
cells seem to be required for initial epidermal overexpression of TSLP. Once sustained overexpression of
TSLP is established, mast cells may play a more important role in persistent dermatitis than TH2 cells. PLC-β3
can regulate activities of the cellular elements of thisnetwork, such as proliferation of mast cells, periostin
production/secretion by fibroblasts and TSLP production/secretion by keratinocytes. Our data also suggest
the presence of a feedback loop for inhibition of fibroblast periostin production by TSLP, and that PLC-β3 infibroblasts is required for this feedback inhibition. DC,
dendritic cell; TH0, naïve CD4+ T cells.

Recent Major Publications
Ando T, Matsumoto K, Namiranian S, Yamashita H,
Glatthorn H, Kimura M, Dolan BR, Lee JJ, Galli SJ,
Kawakami Y, Jamora C, Kawakami T. Mast cells are
required for full expression of allergen/SEB-induced
skin inflammation. J Invest Dermatol 133, 2695-705(2013)
Kawakami T, Ando T. Salmonella’s masterful skill inmast cell suppression. Immunity 39, 996-8 (2013)
Ando T, Xiao W, Gao P, Namiranian S, Matsumoto K, Tomimori Y, Hong H, Yamashita H, Kimura M, Kashiwakura JI, Hata TR, Izuhara K, Gurish MF, Roers A, Rafaels
NM, Barnes KC, Jamora C, Kawakami Y, Kawakami T.
Critical role for mast-cell Stat5 activity in skin inflammation. Cell Rep 6, 366-76 (2014)

Invited Presentations
Kawakami T. Phospholipase C-beta3 in allergic diseases. 2nd Allergy & Respiratory Drug Discovery Conference, San Diego, California, USA. February 1st, 2013.
Kawakami T. Phospholipase C-beta3 in immune cells.
2013 International Symposium: PI-PLC Activity and
Signaling, Ulsan, Korea. July 19th, 2013.
Kawakami T. Hospholipase C-beta3 in atopic dermatitis. 7th International Leukocyte Signal Transduction
Conference, Kos, Greece. September 11th, 2013.
Kawakami T. Phospholipase C-β3 as a tumor suppressor and a guardian of the immune system. The 5th
LJI & IMS-RCAI Workshop, Yokohama, Japan. October
30th, 2013.
Kawakami T. Phospholipase C in immune cells. NHRI &IBMS Joint Conference: 2013 International Conferenceof Inflammation, Cancer and Metabolic Disorders,
Zhunan, Miaoli, Taiwan. November 4th, 2013.

42

Role of histamine-releasing factor (HRF) in allergic diseases
HRF is a cytokine-like protein that can stimulate histamine release and cytokine(IL-4 and IL-13) production/secretion from IgE-sensitized basophils and mast
cells. HRF-like ac­tivities are found in bodily fluids during the late phase of allergic
reactions. We recently demonstrated that some, but not all, IgE and IgG moleculesinteract with HRF with low affinity. By mapping the binding sites on both HRF
and IgE/IgG molecules, we developed competitive inhibitors of HRF-IgE (or IgG)
interactions. Using these inhibitors, we showed that HRF promotes allergic inflammation in mouse models of anaphylaxis and asthma (Kashiwakura et al., 2012). We
are now investigating the role of HRF in food allergy. Using a standard food allergy
model, which is mast cell-, IgE-, and FcεRI-dependent, our initial experiments suggest that HRF is involved in the promotion of the allergic reactions (unpublished).
We have been measuring HRF and HRF-reactive IgGs in sera of food allergy patients before and after the rapid oral induction of tolerance (ROIT). Another areaof research is on HRF-related human mRNA sequences (unpublished). We are now
studying whether the HRF-related proteins encode by these transcripts affect HRF
functions.
Pathogenic mechanisms of atopic dermatitis (AD)AD is a chronic pruritic inflammatory skin disease. In the AD project, we havebeen studying the cellular and molecular mechanisms using our previously established in vivo AD induction model and the spontaneously occurring AD-like skinlesions in phospholipase C (PLC)-β3-deficient mice. As we recently showed the importance of PLC-β3-mediated Stat5 regulation in mast cells in causing AD (Andoet al., 2014; Fig.), we are now studying whether Jak family members as upstream
regulators are involved in the regulation of Stat5 in mouse and human mast cells.

Lab activities

RIKEN-TORII
Joint Research Team
Team Leader : Masaru Taniguchi

Figure: Subcutaneous vaccination significantly
inhibits the increase of Cry j 2-specific IgE after
Cry j 2 intranasal sensitization.
The recombinant vaccine was administered subcutaneously into Cry j 2-sensitized mice three times andthe mice were again sensitized with Cry j 2 intranasally
after the vaccination. The increase of Cry j 2-specific IgEafter the last challenge with Cry j 2 was significantly
attenuated in the vaccinated mice compared with thecontrol mice with no-vaccination (saline). This figure
shows the mean Cry j 2-specific IgE titer in each group.
*: p<0.05 by Mann-Whitney U-test.

Recent Major Publications
Fujimura T, Okamoto Y, Taniguchi M. Therapeutic effects and biomarkers in sublingual immunotherapy: areview. J Allergy (Cairo) 2012, 381737 (2012)J

apanese cedar pollinosis is a common allergy in Japan. Antigen-specific immunotherapy (SIT) is considered to be the only curative treatment for allergy, andonly a crude extract from Japanese cedar pollen has been approved for clinical use
by the Ministry of Health, Labour and Welfare in Japan. Vaccines using allergoids
and modified Cry j 1, a major allergen of Japanese cedar pollen, have been developed and used in pre-clinical trials; however, none of them has been commerciallyprovided for medical use. This situation is due to poor clinical outcomes at laterstage clinical trials or the inability to find a cooperative pharmaceutical company
that will introduce them onto the market. To fill the critical gap between basic research and the later stage of drug development, IMS-RCAI established a program
named ‘exchange zone’, where RIKEN, universities, hospitals, and pharmaceuticalcompanies work together for drug development, including allergy vaccines especially for Japanese cedar pollinosis. Based on this program, RIKEN and TORII
pharmaceutical Co., Ltd. set up a joint research laboratory in RCAI in May 2010
and began to develop the SIT vaccine.
For the vaccine, recombinant technology is used to conjugate two major allergens from Japanese cedar pollen, namely Cry j 1 and Cry j 2, and then the protein
is further modified with polyethylene glycol to prevent binding with immunoglobulin E (IgE) and to improve its solubility.
Systemic injections of the vaccine into Cry j 1 or Cry j 2-sensitized mice prevented the increase of serum antigen-specific IgE after systematic or local sensitization with native Cry j 1 or Cry j 2 in a dose dependent manner. To improve thetherapeutic effects of the vaccine, elucidation of the therapeutic mechanisms andidentification of biomarkers are important issues. Therefore, we are looking forbiomarkers to monitor therapeutic responses during or after the vaccination.

43

Lab activities

Drug Discovery
Antibody Platform Unit
Unit Leader : Toshitada Takemori

Figure: ADCC activity estimated using the KHYG
NK cell line
ADCC activity by Rituximab (as a positive control) andmouse and chimeric anti-X mAbs against cancer cells
measured by utilizing NK cells from spleens of SCID
mice and NK cell lines as effectors.

Recent Major Publications
Ato M, Takahashi Y, Fujii H, Hashimoto S, Kaji T, Itamura
S, Horiuchi Y, Arakawa Y, Tashiro M, Takemori T. Influenza A whole virion vaccine induces a rapid reduction of
peripheral blood leukocytes via interferon-α-dependent
apoptosis. Vaccine 31, 2184-90 (2013)
Kometani K, Nakagawa R, Shinnakasu R, Kaji T,
Rybouchkin A, Moriyama S, Furukawa K, Koseki H,
Takemori T, Kurosaki T. Repression of the transcription
factor Bach2 contributes to predisposition of IgG1
memory B cells toward plasma cell differentiation. Immunity 39, 136-47 (2013)
Kaji T, Furukawa K, Ishige A, Toyokura I, Nomura M,
Okada M, Takahashi Y, Shimoda M, Takemori T. Bothmutated and unmutated memory B cells accumulate
mutations in the course of the secondary response anddevelop a new antibody repertoire optimally adapted
to the secondary stimulus. Int Immunol 25, 683-95(2013)

Invited Presentations
Takemori T. CD4 memory T cell development is licensed
by Bcl6 and accomplished by expression of a groupof genes mediated by cognate B cell interactions. The
36th Annual Meeting of the Molecular Biology Societyof Japan, Kobe, Japan. December 5th, 2013.

44

T

he use of monoclonal antibodies (mAbs) for cancer therapy has achieved significant success in recent years. Our laboratory, linked to the RIKEN Program for Drug Discovery and Medical Technology Platforms (DMP), is preparing
new mAbs for cancer and other diseases by collaborations within and outside of
RIKEN. Along with mAb production, we clarify the activity of mAbs obtained in
our laboratory in terms of whether they could be applicable in medical therapies
by using several model systems, prior to further analysis on the clinical side.
Tumor cell killing is mediated through direct action of the antibody (throughreceptor blockade or agonist activity), complement-dependent cytotoxicity(CDC) and/or antibody-dependent cellular cytotoxicity (ADCC). The antibody
Fc is essential for mediating tumor cell killing through CDC and ADCC, but theantibody V-region is also important for the killing activity with respect to the recognition sites and affinity for the target molecules.
In 2013, in order to isolate a large number of candidate mAb with ADCC activity from a hybridoma screening, we prepared a human NK cell line that expresses
a mouse or human FCγΙΙΙ receptor as the effector cells for the assay (Fig.). This
strategy is very efficient as it enables us to measure ADCC activity by a minuteamount of Abs in the culture supernatant at any time point without a complex
process. Accordingly we are able to preferentially select ADCC-positive clones
during the initial hybridoma culture, prior to the antibody purification. Furthermore, we established a mouse model to evaluate the anti-cancer activity of mAbs
selected by in vitro analysis.
The successful development of candidate antibodies for the clinic involves acomplex process of scientific and preclinical evaluations, with a convergence ofcancer biology and immunology. We hope to develop antibodies for the clinic,
particularly leukemic cancers, in collaboration with clinical researchers.

Central facilities

Central Facilities
C

entral Facilities in IMS provide all researchers in the Centerwith access to the most advanced equipment and technologies. Central Facilities consist of four sections; the FACS Laboratory
managed by Dr. Takashi Saito, the Confocal Laboratory managed

by Dr. Takaharu Okada, the Genomics Laboratory managed by Dr.
Osamu Ohara, and the Animal Facility managed by Dr. Haruhiko
Koseki.

FACS Laboratory

The FACS Lab provides a range of support for flow cytometry
and cell sorting, procedures that are essential for not only immunological experiments but also a wide variety of biological experiments. The FACS Lab has added new FACS Aria and reduced
the number of FACS Vantages. In addition to FACS, the lab installed ImageStreamX, a device that combines flow cytometry withthe visual detail of microscopy in a single platform and CyTOF, a
mass-spectometry-based cytometer that has potential for analyzing
more than 30 markers simultaneously with metal-labelled antibodies.
For the users of the FACS machines (cell analyzers and cell sorters), Tomomi Aoyama and Noriko Yoza cover various services:(1) Technical support and training: In 2013, the facility offered 16
technical courses (8 for cell sorting and 8 for cell analysis). Courses were held at 3 different levels, Calibur basic, Canto II and Aria
basic. A total of 66 researchers took the courses in 2013. (2) Cellsorting 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 2013, 293 such operation
services were provided by the lab. (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 inadvance through an internal website. In addition to the in-house
FACS Lab staff, engineers from Becton Dickinson visit once a week
to provide maintenance and technical support.
Table: Instruments in the FACS Lab.

Machine types

Machines# of machines

FACS cell analyzer

Calibur
Cantoll

42

FACS cell sorter

Aria l/II
Aria III
Vantage (Diva)

3
2
1

Mass-cytometer

CyTOF

1

Imaging flow cytometer

ImageStreamX

1

Confocal Laboratory
The Confocal Laboratory provides equipment for cell and tissue
imaging, and coordinates technical support by Leica Microsystems.
There are seven laser-scanning fluorescence microscopes available
to IMS researchers.
1. Inverted SP2 system with visible lasers for single-photon excitation and a femtosecond Ti:Sa laser for two-photon excitation.
2. Inverted SP2 system with visible lasers for single-photon excitation including a 405 violet laser. This microscope is equippedwith a chamber system that controls CO2 concentration, temperature and humidity for live cell imaging.
3. Inverted SP5 system with visible lasers for single-photon excitation including a 405 violet laser.
4. Upright SP2 system with visible and UV lasers for single-photon
excitation.
5. Inverted SP8 system with visible lasers for single-photon excitation. SP8 is Leica’s newest system with improved optics.
6. Upright SP5 system with twin femtosecond Ti:Sa lasers for
two-photon excitation. This system utilizes resonant scanners

that enable high-speed acquisition of large z-stacks for live tissue
imaging.
7. Inverted SP5 II system with a femtosecond Ti:Sa laser for
two-photon excitation. This system utilizes resonant scanners.
The laser system is equipped with an optical parametric oscillator (OPO) that enables two-photon imaging by long wavelength
excitation (1100-1300 nm).

Figure: Left: SP5 confocal microscope (#3). Right: SP5 II two-photon microscope with a femtosecond Ti:Sa laser plus an OPO (#7).

45

Central facilities

Genomics LaboratoryThe Laboratory for Integrative Genomics also serves as a technical support service lab which provides genome- and proteome-wide analyses for the scientific research community in theCenter for Integrative Medical Sciences (IMS). We offer a variety
of services to suit the needs of laboratories in various biologicalfields. These services include DNA sequencing, proteomics analysis, multiplex suspension array (Luminex technology), DNAmicroarray (Affymetrix /Agilent), cDNA/genomic clone distribution, and Primer/labeled probe distribution for qRT-PCR analysisof immune cells (see Table). As part of our advanced technologies
on demand, we provide comprehensive interrogation of the nucleic-acid based information in a cell at single-base resolution with
Illumina HiSeq1500 and proteomic approaches using AB SCIEX
TripleTOF 5600. With the unbiased approach using this sequencer,
we have been identifying transcription units, alternative splice sites
and transcription factor binding sites, as well as performing mapping/genome annotation. Our mass spectrometry system will makeit possible to take quantitative proteomic approaches in various
immunological studies. These technologies will help to illuminate
additional hidden features of the dynamic genomic and proteomic
landscape that are regulated by both genetic and epigenetic pathways in all organisms. Together with the Laboratory for Integrated Bioinformatics, we also carry out primary processing of large
amounts of data from DNA sequencers and mass spectrometers for
IMS users.

Table: Services provided by the Genomics Lab in 2013

# of samples# of teams

Next-generation DNA sequencing
Illumina HiSeq1500

731

20

Proteimics
Two-dimentional electrophoresis
Mass Spectrometry Analysis

1
2

1
2

2,802

11

90
153
243

2
11
11

9,068
5,610
14,67818
1533

cDNA clone delivery

11

4

Primer/labeled probe delivery

58

2

Multiplex suspension array
Affymetrix Genechip(Exon array, Gene array, miRNA array)
Human
Mouse
Total
Sanger DNA sequencing
36cm capillary
50cm capillary
Total

Animal FacilityWe have been maintaining over 45,000 mice in the SPF area,
1,750 mice in an isolated area and several germfree mice.
We have newly introduced mouse lines into the SPF area by a combination of in vitro fertilization (IVF) and embryo transfer andgenerated cryostocks of genetic resources for 642 lines. We also
maintain relatively large colonies of several commonly used strains
such as NOD/SCID/γCKO mice, Rag1KO and cre deleters andprovided them to users on demand. We have also provided technical assistance to generate knockouts (126 lines) and transgenic
mice (6 lines). We have been undergoing renovation of an internally available database for genetic resources.In addition, we havelaunched a new activity to improve the efficacy of transplantationof human hematopoietic stem cells into NOD/SCID/γCKO miceby “humanizing” the host strain. For this purpose, we have introduced large genomic fragments containing human genes encoding
MHC, cytokines, adhesion molecules, virus receptors and others
into NOD/SCID/γCKO mice. Up to now, we have established 15
BAC transgenics and confirmed the expression of human genes on
a C57BL/6 background and begun back-crossing these mice onto
the NOD/SCID/γCKO mice using the speed-congenic method. To
adapt to increasing demands for the use of humanized animals, wehave started a new facility primarily for humanized mice, which is
fully equipped with autoclaves and cage washing systems on the 1st
floor of the north building. In this new facility, we can maintain up
to 900 cages (up to 4,500 mice) in either isolators or bio-bubbles.
More recently, we have added a new activity to create and maintain46

germ-free and gnotobiote mice in collaboration with Dr. Hiroshi
Ohno (Laboratory for Intestinal Ecosystem), Dr. Sidonia Fagarasan(Laboratory for Mucosal Immunity) and Dr. Kenya Honda (Laboratory for Gut Homeostasis). We performed sanitization of standard
strains and knockout mice at a pace of 1-2 strain(s) per month.

Photo: New animal facility for humanized miceYoung Chief
Investigator Program

YCI programT

he Young Chief Investigator Program aims 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 an access to mentoring by multiple senior specialists in related research
fields. Mentors provide guidance for experimental design, preparation of papers and presentations, promotion of international visibility, and obtaining research funding. The YCI laboratory will also
share space, equipment and facilities with a host laboratory in IMS(Fig.) The YCI Program Committee considers necessary changes in
the Center’s support for each YCI and discusses the relevance andvalue of each YCI research project as part of the core research projects at IMS.
There will be an initial 5-year appointment with the possibility ofextending for an additional 2 years after evaluation by the Director
and an internal committee. At that point, a Young Chief Investigator can leave IMS to take a position at another institution or bepromoted to another type of position within IMS.

YCI Laboratory forMathematical Modeling of
Immune System
Young Chief Investigator: Shinji NakaokaIn our laboratory, we undertake several mathematical works thatare indispensable for construction of multi-scale mathematicalmodels. In this year, we especially focused on a quantitative studyof T cell population dynamics (Fig.). On the basis of label-based
measurement data, such as BrdU, to track cell division and death,
we proposed a general framework that allows incorporating general
distributions for the timing of cell division and death. Specifically,
we proposed a mechanistic mathematical model, called a generation
progression model (GPM), that is described by a system of Volterra
integral equations. Formulation by ODEs corresponds to a special
case of GPM with an exponentially distributed kernel. A formulation developed in mathematical demography is employed to track
the history of cell divisions. We newly defined a quantity called
generation progression ratio (GPR) which provides a quantitative
measure for the expected number of daughter cells generated fromtheir survived mother cell. As an application of GPM and GPR, atime-course measured data for the transient growth of lymphocytes
at the single cell level was used to quantitatively characterize theexpansion and contraction phase.
Figure: A scheme of a generation progression model and a generation progression ratio

47

YCI program

YCI Laboratory for

Stem Cell Competency
Young Chief Investigator : Hayato Kaneda

A

Figure: Restoration of neurogenic potential in development-progressed
gliogenic NSPCs by overexpression of miR-17.
Embryonic stem cell-derived NSPCs can sequentially generate specific types of neurons
and glial cells in vitro in their proper in vivo order. Overexpression of miR-17 inhibitedthe acquisition of gliogenic competence and forced stage-progressed NSPCs to regain
neurogenic competence.

dult tissue stem cells (TSCs) are impaired in their functions
with age. TSC dysfunction and decreased regenerative capacity are involved, at least in part, in disturbances of tissue homeostasis, e.g., inefficient muscle repair, reduced bone mass, neurodegenerative diseases, and dysregulation of hematopoiesis. Therefore, therestoration of TSC functions is expected to contribute to recovery oftissue homeostasis and improvements in our health.
Previously, we identified the “competence change”, which is responsible for the responsiveness of neural stem/progenitor cells(NSPCs) to extrinsic signals (Naka H et. al. Nat Neurosci 2008).
Moreover, further investigation revealed that the competence regulation enabled us to control neurogenic-to-gliogenic transition andrestore neurogenic potential in developmentally-progressed gliogenic NSPCs (Fig.) (Naka-Kaneda H et. al. Proc Natl Acad Sci USA
2014). Based on these findings, we have been investigating stem cellaging. Competence regulation is also involved in the aging of other
TSCs, such as the decline in lymphopoiesis by hematopoietic stem
cells and in the osteogenesis ability of mesenchymal stem/stromalcells. We aim to elucidate the central molecular machinery of stem
cell aging and its influence on tissue homeostasis and in turn todevelop a method for functional recovery of aged stem cells andrestoration/maintenance of tissue homeostasis.

YCI Laboratory for

Immune Regeneration
Young Chief Investigator : Tomokatsu Ikawa

T,

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

48

B and NK lymphocytes are generated from pluripotent hematopoietic stem cells (HSCs) through a successive series of lineage
restriction processes. Many regulatory components, such as transcription factors, cytokines/cytokine receptors, and signal transduction molecules orchestrate the cell fate specification and determination. Above
all, transcription factors play a key role in regulating lineage-associatedgene programs. Although many essential transcription factors, such as
PU.1, Ikaros, GATA3, TCF-1, Bcl11b, E2A, EBF1 and Pax5 have been implicated in regulating the cell fate choice of lymphoid lineages, molecular mechanisms underlying the generation of these patterns during cellfate determination remain unexplored because of an absence of suitable
experimental systems. We have recently established an ideal system inwhich we can examine gene regulatory networks during lymphoid lineage
specification from HSCs (Fig.). This novel system enabled the analysis ofa large set of regulatory molecules that control the generation of T and
B lymphocytes. It can also be applied for ex vivo expansion of humanhematopoietic stem/progenitors, which will be required for immune celltherapy or transplantation of HSCs. Thus, the aims of our study are 1)
from a basic science perspective, to elucidate the mechanisms that orchestrate cell fate specification, commitment and differentiation during
lymphocyte development and 2) from a clinical medicine perspective, toestablish a novel method to expand human hematopoietic stem/progenitors for the development of HSC transplantation as a clinical strategy.

YCI program

YCI Laboratory for

Integrative Genomics
Young Chief Investigator: Katsuyuki Shiroguchi

Q

Figure: Detection of homeostasis destruction by genomics study
The destruction of homeostasis can be detected by accurate counting of RNA moleculesin single cells. Each symbol corresponds to a single cell.

uantitative Genomics at the Single Cell & Single Molecule
Level
In order to investigate at the molecular level how homeostasis
is destroyed at the beginning of a disease and how compensation
occurs while responding to an external perturbation before thebreak, we wish to visualize the distribution of cell states by accurate
system-wide measurements with single molecule and/or single cellresolution. To visualize the distribution and its shift, we are working
on multiple projects with development of highly accurate quantification methods. One of them is to digitally count the copy numberof RNA molecules genome-wide using our technique under development and a next generation sequencer. This method provides
highly reproducible quantification even for low-expressed RNAs
and the absolute RNA copy number per cell, which should allow
us to find small differences between samples whose phenotypes are
only slightly different. In addition, I believe that the absolute counting of cellular molecules or rates of biological events will become
more important, especially in a coming era, since it makes possible
the integrative interpretation with other data obtained by different
measurements. We will work on developing such a technique to"see" something unknown, which may contribute to understanding
biological systems and diseases.

YCI Laboratory for

Cellular Bioenergetic Network
Young Chief Investigator: Toshimori KitamiT

he overarching goal of our laboratory is to understand the roleof cellular metabolism in the pathogenesis of complex diseases. Research over the past decades has shown that monogenic mutations in metabolic pathways cause a wide variety of human diseases.
However, more recent studies have highlighted the role of cellular
metabolism in the development of a wide variety of complex human diseases. Our laboratory in particular has been studying thefunction of mitochondrial energy metabolism, which is associatedwith neurodegenerative diseases, cardiovascular disease, type 2 diabetes, and aging. We hope to identify novel pathways that restore
or improve mitochondrial function through genetic and chemical
screens and to examine their potential therapeutic value using genetically engineered mouse models and unique chemical probes.
Our new laboratory, established in FY2013, hopes to take advantage of the immunology expertise of IMS-RCAI and our expertisein mitochondrial biology, chemical screening, and genomics to explore the interface between immunity, mitochondria, and complex
diseases.
Figure: Schematic of image-based assay for mitochondria using automatedmicroscopy
High-throughput automated microscopy is one of many cutting-edge technologies
available at IMS-RCAI.

49

RIKEN International Program Associate
(IPA)

I

MS accepted four international students as RIKEN International Program Associates (IPA). Under this IPA program, IMS
lab heads host international students from collaborating graduate
schools and supervise their Ph.D. program as Joint Supervisors. The
students receive a daily living allowance and housing costs for up toa maximum of three years.
The IPA students who studied at IMS in 2013 were

Other programs

Yue Ren (Jilin University, China) studied in the Laboratory for Immune Regulation.
Mohamed El Sherif Gadelhaq Gadelhaq Badr (Tokyo Medical
and Dental University) from Edypt studied in the Laboratory for
Cell Signaling.
Mei Suen Kong (Universiti Sains Malaysia, Malaysia) studied in the
Laboratory for Cell Signaling.
Cheng Chung Yong (Universiti Sains Malaysia, Malaysia) studied
in the Laboratory for Intestinal Ecosystem.

RIKEN Foreign Postdoctoral Researcher (FPR) ProgramT

he RIKEN Foreign Postdoctoral Researcher (FPR) program offers aspiring young foreign researchers with creative ideas andwho show promise of becoming internationally active in the future
the opportunity to pursue innovative research at RIKEN under the
direction of a RIKEN laboratory head. The FPR program is one of

RIKEN’s initiatives to open up its facilities and resources to the forefront of global science and technology.
In 2013, Wooseok Seo studied in the Laboratory for Transcriptional Regulation as a RIKEN FPR.

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, senior 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 thedevelopment of these young scientists while also energizing RIKEN
with their innovative thinking.
This year, 20 JRA students studied in IMS.
Norihiko Inoue (Laboratory for Integrated Cellular Systems)
Chie Kano (Laboratory for Intestinal Ecosystem)
Misao Hanazato (Laboratory for Intestinal Ecosystem)
Masanaka Sugiyama (Laboratory for Inflammatory Regulation)
Hisashi Wada (Laboratory for Transcriptional Regulation)

Takashi Ikeno (Laboratory for Tissue Dynamics)
Satoshi Koga (Laboratory for Immune Cell System)
Ryuichi Murakami (Laboratory for Immune Homeostasis)
Yujiro Yamamoto (Laboratory for Genome Sequencing Analysis)
Yuji Nagano (Laboratory for Gut Homeostasis)
Akihiko Kimura (Laboratory for Immune Homeostasis)
Akemi Fujiwara (Laboratory for Intestinal Ecosystem)
Yuuhou Najima (Laboratory for Human Disease Models)
Rikiya Ishikawa (Laboratory for Intestinal Ecosystem)
Yusuke Sato (Laboratory for Immunotherapy)
Tomohiro Miyai (Laboratory for Immune Cell System)
Rumiko Ono (Laboratory for Inflammatory Regulation)
Takaharu Sasaki (Laboratory for Immune Cell System)
Yuma Sakamoto (Laboratory for Bone and Joint Diseases)
Atsushi Ono (Laboratory for Digestive Diseases)

RIKEN Special Postdoctoral Researcher (SPDR) Program

R

IKEN’s Special Postdoctoral Researcher Program was instituted to provide young and creative scientists the opportunity to
be involved in autonomous and independent research in line withRIKEN objectives and research fields. The positions are competitive, but if selected, researchers receive salaries and research budgets (1 million yen) from RIKEN and they are able to conduct theirresearch at one of its laboratories.

50

This year, 4 postdocs conducted their research at IMS throughthe SPDR program.
Shimpei Kawamoto (Laboratory for Mucosal Immunity)
Saya Moriyama (Laboratory for Tissue Dynamics)
Shinsuke Ito (Laboratory for Developmental Genetics)
Hirokazu Tanaka (Laboratory for Transcriptional Regulation)

Award winners 2013

Award winners

Encouragement Award in November 2013.

Photo 1

Photo 4

Photo 7

Photo 2

Photo 5Photo 3

Photo 6

Photo 8Ichiro Taniuchi (photo 1), Group Director of the Laboratory
for Transcriptional Regulation, received the 16th JapaneseSociety for Immunology (JSI) Award for his study, “Transcriptional control of T cell development”.
Hidewaki Nakagawa (photo 2), Team Leader of the Laboratory for Genome Sequencing Analysis, received the Princess Takamatsu Cancer Research Fund Award for his study,“Whole genome sequencing analysis of intrahepatic cholangiocarcinoma”.

Sidonia Fagarasan (photo 6), Team Leader of the Laboratory for Mucosal Immunity, and Kenya Honda (photo7), Team Leader of the Laboratory for Gut Homeostasis,received the NISTEP Award from the National Institute of
Science and Technology Policy (NISTEP), under the direct
jurisdiction of the Ministry of Education, Culture, Sports,
Science and Technology (MEXT). They were selected fortheir outstanding contributions to elucidating the interactions between the immune system and microbes at the mucosal barrier.
Koji Ueda (photo 8), Senior Research Scientist in the Laboratory for Genome Sequencing Analysis, received the Japan
Human Proteome Organization Young Researcher’s Awardfor his study, “Glycol-proteomics technology and tumor
marker research by glycol-proteomics”.
Hidewaki Nakagawa, Team Leader of the Laboratory for
Genome Sequencing Analysis, received the Takeda Science
Foundation Award for his study, “Identification of prostatecancer-susceptibility genes /variants and their association
with diet”.
Kohei Kometani, Research Scientist in the Laboratory for
Lymphocyte Differentiation, received the Japanese Society
for Immunology Young Investigator Award for his study,"Unveiling molecular mechanism of B cell activation andsubsequent plasma cell differentiation".
Takashi Kanaya, Research Scientist in the Laboratory for
Intestinal Ecosystem, received the Research Encouragement
Award from the Japan Bifidus Foundation on June 14th,
2013.

Fumihiko Ishikawa (photo 3), Group Director of the Laboratory for Human Disease Models, received the JSPS (Japan
Society for the Promotion of Science) Prize for his study,“Development of Humanized Mouse System Enabling invivo Investigation of Human Leukemia and Therapeutic Approach”.

Naoka Ito-Nagato, Research Scientist in the Laboratory for
Immune Regulation, and Tomomitsu Hirota, Research Scientist in the Laboratory for Respiratory and Allergic Diseases, received the Abstract Award at the European Academy ofAllergy and Clinical Immunology –World Allergy Organization (EAACI-WAO) Congress 2013 held in Milan, Italy,
June-July 2013.

Atsushi Takahashi (photo 4), Team Leader of the Laboratory for Statistical Analysis, and Yuta Kochi (photo 5), SeniorResearch Scientist of the Laboratory for Autoimmune Diseases, received the Japan Society of Human Genetics (JSHG)

Mari Tenno, Research Scientist in the Laboratory for Transcriptional Regulation, received the poster award for her
research on “Unique role of Cbfb2 variant in Langerhans
cell development” at the 13th International Workshop on

51

Award winners

Langerhans Cells, held on October 10th-13th, 2013, in Amsterdam, the Netherlands.
Shintaro Hojyo, Research Scientist in the Laboratory forLaboratory for Homeostatic Network, received the Suzuki
Koichi Memorial Award at the 86th Annual Meeting of theJapanese Biochemical Society in October 2013.
Tomohiro Miyai, Junior Research Associate in the Laboratory for Homeostatic Network, received the excellent
presentation award at the meeting of the Japanese ResearchGroup for Studies of Metalbioscience 2013 on September
27th.
Siew-Kee Low, Postdoctoral Researcher in the Laboratory
for Statistical Analysis, was selected as a European Societyof Human Genetics (ESHG) Poster Award Finalist in June2013.
Shimpei Kawamoto, Special Postdoctoral Researcher inthe Laboratory for Mucosal Immunity, Takashi Kanaya, Research Scientist in the Laboratory for Intestinal Ecosystem
and Raoul Eduardo Vizcardo Sakoda, Research Scientist

The IMS Excellent Paper of the Year 2013 award wasgiven to the following four papers:
Dr. Yukihiro Furusawa, Mr. Yuuki Obata, Dr. Shinji Fukuda,
Dr. Koji Hase and Dr. Hiroshi Ohno Laboratory for Intestinal Ecosystem “Commensal microbe-derived butyrate
induces colonic regulatory T cells.” Nature Vol. 504, pp.446450, 2013.
Dr. Yoriko Saito and Dr. Fumihiko Ishikawa Laboratory for
Human Disease Models “A pyrrolo-pyrimidine derivative
targets human primary AML stem cells in vivo.” Science52

in the Laboratory for Developmental Genetics received theTadamitsu Kishimoto International Travel Award for theirpresentations at the 15th International Congress of Immunology held in Milan, Italy.
Koji Atarashi, Senior Research Scientist in the Laboratory
for Gut Homeostasis, and Tomomitsu Hirota, Research Scientist in the Laboratory for Respiratory and Allergic Diseases, received RIKEN Research Incentive Awards. Tomoyuki
Ishikura, Technical Staff member in the Laboratory for Developmental Genetics received a RIKEN Technology Incentive Award in March, 2014.
Two graduate students studying at IMS won Best Poster
Awards at the RIKEN Noyori Summer School held in Kobeon September 6th-7th, 2013. Yuho Najima, Junior Research
Associate in the Laboratory for Human Disease Models, received the Medical Science Prize for his presentation “Induction of human WT1-specific CD8+ T cells in an HLA class I
TG NSG mice”. Takaharu Sasaki, Junior Research Associate in the Laboratory for Immune Cell System, received the
Biology Prize for his presentation “Role of Natural Helper
Cells in Obesity.”

Translational Medicine Vol. 5, 181ra52, 2013.
Dr. Koji Atarashi, Dr. Takeshi Tanoue and Dr. Kenya Honda Laboratory for Gut Homeostasis “Treg induction by arationally selected mixture of Clostridia strains from the human microbiota.” Nature Vol. 500, pp.232-236, 2013.
Dr. Ikuyo Kou, Dr. Yohei Takahashi and Dr. Shiro Ikegawa
Laboratory for Bone and Joint Diseases “Genetic variants in
GPR126 are associated with adolescent idiopathic scoliosis.”Nature Genetics Vol. 45, pp.676-679, 2013.

Part 2
Research
Projects

Intestinal Bacteria Show ‘Community Spirit’
The collaborative effects of multiple bacterial
strains in the gut may help prevent the onset ofcertain inflammatory diseases

Research highlights

Figure: Inflammatory damage to the intestinal
epithelium in a mouse model of colitis (left) is
greatly reduced after oral administration of a
mixture of 17 Clostridia strains (right).
Reproduced from Ref. © 2013 K. Atarashi et al.

At first, it may sound alarming to learn that a population of bacteria in your gut is conspiring to suppress
your immune system—however, this is actually good news.
By identifying the strains responsible, a research team led by
Kenya Honda (Laboratory for Gut Homeostasis) may haveuncovered a promising avenue of treatment for certain inflammatory disorders.
Immune cells known as regulatory T (Treg) cells are inpart responsible for preventing the immune system fromoverreacting to foreign molecules or attacking healthy tissue. It is well established that immune function is affected by
the diverse microbial community within the digestive tract,and Honda’s team previously discovered that bacteria belonging to the genus Clostridium act on this particular immune pathway in mice to exert a strong anti-inflammatory
effect.
“We showed that they were responsible for triggering
production of Treg cells in the colon of mice,” says Honda,“and that oral administration of these strains protected miceagainst colitis and systemic allergic responses.”
Honda and his colleagues have now verified the existence of an equivalent bacterial population in humans. Theyobtained a stool sample from a healthy volunteer and subjected it to the same purification regimen that yielded theClostridia subpopulation identified in mice. When theseThis article was reproduced from RIKEN Research withpermission
http://www.rikenresearch.riken.jp/eng/research/7489

54

bacteria were transplanted into the colons of ‘germ-free’
mice, in which the normal population of gut bacteria is entirely absent, they exerted a potent immunomodulatory effect. Through systematic analysis of this microbial cohort,the researchers zoomed in on a specific subset of 17 distinct
Clostridia strains.
These strains collectively secrete a host of signaling molecules that promote Treg cell activation. “None of the organisms alone were nearly as potent as when they were inconsortium,” says Honda. “This suggests that cooperation
between the strains is essential to their therapeutic effects.”
The collective benefit also appears to pertain to humans;
analysis of the gut ‘microbiome’ in healthy patients versus
individuals with ulcerative colitis revealed that all 17 strains
were present at significantly lower levels in the latter group.
Accordingly, oral administration of this 17-microbe‘cocktail’ greatly mitigated intestinal inflammation in mousemodels of allergic diarrhea and ulcerative colitis (Fig. 1),
suggesting the potential for a more ‘natural’ treatment ofsuch conditions in humans. “A substantial number of patients don’t benefit from existing drugs, which also haveconsiderable adverse effects,” says Honda. “We want to clinically test our hypothesis that reconstituting these bacteria tonormal levels in patients may help restore immune tolerance
and resolve chronic inflammatory processes.”

Original paper
Atarashi, K., Tanoue, T., Oshima, K., Suda, W., Nagano,Y., Nishikawa, H., Fukuda, S., Saito, T., Narushima, S.,
Hase, K., Kim, S., Fritz, JV., Wilmes, P., Ueha, S., Matsushima, K., Ohno, H., Olle, B., Sakaguchi, S., Taniguchi,T., Morita, H., Hattori, M., Honda, K. Treg induction by arationally selected mixture of Clostridia strains from thehuman microbiota. Nature 500, 232–236 (2013).

Friends in Low Places Preserve Gut Health
Bacteria living in the mammalian intestine
help to digest dietary fiber, generating
metabolites that also control gut
inflammation

Research highlights

Figure: Commensal bacteria belonging to the
Clostridiales order help to control inflammationin the mammalian gut.
Image courtesy of the Centers for Disease Control andPreventionT

he bacterial communities that live in our intestine
should not be considered freeloaders—they contribute substantially to our well-being in a number of ways,
including assisting in the breakdown of otherwise indigestible dietary fiber. Hiroshi Ohno (Laboratory for Intestinal
Ecosystem) and colleagues have discovered a mechanism bywhich this digestive assistance also helps to prevent gut inflammation.
Bacteria belonging to the order Clostridiales are known tometabolize indigestible dietary fiber to produce metabolites,such as short-chain fatty acids (SCFAs). Ohno’s team foundthat mice lacking gut commensal bacteria including Clostridiales exhibit bloating within the cecum—the start of thelarge intestine—after consuming a high-fiber diet. However,this problem could be repaired by transplanting a Clostridiales-enriched gut bacterial community (Fig.).
In other recent work, Honda and his colleagues demonstrated that these same bacteria promote the differentiationof regulatory T (Treg) cells, which specifically prevent theimmune system from overreacting (detailed in the previouspage). Ohno suspected that this and his most-recent observation might be connected. “This led us to hypothesize thatbacterial metabolism of dietary fiber may be the cause of Treg
induction,” he says.
After searching for metabolic products that were elevated following consumption of a high-fiber diet, the researchThis article was reproduced from RIKEN Research withpermission
http://www.rikenresearch.riken.jp/eng/research/7607.
html

ers focused on one SCFA, butyrate, as a likely candidate.
Butyrate proved capable of converting immature ‘naive’ T
cells into Treg cells in culture, and a maize starch diet that had
been chemically enriched with butyrate invoked a similarly
potent Treg differentiation within the mouse colon. The same
butyrate-enriched maize starch diet failed to elicit Treg differentiation in mice lacking commensal microbes, suggesting that in addition to butyrate, bacterial components are
required as an antigen to be recognized by naive T cells.
Butyrate modulates the activity of histone deacetylase tomodify epigenetic status of chromatin via histone acetylation, which can dramatically affect gene expression, andOhno and his colleagues found the upregulation of gene encoding the Foxp3 transcription factor essential for Treg differentiation. “Bacterial butyrate affected the epigenetic statusof naive T cells to propel their differentiation into Treg cells
within the colonic tissue,” he says. This resulted in a strong
protective effect, and the increased numbers of Treg cells thatdeveloped following consumption of a butyrate-enricheddiet ameliorated inflammation in a mouse model of colitis.
These findings may thus reveal a critical component of
the pathology of human inflammatory bowel diseases as well
as a potential means for treatment. Ohno and his colleagues
now hope to explore whether the same mechanism is also
relevant in the inflammatory response to food allergies.

Original paper
Furusawa, Y., Obata, Y., Fukuda, S., Endo, T. A., Nakato,G., Takahashi, D., Nakanishi, Y., Uetake, C., Kato, K.,
Kato, T. Takahashi, M., Fukuda, N., Murakami, S., Miyauchi, E., Hino, S., Atarashi, K., Onawa, S., Fujimura, Y.,
Lockett, T., Clarke, JM., Topping, DL., Tomita, M., Hori,
S., Ohara, O., Morita, T., Koseki, H., Kikuchi, J., Honda,
K., Hase K., Ohno, H. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T

cells. Nature 504, 446–450 (2013)55

Rheumatoid Arthritis Research Shows the
Potential of Large-scale Genetic Studies
for Drug DiscoveryThe study, conducted by Dr. Robert M. Plenge from the Harvard Medical School and the Broad Institute in the USA and
Dr. Yukinori Okada from the Laboratory for Autoimmune Diseases, collaborating with colleagues from 70 institutions worldwide,is published in the journal Nature.
Genome-wide association studies are a method employed byscientists to identify the genes contributing to human disease.
The current Nature study is the first to demonstrate that integrating the information provided by genome-wide association studies with existing datasets of genomic and biological information,such as drug targets, can assist in the discovery of drugs to curehuman disease.
Rheumatoid arthritis is an autoimmune disease leading to
inflammation of the joints and affecting 0.5-1% of adults in thedeveloped world. The disease is thought to be caused by a complex combination of genetic and environmental factors and several genes have been shown to be associated with the disease.
However, most of the findings were based on single population
studies, and no large-scale trans-ethnic study had been carriedout to date.
The international team performed a genome-wide association
study meta-analysis on a total of over 100,000 subjects of European and Asian descent - 29,880 rheumatoid arthritis patients and73,758 controls – by analysing around 10 million genetic variants
called single nucleotide polymorphism (SNPs). They identified 42

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56

Research highlights

new regions in the genome (loci) that are associated with rheumatoid arthritis, bringing the total number of known rheumatoid
arthritis loci to 101.
By conducting bioinformatics studies integrating existing datasets with this new information, the researchers were able to pinpoint 98 genes in these 101 loci that could potentially contributeto the onset of rheumatoid arthritis. By integrating their findings
with existing drug databases they demonstrated that these genes
indeed possess many overlapping regions with the genes targeted by approved rheumatoid arthritis drugs – although this wasn’t
known when the drugs were developed. The team identified existing drugs used to treat cancer that also target rheumatoid arthritis genes and could potentially be used as therapy for the disease,such as CDK4/6 inhibitors.
The bioinformatics study also reveals that there is significant
overlap between the genes involved in rheumatoid arthritis, human primary immunodeficiency disorders and blood cancers.
“This study sheds light on the fundamental genes, pathways
and cell types that contribute to the onset of rheumatoid arthritis
and provides evidence that the genetics of rheumatoid arthritis
can provide important information for drug discovery,” conclude
the authors.“While there are previous anecdotal examples, ourstudy provides a systematic approach by which human genetic
data can be efficiently integrated with other biological information to derive biological insights and drug discovery,” they add.

Original paper
Okada, Y. Wu, D., Trynka, G., Raj, T., Terao, C., Ikari, K.,
Kochi, Y., Ohmura, K., Suzuki, A., Yoshida, S., Graham,
RR., Manoharan, A., Ortmann, W., Bhangale, T., Denny,JC., Carroll, RJ., Eyler, AE., Greenberg, JD., Kremer, JM.,
Pappas, DA., Jiang, L., Yin, J., Ye, L., Su, DF., Yang, J.,
Xie, G., Keystone, E., Westra, HJ., Esko, T., Metspalu,A., Zhou, X., Gupta, N., Mirel, D., Stahl, EA., Diogo, D.,
Cui, J., Liao, K., Guo, MH., Myouzen, K., Kawaguchi, T.,

Coenen, MJ., van Riel, PL., van de Laar, MA., Guchelaar,
HJ., Huizinga, TW., Dieudé, P., Mariette, X., Bridges SL
Jr., Zhernakova, A., et al. Genetics of rheumatoid arthritis contributes to biology and drug discovery. Nature
506, 376-381, 2013

Connecting the Genetic Dots inConnective Tissue Disorders
Scientists identify a mutated gene responsible
for a spectrum of skeletal and connective
tissue disorders

Research highlights

Figure: Spinal x-ray of an SEMD-JL1 patient.
© 2013 Shiro Ikegawa, RIKEN Center for Integrative
Medical SciencesT

he disorder known as spondyloepimetaphyseal dysplasia with joint laxity type 1, or SEMD-JL1, is characterized by skeletal abnormalities and loose ligaments that resultin spinal misalignment and respiratory problems. The genetic basis of SEMD-JL type 2, a related disorder, was recently determined, but the genetic underpinnings of the type 1
form of the disease remain unknown.
By studying the genomes of seven people with SEMDJL1, a large international research team led by Shiro Ikegawa
from the Laboratory for Bone and Joint Diseases has now
identified a gene that, when mutated, is responsible not only
for SEMD-JL1 but also a range of other bone and connective
tissue defects.
Noriko Miyake, who joined Ikegawa’s team from Yokohama City University, discovered the gene by sequencing theentire protein-coding region of the genomes of seven Japanese people with SEMD-JL1 from six unrelated families.
Using targeted sequencing to confirm initial ‘hits’, Masahiro Nakajima, a member of Ikegawa’s lab, showed that all of
the subjects, in addition to an eighth Vietnamese individualwith SEMD-JL1, had mutations in a gene called B3GALT6
on the short arm of chromosome 1. This gene codes for a
type of enzyme known as a galactosyltransferase II, whichis involved in the synthesis of the proteoglycan linker region
that helps form the structural cement of connective tissue.

This article was reproduced from RIKEN Research withpermission
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The researchers found that mutations in B3GALT6 caused
disease in a recessive fashion, meaning that all of the affectedindividuals had inherited two faulty copies of the gene.
The researchers then noticed that some of the individuals with SEMD-JL1 in the study exhibited many of thesame clinical characteristics as those found in people withanother connective tissue disorder—the progeroid form ofEhlers-Danlos syndrome (EDS), which is characterized bya defect in the synthesis of collagen. To investigate this further, they sequenced the B3GALT6 gene in four people withprogeroid-form EDS of unknown genetic cause. All four
subjects carried mutations in the B3GALT6 gene.
“B3GALT6 enzyme deficiency results in a wide variety ofdisorders,” says Ikegawa. “These diseases have been considered to belong to totally different categories of disease, but
actually are a spectrum of disorders affecting bone, cartilage,
muscle, tendon, ligament, and skin. Our findings will enable
genetic diagnosis of these diseases.” The researchers anticipate that the findings could open the door to future therapies for disorders related to B3GALT6.

Original paper
Nakajima, M., Mizumoto, S., Miyake, N., Kogawa, R.,
Iida, A., Ito, H., Kitoh, H., Hirayama, A., Mitsubuchi,
H., Miyazaki, O. Kosaki, R., Horikawa, R., Lai, A., Mendoza-Londono, R., Dupuis, L., Chitayat, D., Howard,
A., Leal, GF., Cavalcanti, D., Tsurusaki, Y., Saitsu, H.,
Watanabe, S., Lausch, E., Unger, S., Bonafé, L., Ohashi,
H., Superti-Furga, A., Matsumoto, N., Sugahara, K.,
Nishimura, G., Ikegawa, S. Mutations in B3GALT6,

which encodes a glycosaminoglycan linker region enzyme, cause a spectrum of skeletal and connective tissue disorders. Am J Hum Genet. 92, 927–934 (2013)57

Getting Ahead of the Curve
A gene involved in spinal development may
contribute to a common childhood disease
responsible for spinal curvature

Research highlights

Figure: Spinal x-ray of a child with AIS.
© 2013 Shiro Ikegawa, RIKEN Center for Integrative
Medical Sciences

A

dolescent idiopathic scoliosis (AIS) is the most common pediatric skeletal disease, causing complex rotational deformity of the spine in approximately 2% ofschool-age children worldwide. Recent studies have impliedthat AIS may, at least in part, be caused by genetics, but thepathogenesis of AIS still remains poorly understood. Aninternational research team led by Shiro Ikegawa from theLaboratory for Bone and Joint Diseases has now identified
another gene that may contribute to AIS by altering spinal
development.
Ikegawa’s group previously identified a gene associated with AIS in Japanese populations. To further reveal thegenetics underlying the disease, Ikuyo Kou from Ikegawa’s
lab conducted a genome-wide association study including
over 27,000 Japanese individuals. In this study, Kou identified another single nucleotide polymorphism (SNP) that issignificantly associated with AIS in Japanese subjects. Additional testing showed that the association was replicated in
Han Chinese and European populations, marking the firsttime a SNP associated with AIS has been identified in distinct populations.
Kou also determined that the SNP is located in thegene GPR126, which encodes a receptor protein. GPR126 is
known to be associated with the development of the sheaths
that insulate nerve fibers, but its other functions are large-

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html

58

ly unknown. By examining the tissue-specific expression of
GPR126, the researchers found that it is expressed in spinal
cartilage, implying a role in the development of the spine.
Using zebrafish, Long Guo and Chisa Shukunami who
joined Ikegawa’s team from Kyoto University knocked down
GPR126 expression to investigate the role of the gene in vertebrate development. They found that zebrafish with lower
levels of the GPR126 protein had shorter bodies and theirvertebrae showed delayed bone formation, or osteogenesis. “Our zebrafish knockdown studies indicate the importance of GPR126 in bone tissue growth and formation, andraise the possibility that abnormal spinal development andgrowth induce AIS,” explains Kou.
GPR126 is also known to be associated with shortertrunk length and reduced height in European populations,which coincides with the new findings. “Their associations
are in the same direction; the susceptibility allele is the samefor both AIS and shortened trunk,” says Kou.
Although the identification of GPR126 sheds new lighton the pathogenesis of AIS, the two genes implicated so far
explain only ~1% of the variation in AIS traits. Further studies therefore remain necessary to determine precisely how
mutations of GPR126 lead to AIS in humans.

Original paper
Kou, I., Takahashi, Y., Johnson, T. A., Takahashi, A., Guo,
L., Dai, J., Qiu, X., Sharma, S., Takimoto, A., Ogura, Y.
Jiang, H., Yan, H., Kono, K., Kawakami, N., Uno, K., Ito,
M., Minami, S., Yanagida, H., Taneichi, H., Hosono, N.,
Tsuji, T., Suzuki, T., Sudo, H., Kotani, T., Yonezawa, I.,
Londono, D., Gordon, D., Herring, JA., Watanabe, K.,
Chiba, K., Kamatani, N., Jiang, Q., Hiraki, Y., Kubo, M.,
Toyama, Y., Tsunoda, T., Wise, CA., Qiu, Y., Shukunami,C., Matsumoto, M., Ikegawa, S. Genetic variants in
GPR126 are associated with adolescent idiopathic scoliosis. Nat Genet. 45, 676–679 (2013)

Compound that Could Prevent Acute
Blood Cancer Relapse Identified

Research highlights

Figure: Eradication of
AML cells from bonemarrow (in brown)
following treatment
with small molecule
RK-20449 (lower
panels).

T

he research group led by Fumihiko Ishikawa (Laboratory for
Human Disease Models) identified a compound that could
be used as a new treatment to prevent relapse in acute myeloid
leukemia patients.
In a study published in Science Translational Medicine, they
showed that this compound reduces the risk of relapse in a mousemodel of the human disease. They report that this compound
could be most active in patients that carry a mutation lowering
their chances of recovery.
Acute myeloid leukemia (AML) is an acute type of bloodcancer that starts in the blood-forming cells in the bone marrow.
AML is the most common type of acute leukemia in adults.
While many patients are able to fight off the disease at firstwith conventional chemotherapy, long-term outcomes in the majority of patients are poor due to disease relapse.
“To improve patient outcomes, it is crucial to understand themechanisms of AML relapse and to develop effective treatment
strategies to reduce AML relapse,” explains Ishikawa.
Over the last decade, bone marrow cells called leukemia stem
cells (LSC) have been recognized as key players in human AML
pathogenesis as well as in chemotherapy resistance and relapse.
Previous studies have suggested that LSCs might cause relapse if
they are not properly eliminated by conventional chemotherapy.
By transplanting LSCs obtained from AML patient samples
into immune-deficient newborn mice, Ishikawa and his team developed a mouse model for AML, which they used to study AML
and LSCs.
Using this model, they were able to identify a protein (HCK)present in higher quantities in human AML LSCs than in normal

This article was reproduced from RIKEN Press Releasewith permission
http://www.riken.jp/en/pr/press/2013/20130418_1

blood-forming stem cells, and that could be used as a target fortherapeutic agents against human AML LSCs.
In the present study, the researchers screened a library oftens of thousands of small molecules that could act as therapeutic agents by specifically inhibiting HCK. They isolated one small
molecule that was highly active against patient-derived AML
LSCs grown in culture. To assess the potential of this molecule
for therapeutic development, they administered it to their mousemodel of AML. They found that administration of this molecule
resulted in a significant reduction of human AML cells in the
blood of the mice, as well as a reduction of human AML LSCs in
the bone marrow of the mice (Fig.).
In particular, in mice engrafted with human AML derived
from patients with the FLT3-ITD mutation, one of the mutations
associated with worse clinical outcomes, the administration of thesmall molecule led to nearly complete elimination of both AML
LSCs and non-stem AML cells in the bone marrow of multiple
bones (femur, tibia, sternum and spine) as well as the spleen andperipheral blood.
“These findings suggest that treatment with this small molecule may help reduce relapse in AML patients,” conclude theauthors.
“However, more work is needed before this small moleculecan be delivered to patients as a therapeutic agent. We now plan
to proceed with a more in-depth biochemical and pharmacologic
characterization of this compound in the lab, to find out whether
it is safe and to determine which subset of AML patients could
benefit from it. Ultimately, we hope to develop a drug that can beused in the clinic,” adds Ishikawa.

Original paper
Saito, Y., Yuki, H., Kuratani, M., Hashizume, Y., Takagi,
S., Honma, T., Tanaka, A., Shirouzu, M., Mikuni, J.,
Handa, N. et al. A pyrrolo-pyrimidine derivative targets

human primary AML stem cells in vivo. Sci Transl Med.
5, 181ra52 (2013).

59

Genetic Variations May Help Identify Best
Candidates for Preventive Breast Cancer
Drugs

N

ewly discovered genetic variations may help predict
breast cancer risk in women who receive preventive
breast cancer therapy with the selective estrogen receptor
modulator drugs tamoxifen and raloxifene, a Mayo Clinic-led study has found. The study is published in the journal
Cancer Discovery.
“Our findings are important because we identified genetic factors that could eventually be used to select women who
should be offered the drugs for prevention,” said James Ingle,
M.D., an oncologist at Mayo Clinic.
Dr. Ingle and collaborators at the National Surgical Adjuvant Breast and Bowel Project (NSABP) and the RIKEN
Center for Integrative Medical Sciences (IMS) conducted agenome-wide association study involving 592 patients whodeveloped breast cancer while receiving preventive therapy and 1,171 matched controls. Participants were selectedfrom 33,000 women enrolled in the NSABP breast cancer
prevention trials. This research was supported by a Pharmacogenomics Research Network grant from the National Institute of General Medical Sciences and the National Cancer
Institute.
The researchers analyzed participants’ DNA to identify
variations in their genetic makeup and identified two genetic variations, or single nucleotide polymorphisms (SNPs),
that were associated with breast cancer risk in or near thegenes ZNF423 and CTSO.
They discovered that women with favorable variations inthese genes were more likely to respond to preventive therapy with the drugs while women with unfavorable variations
may not. In addition, women with unfavorable variations
had a five-fold increased risk of developing breast cancer.
Dr. Ingle says the recent guidelines by the U.S. Preventive
Services Task Force emphasize that selective estrogen receptor modulators (SERM) therapy with tamoxifen and raloxifene can lower a woman’s risk for developing breast cancer.

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60

Research highlights

However, there currently is no way to know which women
will benefit from the therapy.
“This is a major step toward truly individualized prevention of breast cancer,” says Dr. Ingle. “Our findings provide
clear direction as to which women are likely and which areunlikely to benefit from tamoxifen or raloxifene.” Dr. Ingle
says the findings provide the basis for a reinvigoration of research efforts in breast cancer prevention.
The researchers also studied breast cancer cell lines withthe most common variation and the less common variationof the SNPs. They found that in cells with the most common
variation of the SNPs, estrogen increased expression of bothZNF423 and CTSO and the expression of BRCA1, a geneassociated with breast cancer risk. Estrogen did not increase
expression of these genes in cells that had the less common
form of the SNPs. Importantly, however, when tamoxifen or
raloxifene were added to estrogen, there was a striking reversal in the patterns of expression of ZNF423 and BRCA1.
In cells with the less common ZNF423 SNP, expression of
ZNF423 and BRCA1 rose dramatically. This reversal in expression patterns provides a potential explanation for thedecreased occurrence of breast cancer in women undergoing
SERM therapy who carry this SNP.
NIH’s National Institute of General Medical Sciences
and National Cancer Institute funded this research throughgrants U19GM61388, P50CA116201, U10CA37377,
U10CA69974, U24CA114732 and U01GM63173.

Original paper
Ingle JN, Liu M, Wickerham DL, Schaid DJ, Wang L,
Mushiroda T, Kubo M, Costantino JP, Vogel VG, Paik S,
Goetz MP, Ames MM, Jenkins GD, Batzler A, Carlson
EE, Flockhart DA, Wolmark N, Nakamura Y, Weinshilboum RM. Selective Estrogen Receptor Modulators andPharmacogenomic Variation in ZNF423 Regulation of
BRCA1 Expression: Individualized Breast Cancer Prevention. Cancer Discov. 3, 812-825 (2013)

Drug Treatment Improves Survival of
Insulin-producing Cells
Pretreatment with a calcium-blocking
drug improves the effectiveness of islet
transplantation for diabetes in mice

Research highlights

Figure: The process of clinical islet transplantation.
Reproduced from PLoS Medicine 1, e58 (2004). Licensed under CC by 2.5 at dx.doi.org/10.1371/journal.
pmed.0010058. © 2004 M. A. Naftanel and D. M.
HarlanT

he transplantation of insulin-producing islet cells froma donor pancreas could help diabetes sufferers avoid
the need for daily insulin injections. However, the use ofthis experimental procedure is hampered by an immune
response in the recipient that often rejects the transplanted
islet cells. Masaru Taniguchi from Laboratory for Immune
Regulation and Yohichi Yasunami from Fukuoka University
have now led research that has improved the efficiency ofthe procedure by pretreating the islet cells with a drug thatblocks the sodium–calcium exchanger (NCX) protein.
Islet transplantation often involves the use of islet cells
from two or three different donors in order to achieve sufficient cell engraftment. “This low efficiency has been a major
obstacle facing clinical islet transplantation,” says Taniguchi.
Taniguchi, Yasunami and their respective lab groups set
out to improve the islet transplantation procedure by pretreating islet cells prior to transplantation with a drug thatblocks the NCX protein. This pretreatment protected thecells from innate immune responses in the liver, the site ofislet transplantation, which led to longer-term survival of
the cells in mouse models of diabetes.
“Pretreatment of donor islets with an NCX inhibitor prior to transplantation prevents early loss of transplanted islets
and affords a new strategy to improve the efficiency of islet
transplantation,” says Taniguchi. Notably, the method allows

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for improved engraftment efficiencies without requiringtransplant recipients to take any additional anti-rejection
drugs. “Our new strategy to target donor islets does not add
further risks to recipients,” notes Taniguchi.
The drug pretreatment is thought to work by stopping
NCX from boosting the intracellular levels of calcium ions.
Normally, this calcium influx leads to low oxygen conditions,which trigger the release of a protein called high-mobility
group box 1 (HMGB1) from islets soon after their transplantation. HMGB1 in turn activates immune cells, causing theearly loss of transplanted islets. The NCX-blocking drug,
called SEA0400, can prevent this cascade of events.
After transplanting pretreated and untreated islets into
diabetic mice, the research team found that mice receiving
SEA0400-treated islets displayed normal blood sugar control. In contrast, the control animals, which received thesame number of untreated islets, experienced elevated blood
sugar levels due to a lack of functional insulin-producing
cells, which presumably had been attacked by the immune
system. This same effect was seen whether the transplanted
islets were human or murine in origin.

Original paper
Mera, T., Itoh, T., Kita, S., Kodama, S., Kojima, D.,
Nishinakamura, H., Okamoto, K., Ohkura, M., Nakai, J.,
Iyoda, T., Iwamoto, T., Matsuda, T., Baba, A., Omori, K.,
Ono, J., Watarai, H., Taniguchi, M., Yasunami, Y. Pretreatment of donor islets with the Na+/Ca2+ exchanger
inhibitor improves the efficiency of islet transplantation. Am J Transplant. 13, 2154-2160 (2013)

61

Fighting Asthma Drug Resistance
The poor efficacy of corticosteroid
treatments in some patients with severe
asthma could be overcome by blocking theaction of an inflammatory protein

Research highlights

Figure: Asthmatic airway inflammation in miceinduced by IL-33 and TSLP (bright pink, left)
cannot be reduced by steroid treatment (center)but can be reduced when steroid treatment isaugmented with the STAT5 inhibitor pimozide(right).
Reproduced, with permission, from Ref. © 2013 H.
Kabata et al.

Current asthma treatments include drugs that open up
the tubes of the lungs and corticosteroids that fightlung inflammation. Some patients, however, are stubbornly
resistant to corticosteroids, limiting the therapies available
to them. Shigeo Koyasu and Kazuyo Moro from the Laboratory for Immune Cell System, in collaboration with Hiroki
Kabata of the Keio University School of Medicine and Koichiro Asano of Tokai University and co-workers, have now
found that the inflammatory protein called thymic stromal
lymphopoietin (TSLP) and its downstream signaling molecules play key roles in the resistance of natural helper (NH)cells, a member of group 2 innate lymphoid cells (ILC2s),
to the anti-inflammatory effects of corticosteroid treatment.
Asthma is a chronic disease of the airways characterized
by persistent lung inflammation. The condition is known tobe driven by the production of type 2 cytokines, such as interleukin (IL)-5 and IL-13, by NH cells.
The researchers developed a mouse model for this condition by administering an allergen and the pro-inflammatory
cytokine IL-33 to mice. The treatment caused an increase in
NH cells and mucus production in the lung, similar to the
response observed in the lungs of asthmatic patients. Micetreated with corticosteroids did not show any improvement,
indicating that the condition was resistant to the drug.
Koyasu and his colleagues found that the lungs of theThis article was reproduced from RIKEN Research withpermission
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62

mice that received the allergen and IL-33 also had higher
levels of TSLP. Separately, they found that the addition of
TSLP to NH cells cultured in the presence of IL-33 induced
the proliferation of NH cells, even when corticosteroids were
present. These findings suggest that TSLP causes NH cells to
become resistant to the anti-inflammatory effects of corticosteroid treatment.
In other cell culture experiments the researchers foundthat in the absence of TSLP, NH cells died more readily under corticosteroid treatment, indicating that an antibody
that blocks TSLP could eliminate corticosteroid resistance.
The team then discovered that in the presence of TSLP,
NH cells activate the intracellular signaling protein STAT5.
Pimozide, widely used as an anti-psychotic drug, is known
to inhibit STAT5. Experiments confirmed that pimozide did
indeed restore the susceptibility of NH cells to corticosteroids in cell culture (Fig.)
The research provides strong evidence that pimozide
could be used to eliminate resistance to corticosteroids forthe treatment of asthma. “Because pimozide has alreadybeen in clinical use for a while, this drug could be applied
to patients with corticosteroid-resistant severe asthma after
appropriate clinical studies are conducted,” explains Koyasu.

Original paper
Kabata, H., Moro, K., Fukunaga, K., Suzuki, Y., Miyata,J., Masaki, K., Betsuyaku, T., Koyasu, S., & Asano, K.
Thymic stromal lymphopoietin induces corticosteroid
resistance in natural helper cells during airway inflammation. Nature Commun. 4, 2675 (2013).

Rapid Recall to Fight Familiar Foes
By tinkering with immune cell development,
researchers learn how the body mounts anaccelerated response to recurring threats

Research highlights

Figure: Memory B cells quickly recognize threats
previously encountered by the immune system tomount a rapid and robust antibody response.
© Hemera/ThinkstockT

he immune system’s first encounter with a potential
threat is a valuable learning experience. Through aprocess of genetic recombination, our immune B cells can
produce a wide array of B cell receptor (BCR) molecules,
each recognizing a distinct molecular target. When a naive
B cell bumps into its specific target, it initiates an immune
response that yields antibodies with the same specificity as
its BCR, but also gives rise to ‘memory cells’ that can quickly recognize the target if it appears in the future. Working
with genetically modified mice, Tomohiro Kurosaki’s team,Laboratory for Lymphocyte Differentiation, has now gained
insight into how memory cells mobilize.
“Memory B cells achieve rapid and robust antibody production during a secondary immune response, but its molecular mechanism was unknown,” explains Kohei Kometani, aresearcher in Kurosaki’s lab and lead author of the study.
Naive B cells typically produce BCRs and antibodies thatbelong to the immunoglobulin M (IgM) class of proteins. In
contrast, memory B cells produce an alternative immunoglobulin G (IgG) subtype of BCRs and antibodies throughan additional gene recombination process called ‘classswitching’. Some researchers believe that the IgG form of
BCR contains structural elements that stimulate the memory B cell response, while others have favored cellular mechanisms besides IgG-induced signaling.

This article was reproduced from RIKEN Research withpermission
http://www.rikenresearch.riken.jp/eng/research/7431

In initial experiments, the researchers worked with IgMnaive and IgG memory cells that specifically recognize aknown antigen, 4-hydroxy-3-nitrophenylacetyl (NP). As
expected, the former cells proliferated in response to NP,
while the latter promptly developed into antibody-secreting
plasma cells, as occurs in a typical secondary immune response. To determine whether IgG is specifically responsible, Kurosaki’s group generated cloned mice derived from an
IgG memory cell that recognizes NP.
The resulting animals produced NP-specific IgG naive B
cells, which do not normally occur in nature. Remarkably,
these cells responded to NP by proliferating in essentially the same fashion as IgM naive cells, suggesting that IgG
alone does not drive the memory response. A comparative
analysis of gene expression revealed an alternative mechanism, controlled by a protein called Bach2. “We found that
Bach2 is reduced in memory B cells, and is important for
their enhanced antibody production,” says Kometani.
By revealing this IgG-independent mechanism, theseresults should help resolve the long-standing debate overmemory cell function. However, this finding is just a starting
point, and Kurosaki’s group is now engaged in exploring the
upstream factors that switch off Bach2 production in memory cells.

Original paper
Kometani, K., Nakagawa, R., Shinnakasu, R., Kaji, T.,
Rybouchkin, A., Moriyama, S., Furukawa, K., Koseki,
H., Takemori, T. & Kurosaki, T. Repression of the transcription factor Bach2 contributes to predisposition of
IgG1 memory B cells toward plasma cell differentiation.
Immunity 39, 136–147 (2013).

63

Helping Cells Make a Commitment
Multiple levels of gene inhibition help
determine and maintain the developmental
destiny of immune cells

Research highlights

Figure: Researchers at RIKEN have investigated
the role of the Thpok gene in the development of
the two classes of immune T cells.
© iStockphoto/ThinkstockI

mmune T cells can be broadly categorized into two classes: cytotoxic T lymphocytes (CTLs), which directly kill
infected or cancerous cells, and helper T cells, which coordinate the overall immune response. Immature T cells commit
to one or the other lineage via a process called positive selection, and generally remain locked into their developmental
fate from that point onward. The Laboratory for Transcriptional Regulation led by Ichiro Taniuchi has now gained insight into how these cells remain dedicated to their careerpath.
It is known that the Thpok gene is a critical activator ofhelper T cell development, and gets switched off in developing CTLs by a regulatory region called the silencer. However,Taniuchi’s team determined that the silencer only provides
the initial deactivation signal, and requires further support
to make its orders ‘official’.
Many genes undergo what is known as epigenetic regulation through the addition of various chemical modifications
to the chromosome. For instance, different patterns of ‘histone methylation’ can either activate or repress nearby genes.
The researchers determined that while the silencer facilitates
the accumulation of repressive methylation patterns at theThpok gene in CTL precursors, helper T cell precursors exhibit activating methylation patterns. When the researchers
selectively deleted the silencer in cells that had already com-

This article was reproduced from RIKEN Research withpermission
http://www.rikenresearch.riken.jp/eng/research/7324

64

mitted to CTL development, Thpok remained repressed, indicating that these marks are critical to long-term inhibitionof this gene.
Taniuchi and his colleagues found that by engineering additional copies of the silencer region into the Thpok
gene, they could induce repression in helper T cells where
the gene would otherwise be active. If introduced into T cell
precursors, these additional silencer copies caused the accumulation of repressive histone methylation marks normallyobserved in CTLs, and disrupted normal helper T cell development. This demonstrates the critical role of the silencer
in establishing T cell fate.“Our finding that the epigenetic
processes that stably silence Thpok can occur independentlyof commitment to the cytotoxic-lineage is striking,” says Hirokazu Tanaka, lead author of the study.
Importantly, their work also suggests how these instructions might be overridden. Previous studies have suggested that prolonged signaling via the T cell receptor (TCR)
specifically promotes helper T cell development, and theseprolonged signals may work by preventing inhibitory modifications that would otherwise accumulate through the influence of the Thpok silencer. “Our findings provide for the
first time an epigenetic view as to why persistent TCR signals
are necessary,” says Tanaka.

Original paper
Tanaka, H., Naito, T., Muroi, S., Seo, W., Chihara, R.,
Miyamoto, C., Kominami, R., Taniuchi, I. Epigenetic
Thpok silencing limits the time window to choose
CD4+ helper-lineage fate in the thymus. EMBO J. 32,
1183–1194 (2013)

Building Blocks Help Silence Genes
Head-to-tail connections in gene-repressing
complexes maintain proper DNA regulation
during embryo development and tumor
formation

Research highlights

Figure: A functional SAM domain is needed to facilitate PRC1 clustering and PcG-mediated genesilencing.
© 2013 ElsevierP

olycomb-group (PcG) proteins play an important rolein controlling gene expression. Complexes containing
PcG proteins are thought to inhibit or ‘silence’ gene activity
by localizing to specific targets in the genome and remodeling how DNA is wound up into chromosomes, but the exact
mechanism by which these complexes repress gene activity
remains poorly understood. Kyoichi Isono, Haruhiko Koseki
and colleagues from the Laboratory for Developmental Genetics have now pinpointed the part of a critical PcG protein complex that is essential for maintaining a robust yet
reversible gene repression program during both mammaliandevelopment and cancer progression.
Isono, Koseki and their colleagues set out to identify theformation mechanism of a cluster of PcG proteins known
as Polycomb-group repressive complex-1 (PRC1). They focused their attention on a particular domain within one of
the molecules in the PRC1 complex: the sterile alpha motif(SAM) of polyhomeotic-like protein 2 (Phc2). The SAM domain helps to keep Phc2 proteins in the same orientation,
facilitating head-to-tail, building-block-like linking of repeated copies of the PRC1 complex (Fig.)
The team created human cells designed to express Phc2
carrying a mutation in the SAM domain. This mutation prevented PRC1 binding but did not affect the basic assemblyof each complex. Nonetheless, the researchers observed aThis article was reproduced from RIKEN Research withpermission
http://www.rikenresearch.riken.jp/eng/research/7547.
html

substantial reduction in PRC1 cluster or ‘body’ formation in
the cell nucleus, indicating that the construction of linked
repeats of PRC1, driven by SAM domain polymerization,
may be necessary for proper functioning of the complex. In
embryonic mice genetically engineered to possess a mutant
SAM domain, Isono, Koseki and their team observed defects
in the developing skeleton, further supporting the notionthat the aberrant PRC1 bodies were not maintaining proper
gene regulation in the absence of the SAM domain.
The findings illuminate a key aspect of mammalian embryo formation. According to Isono, however, the results
could also have implications that go well beyond the understanding of basic developmental processes. “The repressivefunction of PRC1 has a strong impact on not only pluripotency and differentiation of stem cells but also tumorigenesis,” he says. “The mechanism that underlies SAM polymerization and depolymerization could be useful for regenerative medicine and cancer therapy.”
A particularly exciting potential therapeutic target might
be compounds that destroy the interactions between SAM
domains, which could halt tumor growth in cancer patients.
“I believe we can develop such drugs by conducting microscopic screenings for PcG body morphology,” Isono says.

Original paper
Isono, K., Endo, T. A., Ku, M., Yamada, D., Suzuki, R.,
Sharif, J., Ishikura, T., Toyoda, T., Bernstein, B. E. &Koseki, H. SAM domain polymerization links subnuclear
clustering of PRC1 to gene silencing. Dev Cell. 26,
565–577 (2013).

65

iPS ProjectI

nduced pluripotent stem (iPS) cells possess tremendous therapeutic potential in the fields of both regenerative medicine andimmune therapy. We have started an activity to apply iPS technology
to mouse and human immunology research and therapeutic development. On a collaborative basis with individual research activities
in RCAI, the core facility for iPS research is engaged in developing
efficient protocols to reprogram various lymphocytes and induce
differentiation of iPS cells into lymphoid lineage cells. This activity
is partly supported by JST.
This year, the facility established iPS cells from mature cytotoxic
T cells specific for the melanoma epitope MART-1. JKF6 cells are
long-term cultured tumor infiltrating lymphocytes that were originally derived from a melanoma patient and have been maintainedat the Surgery Branch of the National Cancer Institute. JKF6 cells
are specific for the complex of MART-1-peptide and HLA-A*02:01,
and can be visualized as MART-1-tetramer+ cells by flow cytometry.
These MART-1-specific T cells were transduced with Yamanaka factors, after which they established clones that form colonies with human ESC-like morphology (Vizcardo R., et al., 2013). When co-cul-

Projects

tured with OP9/DLL1 cells, the MART-1-iPS cells efficiently generated CD8+ T cells, and more than 90% of these cells were specific forthe original MART-1 epitope. Stimulation of these CD8+ T cells with
HLA-A*02:01-expressing cells pulsed with MART-1-peptide resulted in the secretion of IFNγ. The present study thus provides a novel
method for cloning and expanding functional CD8+ T cells specificfor a given antigen, which can potentially be applied for immune celltherapy against cancer.

Figure: iPS based approach for regeneration of functional antigen-specific
CTLs.
MART-1 derived iPSCs were separated into small clumps and plated on OP9 feeder
cells. On day 13, cells were transferred to co-culture with OP9-DLL1 feeder cells. On
day 35, cells were stimulated by anti-CD3 mAb for inducing CD8+ cells. After stimulation, CD8+ CD3+ MART-1 tetramer+ cells were sorted for an INFγ production assay.
They were co-cultured with a human EBV-transformed lymphoblastoid cell line expressing HLA-A*02:01 with or without MART-1 peptide for 24hr. These cells produced
a substantial amount of INFγ in the presence of the specific peptide.

Identification of Novel Causative
Genes for Common Variable Immunodeficiency

To understand the mechanisms responsible for homeostasis
in the human immune system, we sought to identify novel
causative genes for common variable immunodeficiency (CVID),
through a collaboration with Tokyo Medical and Dental University(TDMU). CVID is the most frequent symptomatic primary immunodeficiency encountered in adults.
To this end, we have carried out whole exome analysis (WEA)
of DNA samples obtained from five patients with CVID and theirfamily members. After conventional WEA with HiSeq 2000, variant
calling, gene annotation, and elimination of single nucleotide variants at IMS, we identified one to eight potential somatic changes
for each patient. One of the genes identified as disease-causing wasa gene that has been reported as responsible for a severe type of
CVID.

66

We also identified a few candidate heterologous mutations thatwere each only found in a single patient or in a patient and themother. These mutations have not been reported in the literature,and their function in the human immune system is yet to be explored. TMDU will sequence the identified candidate genes in the>200 CVID samples in their cohort, and IMS investigators are in
the process of determining their functions in the immune system
by using different models.

Modeling Skin Diseases
Laboratory for Immunogenetics
Laboratory for Integrative Genomics
Laboratory for Disease Systems Modeling
Laboratory for Integrated Bioinformatics
Laboratory for Developmental Genetics

Projects

Laboratory for Metabolomics
Laboratory for Immune Homeostasis
Laboratory for Cytokine Regulation
Laboratory for Respiratory and Allergic DiseasesThe pathogenesis of human diseases is a highly complex process
because of the complexity of not only pathogenetic insults and homeostatic responses but also of the structures of organs and tissues that
include many cells of different origins. To tackle such complexity, we haveinitiated center-wide projects to understand the pathogenesis of atopic
dermatitis (AD), autoimmune diseases, primary immunodeficiency andothers, in which multiple research groups work interactively and synergistically to achieve their common objectives. In each project, we first aim
to understand molecular and cellular networks underlying homeostasis ofeach organ/tissue. We have initiated this project with skin because GWAS
data for AD and related diseases and several unique dermatitis model animals are already available. Skin is a complex and highly dynamic organ
where there are continuous processes of proliferation, differentiation, migration, and death of cells in addition to constant surveillance by immune
and neural systems. This implies that highly integrative efforts, from basic
research to clinical applications, are required to model skin homeostasis.

In order to unify the expertise of the various IMS laboratories in a coordinated manner, it is important to thoroughly considered project planningwith specific milestones. Our primary goal in the AD project, and hence
the initial milestone, is to use a combined computational and experimental approach to investigate the molecular interaction network leading tothe onset of AD, its underlying dynamics and how they play a role in the
progression of skin dysfunction in AD.

Figure: Molecular and cellular events preceding atopic dermatitis onset inmouse skin tissue
A genetic factor, a point mutation in the Jak1 signal transduction molecule, induces
protease over-expression in the epidermis and induces sequential events in various
cells in skin tissue under the influence of environmental factors and ultimately resultsin atopic dermatitis.

Collaboration with the SardiNIA ProjectT

he SardiNIA project was originally launched by the National
Institute on Aging (NIA) in NIH (US) and Italian investigators(https://sardinia.irp.nia.nih.gov/) to unravel the genetic processes
involved in age-related traits and diseases in people living on the
Mediterranean island of Sardinia. As an extension of the SardiNIA
project, they have focused on immune diseases and recently elucidated human genetic variations regulating immune cell levels in
health and disease (Cell, 2013, 155(1):242-256). However, how variations in immune cell levels eventually result in immune diseases,
e.g. autoimmunity, still remains unclear. Thus, to bridge the gap
between immune cell levels and disease etiology, we have begun tocollaborate with the SardiNIA group through generation of micewith genetic variations corresponding to those found in humans:
Based on the human genetic variations identified by the SardiNIA group, genetically engineered mice have been designed and areunder development at IMS. In addition, we have performed protein-protein interaction assays to evaluate the impact of the genetic
variation(s) in the protein-coding region identified by the SardiNIA
genome cohort at the molecular level. In this regard, the collaboration with the SardiNIA group serves as an extramural version of the
on-going IMS projects, which aim to fill the gap between research inhumans and mice. Thus, we have very good reasons to collaborate
with the SardiNIA group toward our shared goal. We expect thistri-lateral collaboration will considerably enhance the international
visibility of our “integrative medical science (IMS)” approach using
animal models.

Figure: IMS Approach in a Tri-Lateral Collaboration with the SardiNIA Project67

Projects

Linkage to RIKEN Program for
Drug Discovery and Medical Technology Platforms
(DMP)

I

MS aims to contribute to the identification of new treatments forcancer and other diseases by promoting collaboration with DMP.
The DMP was founded in RIKEN in 2010 in order to support allphases of development of new therapeutics, from the discovery ofpromising targets to the identification of potential lead compounds
such as small molecules and antibodies, and the acquisition of intellectual property rights to drugs and technologies that can thenbe brought to the development phase. The implementation of drug
discovery requires different technology, thus DMP established several medical technology platforms that promote research and development. IMS contributes to this effort in several ways, including

by setting up a facility for development of antibody drugs (DrugDiscovery Antibody Platform Unit lead by Toshitada Takemori).
IMS now has five programs in association with DMP, including cancer treatment with NKT cells (Masaru Taniguchi), allergy
prophylaxis (Masaru Taniguchi), Artificial adjuvant vector cells(Shin-ichiro Fujii), Leukemia treatment drugs targeting leukemicstem cells (Fumihiko Ishikawa), and a Mucosal Vaccine delivery
System (Hiroshi Ohno). Some of programs will provide the transferof potential drug candidates to pre-clinical and clinical phases ofdrug development.

Figure: IMS links to the DMP project

Humanized Mouse ResearchWe have created humanized mice by injecting human cord
blood hematopoietic stem cells (HSCs) into immune-deficient NOD/SCID/Il2rgKO (NSG) newborns. Though the NSG humanized mouse model has enabled us to achieve high levels of human leukocyte engraftment in multiple organs, limitations remain
due to species barriers between human immune cells and the mousemicroenvironment. In the last few years, in collaboration with Dr.
Leonard Shultz at the Jackson Laboratory, the IMS Laboratories for
Human Disease Models, Developmental Genetics, and Integrative
Genomics have been trying to develop mice with a humanized microenvironment. By infecting humanized mice with EBV, we haveshown the development of HLA-restricted human CD8+ T cells in
HLA class Iexpressing NSG mice (PNAS 2010). We are currently
evaluating whether a peptide vaccine can elicit an Ag-specific CTL

Figure: Development of NSG mice expressing
human SCF
We have created new strains of NSG mice expressing
two distinct spice variants of stem cell factor (SCF),
membrane-bound form and soluble form. One of thevariants (Variant A: SCF220) lacks the primary cleavage site, therefore human SCF is not secreted into the
BM but remains as a membrane bound form.

68

response in the HLA class I TG NSG mice.
For humanized BM environment, we have developed NSG
mice expressing two distinct splice variants of human SCF, themembrane-bound (SCF220) and soluble (SCF248) forms. In bothstrains, we found significantly higher engraftment of human CD45+
leukocytes in the bone marrow compared with conventional NSG
mice (Blood 2012). In addition, expression of human SCF in themouse environment has overcome human B cell-dominant engraftment in the recipient mouse BM that has been considered as oneof the major differences between humanized mouse BM and native
human BM. We aim to clarify the potentially distinct roles of membrane-bound and soluble forms of SCF in supporting human HSC/
hematopoietic progenitor cell development and in human granulocyte development.

NKT ProjectN

KT cells are known to enhance immune responses. The medical innovation groups in IMS have launched projects aimed
at application of NKT cells to cancer and allergic disease as follows.
Based on previous success using NKT ligand, α-galactosylceramide (α-GalCer)-loaded dendritic cells (DCs) against advanced
lung cancer, we started a new collaboration with the National Hospital Organization of a randomized phase IIa trial in early stage lungcancer and also a phase IIa trial for head and neck cancer, in collaboration with Chiba University. Second, based on two previous studies in which we demonstrated the efficacy of human iPS-derived T
cells (Cell Stem Cell 2013) and mouse iPS-NKT cells (JCI 2010), we
will establish human iPS-NKT cells. This IMS iPS project on NKT
cell-targeted therapy has been accepted as a Center for Clinical Application Research in the Research Center Network for Realizationof Regenerative Medicine, Japan in 2013. Third, we previously established artificial adjuvant vector cells against cancer that are composed of tumor mRNA and α-GalCer leading to activation of both

Projects

innate and adaptive immunity. We have been working on preclinicalstudies with the human artificial adjuvant vector cells and have begun discussions about preclinical studies with the Pharmaceuticals
and Medical Devices Agency (PMDA). Forth, we have developed a
new chemical compound that selectively induces apoptosis of IgE
B cells, resulting in the preferential suppression of IgE production.
A contract between a pharmaceutical company and us for a drug
applicable for asthma, pollinosis or food allergy has been made.

Figure: NKT projects in IMS as translational research
Four different NKT translational research projects have been launched in IMS. Three
projects are for cancer treatment and one project is for treatment of allergic diseases.

Allergy ProjectA

chemical compound that selectively induces suppression of
total IgE production has been developed. This compound carries an α-GalCer analog, RCAI-X, and selectively targets marginal B
cells (MZB), but not DCs or macrophages. These B cells, after taking
up the compound, express IgE mRNA, IL-21 receptor, and CXCL16
to recruit NKT cells. The NKT cells activated by RCAI-X presented
by the MZB cells produce IL-21. The NKT cell production of IL-21
in turn acts on these MZB cells to induce phosphorylation of Bmf,
causing it to detach from the dynein light chain and bind with Bcl2, inhibiting its function and inducing apoptosis of the IgE B cells.

Bmf expression in IgG B cells is very low, therefore, the compound
only affects IgE B cells and not IgG B cells. The RCAI-X compound,even at doses in the 1-100 ng/kg ranges, resulted in significant suppression of not only the antigen-specific secondary IgE but also of
total IgE production (Fig.). A contract with a pharmaceutical company (Kaken Pharmaceutical Co. Ltd.) was completed in 2013 todevelop a drug applicable for asthma, pollinosis or food allergy.
The project is supported by the RIKEN Drug Discovery and Medical Technology Platforms and the Scientific Research Fund from
Health and Welfare.

Figure: Development of a compound mediating
IgE-speciﬁc suppression by RCAI-X-liposomes

69

Influenza Project
Influenza is a major life-threatening infectious disease in our society. Although the current vaccination method effectively reduces
the risk of death, the following issues still remain.
1. What are the host factors that contribute to pathogenesis
during the acute phase of infection?
Pathogenesis developed during infection varies among individuals. Several reports have suggested that dysregulation of immune
responses might contribute to the pathogenesis. To understand thecontribution of immune responses to pathogenesis, we have taken asystems biological approach. Gene expression profiles of subsets oftissue-invaded immune cells over time after infection are generated
and analyzed.With this large dataset, we expect to identify new biomarkers, especially to predict the risk of death. The new biomarkers
should enable us to prevent deaths from influenza by combining
them with existing drugs.

Projects

2. Generating a next-generation vaccine against a broad range
of influenza viruses.
Several groups have succeeded in isolating human and rodent antibodies that are broadly reactive with various influenza virus strains
such as H1N1 and H5N1, findings that point to the feasibility ofdeveloping a universal vaccine. These antibodies mostly recognize
the stem region of the Hemagglutinin (HA) protein, a region thatis well-conserved at the amino acid sequence level. We are designing synthetic peptides to induce B cell antibody responses that canbroadly neutralize influenza virus infections. Three teams from theresearch fields of immunology/biochemistry, computer science andstructural biology are running this project, which aims to generate
a next-generation vaccine for any upcoming pandemics resulting
from antigenic shift and drift (Figure).

Figure: Crafting a universal vaccine for pandemic
Influenza
A) Cross-reactivity of a “Universal” antibody. B) Numerous influenza peptides are designed and their affinities
for “universal” antibody are predicted in silico. C) Selected peptides are subjected to biochemical binding
analyses using the universal antibody. D) Potential peptides for the vaccine will be evaluated for their ability
to generate “universal” antibody in vivo and to protect
animals, and ultimately humans, from infection withvarious influenza virus strains.

PGRN-CGM International Collaborative StudiesT

he U.S. NIH Pharmacogenomics Research Network (PGRN) is
a consortium of research groups funded as individual cooperative agreements by the NIH. PGRN investigators are top researchers
from U.S. academic institutions and conduct studies of variation inhuman genes relevant to drug metabolism, pharmacokinetics andpharmacodynamics, and the relationship of the genetic variation
to drug responses. Principal investigators of the PGRN and RIKEN
Center for Genomic Medicine (now RIKEN IMS Core for Genomic Medicine: CGM) held a series of discussions on the need to accelerate discoveries in pharmacogenomics (PGx) and launched the
Global Alliance of Pharmacogenetics (GAP) in 2008.
In this international collaboration, the PGRN has been successfully assembling an abundance of DNA samples from well-phenotyped patients receiving specific drugs and drug combinations in clinical trials conducted in the U.S. The CGM focuses on

Figure: Pharmacogenomics Research Network (PGRN) - RIKEN IMS Core for
Genomic Medicine (CGM) strategic alliance.
Please visit http://bts.ucsf.edu/pgrn-cgm/

70

high-throughput genome-wide SNP scan with technological andmethodological expertise to identify genetic factors associated withdrug responses, risk of severe adverse drug reactions and non-response to medications. Together, the PGRN-CGM capitalizes onthese strengths to advance discoveries in PGx research. More than
30 collaborative studies for various drug responses are ongoing to
identify genomic biomarkers, which will develop better and safer
medications and realize the dream of global personalized medicine.

Collaboration with
Asian Institutes and SEAPharmO

ne of the aims of the Laboratory of International Allianceon Genomic Research is to promote research collaborations
around the globe. We have established connections with the Institute of Biomedical Sciences at Academia Sinica in Taiwan where wewill explore several diseases and pharmacogenetic studies (PGx)
together. In addition, we will also work closely with the Taiwan
Biobank to study complex diseases and PGx across the two populations. It has been noticed that rare adverse skin reactions called
SJS/TENS occur at a much higher frequency in East Asian andSoutheast Asian populations and that these drug induced adverse
reactions have strong genetic associations. To tackle this problem,
we established the South East Asian Pharmacogenomics Research
Network (SEAPharm) together with five Asian countries (Korea,
Indonesia, Malaysia, Taiwan, and Thailand). The aim of the collaborative effort is to identify significant PGx events important to
the region, so that we can identify genetic markers associated withthese adverse drug reactions, which could then lead to a reductionin these events. Our first project will be to study the genetic associ-Projects

ations of phenytoin-induced SJS/TEN. In addition, we also aim tounderstand how the identified genetic markers lead to the adverse
events. It is hoped that through the discoveries from our collaborative efforts, we will identify useful biomarkers that can be used to
predict drug-induced adverse events, guide drug use and be usefulin disease prediction/diagnosis.

Figure: South East Asian Pharmacogenomics Research Network (SEAPharm)

International Cancer Genome Consortium (ICGC)
Laboratory for Genome Sequencing Analysis
Laboratory for Medical Science Mathematics

I

Laboratory for Digestive Diseases

CGC has been organized to launch and coordinate a large number of research projects that have the common aim of comprehensively elucidating the genomic changes present in many types ofcancers. Its primary goals are to generate comprehensive cataloguesof genomic abnormalities in different cancer types and to makethe data available to the entire research community with minimal
restrictions. At the end of 2013, 71 cancer genome projects across
16 countries and the EU were ongoing, and the ICGC released the
genomic data from 8,532 cancer samples as Release 14 (September
26th, 2013). Our RIKEN group has been involved with the genome
project of liver cancer, which is one of the most common and deadly
cancers worldwide, especially in Asia. We performed whole genome
sequencing (WGS) of 270 liver cancers and called somatic mutations by using our in-house pipeline. We deposited WGS data of
153 liver cancers so far, and released them as a Japanese ICGC project. As an internal working group, we are involved with benchmark
comparison studies, where eleven genome centers analyze the sameraw sequence data or the same DNA by each of their pipelines andsequencing platforms to compare their results of somatic mutation
identification. ICGC has launched a “pan-cancer” genome project,
where 2000~ cancer WGS data will be analyzed in the same pipeline by a world-wide collaboration. We are contributing to this ambitious project as a member of technical working group and data
center, as well as by providing 300 WGS data.

Figure: ICGC Data Release 14 (September 26 th, 2013)71

Commissioned
research

The BioBank Japan ProjectT

he BioBank Japan project was started as a leading project of the
Ministry of Education, Culture, Sports, Science and Technology (MEXT) in 2003 [Project leader: Yusuke Nakamura (FY20032011), Michiaki Kubo (FY2011-present)]. In the first 5-year period(FY2003-2007), this project constructed the BioBank Japan, whichcollected DNA, serum and clinical information from 200,000 patients who suffered from at least one of the 47 target diseases. In
the second 5-year period (FY2008-2012), this project performed
GWAS for various diseases using the samples stored in the BioBank
Japan and identified many susceptibility genes for various diseases and drug responses. In the third period of 5 years from 2013,
this project is expanding the BioBank infrastructure to collect DNA
and clinical information from an independent cohort of 100,000
patients who suffer from at least one of 38 target diseases, including kidney cancer, dementia and depression as new disease targets.
In collaboration with other national projects, BioBank Japan willfurther promote genomic research and move forward to apply its
findings to clinical research.

Website: http://www.biobankjp.org/index.html
Photo: DNA Storage
Photo: Blood Serum Storage

Genome-guided drug Therapy Optimization
Project (G-TOP)T

he Genome-guided drug Therapy Optimization Project wasstarted in December, 2011 as a top-down project of MEXT tovalidate the clinical utility, clinical efficacy and cost-effectiveness ofpharmacogenomic research findings by clinical intervention study
and to implement pharmacogenomic testing into clinical use [Project leader: Michiaki Kubo (FY2011-present)]. Currently, this project conducts three clinical interventional studies for the preventionof Carbamazepine-induced skin rush (GENCAT), genome-guided
dose adjustment of Warfarin for safer anticoagulation (GENCAT),and genome-guided dose adjustment of tamoxifen for breast cancer
therapy (TARGET-1). The Core for Genomic Medicine and the BioBank Japan organize all three projects in collaboration with many
universities and hospitals in Japan. The Research Group for Pharmacogenomics (Group Director: Mushiroda) also manages thisproject.

Website: http://www.biobankjp.org/pgx/index.html
Figure: Website of the Genome-guided drug Therapy Optimization
Project (G-TOP)

72

Part 3
Events

Events

RIKEN
IMS
Summer
Program (RISP)
2013

The 8
IMS-JSI
International
Symposium on
Immunology
2013

I

T

MS was delighted to continue the RISP, now RIKEN IMS
Summer Program, in 2013 after starting our new institute.
RISP started in 2006 with the goal of offering training in current
topics in immunology as well as providing networking opportunities on an international scale for young immunologists. It wasalso geared to encourage future collaboration and postdoctoral
training experiences in Japan, so that RISP has included an internship program in which the participants perform researchin the IMS laboratories. RISP is now co-organized by the Chiba
University Leading Graduate School Program.
In RISP 2013, forty-seven graduate students/postdoctoral fellows from 18 countries and six students from Chiba University
gathered at Yokohama from June 21st to 28th. The RISP 2013
program started with a tour of IMS research facilities, which include advanced two-photon microscopes, HILO microscope forsingle molecule imaging and conventional cell sorting instruments as well as a new CyTOF mass spec-based sorter. This wasfollowed over the next four days by 12 lectures by Japanese andforeign distinguished scientists and oral and poster presentations
by RISP students. The RISP program ended with participation inan annual international symposium, co-organized by IMS-RCAIand the Japanese Society for Immunology.
RISP 2013 was a success, as all of the students indicated in the
RISP evaluation survey that they would recommend the program
to colleagues and would encourage them to apply for a position
at IMS-RCAI. IMS is planning to continue RISP, updating someof the lecture topics as necessary to keep pace with recent developments in this rapidly moving field of life science.

74th

he 8th IMS-JSI International Symposium on Immunology, hosted by the RIKEN Center for Integrative Medical
Sciences (IMS), in conjunction with the Japanese Society for
Immunology (JSI), was held on June 27-28 in Yokohama. This
was the first international event since the launch of IMS in April
2013, which was in transition from RCAI, Research Center forAllergy and Immunology (2001-2013). Eighteen internationally-recognized speakers presented their cutting-edged researchat the symposium entitled “Interface between Immune System
and Environment”, which attracted more than 350 participants.
The symposium consisted of five sessions: Interface between gut
and immune system; Interface between the lung and immune
system; Interface between the skin and immune system; Molecular mediator between environment and immune system; Metagenomic analysis of the human immune system. Many different
subpopulations of immune cells are active on the front line at
the barriers of our body, such as skin, intestinal and bronchial
mucosa. Microbiota and their metabolites affect these immunological actions relevant to both innate and acquired immunity,and might be linked to human diseases. Structural microenvironments established at the barriers, such as mucosa-associatedlymphoid tissues, also appear to play a role in immunologicalmemory functions of the acquired immune system. The research
field of “Interface between Immune System and Environment”
brings us a novel view to better understand the immune systemand human diseases. Furthermore, expansion of large datasetsfrom metagenomic analysis will be a powerful resource for the
research community in this field.

Events

The 8
PGRN-RIKEN
Strategic
Alliance
Meeting

LJI &IMSWorkshopTTthhe Global Alliance for Pharmacogenomics (GAP), a collaborative program between the former RIKEN Center for
Genomic Medicine (now Core for Genomic Medicine, RIKEN
IMS) and the US National Institute of Health (NIH) Pharmacogenomics Research Network (PGRN), was started in 2008 with theobjective of identifying the relationship between genetic variants
and individual responses to drugs, including efficacy/side effects.
Its strategic alliance meetings, held alternately in Japan and United States, allow for face-to-face discussions about the progressof ongoing projects and future directions for the PGRN-RIKEN
CGM collaboration.
On June 19-20, 2013, CGM hosted the 8th PGRN-RIKEN
Strategic Alliance Meeting at the Business Support Floor of theLandmark Tower Yokohama, in Yokohama, Japan. Some 40 participants attended this meeting, and it provided a valuable forum
for exchanging information on ongoing collaborative activities:
Aromatase Inhibitors and Bone Fractures, Adverse Cardiovascular Outcomes and New-onset Diabetes after Antihypertensive
Therapy, Pharmacogenetics of Prostacyclin Dosing in Pulmonary Arterial Hypertension, Colorectal Cancer Pharmacogenetics, and SSTI Pharmacogenomics. Participants from PGRN and
RIKEN also had in-depth discussions and shared ideas about
their research projects. In addition, participants explored newcollaborative research proposals as well as data analysis issues.
Finally, it was agreed to hold this meeting on a regular basis tofurther enhance collaborations between PGRN and CGM, andthe meeting successfully concluded.

he 5th joint workshop between LJI (La Jolla Institute of Allergy and Immunology, formerly abbreviated as LIAI) and
IMS “New Horizon in Immune Regulation towards Disease Intervention” was held on October 30-31 in the RIKEN Yokohama
campus. The workshop featured 11 speakers from both institutes.
The acting director of IMS, Dr. Koyasu started by introducing
the outline of IMS, and this was followed by the introduction of
LJI by the director Dr. Kronenberg. Both speakers stressed theimportance of continuation the mutual interactions and collaborations. The workshop had four scientific sessions on the 1st day:Development and function of T cells, Allergy and inflammation
I, Regulation and intervention of diseases, Signal regulation of
diseases, and three sessions on the 2nd day: Mucosal regulationand microbiota, Allergy and inflammation II, Systems and Immunology.
The topics covered broad aspects in lymphocyte development,
signal transduction, mucosal regulation, allergy and inflammation, and integrative approaches to understand immune responses. One of the most cutting edge topics was the mucosal regulation of immune responses by the microbiota. In addition, thespeakers from both institutes emphasized the study of diseases,
particularly allergic skin diseases, inflammation, cancer, and diabetes.
Based of the facts that both institutes have similar goals,
targets, and aspects of research and that two LJI investigators(Cheroutre and Kawakami) are now also PIs in IMS beginning
this year, the two institutes promised to develop further strong
relationships and collaborations.

75

Events

Harvard
SummerSchool2013

Adjunct
Professorship
Programs

I

I

MS offers a summer internship program for undergraduate
students from Harvard University. In this program students
do a research internship in the IMS laboratories and have basic immunology lectures by IMS PIs and a Japanese language
course. They also participate in the RIKEN IMS Summer Program (RISP) and the International Symposium on Immunology,
organized by IMS and JSI. The participants receive course credit from Harvard. In 2013, we had three students from Harvard
University (James Carey, Albert Li and Kathleen Wallace), andone from Brown University (Marjorie Palmeri) from June 3 toAugust 12.
Carey conducted his research project in the Laboratory for
Immune Regulation (Dr. Taniguchi), Li in the Laboratory for
Immunogenetics (Dr. Yoshida), Wallace in the Laboratory for
Vaccine Design (Dr. Ishii), and Palmeri in the Laboratory for
Cytokine Regulation (Dr. Kubo). During their internships, thestudents had numerous discussions with IMS researchers and, atthe end of the program, they gave oral presentations describing
their scientific results.
In addition, they visited the Science Frontier High School
and met with students who want to be scientists. On this occasion, they experienced Japanese culture and learned Sado (teaceremony) from the local high school students.

Photo: Cultural Exchange at Science Frontier High School76

MS collaborates with and accepts graduate students from 11
domestic university graduate schools. There are now a totalof 35 adjunct professors/associate professors in IMS (Table),and 118 students studied at IMS in 2013. On May 25, IMS held abriefing session on adjunct graduate school programs. Thirteen
students participated from Kyushu, Kyoto, Kanagawa, Tokyo,
Saitama, and even from Ireland. The session provided an opportunity for students to visit and talk directly with lab leaders andto consider their future directions.

Table: Adjunct professorship programs
Graduate Program

Affiliated RCAI Investigator

Graduate School of Frontier Bioscience,
Osaka University

Tomohiro Kurosaki (Professor), Ichiro
Taniuchi (Visiting Professor), Keigo Nishida(Visiting Associate Professor)

Graduate School of Medicine,
Osaka University

Takashi Saito (Visiting Professor), Toshiyuki
Fukada (Visiting Associate Professor)

Department of Immunology,
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 Pharmaceutical Sciences, Chiba University

Osamu Ohara (Visiting Professor)

School of Biomedical Science,
Tokyo Medical and Dental University

Takashi Saito (Visiting Professor)

Graduate School of Medicine, Yokohama
City University

Michiaki Kubo (Visiting Professor), Shiro
Ikegawa (Visiting Professor), Mayumi Tamari(Visiting Professor), Tatsuhiko Tsunoda (Visiting Professor), Hidewaki Nakagawa (Visiting
Professor), Shiro Maeda (Visiting Professor),
Taisei Mushiroda (Visiting Professor), Atsushi
Takahashi (Visiting Professor)

Graduate School of Medical Life Science,
Yokohama City University

Hiroshi Ohno (Visiting Professor), Haruhiko Koseki (Visiting Professor), Mariko
Okada (Visiting Professor), Takaharu Okada(Visiting Associate Professor), Kazuyo Moro
(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 Medicine, Kyoto
University

Fumihiko Ishikawa (Visiting Associate
Professor)

Graduate School of Medicine, Keio
University

Masayuki Amagai (Professor), Shigeo Koyasu(Visiting Professor), Haruhiko Koseki (Visiting
Profefssor)

Part 4
Data andStatistics

Publlications 2013

1 Abe, H., Hayes, C. N., Hiraga, N., Imamura, M., Tsuge, M., Miki, D., Takahashi,S., Ochi, H. & Chayama, K. A translational study of resistance emergence using
sequential direct-acting antiviral agents for hepatitis C using ultra-deep sequencing. Am J Gastroenterol Sep. 2013, 1464-1472.
2 Ando, T., Matsumoto, K., Namiranian, S., Yamashita, H., Glatthorn, H., Kimura,M., Dolan, B. R., Lee, J. J., Galli, S. J., Kawakami, Y., Jamora, C. & Kawakami, T.
Mast cells are required for full expression of allergen/SEB-induced skin inflammation. J Invest Dermatol Dec. 2013, 2695-2705.

Table: IMS Publlications Jan-Dec, 2013
Journals(2012)Factor
ImpactIF(2012)

Lancet39.06

1

Nature38.60

5

Nat Genet

35.21

5

Nat Rev Immunol

33.13

1

Science31.03

1

Nat Immunol

26.99

4

Cell Stem Cell

25.32

2

Nat Med24.30

1

Immunity

19.80

7

Mol Cell

15.28

1

J Exp Med

13.21

1

Dev Cell

12.86

2

Gastroenterology

12.82

1

J Clin Invest

12.81

3

Cell Host Microbe

12.61

1

J Allergy Clin Immunol

12.05

3

Am J Hum Genet

11.20

1

Sci Transl Med

10.76

1

Gut

10.73

1

Cancer Discov

10.14

1

Nat Commun

10.02

2

Proc Natl Acad Sci U S A

9.74

7

Annu Rev Genomics Hum Genet

9.50

1

EMBO J

9.49

2

Blood

9.06

3

Cancer Res

8.65

1

PLoS Genet

8.52

2

Diabetes

7.90

1

Hum Mol Genet

7.69

1

Am J Gastroenterol

7.55

1

Oncogene

7.36

1

Clin Pharmacol Ther

6.85

3

Diabetologia

6.49

2

J Clin Endocrinol Metab

6.43

1

Philos Trans R Soc Lond B Biol Sci

6.23

1

Development

6.21

1

Int J Cancer

6.20

1

J Invest Dermatol

6.19

1

Am J Transplant

6.19

1

Haematologica

5.94

1

Allergy

5.88

1

Breast Cancer Res

5.87

1

FASEB J

5.70

2

J Med Genet

5.70

1

J Immunol

5.52

7Neoplasia

5.47

1

Hum Mutat

5.21

1

2013
NumberofPapersOther Journals

98

Total

189

78

3 Ando, T., Xiao, W., Gao, P., Namiranian, S., Matsumoto, K., Tomimori, Y., Hong,
H., Yamashita, H., Kimura, M., Kashiwakura, J., Hata, T. R., Izuhara, K., Gurish, M.
F., Roers, A., Rafaels, N. M., Barnes, K. C., Jamora, C., Kawakami, Y. & Kawakami,
T. Critical role for mast cell stat5 activity in skin inflammation. Cell Rep Jan 30.
2014, 366-376.
4 Arataki, K., Hayes, C. N., Akamatsu, S., Akiyama, R., Abe, H., Tsuge, M., Miki,
D., Ochi, H., Hiraga, N., Imamura, M., Takahashi, S., Aikata, H., Kawaoka, T.,
Kawakami, H., Ohishi, W. & Chayama, K. Circulating microRNA-22 correlates withmicroRNA-122 and represents viral replication and liver injury in patients withchronic hepatitis B. J Med Virol May. 2013, 789-798.
5 Atarashi, K., Tanoue, T., Oshima, K., Suda, W., Nagano, Y., Nishikawa, H., Fukuda,S., Saito, T., Narushima, S., Hase, K., Kim, S., Fritz, J. V., Wilmes, P., Ueha, S., Matsushima, K., Ohno, H., Olle, B., Sakaguchi, S., Taniguchi, T., Morita, H., Hattori, M.
& Honda, K. Treg induction by a rationally selected mixture of Clostridia strainsfrom the human microbiota. Nature Aug 8. 2013, 232-236.
6 Ato, M., Takahashi, Y., Fujii, H., Hashimoto, S., Kaji, T., Itamura, S., Horiuchi, Y.,
Arakawa, Y., Tashiro, M. & Takemori, T. Influenza A whole virion vaccine induces
a rapid reduction of peripheral blood leukocytes via interferon-alpha-dependent
apoptosis. Vaccine Apr 19. 2013, 2184-2190.
7 Betcheva, E.T., Yosifova, A.G., Mushiroda, T., Kubo, M., Takahashi, A., Karachanak,
S. K., Zaharieva, I. T., Hadjidekova, S. P., Dimova, II, Vazharova, R. V., Stoyanov, D.
S., Milanova, V. K., Tolev, T., Kirov, G., Kamatani, N., Toncheva, D. I. & Nakamura, Y.
Whole-genome-wide association study in the Bulgarian population reveals HHAT
as schizophrenia susceptibility gene. Psychiatr Genet Feb. 2013, 11-19.
8 Capietto, A. H., Kim, S., Sanford, D. E., Linehan, D. C., Hikida, M., Kumosaki, T.,
Novack, D. V. & Faccio, R. Down-regulation of PLCgamma2-beta-catenin pathway
promotes activation and expansion of myeloid-derived suppressor cells in cancer.
J Exp Med Oct 21. 2013, 2257-2271.
9 Choi, D. W., Na, W., Kabir, M. H., Yi, E., Kwon, S., Yeom, J., Ahn, J. W., Choi, H. H.,
Lee, Y., Seo, K. W., Shin, M. K., Park, S. H., Yoo, H. Y., Isono, K., Koseki, H., Kim,
S. T., Lee, C., Kwon, Y. K. & Choi, C. Y. WIP1, a homeostatic regulator of the DNA
damage response, is targeted by HIPK2 for phosphorylation and degradation.
Mol Cell Aug 8. 2013, 374-385.
10 Chou, C. H., Lee, C. H., Lu, L. S., Song, I. W., Chuang, H. P., Kuo, S. Y., Wu, J. Y.,
Chen, Y. T., Kraus, V. B., Wu, C. C. & Lee, M. T. Direct assessment of articular cartilage and underlying subchondral bone reveals a progressive gene expression
change in human osteoarthritic knees. Osteoarthritis Cartilage Mar. 2013,450-461.
11 Chou, C. H., Wu, C. C., Song, I. W., Chuang, H. P., Lu, L. S., Chang, J. H., Kuo, S. Y.,
Lee, C. H., Wu, J. Y., Chen, Y. T., Kraus, V. B. & Lee, M. T. Genome-wide expression
profiles of subchondral bone in osteoarthritis. Arthritis Res Ther Nov 15. 2013,
R190.
12 Chung, S., Low, S. K., Zembutsu, H., Takahashi, A., Kubo, M., Sasa, M. & Nakamura, Y. A genome-wide association study of chemotherapy-induced alopecia inbreast cancer patients. Breast Cancer Res 2013, R81.
13 Cornejo-Garcia, J. A., Liou, L. B., Blanca-Lopez, N., Dona, I., Chen, C. H., Chou,
Y. C., Chuang, H. P., Wu, J. Y., Chen, Y. T., Plaza-Seron Mdel, C., Mayorga, C.,
Gueant-Rodriguez, R. M., Lin, S. C., Torres, M. J., Campo, P., Rondon, C., Laguna,
J. J., Fernandez, J., Gueant, J. L., Canto, G., Blanca, M. & Lee, M. T. Genome-wide
association study in NSAID-induced acute urticaria/angioedema in Spanish and
Han Chinese populations. Pharmacogenomics Nov. 2013, 1857-1869.
14 Daneshjou, R., Tatonetti, N. P., Karczewski, K. J., Sagreiya, H., Bourgeois, S., Drozda, K., Burmester, J. K., Tsunoda, T., Nakamura, Y., Kubo, M., Tector, M., Limdi, N.
A., Cavallari, L. H., Perera, M., Johnson, J. A., Klein, T. E. & Altman, R. B. Pathway
analysis of genome-wide data improves warfarin dose prediction. BMC Genomics 2013, S11.

15 Dieterle, A. M., Bohler, P., Keppeler, H., Alers, S., Berleth, N., Driessen, S., Hieke,N., Pietkiewicz, S., Loffler, A. S., Peter, C., Gray, A., Leslie, N. R., Shinohara, H.,
Kurosaki, T., Engelke, M., Wienands, J., Bonin, M., Wesselborg, S. & Stork, B.
PDK1 controls upstream PI3K expression and PIP generation. Oncogene Jul 29.
2013.
16 Ellinghaus, D., Baurecht, H., Esparza-Gordillo, J., Rodriguez, E., Matanovic, A.,
Marenholz, I., Hubner, N., Schaarschmidt, H., Novak, N., Michel, S., Maintz,
L., Werfel, T., Meyer-Hoffert, U., Hotze, M., Prokisch, H., Heim, K., Herder, C.,
Hirota, T., Tamari, M., Kubo, M., Takahashi, A., Nakamura, Y., Tsoi, L. C., Stuart,P., Elder, J. T., Sun, L., Zuo, X., Yang, S., Zhang, X., Hoffmann, P., Nothen, M. M.,
Folster-Holst, R., Winkelmann, J., Illig, T., Boehm, B. O., Duerr, R. H., Buning, C.,
Brand, S., Glas, J., McAleer, M. A., Fahy, C. M., Kabesch, M., Brown, S., McLean,
W. H., Irvine, A. D., Schreiber, S., Lee, Y. A., Franke, A. & Weidinger, S. High-density genotyping study identifies four new susceptibility loci for atopic dermatitis.
Nat Genet Jul. 2013, 808-812.
17 Fritsche, L. G., Chen, W., Schu, M., Yaspan, B. L., Yu, Y., Thorleifsson, G., Zack,
D. J., Arakawa, S., Cipriani, V., Ripke, S., Igo, R. P., Jr., Buitendijk, G. H., Sim, X.,
Weeks, D. E., Guymer, R. H., Merriam, J. E., Francis, P. J., Hannum, G., Agarwal,
A., Armbrecht, A. M., Audo, I., Aung, T., Barile, G. R., Benchaboune, M., Bird, A.
C., Bishop, P. N., Branham, K. E., Brooks, M., Brucker, A. J., Cade, W. H., Cain, M.
S., Campochiaro, P. A., Chan, C. C., Cheng, C. Y., Chew, E. Y., Chin, K. A., Chowers,
I., Clayton, D. G., Cojocaru, R., Conley, Y. P., Cornes, B. K., Daly, M. J., Dhillon,
B., Edwards, A. O., Evangelou, E., Fagerness, J., Ferreyra, H. A., Friedman, J. S.,
Geirsdottir, A., George, R. J., Gieger, C., Gupta, N., Hagstrom, S. A., Harding, S. P.,
Haritoglou, C., Heckenlively, J. R., Holz, F. G., Hughes, G., Ioannidis, J. P., Ishibashi, T., Joseph, P., Jun, G., Kamatani, Y., Katsanis, N., C, N. K., Khan, J. C., Kim, I.
K., Kiyohara, Y., Klein, B. E., Klein, R., Kovach, J. L., Kozak, I., Lee, C. J., Lee, K. E.,
Lichtner, P., Lotery, A. J., Meitinger, T., Mitchell, P., Mohand-Said, S., Moore, A.
T., Morgan, D. J., Morrison, M. A., Myers, C. E., Naj, A. C., Nakamura, Y., Okada,Y., Orlin, A., Ortube, M. C., Othman, M. I., Pappas, C., Park, K. H., Pauer, G. J.,
Peachey, N. S., Poch, O., Priya, R. R., Reynolds, R., Richardson, A. J., Ripp, R.,
Rudolph, G., Ryu, E., Sahel, J. A., Schaumberg, D. A., Scholl, H. P., Schwartz, S. G.,
Scott, W. K., Shahid, H., Sigurdsson, H., Silvestri, G., Sivakumaran, T. A., Smith,
R. T., Sobrin, L., Souied, E. H., Stambolian, D. E., Stefansson, H., Sturgill-Short,
G. M., Takahashi, A., Tosakulwong, N., Truitt, B. J., Tsironi, E. E., Uitterlinden, A.
G., van Duijn, C. M., Vijaya, L., Vingerling, J. R., Vithana, E. N., Webster, A. R.,
Wichmann, H. E., Winkler, T. W., Wong, T. Y., Wright, A. F., Zelenika, D., Zhang,
M., Zhao, L., Zhang, K., Klein, M. L., Hageman, G. S., Lathrop, G. M., Stefansson,
K., Allikmets, R., Baird, P. N., Gorin, M. B., Wang, J. J., Klaver, C. C., Seddon, J. M.,
Pericak-Vance, M. A., Iyengar, S. K., Yates, J. R., Swaroop, A., Weber, B. H., Kubo,
M., Deangelis, M. M., Leveillard, T., Thorsteinsdottir, U., Haines, J. L., Farrer, L. A.,
Heid, I. M. & Abecasis, G. R. Seven new loci associated with age-related maculardegeneration. Nat Genet Apr. 2013, 433-439, 439e431-432.
18 Fujii, S. I. & Shimizu, K. Immunotherapy with artificial adjuvant vector cells:
Harnessing both arms of the immune response. Oncoimmunology Apr 1. 2013,
e23432.
19 Fujii, S. I., Shimizu, K., Okamoto, Y., Kunii, N., Nakayama, T., Motohashi, S. & Taniguchi, M. NKT Cells as an Ideal Anti-Tumor Immunotherapeutic. Front Immunol
2013, 409.
20 Fukada, T., Hojyo, S. & Furuichi, T. Zinc signal: a new player in osteobiology. J
Bone Miner Metab Mar. 2013, 129-135.
21 Fukata, M., Ishikawa, F., Najima, Y., Yamauchi, T., Saito, Y., Takenaka, K., Miyawaki, K., Shimazu, H., Shimoda, K., Kanemaru, T., Nakamura, K., Odashiro,K., Nagafuji, K., Harada, M. & Akashi, K. Contribution of bone marrow-derived
hematopoietic stem/progenitor cells to the generation of donor-marker(+) cardiomyocytes in vivo. PLoS One 2013, e62506.
22 Fukuda, H., Imamura, M., Tanaka, Y., Iwata, M., Hirose, H., Kaku, K., Maegawa,
H., Watada, H., Tobe, K., Kashiwagi, A., Kawamori, R. & Maeda, S. Replication
study for the association of a single-nucleotide polymorphism, rs3746876, within
KCNJ15, with susceptibility to type 2 diabetes in a Japanese population. J Hum
Genet Jul. 2013, 490-493.
23 Furusawa, J., Moro, K., Motomura, Y., Okamoto, K., Zhu, J., Takayanagi, H., Kubo,
M. & Koyasu, S. Critical role of p38 and GATA3 in natural helper cell function. J
Immunol Aug 15. 2013, 1818-1826.

24 Furusawa, Y., Obata, Y., Fukuda, S., Endo, T. A., Nakato, G., Takahashi, D., Nakanishi, Y., Uetake, C., Kato, K., Kato, T., Takahashi, M., Fukuda, N. N., Murakami, S.,
Miyauchi, E., Hino, S., Atarashi, K., Onawa, S., Fujimura, Y., Lockett, T., Clarke, J.
M., Topping, D. L., Tomita, M., Hori, S., Ohara, O., Morita, T., Koseki, H., Kikuchi,J., Honda, K., Hase, K. & Ohno, H. Commensal microbe-derived butyrate induces
the differentiation of colonic regulatory T cells. Nature Dec 19. 2013, 446-450.
25 Ghosh, S., Matsuoka, Y., Asai, Y., Hsin, K. Y. & Kitano, H. Toward an integrated
software platform for systems pharmacology. Biopharm Drug Dispos Dec.
2013, 508-526.
26 Hara, K., Fujita, H., Johnson, T. A., Yamauchi, T., Yasuda, K., Horikoshi, M., Peng,
C., Hu, C., Ma, R. C., Imamura, M., Iwata, M., Tsunoda, T., Morizono, T., Shojima,N., So, W. Y., Leung, T. F., Kwan, P., Zhang, R., Wang, J., Yu, W., Maegawa, H.,
Hirose, H., Kaku, K., Ito, C., Watada, H., Tanaka, Y., Tobe, K., Kashiwagi, A., Kawamori, R., Jia, W., Chan, J. C., Teo, Y. Y., Shyong, T. E., Kamatani, N., Kubo, M.,
Maeda, S. & Kadowaki, T. Genome-wide association study identifies three novelloci for type 2 diabetes. Hum Mol Genet Jan 1. 2014, 239-246.
27 Hariharan, R., Simon, R., Pillai, M. R. & Taylor, T. D. Comparative analysis of DNA
word abundances in four yeast genomes using a novel statistical backgroundmodel. PLoS One 2013, e58038.
28 Hase, T., Ghosh, S., Yamanaka, R. & Kitano, H. Harnessing diversity towards thereconstructing of large scale gene regulatory networks. PLoS Comput Biol Nov.
2013, e1003361.
29 Hayashi, M., Hirota, T., Saeki, H., Nakagawa, H., Ishiuji, Y., Matsuzaki, H., Tsunemi, Y., Kato, T., Shibata, S., Sugaya, M., Sato, S., Tada, Y., Doi, S., Miyatake, A.,
Ebe, K., Noguchi, E., Ebihara, T., Amagai, M., Esaki, H., Takeuchi, S., Furue, M. &Tamari, M. Genetic polymorphism in the TRAF3IP2 gene is associated with psoriasis vulgaris in a Japanese population. J Dermatol Sci Mar. 2014, 264-265.
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Y. S., Tai, E. S., Kelly, M. A., Chambers, J. C., Kooner, J. S., Kadowaki, T., Deloukas,P., Rader, D. J., Danesh, J. & Sanghera, D. K. Genome-wide association study
identifies a novel locus contributing to type 2 diabetes susceptibility in Sikhs of
Punjabi origin from India. Diabetes May. 2013, 1746-1755.
135 Schlitzer, A., McGovern, N., Teo, P., Zelante, T., Atarashi, K., Low, D., Ho, A. W.,
See, P., Shin, A., Wasan, P. S., Hoeffel, G., Malleret, B., Heiseke, A., Chew, S.,
Jardine, L., Purvis, H. A., Hilkens, C. M., Tam, J., Poidinger, M., Stanley, E. R.,
Krug, A. B., Renia, L., Sivasankar, B., Ng, L. G., Collin, M., Ricciardi-Castagnoli, P.,
Honda, K., Haniffa, M. & Ginhoux, F. IRF4 transcription factor-dependent CD11b+
dendritic cells in human and mouse control mucosal IL-17 cytokine responses.
Immunity May 23. 2013, 970-983.
136 Semba, K., Araki, K., Matsumoto, K., Suda, H., Ando, T., Sei, A., Mizuta, H., Takagi, K., Nakahara, M., Muta, M., Yamada, G., Nakagata, N., Iida, A., Ikegawa,S., Nakamura, Y., Araki, M., Abe, K. & Yamamura, K. Ectopic expression of Ptf1a
induces spinal defects, urogenital defects, and anorectal malformations in Danforth's short tail mice. PLoS Genet 2013, e1003204.
137 Sharif, J., Shinkai, Y. & Koseki, H. Is there a role for endogenous retroviruses tomediate long-term adaptive phenotypic response upon environmental inputs?
Philos Trans R Soc Lond B Biol Sci Jan 5. 2013, 20110340.
138 Shi, N., Hiraga, N., Imamura, M., Hayes, C. N., Zhang, Y., Kosaka, K., Okazaki,A., Murakami, E., Tsuge, M., Abe, H., Aikata, H., Takahashi, S., Ochi, H., Tateno-Mukaidani, C., Yoshizato, K., Matsui, H., Kanai, A., Inaba, T., McPhee, F.,
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139 Shigemizu, D., Fujimoto, A., Akiyama, S., Abe, T., Nakano, K., Boroevich, K. A.,
Yamamoto, Y., Furuta, M., Kubo, M., Nakagawa, H. & Tsunoda, T. A practicalmethod to detect SNVs and indels from whole genome and exome sequencing
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140 Shimane, K., Kochi, Y., Suzuki, A., Okada, Y., Ishii, T., Horita, T., Saito, K., Okamoto, A., Nishimoto, N., Myouzen, K., Kubo, M., Hirakata, M., Sumida, T., Takasaki,Y., Yamada, R., Nakamura, Y., Kamatani, N. & Yamamoto, K. An association
analysis of HLA-DRB1 with systemic lupus erythematosus and rheumatoid arthritis in a Japanese population: effects of *09:01 allele on disease phenotypes.
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141 Shimizu, K., Asakura, M., Shinga, J., Sato, Y., Kitahara, S., Hoshino, K., Kaisho, T.,
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142 Shimizu, K., Mizuno, T., Shinga, J., Asakura, M., Kakimi, K., Ishii, Y., Masuda, K.,
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143 Shiozaki, M., Tashiro, T., Koshino, H., Shigeura, T., Watarai, H., Taniguchi, M. &Mori, K. Synthesis and biological activity of hydroxylated analogs of RCAI-80.
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144 Shiozaki, M., Tashiro, T., Koshino, H., Shigeura, T., Watarai, H., Taniguchi, M. &Mori, K. Synthesis of RCAI-172 (C6 epimer of RCAI-147) and its biological activity.
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145 Shirakawa, T., Yaman-Deveci, R., Tomizawa, S., Kamizato, Y., Nakajima, K., Sone,
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146 Shirasaki, Y., Yamagishi, M., Shimura, N., Hijikata, A. & Ohara, O. Toward an understanding of immune cell sociology: real-time monitoring of cytokine secretionat the single-cell level. IUBMB Life Jan. 2013, 28-34.

84

147 Song, Y. Q., Karasugi, T., Cheung, K. M., Chiba, K., Ho, D. W., Miyake, A., Kao, P.
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149 Suenaga, K., Takasawa, H., Watanabe, T., Wako, Y., Suzuki, T., Hamada, S. & Furihata, C. Differential gene expression profiling between genotoxic and non-genotoxic hepatocarcinogens in young rat liver determined by quantitative real-time
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150 Suna, S., Sakata, Y., Nakatani, D., Okuda, K., Shimizu, M., Usami, M., Matsumoto,S., Hara, M., Ozaki, K., Mizuno, H., Minamino, T., Takashima, S., Nishino, M.,
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151 Takahashi, M., Izawa, K., Kashiwakura, J., Yamanishi, Y., Enomoto, Y., Kaitani, A.,
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152 Takamoto, I., Kubota, N., Nakaya, K., Kumagai, K., Hashimoto, S., Kubota, T.,
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153 Takase, M., Iida, R., Maruya, M., Sakaue-Sawano, A., Miyawaki, A., Wakayama,T., Nishigami, S., Fagarasan, S. & Kanagawa, O. Nuclear transferred embryonicstem cells for analysis of B1 B-lymphocyte development. Int Immunol Mar.
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154 Tamaki, M., Fujitani, Y., Hara, A., Uchida, T., Tamura, Y., Takeno, K., Kawaguchi,M., Watanabe, T., Ogihara, T., Fukunaka, A., Shimizu, T., Mita, T., Kanazawa, A.,
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155 Tamari, M., Tanaka, S. & Hirota, T. Genome-wide association studies of allergic
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156 Tanaka, H., Naito, T., Muroi, S., Seo, W., Chihara, R., Miyamoto, C., Kominami,
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157 Tanaka, S., Hirota, T., Kamijo, A., Ishii, H., Hatsushika, K., Fujieda, S., Ishitoya, J.,
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158 Taniuchi, I. Stage-specific epigenetic gene silencing during thymocyte lineage
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159 Taniue, K., Kurimoto, A., Sugimasa, H., Nasu, E., Nagashima, T., Okada-Hatakeyama, M. & Akiyama, T. Functional analysis of a novel long ncRNA involvedin the tumorigenicity of colorectal cancer. Proc of AACR 104th Annual Meeting
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160 Tashiro, T., Nakagawa, R., Shigeura, T., Watarai, H., Taniguchi, M. & Mori, K.
RCAI-61 and related 6'-modified analogs of KRN7000: their synthesis and bioactivity for mouse lymphocytes to produce interferon-gamma in vivo. Bioorg Med
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161 Tashiro, T., Shigeura, T., Shiozaki, M., Watarai, H., Taniguchi, M. & Mori, K. RCAI133, an N-methylated analogue of KRN7000, activates mouse natural killer T
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162 Tateno, C., Miya, F., Wake, K., Kataoka, M., Ishida, Y., Yamasaki, C., Yanagi, A.,
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163 Terada, A., Okada-Hatakeyama, M., Tsuda, K. & Sese, J. Statistical significance ofcombinatorial regulations. Proc Natl Acad Sci U S A Aug 6. 2013, 12996-13001.
164 Terao, C., Ohmura, K., Kawaguchi, Y., Nishimoto, T., Kawasaki, A., Takehara,
K., Furukawa, H., Kochi, Y., Ota, Y., Ikari, K., Sato, S., Tohma, S., Yamada, R.,
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165 Tetsuka, S., Morita, M., Iida, A., Uehara, R., Ikegawa, S. & Nakano, I. ZNF512B
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166 Toker, A., Engelbert, D., Garg, G., Polansky, J. K., Floess, S., Miyao, T., Baron, U.,
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167 Tomita, K., Sakashita, M., Hirota, T., Tanaka, S., Masuyama, K., Yamada, T., Fujieda, S., Miyatake, A., Hizawa, N., Kubo, M., Nakamura, Y. & Tamari, M. Variants in
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168 Tsuge, M., Murakami, E., Imamura, M., Abe, H., Miki, D., Hiraga, N., Takahashi,S., Ochi, H., Nelson Hayes, C., Ginba, H., Matsuyama, K., Kawakami, H. & Chayama, K. Serum HBV RNA and HBeAg are useful markers for the safe discontinuation of nucleotide analogue treatments in chronic hepatitis B patients. J Gastroenterol Oct. 2013, 1188-1204.
169 Tsukumo, S., Unno, M., Muto, A., Takeuchi, A., Kometani, K., Kurosaki, T., Igarashi, K. & Saito, T. Bach2 maintains T cells in a naive state by suppressing
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170 Tsuruoka, N., Arima, M., Yoshida, N., Okada, S., Sakamoto, A., Hatano, M., Satake, H., Arguni, E., Wang, J. Y., Yang, J. H., Nishikura, K., Sekiya, S., Shozu, M. &Tokuhisa, T. ADAR1 protein induces adenosine-targeted DNA mutations in senescent Bcl6 gene-deficient cells. J Biol Chem Jan 11. 2013, 826-836.
171 Vinuesa, C. G., Fagarasan, S. & Dong, C. New territory for T follicular helper cells.
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172 Vizcardo, R., Masuda, K., Yamada, D., Ikawa, T., Shimizu, K., Fujii, S., Koseki, H. &Kawamoto, H. Regeneration of human tumor antigen-specific T cells from iPSCs
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173 Wada, T., Muraoka, M., Toma, T., Imai, T., Shigemura, T., Agematsu, K., Haraguchi, K., Moriuchi, H., Oh-Ishi, T., Kitoh, T., Ohara, O., Morio, T. & Yachie, A. Rapid
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177 Wu, A. C., Himes, B. E., Lasky-Su, J., Litonjua, A., Peters, S. P., Lima, J., Kubo,
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178 Xiao, W., Kawakami, Y. & Kawakami, T. Immune regulation by phospholipase
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179 Yamagishi, J., Okimoto, N., Kasai, T., Suenaga, A., Okada, M., Imamoto, A. & Taiji,
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180 Yamagishi, M., Shirasaki, Y. & Funatsu, T. Single-molecule tracking of mRNA inliving cells. Methods Mol Biol 2013, 153-167.
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85

Guest Lectures 2013
Table: Guest Lectures Jan-Dec, 2013
Date

Speaker

Affiliation

Country

Title

23 - Jan -13

Dr. Shinichi Nishikawa

Center for Developmental Biology, RIKENJapan

Development of Hematopoietic Stem Cell: A final
scenario

28 - Jan -13

Dr. Becca Asquith

Imperial College London

UK

Efficiency of the CD8+ T cell response to persistent viral
infection

15 - Feb -13

Dr. Wilfred T.V. Germeraad

GROW School for Oncology and Developmental Biology
Maastricht University

Neth.

Development of new Immunotherapies for cancer

07- Mar -13

Dr. Denis Duboule

Department of Genetics and Evolution, University ofGenevaItaly

Long range regulation of HoxD genes during mammalian development

07- Mar -13

Dr. Yusuke Miyanari

Institute of Genetics and Molecular and Cellular Biology (IGBMC)

FranceReprogramming and nuclear dynamics

13 - Mar -13

Dr. Eugene Oltz

Washington University School of Medicine

USA

Defining the Malignant Epigenome in Follicular Lymphoma

02 - Apr -13

Dr. Akira Imamoto

The Ben May Department for Cancer Research,The
University of Chicago

USA

The CRK gene family: A tale of a small family businessfor mesenchymal property management

10 - Apr -13

Dr. Koichi S Kobayashi

College of Medicine, Texas A&M Health Science Center

USA

Regulation of MHC class I and class II pathways by NLR
family Proteins15 - Apr -13

Dr. Tsvee Lapidot

Dept. of Immunology, The Weizmann Institute of
Science

IsraelStem cell interactions with the BM microenvironment

27- May -13

Dr. Yumi Nakamura

Department of Pathology and Comprehensive Cancer
Center, University of Michigan Medical School

USA

Understanding the link between Staphylococcus aureus
colonization and atopic dermatitis

05 - Jun -13

Dr. Fubito Nakatsu

Howard Hughes Medical Institute, Yale University
School of Medicine

USA

Phosphoinositide metabolism at the plasma membrane: From signaling to membrane dynamics

07- Jun -13

Dr. Yoshiyuki Asai

Okinawa Institute of Science and Technology Graduate
University

Japan

Tutorial of PhysioDesigner and Flint

13 - Aug -13

Dr. Takeshi Egawa

Washington University School of Medicine

USA

Requirements for transcription factors in regulation of
T cell activation

05 - Sep -13

Dr. Patrick Varga-Weisz

Babraham Institute

UK

Chromatin Dynamics and Epigenetic Stability

27- Sep -13

Dr. Naoto Kubota

Graduate School of Medicine, The University of TokyoJapan

The roles of IRS-1 and IRS-2 in the regulation of glucose
and lipid metabolism

07- Oct -13

Dr. David Scott

Department of Medicine, Uniformed Services University
for the Health Sciences

USA

Gene therapy for tolerance, IgG fusion proteins andspecific Tregs

11- Oct -13

Dr. Masahira Hattori

Graduate School of Frontier Sciences, The Universityof TokyoJapan

Ethnic variances of the human gut micro biome

16 - Oct -13

Dr. Huh, Jun R

University of Massachusetts Medical School

USA

Small molecule inhibitors of RORγt: their development
to study the function of inflammatory immune cells

17- Oct -13

Dr. Toshiaki Maruyama

Abwiz Bio Inc.

Japan

The use of phage display for the development of monoclonal antibodies from immune libraries as research
reagents, diagnostics and therapeutics

21- Oct -13

Dr. Kaoru Saijo

School of Medicine, University of California, San Diego

USA

Nuclear Receptor-mediated Regulation of Neuroinflammation

14 - Nov -13

Dr. Masatsugu Ohora

Medical Institute of Bioregulation, Kyushu University

Japan

Calcium signaling and T cell development

09 - Dec -13

Mr. Minoru Yano

IDT-MBL KK.

Japan

Improved methods/reagents for target enrichment in
NGS

18 - Dec -13

Dr. Takashi Ebihara

Howard Hughes Medical Institute, Washington University Medical CenterUSA

NK cell licensing and development: How do NK cells
acquire functional competence?

86Budget, Personnel and Patents
Personnel FY2013
IMS Budget FY2013

Government funding for operations

JPY Million

Category

Number

3,963

Acting Director

1

Commissioned research

921

Special Advisor

2

External competitive funding

917

Deputy Director

4

5,801

Group Director

8Team Leader

26

Deputy Team Leader

1

Coordinator

2

Senior Scientist

30

Postdoctoral Researcher

18

Research Scientist

44

Research Associate

13

Research Fellow

8Special Postdoctoral Researcher

4

Foreign Postdoctoral Researcher

1

International Program Associate

4

Junior Research Associate

20

Student Trainee

94

Senior Technical Scientist

2Technical Scientist

6

Total

An additional supplementary budget (4,400 million yen) was provided in 2013 forcommissioned research “Order-made Medicine Realization Program”.

Technical Staff

137

Assistant

39

Consultant

10

Senior Visiting Scientist

9

Visiting Scientist

137

Visiting Technician

33

Temporary Employment

11

Total

664

Patents 2013
There were 19 patents filed from January to December,2013.