RIKEN
Research Center forAllergy and Immunology ( RCAI )Organization
Special Advisor
Kimishige Ishizaka M.D., Ph.D.

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

Group

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

Team

Lab. for Cell Signaling Takashi Saito Ph.D.

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

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

Lab. for Signal Network Masato Kubo Ph.D.

Lab. for Immunogenomics Osamu Ohara, Ph.D.

Lab. for Immunochaperones Hei-ichiro Udono M.D., Ph.D.

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

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

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

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

Lab. for Autoimmune Regulation Osami Kanagawa M.D., Ph.D.

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

Lab. for Cytokine Signaling Toshio Hirano M.D., Ph.D.

Lab. for Innate Cellular Immunity Masato Tanaka M.D., Ph.D.

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

Lab. for Host Defense Tsuneyasu Kaisho M.D., Ph.D.
Lab. for Dendritic Cell Immunobiology Katsuaki Sato Ph.D.
Lab. for Infectious Immunity Satoshi Ishido M.D., Ph.D.
Lab. for Transcriptional Regulation Ichiro Taniuchi M.D., Ph.D.
Lab. for Immunogenetics Hisahiro Yoshida M.D., Ph.D.
Lab. for Vaccine Design

Yasuyuki Ishii Ph.D.

Lab. for Cellular Systems Modeling

Mariko Okada Ph.D.

Research Coordination Office
Toshitada Takemori M.D., Ph.D.
Haruka Iwano D.V.M., Ph.D.
Peter Burrows Ph.D.

International Advisory Council
Max D. Cooper, Chair
Rudolf Aebersold
Antonio Coutinho
Alain Fischer
Paul W. Kincade

Tadamitsu Kishimoto
Bernard Malissen
Diane Mathis
Ruslan Medzhitov
Masayuki Miyasaka

Unit

Hiromitsu Nakauchi
William E. Paul
Susan K. Pierce
Klaus Rajewsky
Takehiko Sasazuki

Ralph M. Steinman
Kiyoshi Takatsu
Dale Umetsu
Arthur Weiss

International Research Unit

Unit for Molecular Systems Immunology Makio Tokunaga Ph.D.

Unit for Thymic Environment

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

Unit for Immunogenetics Miguel Vidal Ph.D.

Willem van Ewijk Ph.D.

Unit for Immune Homeostasis Shohei Hori Ph.D.
Unit for Cellular Immunotherapy Shin-ichiro Fujii M.D., Ph.D.
Unit for Immunoinformatics S. Sujatha Mohan Ph.D.
Unit for Lymphocyte Cloning Andrei Rybouchkin Ph.D.
Unit for Single Molecule Imaging Kumiko Sakata-Sogawa Ph.D.
Unit for Therapeutic Model Kanako Shimimzu M.D., Ph.D.
Unit for Inflammatory Regulation Takashi Tanaka M.D., Ph.D.
Unit for Immunodynamics Takaharu Okada Ph.D.

i

i  .  .  . Organization

Laboratory Activitiesii  .  . Contentsiv  .  .  . Director's Report22  .  .  . Laboratory for Developmental
Genetics
24  .  .  . Laboratory for Lymphocyte
Development

Research Highlights

26  .  .  . Research Unit for Lymphocyte
Cloning

Contents

2  .  .  . Sights set on immunization target

28  .  .  . Laboratory for Transcriptional
Regulation

3  .  .  . Helping cells reach out

30  .  .  . Laboratory for Cell Signaling

4  .  .  . Bringing leukemia out of hiding

32  .  .  . Research Unit for Single Molecule
Imaging5  .  .  . Tipping the balance of immune
response
6  .  .  . A zinc transporter is selectively
required for one type of mast cellmediated allergic response
7  .  .  . A novel gene essential for thedevelopment of single positive
thymocytes
8  .  .  . Natural suppressors of a treatmentinduced disease
9  .  .  . Generation of functional NKT cells
in vitro from embryonic stem cells

34  .  .  . Laboratory for Lymphocyte
Differentiation
36  .  .  . Laboratory for Immune Diversity
38  .  .  . Laboratory for Immunological
Memory40  .  .  . Laboratory for Epithelial
Immunobiology
42  .  .  . Laboratory for Mucosal Immunity
44  .  .  . Laboratory for Host Defense
46  .  .  . Laboratory for Infectious Immunity

10  .  .  . Outstanding Contribution of theYear 200914  .  .  . Toshio Hirano awarded the
Crafoord Prize 200948  .  .  . Laboratory for Innate Cellular
Immunity
50  .  .  . Research Unit for Inflammatory
Regulation

16  .  .  . Other Prize Winners 200918  .  .  . Excellent Paper of the Year 200952  .  .  . Research Unit for TherapeuticModel19  .  .  . Excellent Poster Award 2009

54  .  .  . Laboratory for Immune Regulation

20  .  .  . RIKEN Special Postdoctoral
Researcher (SPDR) and JuniorResearch Associate (JRA)56  .  .  . Laboratory for Dendritic Cell
Immunobiology
58  .  .  . Laboratory for Immunochaperones
60  .  .  . Research Unit for Immune
Homeostasis
62  .  .  . Laboratory for Autoimmune
Regulation
64  .  .  . Laboratory for Cytokine Signalingii

66  .  .  . Laboratory for Signal Network

99  .  .  . Kyoto T cell Conference (KTCC)

68  .  .  . Laboratory for Immunogenetics

100  .  .  . The 2nd Symposium for PID in Asia

70  .  .  . Laboratory for Vaccine Design102  .  .  . International Research
Collaboration Award

72  .  .  . Research Unit for Human Disease
Models74  .  .  . Research Unit for Cellular
Immunotherapy

Events76  .  .  . Laboratory for Cellular System
Modeling

104  .  .  . RCAI International Summer
Program 200978  .  .  . Laboratory for Immunogenomics

106  .  .  . RCAI-JSI International Symposium
on Immunology 200980  .  .  . Research Unit for
Immunoinformatics

106  .  .  . Cellular and Genetic View on
Autoimmunity

82  .  .  . Research Unit for Immunodynamics
84  .  .  . Research Unit for Molecular
Systems Immunology
86  .  .  . Research unit for Thymic
Microenvironments
88  .  .  . Reseaerch Unit for
Immunoepigenetics

108  .  .  . US-Japan International Cancer
Systems Biology Meeting110  .  .  . Briefing session for the RCAI
adjunct
graduate school programs111  .  .  . RIKEN Yokohama Open Campus
111  .  .  . RCAI Retreat

90  .  .  . iPS Project Team112  .  .  . Super Science High School
92  .  .  . Central Facilities

Collaborative Networks

Data and Statistics
115  .  .  . Publications

96  .  .  . RCAI and Torii Collaborate for theAllergy Vaccine
96  .  .  . Open Laboratory for Allergy
Research

119  .  .  . Invited Presentations
121  .  .  . RCAI Seminars
123  .  .  . Budget, Personnel and Patents

97  .  .  . Multidisciplinary Research Projects
97  .  .  . RCAI and the Univ . of Palermo Signa Cooperative Agreement

124  .  .  . Cover and Section Heading Photo
Legends

98  .  .  . Harvard Summer School Program at
RCAI
98  .  .  . RIKEN Joint Graduate School
Program98  .  .  . International Program Associateiii

Director’s ReportT

his is the sixth annual report of the RIKEN Research Center for Allergy and Immunology (RCAI).
Six years have passed since all research groups shifted in 2004 from universities into a newbuilding on the RIKEN Yokohama campus. As we move into the second half of our initial ten year
term, significant changes have occurred that have necessitated strategic changes. There has been
an ongoing decline in government support, but, as I have summarized in this report, the Center
nevertheless has continued to be successful in its core mission – cutting edge immunology
research.

Change of the government affects RIKEN/RCAIIn August, 2009, the Democratic Party of Japan (DPJ) won the Lower House election and secured asignificant majority. The new government, lead by Prime Minister Yukio Hatoyama, has focused on
the more efficient use of the government budget. To eliminate needless or excessive projects, a
special Government Revitalization Unit was formed in November, 2009 to reevaluate 220 alreadyfunded government projects. These included a number of projects at RIKEN institutes: the Super
Computer Project, the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, the Bioresource Project, and several Plant Science projects. This government initiative reminded us of how essential it is to explain the importance of scientific research to non-scientists.
In order to have the support of the people, we have to help them become more familiar with the scientific approach to questions, and also to make it very clear that even basic science research canhave unexpected long term practical benefits for human health and society.
The new government’s strategies focus on innovation and technology that promote environmental conservation, health care and the economy. Based on this emphasis, RCAI is facing difficulties in
obtaining funding for our basic science programs. The government also aims to reduce or eliminate
any overlap in projects being carried out at institutions, universities and ministries. Overlap, in termsof scientific projects, may be difficult to quantify. Because of the importance of a particular area,
several groups may be working on seemingly similar projects, but be utilizing many different experimental approaches. It will be important for our future that we make this feature of the scientificresearch endeavor very clear to the various governmental agencies.

iv

The future direction of RCAI continues to be the focus of intensive discussions.
Last year we formulated a new organizational plan, and the basic premises and the new research
directions proposed in the plan were supported by the RCAI External Advisory Council in December,2008. Three areas were given the highest priority: molecular and cellular movement (single molecule
imaging and in vivo imaging), human immunology (humanized mouse models, artificial lymph nodes
and immune iPS cells), and systems biology. Following this direction, the Center began a reorganization process in 2009. This process will continue until 2013, when RCAI celebrates its 10th anniversary and enters its new term.

Platforms for Translational Research and Drug Development
In 2009, we established a platform for bridging basic and translational, patient-oriented research.
This model aims to support the development of orphan drugs or drugs that private companies would
otherwise hesitate to risk investing in. This is a two stage plan: 1) RIKEN, in collaboration with universities, will analyze the mechanisms and pharmacological effects of a particular new drug, and conduct
preliminary studies of efficacy and safety as translational research. 2) Based on promising results inthe phase 1/2 clinical studies, companies will be invited to develop the drug and conduct clinical trials. As an example of this platform in action, in December, 2009, RIKEN and Torii Pharmaceutical Co.,
Ltd. agreed to collaborate in the development of cedar pollen allergy vaccines.

Collaborative projects
With the aim of developing new paradigms in immunology, in 2009 RCAI launched a support programfor multidisciplinary projects. Five out of eight collaborative projects were selected. This programsupports several collaborations between “wet” and “dry” laboratories, and we hope it will be the firststep in integrating systems biology approaches with experimental immunology.
We also launched the “Open Laboratories for Allergy Research” projects. A patient cohort study
project combined with humanized mouse studies was started in collaboration with Chiba University
and the National Center for Child Health and Development. A project on oral tolerance for the treatment of allergy will be performed in collaboration with Advanced Industrial Science and Technologies
and also with the research group supported by the Ministry of Health, Labour and Welfare.

Coordination Office
In 2009, we reorganized the RCAI administrative staff and established the coordination office. All theassistants will now work together as a team to support RCAI and the laboratories. This was not atrivial reorganization, but became necessary because of the austere and uninterrupted budget cuts
faced by the Center. Using this centralized approach, we hope to be able to provide sufficient administrative support uniformly to all RCAI laboratories under these difficult circumstances. The new officewill begin its activity in April, 2010.

Significant research achievements
Several high impact publications since the last annual report are grounded in fundamental research
but have significant translational implications.
Two of these publications deal with a newly designated research priority, the humanized mousemodel. These studies are also potentially highly translational in nature, since they deal with new
strategies for the cure of human leukemia. Drs. Saito and Kitamura were able to identify importanttherapeutic targets in human acute myelogenous leukemia (AML). The leukemic stem cells (LSC) inthis disease are quiescent, and therefore resistant to most forms of chemotherapy. Unfortunately, it isthese LSC that are responsible for disease relapse and the poor prognosis in AML (Science Translational Medicine). Drs. Saito and Ishikawa were able to induce the LSC to enter cell cycle by treatment
with cytokines, thereby promoting their elimination (Nature Biotechnology).

v

Since many pathogens, including HIV and the influenza virus, gain entry through mucosal surfaces, the development of effective mucosal vaccines has been a long sought but elusive goal of
immunologists. Drs. Hase, Kawano and Ohno have identified a molecule, Glycoprotein 2 (GP2),
which may be a very important new target for mucosal vaccines. GP2 is expressed on the M cells
that overlay Peyer’s patches, structures that behave somewhat like lymph nodes for mucosal immunity. These investigators found that the fimbriae on certain pathogenic bacteria are specifically recognized by GP2, initiating a potent mucosal immune response (Nature). This group also discovered
a novel method of intercellular communication via tunneling nanotubes (Nature Cell Biology).
The therapeutic efficacy of NKT cells has been verified in many experimental settings, but there
are practical problems in applying this therapy to human disease. Among the most vexing is the low
frequency of these cells and the fact that they may be functionally impaired in diseases like cancer.
Drs. Watarai, Rybouchkin, Hongo and Taniguchi have found that large numbers of functional NKT
cells can be generated in vitro. The precursors of these cells were initially derived from somatic cell
nuclei that were then converted to embryonic stem cells, thereby bypassing important ethical issues(Blood).
As described in last year’s report, Drs. Nishida, Hasegawa and Hirano have been studying therole of zinc in allergic diseases and embryonic development. Since then, they have made an important discovery, that one of the Zinc transporters, Znt5/Slc30a5, is required for the mast cell-mediated delayed-type allergic reaction, but not the immediate-type reaction. These two phases of theallergic response were known to have different, but unknown mechanisms, and to require different
therapies. Znt5 is thus a new target for allergy therapy (J. Exp. Med.).
The International Collaboration Award, which teams RCAI and foreign researchers, was established at the start of RCAI and has lead to many significant publications. The tradition continued thisyear, with the finding by Dr. Bix (St. Jude Children’s Research Hospital, Memphis, TN, USA) and Dr.
Kubo (RCAI) that Mina, an Il4 transcriptional repressor, controls T helper type 2 bias, an issue thathas puzzled immunologists for decades (Nature Immunology).
The ENU mutagenesis project is a long term, labor intensive effort to identify genes importantin immunological diseases. Several genes have already been identified and this year there wasanother milestone paper. Drs. Kakugawa, Yasuda, Kawamoto, and Yoshida discovered a geneessential for the development of single positive thymocytes (Mol. Cell. Biol.). The conversion of
double positive to single positive thymocytes is crucial for the development of functional T cells, andthese studies are likely to shed new light on human primary immunodeficiency diseases, anotherfocus of RCAI translational studies.
The quality of research at RCAI depends on a number of factors, the creativity of the investigators, the scientific interactions with their colleagues at RCAI and other institutions in Japan andabroad, as well as financial and administrative support. Also important is the work of the RCAI
technical staff. This effort is clearly indicated in a publication in Blood from Dr. Sato’s laboratory that
had two technical staff members, Ms. Kaori Sato and Ms. Kawori Eizumi, as sole co-authors. Thispublication described naturally occurring regulatory dendritic cells and their regulation of cutaneous
chronic graft-versus-host disease, a severe clinical problem in human bone marrow transplant
recipients.

Masaru Taniguchi
March, 2010

vi

2009

Research Highlights

Sights set on immunization target
Hiroshi Ohno

Figure :A schematic diagram depicting the role of
GP2 in the immune response of the gut.

A

RIKEN-led research team has unraveled themolecular details of a key mechanism of theimmune system in the gut. The work opens the way to
new possibilities for developing versatile, inexpensive
vaccines that are swallowed, rather than injected.
Ohno and colleagues from Laboratory for Epithelial
Immunobiology, in collaboration with biologists from
Yokohama City University, several other Japanese universities and Stanford University in the US, uncovereda particular receptor molecule on M cells that stimulates the immune response by binding to a protein on
the hair-like projections or pili of bacteria. The epithelial
M cells engulf foreign bodies in the gut cavity, surrounding them with the cell membrane. These membrane-sealed packages or vesicles are then passed
through the body of the M cell to waiting immune system cells in a pocket on the underside (Fig.).
The researchers speculated that the M cells may
have specific molecular receptors to bind to antigen
targets. So, working in mice, Ohno and colleagues
used the microarray technology to scan the genome
for receptor molecules specific to M cells. Glycoprotein
2 (GP2) was one such molecule and the microarray
analysis showed it was highly associated with epithelium close to Peyer’s patches. When they stained both
GP2 and M cells, they discovered that not only was
GP2 restricted to M cells on Peyer’s patches in the gut,
but that it was also found on M cells associated withimmune system tissues in other parts of the body inhumans as well as mice. In fact, GP2 is a universal
marker for M cells.
Moving in closer, the researchers employed electron microscopy to determine where GP2 was distributed on the M cells. They found it was localized on themembrane facing the gut cavity. By using an antibody
that binds only to GP2, they discovered that some wasincorporated into vesicular structures in the body of the
M cells. This provided further evidence that the receptors were involved in the mechanism for transporting
antigens to cells of the immune system beneath.
The next step was to determine what compoundor compounds linked to the GP2 receptor. Ohno andcolleagues experimented by mixing GP2 with E. coli,
and found that it bound to the protein FimH on thebacterial pili. In fact, GP2 binds only to bacteria thatcarry FimH, and not to bacteria that lack this protein,such as Pseudomonas and Listeria.
Using an intact mouse intestine, the researchers
tracked E. coli expressing green fluorescent protein.
GP2 accumulated around the bacteria, which thencould be followed moving inside the M cells. More than
90% of the bacteria transported through the M cells
are captured by dendritic cells. They also showed that
this process was severely hampered in E. coli lacking
FimH or mice lacking GP2.
“It is reasonable to assume there are other molecules on the M-cell surface responsible for binding anduptake of bacteria lacking FimH,” Ohno says.
“This is one of the projects we are now working
on. Another is to screen
for small compounds
that bind tightly to GP2.
We can then use thesemolecules to target to
GP2 on M cells in an
Masumi Oomae (left) andeffort to develop efficient
Koji Hase (right)
oral vaccines.”

ORIGINAL RESEARCH PAPER
Hase, K., Kawano, K., Nochi, T., Pontes, G.S., Fukuda, S., Ebisawa, M., Kadokura, K., Tobe, T., Fujimura, Y., Kawano, S., et al. Uptake throughglycoprotein 2 of FimH+ bacteria by M cells initiates mucosal immune response. Nature 462, 226 – 232 (2009).

2

This article is abstracted from RIKEN RESEARCH with permission

http://www.rikenresearch.riken.jp/eng/hom/6148

Helping cells reach out
An unexpected result leads to new insights about a poorly
understood mode of communication between cells
Hiroshi OhnoR

emarkably little is known about M cells, which
reside in the intestine and participate in immune
surveillance of potential pathogens. However, Hiroshi
Ohno was taken aback when Koji Hase, a staff scientist in his Laboratory for Epithelial Immunobiology,
RCAI, identified a highly active M cell gene that triggered the formation of unusually long and thin membrane projections when expressed in other cell types.
“I thought it was an artifact,” recalls Ohno, “butKoji insisted on its importance.” Subsequent experiments proved Hase correct; a mammalian protein
called M-Sec appears to drive formation of an enigmatic class of intercellular connections known as ‘tunneling nanotubes’ ( TNT ). TNTs are known to participate
in the trafficking of a variety of cargoes, ranging fromions to proteins to cargo-laden membrane vesicles —and, less beneficially, viruses and prions. However,these findings offer the first real insights into the underlying mechanisms of TNT growth.
In a series of experiments using mouse immune
cells known as macrophages that also express endogenous M-Sec, Ohno’s group showed that formation of
TNTs was inhibited upon knockdown of M-Sec expression (Fig.). These TNTs were also shown to be physiologically active, enabling signals to be transmitted fromcell to cell via the movement of calcium ions.
Cells rely on a protein-based framework known as
the cytoskeleton to provide infrastructure, and major
morphological changes typically involve cytoskeletal
rearrangements mediated by molecular switches
known as small GTPases. Ohno and colleagues foundthat M-Sec associates with RalA, a small GTPase, anda membrane-bound protein complex known as the
exocyst, and that these interactions are essential to theformation and growth of membrane protrusions thatwill ultimately form TNTs.
The researchers are now delving deeper into thebigger picture of M-Sec function by studying mice thathave been genetically engineered to lack this protein,
but are also exploring more direct ways to target M-Sec
activity. “We have started screening M-Sec-binding

Figure :The formation of TNTs connecting mouse macrophagecells (left) is blocked when M-Sec is depleted byknockdown of gene expression (right). (Scale bar, 20 µm).

small-molecular-weight chemicals that could inhibit
M-Sec-mediated TNT formation,” says Ohno.
He suggests that such drugs could ultimately be
used in vivo to block the transmission of viruses via
these intercellular conduits, but sees the most immediate benefits in simply understanding why these connections exist in the first place. “Virtually nothing is
known about their in vivo relevance,” he says. “In thisregard, the discovery of M-Sec as a promoter of TNT
formation will accelerate the understanding of their
physiological role.”

Yumiko Fujimura (left) and Koji Hase (right)

ORIGINAL RESEARCH PAPER
Hase, K., Kimura, S., Takatsu, H., Ohmae, M., Kawano, S., Kitamura, H., Ito, M., Watarai, H., Hazelett, C.C., Yeaman, C. & Ohno, H. M-Sec
promotes membrane nanotube formation by interacting with Ral and the exocyst complex. Nature Cell Biology 11, 1427–1432 (2009).

This article is reproduced from RIKEN RESEARCH with permission.

http://www.rikenresearch.riken.jp/eng/research/6167

3

Bringing leukemia out of hiding
Two new strategies target a deadly cancer that eludes
conventional chemotherapy
Fumihiko Ishikawa

Figure :AML (left) can initially be driven into remission withchemotherapy, but this leaves behind a subpopulation of resistant
LSCs (middle), with a high potential for initiating a fatal relapse(top right). Complete elimination of AML from patients may
eventually be possible by directly targeting LSCs through more
effective chemotherapeutic strategies or the identificationof clinically useful LSC-specific markers (bottom right).

Acute myeloid leukemia (AML) is the most common
form of adult leukemia, and with an estimated fiveyear survival rate of 20%, the long-term prognosis formany patients is relatively grim.
“Current treatments for AML can initially reduce thenumber of AML cells to undetectable levels, a statereferred to as ‘complete remission’,” says Fumihiko
Ishikawa of RCAI’s Research Unit for Human Disease
Model. “Unfortunately, in a substantial proportion ofthese patients, AML eventually comes back— andmany that relapse succumb to the disease.” The needfor an improved arsenal to fight AML has guided muchof Ishikawa’s work, and two recently published articles
from his team present promising strategies for tackling
this dreaded cancer.
AML originates in bone marrow, and relapse is initiated from small pockets of chemotherapy-resistant‘leukemia stem cells’ (LSCs) within the marrow, which
can be identified by their distinctive profile of cell-surface markers. In an effort to identify other features of
LSCs that might offer useful therapeutic targets,
Ishikawa, Yoriko Saito and team performed a thoroughcomparative analysis between LSCs and normal blood
stem cells to identify genes with functional characteristics pertinent to cancerous growth whose expression is
specifically elevated in LSCs.

Their analysis revealed two candidate cell-surface
proteins, CD25 and CD32; both of these are commonly
overexpressed in chemotherapy-resistant LSCs, but
can also be therapeutically targeted without negatively
affecting blood cell development from healthy
hematopoietic stem cells, making them potentiallypromising targets for thwarting relapse.
In parallel, Ishikawa and colleagues have also
explored methods for boosting the efficiency of chemotherapy. Standard AML drugs such as cytosine arabinoside (Ara-C) work by targeting actively dividing cells,and LSCs are believed to elude chemotherapy byentering a quiescent, non-dividing state. The researchers hypothesized that LSCs could be made more
vulnerable to Ara-C via simultaneous treatment withcytokines — naturally- occurring cell signaling molecules — that stimulate them into active division. In fact,
this two-pronged treatment led to a ten-fold increase insurvival rate relative to chemotherapy alone for micethat had been transplanted with human LSCs.
Previous studies have suggested that despitesome risk of toxicity, cytokine treatment is relatively safefor patients, and Ishikawa’s team is actively investigating the practicality and safety of interventions based on
both of their recent discoveries (Fig.). “We have been
putting our best effort into the translation of thesefindings into medicine,” he says. “At the same time, weare continuing to try to identify unknown aspects ofhuman AML and AML stem cells.

Yoriko Saito ( left), Mariko Murasawa (middle)
and Hiroshi Kitamura (right)
ORIGINAL RESEARCH PAPER
Saito, Y., Kitamura, H., Hijikata, A., Tomizawa-Murasawa, M., Tanaka, S., Takagi, S., Uchida, N., Suzuki, N., Sone, A., Najima, Y. et al. Identificationof therapeutic targets for quiescent chemotherapy-resistant human leukemia stem cells. Science Translational Medicine 2, 17ra9 (2010).
Saito, Y., Uchida, N., Tanaka, S., Suzuki, N., Tomizawa-Murasawa, M., Sone, A., Najima, Y., Takagi, S., Aoki, Y., Wake, A. et al. Induction of cell
cycle entry eliminates human leukemia stem cells in a mouse model of AML. Nature Biotechnology 28, 275–280 (2010).

4This article is reproduced from RIKEN RESEARCH with permission.

http://www.rikenresearch.riken.jp/eng/hom/6234

Tipping the balance of
immune response
Hybrid mice help researchers zoom in on a gene with a potential
role in controlling allergic responses

Figure:W

hen the immune system encounters a potential
threat, antigen-presenting cells deliver chunksof protein from the invading pathogen to naive helper T
cells. These naïve cells respond by differentiating into
one of two classes of mature helper T cells: TH1 cells,
which mobilize the immune system against viruses andother intracellular pathogens, and TH 2 cells, which drive
the response against blood-borne threats, such as theparasitic disease leishmaniasis.
Interleukin-4 (IL-4), one of a class of signalling factors known as cytokines, drives TH 2 differentiation andtriggers secretion of additional IL-4, resulting in a positive feedback loop that fuels TH 2 production while
suppressing TH1 production. The degree of initial IL-4
production varies considerably between individuals
and the resulting ‘TH 2 bias’ can have serious clinicalimplications. “TH 2 bias is thought to be a mirror ofallergic response, because many TH 2 cytokines tightly
associate with pathology of allergy,” says Masato
Kubo, RCAI’s Laboratory for Signal Network.
TH 2 bias also varies between different mousestrains, a fact that Kubo, Mark Bix of St. Jude’s Children’s Research Hospital in Memphis, USA, and colleagues exploited in a recent effort to identify
determinants for this trait. It was known that BALB/cstrain mice have a high TH 2 bias — producing large
quantities of IL-4 following T cell activation— while B10.

Masato Kubo

Schematic showing the role of the Mina protein
in TH2 bias. The extent to which the IL-4
gene is being inhibited by Mina protein (left)
affects the maturation of naïve T lymphocytes(right). Low levels of Mina mean a greater bias
towards TH2 production, as in BALB/c mice,
while higher levels lead to relatively higher
TH1 production, as in B10 mice. Strong TH2
bias is thought to be a risk factor for allergy.

D2 mice have a 50-fold lower bias. However, a hybrid
BALB/c strain generated by Kubo and Bix that contains a chunk of chromosome 16 from the B10.D2
strain also exhibited low bias, suggesting that this segment includes a gene pertinent to this characteristic.
Closer analysis spotlighted the Mina gene as alikely suspect; analysis of various mouse strains
revealed that Mina gene activity and levels of Mina protein were inversely correlated with TH 2 bias. Bix andKubo’s team subsequently determined that Mina
assembles into a larger multi-protein complex that
directly binds to and inhibits the gene encoding IL-4,
supporting a key role for this factor in TH 2 bias (Fig.).
The team’s analysis also identified nearly twodozen sequence variations in Mina that correlate withgene activity levels. These so-called single-nucleotide
polymorphisms (SNPs) could provide useful diagnostic
tools, and Kubo and Bix are now
exploring this potential. “We havealready done large-scale SNP analysis with Japanese and US populations,” says Kubo. “The human Mina
locus has several SNPs, and some ofthem have weak correlation withatopic asthma in the Japanese population, but not in the US [ population].”Mariko Okamoto

ORIGINAL RESEARCH PAPER
Okamoto, M., van Stry, M., Chung, L., Koyanagi, M., Sun, X., Suzuki, Y., Ohara, O., Kitamura, H., Hijikata, A., Kubo, M. & Bix, M. Mina, an
IL4 repressor, controls T helper type 2 bias. Nature Immunology 10, 872–879 (2009).

This article is reproduced from RIKEN RESEARCH with permission.

http://www.rikenresearch.riken.jp/eng/research/6043

5

A zinc transporter is selectively
required for one type of mastcell-mediated allergic response

Toshio Hirano

25
Ear swelling (0.01mm)(Mean+SEM)

WT

20

Znt5-/-

15
10
5
0
0
24
48 72
Hours after the antigen stimulation
antigencontrol

Wild typeFigure:antigencontrol

Znt5 -/-

Contact hypersensitivity response (CHS) in wild type and Znt5
deficient mice. Mice were sensitized with experimental hapten
such as FITC. Ear swelling was measured at the indicated times
after hapten challenge. WT mice developed robust CHS responses
by tissue swelling at the site of hapten challenge (lower left), whichreached a maximum 24 h after antigen stimulation (upper panel).
The Znt5 deficient mice exhibited defective CHS reaction (lower
right). Even 24 h after the antigen challenge, the increase in ear
thickness of the Znt5 deficient mice was <45% of that of WT mice.

There are two types of allergic responses mediatedby allergen-specific IgE antibodies bound to IgE
receptors (FcεRI) on mast cells. The first is rapid,
occurring immediately after the allergen crosslinks the
FcεRI, and is caused by mast cell degranulation andrelease of preformed mediators such as histamine. The
second type of response is delayed since it requiresthat the mast cell synthesize new proteins, in particular
cytokines and chemokines. The symptoms of theimmediate response can be treated with over thecounter medications such as antihistamines, but thedelayed-type response is more intractable. Therefore,
understanding of how these responses are regulated isof great clinical significance and has been the focus ofintense investigation. Toshio Hirano and his colleagues
in the Laboratory for Cytokine Signaling have recently
made the unexpected discovery of an essential role fora zinc transporter in the delayed-type response.
Zinc (Zn) is an essential trace element that is a
co-factor in many enzymes and transcription factors.
Hirano’s group had previously described a novel role
for Zn as an intracellular signaling molecule, akin to
calcium. Furthermore, they also found that treatment
ORIGINAL RESEARCH PAPER

with a Zn chelator inhibited mast cell activation, suggesting an important role for this trace element in allergic responses.
Zn deficiency causes growth retardation, immunodeficiency, and other health problems. Therefore cells
have evolved a complex system to maintain appropriate Zn levels through a combination of transporters for
uptake, intracellular storage, and efflux. In their studies
published in the Journal of Experimental Medicine,
Nishida, et al. performed genetic ablation of one of theZn transporter genes, Znt5/Slc30a5, and found that itis required for the mast cell-mediated allergic responsesuch as contact hypersensitivity response (CHS) (Fig.).
Intriguingly, Znt5/Slc30a5 is only needed for thedelayed-type response; degranulation of the mast cells
from the zinc transporter-deficient mice was normal.
They went on to study possible mechanisms for thedefect in the knockout mice. Protein kinase C (PKC) isa serine/threonine kinase that plays an important rolein multiple signaling cascades. In mast cells, PKC isrequired for cytokine production in the delayed-type
response. The Hirano group found that the zinc transporter is involved in the translocation of PKC to theplasma membrane, a process important for its function. Zinc fingers are protein motifs that coordinate Znions, and PKC contains a zinc-finger-like motif. The
authors found that this motif is required for translocation of PKC to the plasma membrane, thus explaining
the requirement for Zn. They also found a defect in the
nuclear translocation of nuclear factor (NF)-κ B in theknockout mast cells. NF-κ B is a transcription factor
involved in upregulating the expression of proinflammatory cytokine genes such as IL-6 and TNFα. Theirfindings suggests that Znt5/Slc30a5 is a novel playerof PKC / NF-κ B signaling.
These studies shed
new light on the Zn-mediated regulation of the allergic response, specificallyof the delayed type, andmay provide new therapeutic targets for treatmentof allergic diseases.
Keigo Nishida (left) andAiko Hasegawa (right)

Nishida, K., Hasegawa, A., Nakae, S., Oboki, K., Saito, H., Yamasaki, S. & Hirano, T. Zinc transporter Znt5/Slc30a5 is required for the mast cellmediated delayed-type allergic reaction but not the immediate-type reaction. J Exp Med 206, 1351-1364, (2009).

6

A novel gene essential for thedevelopment of single positive
thymocytes

Hiroshi Kawamoto

Hisahiro YoshidaAs they develop in the thymus, T cells undergo a
stepwise progression that can be traced by theirexpression of CD8 and CD4, co-receptors for the
Major Histocompatibility Complex Class I and Class II
molecules, respectively. Prior to rearrangement andexpression of genes encoding the T cell receptor ( TCR)
the cells are CD4-/CD8- (DN, double negative) and thenbecome CD4+CD8+ double positive (DP). As they transition through these stages, the developing T cells
undergo positive selection for recognition of self MHC
and negative selection to eliminate cells that react
too strongly. A critical step during intrathymic T cell
development is the transition of cells to the MHC
I-restricted CD4-CD8+ and MHC II-restricted CD4+CD8single positive (SP) cell stage. The cells that leave thethymus to constitute the T cell arm of the immune
system in the periphery retain this SP phenotype andconsequent recognition of antigens presented by MHC
I or MHC II molecules.
RCAI investigators have now identified a genethat is essential for the critical transition from the DP to
the SP thymocyte stage. This collaborative effort
between the research groups of Hisahiro Yoshida,
Laboratory for Immunogenetics and Hiroshi Kawamoto, Laboratory for Lymphocyte Development was
published by Kiyokazu Kakugawa et al. in Molecular
and Cellular Biology. In order to identify the genetic
basis for immune disorders, Yoshida and his team havebeen screening a pool of mutant mice generated
by random chemical mutagenesis with N-ethyl-Nnitrosourea. Through T cell phenotype-based screening, they established a mouse line in which numbersof CD4 and CD8 SP thymocytes as well as peripheral
CD4 and CD8 T cells were dramatically reduced. They
named this mouse line SPOTR (Single Positive Thymocyte Reduction), and by using linkage analysis andDNA sequencing, they identified a missense point
mutation in a predicted gene, E430004N04Rik, of
unknown function. This orphan gene was shown to be
specifically expressed in DP and SP thymocytes andperipheral T cells, whereas the levels of protein encodedby this gene were drastically reduced in mutant thymo-

Figure:Developmental arrest of single positive thymocytes
in ENU mutant mice. CD4 versus CD8 profiles ofthymocytes from a 8 week old ENU mice (SPOTR)
and wild type C57BL/6 mice are shown.

cytes. Kakugawa et al. next generated E430004N04Rikdeficient mice and their phenotype was essentially
identical to that of the ENU mutant mice. This result
conclusively demonstrated that E430004N04Rik isessential for the development of SP thymocytes.
While the RCAI investigators were in the processof manuscript submission, they became aware of thefact that several other groups had found this gene andhad named it Themis, after the Greek goddess of justice and natural order and, in this case, for “thymusexpressed molecule involved in selection”. The RCAI
group graciously agreed to use Themis as the namefor their new gene. Themis is a member of a small genefamily that contains two other members of unknown
function. Apart from a nuclear localization signal and acarboxy terminal proline-rich sequence, the domains in
the predicted Themis protein do not correspond to
any known motifs and provide no clue as to its function. Further studies will likely reveal novel mechanisms
in T cell development.
Although no mutationsin the human Themis
ortholog have beenreported yet, this gene
will also be a goodcandidate for a causative gene in humanprimary immunodeficiency.

ORIGINAL RESEARCH PAPER

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

7

Natural suppressors of atreatment-induced disease
A naturally occurring population of dendritic cells reduces theincidence and severity of graft-versus-host-disease in mice

Stimulated

Unstimulated

Katsuaki Sato

Figure :Phase contrast microscopy images (original magnification ×400) showing the morphology of non-regulatory (CD11c+)
DCs (left column) and CD200R3+ DCregs (right column)
before and after stimulation with a pro-inflammatory agent.

Researchers in RCAI have shown that mouse dendritic cells (DCs), which can promote or inhibit
inflammation depending on the proteins displayed on
their surface, include a subpopulation that exerts beneficial effects during a treatment for leukemia and othermalignancies. The treatment— known as allogeneic
hematopoietic stem cell transplantation (alloHSCT) —can, in some situations, result in graft-versus-hostdisease (GVHD). Acute and chronic GVHD occurs whendonor immune cells called T lymphocytes recognize
and become activated by proteins present on recipient
but not donor tissues. The resulting T lymphocytedriven immune response can result in severe damage
to the recipient skin, gastrointestinal tract and liver.
Previous work by these researchers described thegeneration of regulatory DCs from mouse bone marrow ( BM-DC regs ) that, when injected after alloHSCT,
reduce the severity and incidence of acute and chronic
GVHD. The team, led by Katsuaki Sato at the Laboratory for Dendritic Cell Immunobiology, has now shownthat naturally occurring counterparts of BM-DC regs exist
and influence the outcome of alloHSCT in mice.
The researchers started by searching for genes
associated with immunosuppressive DC function. A
comparison of the genes expressed in BM-DC regs andnon-regulatory DCs revealed that the gene encoding
the surface protein CD200R3 is expressed exclusively
in BM-DC regs. They found that blockade of CD200R3
impaired the ability of BM-DCregs to suppress proliferation of T lymphocytes, whereas forced expression of
CD200R3 in non-regulatory DCs reduced their ability
to promote T lymphocyte cell division. This indicatesthat CD200R3 contributes to the immunosuppressivefunction of BM-DC regs.
Reasoning that naturally occurring regulatory DCs
might also express CD200R3, the researchers screened
blood and spleen cells for CD200R3 expression. They
identified a small population of CD200R3-expressing
cells that, like BM-DC regs, produced immunosuppressive cytokines, which are regulators of the immune
system, and inhibit T cell proliferation. These CD200R3+
DCs exhibited a different morphology than non-regulatory DCs (Fig.).
When injected after alloHSCT, Sato and colleagues
found that these CD200R3+ DCs — like BM-DC regs —suppressed the onset and severity of GVHD. Recipients of CD200R3 DCs contained lower amounts ofserum proinflammatory proteins, and higher numbersof immunosuppressive regulatory T lymphocytes.
Further highlighting the biological importance ofCD200R3+ DCs, pre-treatment with a CD200R3-blocking antibody prior to alloHSCT exacerbated GVHD.
“The functional identification of naturally occurring
human DCregs, as well as their counterparts generated the laboratory, may provide an advantageousmeans of intervening to prevent chronic GVHD after
alloHSCT,” says Sato.

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

8

This article is reproduced from RIKEN RESEARCH with permission.

http://www.rikenresearch.riken.jp/eng/research/5906

Generation of functional NKT cells in
vitro from embryonic stem cells
Masaru TaniguchiSeveral features of NKT cells make them uniqueamong the T cell subsets. Instead of recognizing
peptide antigens presented by proteins of the Major
Histocompatibility Complex (MHC), NKT cells recognize lipid antigens presented by the MHC Class I-likemolecule CD1d. The prototypical NKT cells are known
as invariant NKT (iNKT ) cells because they express aninvariant T cell antigen receptor (TCR) α chain. The
response of NKT cells is also unique in that they arepoised to rapidly secrete large amounts of TH1 and/or
TH2 type cytokines. The nature of the response is dictated by the initial cytokine environment: interaction
with IL-10-producing cells induces negative regulatory
Th2 T cell-type NKT cells, while that with IL-12producing cells results in pro-inflammatory Th1-type
responses. As a result of their potent and varied
cytokine profile, iNKT cells can have seemingly paradoxical functions, including enhancement of responses
to pathogens and tumors, suppression of certain autoimmune diseases, induction of transplant tolerance,and promotion of allergy and asthma. These CD1drestricted invariant natural killer T (NKT) cells bridge
innate and acquired immunity and thus play an important role in both protective and regulatory responses,
but may also be pathogenic. Autologous NKT celltherapy has shown promise in treating cancer in clinical
trials. However, full realization of this promise will require
a better understanding of how NKT cells develop, how
to obtain sufficient numbers of these cells for therapeutic use, and how to control their differentiation alongTh1- or Th2-type pathways. This is particularly relevantto the anti-tumor response; NKT cells mediate adjuvant
activity by their production of IFN-γ, which is a Th1type response. Masaru Taniguchi and his colleagues inthe Laboratory for Immune Regulation have developed
a new mouse model system that should help close thegaps in our knowledge of NKT cells.
In a paper published in Blood, Hiroshi Watarai et
al. used a combination of cutting-edge techniques togenerate NKT cells in vitro. They first transferred nuclei
isolated from liver NKT cells into unfertilized eggs andthen derived embryonic stem cell (ES) lines from theresultingblastocyst
stage ofembryonic
developm e n t(Fig.). The
use of thisFigure : Generation of NKT cells from cloned ES cells
approachmeant that
all of the ES cells (NKT-ES cells) already had productively rearranged TCRα and TCRβ chain genes to
encode the invariant NKT cell TCR. Because of their
genotype, the cells were expected to preferentiallygenerate NKT cells in vitro if they could identify theproper culture conditions. They succeeded in achieving this goal. They were able to identify cells with NKTcell potential as well as to obtain NKT cells with different
cytokine profiles. Early NKT progenitors first appearedon day 11 in cocultures of NKT-ES cells with OP9 cells
expressing a Notch ligand, delta-like. In the continued
presence of Notch signals, the NKT-ES cells differentiated only into thymic-type CD44lo CD24hi NKT cells
producing mainly interleukin-4 (IL-4), a Th2 phenotype.
By contrast, NKT cells resembling CD44hi CD24 lo liver
NKT cells producing mainly IFN-γ, a Th1 phenotype,developed in switch cultures starting at day 14 in theabsence of Notch signaling. These Th1-like cells also
exhibited strong adjuvant activity in vivo, one of thehallmarks of naturally occurring Th1-type NKT cells.
This newly developed cloned ES cell culture system
offers a new opportunity for the elucidation of themolecular events during NKT-cell development. It will
also allow the establishment of an animal model for
NKT-cell therapy, which may ultimately be translatable
into the clinic.

Hiroshi Watarai ( left ), Andrei Rybouchkin (middle)
and Naomi Hongo (right )ORIGINAL RESEARCH PAPER
Watarai, H., Rybouchkin, A., Hongo, N., Nagata, Y., Sakata, S., Sekine, E., Dashtsoodol, N., Tashiro, T., Fujii, S., Shimizu, K., Mori, K.,
Masuda, K., Kawamoto, H., Koseki, H. & Taniguchi, M. Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Valpha14-Jalpha18 TCRalpha gene. Blood 115, 230-237, (2010).

9

Outstanding Contribution of the Year 2009
R

IKEN RCAI’s Outstanding Contribution of theYear Award was established in 2006 to recognize staff members who have made outstanding
contributions to the Center. The awardees are
determined by the Director and receive trophies
and a monetary prize. The awards ceremony washeld on March 26, 2010. Koji Hase and Hiroshi
Ohno (Laboratory for Epithelial Immunobiology),
Yoriko Saito and Fumihiko Ishikawa (Research
Unit for Human Disease Models), and Takanori
Hasegawa and his team (Animal Facility, Laboratory for Developmental Genetics) received theaward.

that recapitulate human AML in mice, they found thatleukemia stem cells are cell cycle quiescent and thus
exhibit chemotherapy resistance in the bone marrowniche, whereas non-stem AML cells can be eliminatedby conventional chemotherapy. It is these chemotherapy-resistant leukemic stem cells (LSCs) that eventually lead to disease relapse (Ishikawa, F., et al. 2007).
Therefore, the researchers decided to explore effective anti-LSC therapy to improve patient outcomes. To
target LSCs, they took a multi-faceted approach. First,
they looked for ways of interfering with LSC function

Yoriko Saito (Photo 1) and Fumihiko Ishikawa
received the award for their contribution to the understanding of human immunity and diseases through thecreation of a humanized mouse system.

Figure 1: Quiescent LSCs within the osteoblast niche in bonemarrow enter the cell cycle after in vivo G-CSF
treatment. (left) At steady state, the LSCs adjacent tothe bone marrow endosteum are cell cycle quiescent(Ki67−), whereas in the perivascular region, CD45+
leukemic cells are Ki67+ (cycling). (Right) After in vivo
G-CSF treatment, LSCs within the bone marrow endosteal
region became Ki67+, indicating that they had entered
the cell cycle. CD45 (red), Ki67 (green), DAPI (blue).
Photo 1 : Yoriko Saito

Their research focuses on studying humanhematopoiesis and immunity by using the humanizedmouse model. By isolating hematopoietic stem cells(HSCs) and transplanting them into immunodeficient
NOD/SCID/IL2r γ KO newborn mice, they succeededin generating mice reconstituted with humanhematopoietic and immune systems ( Ishikawa, F., etal. 2005). Using this system, they created in vivo models of human acute myeloid leukemia (AML). AML is anextremely difficult disease. Typically, a patient presentswith a high level of total body AML burden that istreated by highly toxic combination chemotherapy. In
many cases, this results in what is referred to as complete remission, meaning that AML cells are no longer
detectable in the blood and bone marrow of thepatient. However, in many patients, AML relapses overtime. Therefore, the RCAI investigators thought thatthe humanized mouse would be the key to understandwhat is happening in humans during this period.
In 2007, by establishing a robust in vivo model10

and survival in vivo. They were able to induce cell cyclein LSCs by treatment with granulocyte colony-stimulating factor (G-CSF ). In combination with cell cycle
dependent chemotherapy, G-CSF pretreatment sig-

Figure 2: Two strategies to target LSCs to prevent AML
relapse. The first strategy is interfering with
LSC survival by induction of cell cycles, and thesecond is targeting LSC-specific molecules.

nificantly enhanced the apoptosis and elimination of
LSCs in the humanized mouse ( Fig. 1). Secondly, theylooked for LSC-specific molecules as potential therapeutic targets. In collaboration with Osamu Ohara’s
lab in RCAI, they compared global gene expression in
LSCs and normal HSCs. They succeeded in identifying 25 molecules expressed only in LSCs, including
cell surface markers CD32 and CD25, kinases andtranscription factors. Thus, these two lines of investigation led them to propose two potential therapeuticstrategies that target leukemia stem cells: Cell cyclemodified chemotherapy (Saito, Y., et al. 2010) andLSC-specific molecular targets (Saito, Y., et al. 2010)( Fig. 2, See also Research Highlights).
“We hope that in the near future, we can begin tooffer effective therapies for patients with AML. We
deeply thank the patients and physicians at the Toranomon Hospital for their generous cooperation inproviding samples for these studies.” said Saito.
Koji Hase and Hiroshi Ohno (Photo 2) received
the award for their research on the functions of M

Figure 3: Identification of FAE and M cells. In 1922, a Japanese
doctor reported that bacterial antigens are taken
up by dome-shaped epithelium covering lymphoidfollicle, which is now called follicle-associatedepithelium (FAE). Fifty years later, M cells wereidentified by Max Cooper and Robert Owen.

villi and identified several M cell-specific genes (Hase,K., et al. 2005). One of them was GP-2 ( Fig. 4), which
turned out to be an uptake receptor for E-coli and
Salmonella to initiate the mucosal immune response(see Research Highlights).
However, the road to publication was not easy.
They submitted the paper to Nature, Science andNature Cell Biology, but it kept being immediately
rejected. “So, we decided to add functional data,”
said Ohno. They had been collaborated with Shin-ichi
Fukuoka in Aoyama Gakuin Univ., and started an
additional collaboration with Hiroshi Kiyono in the Univ.
of Tokyo and Anson Lowe in Stanford Univ. After two

Photo 2 : Koji Hase ( left) and Hiroshi Ohno (right)

cells. M cells, specialized epithelial cells in follicleassociated epithelium (FAE), were discovered by Cooper in 1973, and named M cells by Owen in 1974.
Intriguingly, however, 50 years before them, a Japanese physician, Kumagai, had described the follicleassociated epithelium (FAE) how it was responsible
for taking up macromolecules such as bacteria (Fig.
3). When Ohno started his study on M cells in 2002,
almost nothing was known about these cells molecularly. “I studied immunology and worked on membranetraffic at NIH. So I always wanted to combine thesefields. When I heard at an immunology meeting that M
cells were active in transcytosis, I thought this should
be something I could get involved in, a fusion of immunology and cell biology,” Ohno recalled.
When Koji Hase joined Ohno’s lab in 2002, Hase
established the method to isolate FAE from the vast
majority of conventional epithelial cells of the intestinal

Figure 4: Methods to explore M cell-specific genes. FAE
and villous epithelium (VE) were isolated from
Peyer’s Patches. Total RNAs prepared from FAE or
VE were used for microarray analysis. Expressionof the candidate molecules was confirmed byquantitative real-time PCR and in-situ hybridization.

11

years of collaboration, they finally found differences in
the immune responses to oral Salmonella-ToxC in
GP-2 knockout versus wild type mice. “With the efforts
by all these four groups, the paper was finally accepted(Hase, K., et al. 2009). It took nearly three years fromthe first submission,” said Ohno in his speech.

M-sec associates with the small GTPase, RalA, a regulator of actin remodeling. RalA then interacts with two
exocyst subcomplexes, and promotes the full complex
formation, which leads to membrane fusion (Fig. 6).
“We hope to clarify how M cells differentiate, how M
cells work and hopefully, prove that M cells are really
important in gut immunity,” said Ohno, “We appreciate
all the collaborators, and of course we thank the peopleof RCAI for their advice and support.”

Control

M-Sec

Figure 5: Intercellular calcium signalling between remote cells
is observed only in physically connected cell pairs.
HeLa cells stably expressing M-Sec were loaded withfluo-4. Cell pairs were randomly selected and a cell on one
side in each pair was mechanically stimulated to evoke
a calcium activation signal. The propagation of calcium
flux was undetectable between unconnected pairs (upper
panels). In contrast, it was frequently observed between
connected pairs (lower panels). Arrowhead indicates a
TNT interconnecting remote cells. Scale bars, 15 μm.

They also made a surprising discovery of a molecule, named M-sec, which was expressed specificallyin M cells and myeloid cells. When its expression wasforced in other cell types, M-sec triggered the formation of thin tubular connections between remote cells,
called tunneling nanotubes ( TNT ) (see Research Highlights). After stimulation of one of the cells, the calcium
flux was promptly propagated from the stimulated cellto the surrounding cells via interconnecting TNTs (Fig.
5) (Hase, K., et al. 2009). They have further shown that

Sec15

Sec10

Exo84

RalA
Sec5 Sec6

Plasma membrane

Takanori Hasegawa and his Animal Facility teamreceived the award for their continuous and skillful contributions to promote the best quality animal experiments at RCAI. Their contributions cover almost all
aspects of animal experiments, including maintenanceof animals under stable and hygienically well-controlled
conditions, cryopreservation and generation of genetic
resources, compliance and education for animal
experiments, and many others. Moreover, their efforts
enabled very flexible management of the animal facility,
which is not an easy goal to be realized in a research
institution.

2005
(H17)

2003 Chiba University 2004
(H15) ~2003/11/19 (H16)

Membrane vesicle

M-Sec

Photo 3 : Takanori Hasegawa

Sec3 Exo70

Sec8

Exocyst complex
formation

Sec15

Sec10

Exo84
Sec5

Sec6

Sec3 Exo70

Preparation of
cryopreserved
embryos
370 lines
60,000 frozen embryos

2006
(H18)

2007
(H19)

2008
(H20)

Breeding Start
Germ-free isolator

Freeze-Thaw
763 lines
Production ; 15,512 mice

Sec8

Building of environmental
surveillance system

Membrane fusion

Figure 7: Preparations for the start-up of RCAI

Sec15
Exo84

Sec10

TNT formation

Sec5 Sec6
Sec3 Exo70

Sec8

Figure 6: Model for M-Sec to promote TNT formation. M-sec
associates with the small GTPase, RalA, a regulator of actin
remodeling. RalA interacts with two exocyst subcomplexes,
and promotes the full complex formation, which leads tomembrane fusion

12

RCAI started its activity in 2004, but before then,the original team members, Mochizuki, Ishikura,
Matsuda and Hasegawa who were at Chiba University
at that time, had started preparation under Haruhiko
Koseki. Their last day at Chiba University was going to

2009
(H21)

a)

b)

d)

c)

e)

Figure 8: Annual statistics of the animal facility a) Change in the number of cages, b) clean-upof mouse strains by in vitro fertilization, c) cryopreservation of the embryos, d) providing
cleaned-up offspring by thawing the frozen embryos, e) change in the number of users

be Nov. 19, 2003, so in the summer of 2002, they
started cryopreservation of embryos. “We worked like awind-up toy whose spring was tightened to the end,”
Hasegawa recalled. The numbers reached 370 lines or
60,000 embryos. They packed everything and set out
to RIKEN on Nov. 19. Then, at RCAI, they had to do thereverse, but this time, including the mice from otheruniversities, too. They thawed 763 lines and produced
15,512 mice before the launch of RCAI in 2004 (Fig. 7).
“Animal facility members were already so exhausted
when RCAI marked its start in April, 2004,” said
Hasegawa.
“We thought that our work load at the new facility
would increase slowly, but it did not shift like that,”
Hasegawa says. In 2004, the number of cages reached
8,000, and the next year, it already hit the upper limit.
In addition to that, generation of SPF mice, clean-up ofmouse strains by in vitro fertilization, cryopreservationof the embryos and providing cleaned-up offspring by
thawing the frozen embryos, hit a peak in 2005. The
number of the facility users reached its peak in 2005,
too (Fig. 8a-e). To catch up, the number of team members was increased from 5 in 2003 to 20 in 2005.
Besides these services, the team generates transgenic
and chimeric mice, human BAC clone-transgenic mice,
cryopreservation of sperm, and support of the ENU
mutant mice and humanized mice projects. “To someextent, every member of RCAI has profited from theefforts of Hasegawa and his colleagues,” said the
Director Taniguchi at the awarding ceremony.

References
Hase, K., Kawano, K., Nochi, T., Pontes, G. S., Fukuda, S., Ebisawa,
M., Kadokura, K., Tobe, T., Fujimura, Y., Kawano, S., Yabashi, A.,
Waguri, S., Nakato, G., Kimura, S., Murakami, T., Iimura, M., Hamura,K., Fukuoka, S., Lowe, A. W., Itoh, K., Kiyono, H. & Ohno, H. Uptake
through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal
immune response. Nature Nov. 2009; 462, 226-230.
Hase, K., Kimura, S., Takatsu, H., Ohmae, M., Kawano, S., Kitamura,
H., Ito, M., Watarai, H., Hazelett, C. C., Yeaman, C. & Ohno, H. M-Sec
promotes membrane nanotube formation by interacting with Ral andthe exocyst complex. Nat Cell Biol Dec. 2009; 11, 1427-1432.
Hase, K., Ohshima, S., Kawano, K., Hashimoto, N., Matsumoto, K.,
Saito, H. & Ohno, H. Distinct gene expression profiles characterize
cellular phenotypes of follicle-associated epithelium and M cells. DNA
Res 2005; 12, 127-137.
Ishikawa, F., Yasukawa, M., Lyons, B., Yoshida, S., Miyamoto, T.,
Yoshimoto, G., Watanabe, T., Akashi, K., Shultz, L. D. & Harada, M.
Development of functional human blood and immune systems in NOD/
SCID/IL2 receptor {gamma} chain(null) mice. Blood Sep. 2005; 106,1565-1573.
Ishikawa, F., Yoshida, S., Saito, Y., Hijikata, A., Kitamura, H., Tanaka,
S., Nakamura, R., Tanaka, T., Tomiyama, H., Saito, N., Fukata, M., Miyamoto, T., Lyons, B., Ohshima, K., Uchida, N., Taniguchi, S., Ohara,O., Akashi, K., Harada, M. & Shultz, L. D. Chemotherapy-resistanthuman AML stem cells home to and engraft within the bone-marrowendosteal region. Nat Biotechnol Nov. 2007; 25, 1315-1321.
Saito, Y., Kitamura, H., Hijikata, A., Tomizawa-Murasawa, M., Tanaka,S., Takagi, S., Uchida, N., Suzuki, N., Sone, A., Najima, Y., Ozawa, H.,
Wake, A., Taniguchi, S., Shultz, L. D., Ohara, O. & Ishikawa, F. Identification of therapeutic targets for quiescent, chemotherapy-resistanthuman leukemia stem cells. Sci Transl Med Feb. 2010; 2, 17ra19.
Saito, Y., Uchida, N., Tanaka, S., Suzuki, N., Tomizawa-Murasawa,
M., Sone, A., Najima, Y., Takagi, S., Aoki, Y., Wake, A., Taniguchi, S.,
Shultz, L. D. & Ishikawa, F. Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML. Nat Biotechnol
Mar. 2010; 28, 275-280.

13

Toshio Hirano awarded the
Crafoord Prize 2009C

rafoord Prize is a science prize intended to promote international basic research in the disciplines of astronomy and mathematics, geosciences,biosciences, and polyarthritis. The prize was established in 1980 by Anna-Greta and Holger Crafoord
and is administered by the Royal Swedish Academy of
Sciences. The prize is awarded to one discipline eachyear, but a prize for polyarthritis research is only
awarded when a special advance is made. The Crafoord Prize 2009 was awarded to Charles A. Dinarellofor identification of interleukin-1 (IL-1), and Tadamitsu
Kishimoto and Toshio Hirano for the identification ofinterleukin-6 (IL-6) (Photo 1). Both these cytokines arefound in high concentrations in the joints of people
with polyarthritis. Drugs that mitigate the effect of IL-1
are already in use, and a drug against IL-6 has been
approved in Japan and all over the world including
countries in Europe and the US.

Photo 1: The Crafoord Prize 2009 awardees
The Crafoord Prize 2009 was awarded to
Tadamitsu Kishimoto (left), Toshio Hirano(middle) and Charles A. Dinarello (right).

On May 11, Hirano received the award from thehands of His Majesty the King of Sweden (Photo 2).
“We do not know what the view from a mountain top
will be. Life creates many challenges and it is not until
we have reached one summit that we can see whatlies before us. Only the one who stands on the top of
the mountain can know what this view is, how extensive it is, how beautiful it is and what it will lead to.”Hirano explained in his Crafoord Prize lecture.
When Hirano worked at Osaka Prefectural

14

Photo 2 : Hirano received the award from the handsof His Majesty the King of Sweden.

Habikino Hospital in 1978, he saw many patients withtuberculous pleurisy. He found that pleural effusion
cells from these patients produce an active factor thatinduces immunoglobulin production by B cells. He
decided to isolate this factor. The effort continued
although he moved to Kumamoto University Medical
School in 1980 and to Kishimoto’s laboratory in Osaka
University in 1984. After 8 years, on the Sunday
morning of May 25th, 1986, he finally cloned the activation factor. To his surprise, the factor activated not
only B cells, but also a variety of cells and tissues. The
factor, named IL-6, was a multifunctional cytokine thatplays roles in immune and inflammatory responses, aswell as in hematopoiesis and the endocrine and nervous systems (Fig. 1). Then, the receptor for IL-6 was
isolated. It is composed of two subunits, the IL-6
binding subunit and a signal transducer, gp130.
Gp130 was not only part of the IL-6 receptor, but also
a signal transducer for other cytokines such as IL-11,
OSM, LIF, CT-1, CNTF, and IL-27. “The view I saw
was far beyond my expectation,” said Hirano.
That was not the end of the story. They foundthat a benign cardiac tumor, myxoma, produced IL-6
in large quantities. Patients with myxoma have symptoms resembling those of rheumatic diseases: fever
and joint pain, for example, but as soon as the tumor
is surgically removed the symptoms vanish. With this
discovery, they began to suspect that IL-6 is involvedin autoimmune diseases, and in 1988, they also foundoverproduction of IL-6 in the joints of patients with

giving rise to further enhanced production of IL-6. The“IL-6 amplifier” is required for autoimmune arthritis in
F759 mice as well as in experimental autoimmune
encephalomyelitis (EAE) where antigen-specific T cells
are involved. They hypothesize that any event, including autoantigen-specific T cells, virus infection, injury
and physical stimulation capable of activating the“IL-6 amplifier” through either STAT3 activation or
NF-κB activation or both plays a critical role in causing
autoimmune diseases. ( Fig.2).

Figure 1: Multifunction of IL-6

rheumatoid arthritis (RA). Patients with RA show avariety of symptoms: polyclonal plasmacytosis accompanied by production of rheumatoid factor, increase of
acute phase proteins, enhanced bone resorption
activity, and increase of platelets, none of which had
any apparent relationship to each other. However,when one considered the multiple functions of IL-6,
this puzzle was solved, Hirano thought.
In the early 1990s, he proposed a new hypothesis
about the possible mechanisms that operate inchronic inflammatory proliferative diseases (CIPD) like
RA ; The interaction between the immune system andnon-immune tissues plays a role in autoimmune diseases. Hirano and his colleague have extensively
revised this original hypothesis based on extensive
experiments using F759 knockin mice, which expressa mutated gp130 showing enhanced IL-6-induced
STAT3 activation and spontaneously develop RA-likearthritis (F759 arthritis). They demonstrated that theinteraction between non-immune tissues and theimmune system plays a critical role in the pathogenesisof autoimmune F759 arthritis and that this interaction
is mediated at least in part by an “IL-6 amplifier”. IL-17
and IL-6 synergistically induce IL-6 gene expression
through activation of NF-κB and STAT3 in Type I collagen positive non-immune tissue. Enhanced IL-6
production results in enhanced TH17 development,

Figure 2: The model of IL-6 amplifier“Now, I am standing on top of the mountain and I
can finally enjoy the view, the functions of IL-6 that I
could not imagine 30 years ago. For me, IL-6 is a giftfrom patients with tuberculosis. Anti-IL-6 receptor
antibody treatment, which inhibits IL-6 function, hasbeen found to be beneficial for many patients. Thus
our basic studies contributed toward an understanding of the immunological mechanisms of RA andpaved the way to develop a new drug beneficial forthe patients and hopefully other autoimmune
diseases and inflammatory diseases,” Hirano said in
his speech.
RIKEN’s staff celebrated his receiving the award
on Dec. 16, 2009. Sixty people gathered for thecelebration party.

15

Other Prize Winners 2009
Fumihiko Ishikawa, Leader of the Research Unit for Human Disease Model,
received the Minister of Education, Culture, Sports, Science and Technology(MEXT) Prize for Young Investigators, 2009 (photo 1). This prize is awarded toyoung scientists (under 40 years of age) in recognition of creative and original
research and outstanding ability to develop scientific research projects. Ishikawa
was awarded for his research on hematopoietic stem cells and leukemic stem cells
utilizing the newly developed humanized mouse model.

Photo 1: Fumihiko Ishikawa

Tsuneyasu Kaisho (photo 2), Leader of the Laboratory for Host Defense,
received the 12th Japanese Society for Immunology Award during the society’s
annual meeting in December. Kaisho was awarded for his research on molecular mechanisms for the functional regulation of dendritic cells (DC). He discovered a MyD88 independent TLR signaling pathway that causes DC maturation,and found functional abnormalities of dendritic cells that shifted the Th1/Th2
balance towards Th2 in MyD88 KO mice. He later discovered the importanceof IKKα in innate immune responses. He found that inactivation of IKKα inhibits
IFN-α gene induction by nucleic acid sensors, TLR7/9, in plasmacytoid DC,
and he further proved that IKKα actually cooperates with IRF-7 to control IFN-α
gene expression. This award is given to a researcher, age 50 or younger, whose
contribution to the advances in immunology has been original and extraordinary and further research achievements are expected in the future.

Photo 2: Tsuneyasu Kaisho

Hisaaki Shinohara (photo 3), Researcher in the Laboratory for Lymphocyte
Differentiation, received the Research Encouragement Award of the JapaneseSociety for Immunology during its 39 th annual meeting. Shinohara was awarded
for his research on B cell signaling, especially the discovery of TAK1 involvement in
B cell antigen receptor mediated NF-κB activation. His current work on mathematical modeling of NF-κB activation by the B cell antigen receptor is expected todevelop a new field. This award is given to young investigators, age 40 or younger,
who have conducted distinguished immunological studies.
Photo 3: Hisaaki Shinohara

Photo 4: Masahiro Kitano

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Masahiro Kitano (photo 4), Special Postdoctoral Researcher in the Research Unit for
Immunodynamics, received the GE & Science Prize for Young Life Scientists. He wasawarded for his essay “Imaging of Rab5 Activity Identifies Essential Regulators for Phagosome Maturation”. His strong interests in imaging led him to complete a Ph.D. at
Osaka University, where he developed a biosensor to identify regulators of the phagosome maturation process, and he then joined Takaharu Okada’s lab in RCAI to study
dynamics of immune cells in vivo.
The GE & Science Prize for Young Life Scientists was established in 1995 to recognize outstanding Ph.D.s from around the world and reward their research in the field of
molecular biology. Entrants write a 1000-word synopsis of their thesis and submit it to
the journal Science for judging by an executive panel. Prizes are awarded each year to
finalists in four geographic regions, North America, Europe, Japan and other countries.
Each winner receives US$ 5,000 and a trip to Stockholm, Sweden to accept their prize
and meet with the current Nobel laureates.

Toshio Hirano ( photo 5), Leader of the Laboratory for Cytokine Signaling, received
the Suita City Mayor Award on June 15 from the mayor, Yoshio Sakaguchi. He wasawarded for the discovery of Interleukin-6 and the elucidation of IL-6 function in rheumatoid arthritis and other inflammatory diseases.

Photo 5: Toshio Hirano

Koji Hase ( photo 6 ) and Yasuaki Murahashi ( photo 7 )received RIKEN Research and Technology Incentive Awards
2009,. Koji Hase, Researcher in the Laboratory for Epithelial Immunobiology, received the award for his study on the biological significance of intestinal M cells. Yasuaki Murahashi, Technical Scientist
Photo 6: Koji Hase
Photo 7: Yasuaki Murahashi
of RCAI, received the award for construction of ‘PID Japan’, an
integrative database of primary immunodeficiency diseases. RIKEN Research and Technology Incentive Awards were
newly established in FY2009 and are given to young researchers and technicians under age 40 who have contributed
to furthering RIKEN’s ideals by achieving exemplary results in their research or research support activities.

Shinji Fukuda (photo 8), Special Postdoctoral Researcher in the Laboratory for
Epithelial Immunobiology, received The Best Original Paper Award from Nestle Japanfor his PLoS ONE paper “Evaluation and characterization of bacterial metabolic
dynamics with a novel profiling technique, real-time metabolotyping”.

Photo 8: Shinji Fukuda

Shunsuke Kimura (photo 9), Researcher in the Laboratory for Epithelial Immunobiology, was given the CSF Awardof the Year from the Japan Society for Cell Biology for his
paper “Dynein-dependent Movement of Autophagosomes
Mediates Efficient Encounters with Lysosomes” published inCell Structure and Function (CSF ). This annual award is given
to recognize an excellent original paper published in CSF.
Photo 9: Shunsuke Kimura

Gaku Nakato ( photo 10 ), Shinji Fukuda ( photo 8 ) and Koji Hase ( photo 6 ), Laboratory for Epithelial Immunobiology, received The 14th International Congress of Mucosal
Immunology ICMI 2009 travel award.
Photo 10: Gaku Nakato

Masashi Ebisawa ( photo 11), Junior Research Associate in the Laboratory for Epithelial
Immunobiology, received the Encouragement Award from the Hindgut Club Japan for his
presentation “CCR6hi CD11c+ B cells promote M-cell differentiation in Peyer’s Patch”. He also
received the Chancellor’s Encouragement Award from Yokohama City University.
Photo 11: Masashi Ebisawa

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Excellent Paper of the Year 2009T

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

In 2009, 9 excellent papers were selected from 12 candidates for this award.

Koji Hase, Kazuya Kawano and Hiroshi Ohno
Uptake through Glycoprotein 2 of FimH+ bacteria by M cells initiates mucosal immune response
Nature, Vol 462, pp 226-230, 2009

Yoriko Saito and Fumihiko Ishikawa
Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML
Nature Biotechnology, Vol. 28, pp 275-281, 2010

Koji Hase and Hiroshi Ohno
M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex
Nature Cell Biology, Vol. 11, pp 1427-1432, 2009

Mariko Okamoto and Masato Kubo
Mina, an Il4 repressor, controls T helper type 2 bias
Nature Immunology, Vol 10, pp 872-879, 2009

Yoriko Saito and Hiroshi Kitamura
Identification of therapeutic targets for quiescent chemotherapy-resistant human leukemia stem cells
Science Translational Medicine, Vol 2, pp 17ra9, 2010

Keigo Nishida, Aiko Hasegawa and Toshio Hirano
Zinc transporter Znt5 / Slc30a5 is required for the mast cell-mediated delayed-type allergic reaction, but
not the immediate-type reaction
J. Exp. Med., Vol. 206, pp 1351-1364, 2009

Kiyokazu Kakugawa, Takuwa Yasuda, Hiroshi Kawamoto and Hisahiro Yoshida
A novel gene essential for the development of single positive thymocytes
Mol. Cell. Biol, Vol. 29, pp 5128-5135, 2009

Kaori Sato, Kawori Eizumi and Katsuaki Sato
Naturally occurring regulatory dendritic cells regulate murine cutaneous chronic graft-versus-host disease
Blood, Vol. 113, pp 4780-4789, 2009

Hiroshi Watarai, Andrei Rybouchkin, Naomi Hongo and Masaru Taniguchi
Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant
Vα14-Jα18 TCRα gene
Blood, Vol. 115, pp 230-237, 201018

Excellent Poster Award 2009On August 31, RCAI held two poster presentation sessions during its retreat meeting in Chiba. Among ninety posters
presented, nine posters were selected for an “Excellent Poster Award 2009” by vote of laboratory heads.

Awardees of Excellent Poster Award 2009 :Kiyokazu Kakugawa

Kenichi Asano

Hiroshi Watarai( Lab. for Lymphocyte Development )( Lab. for Innate Cellular Immunity )( Lab. for Immune Regulation )Toshiyuki Fukuda

Kohei Kometani

Michio Tomura( Lab. for Cytokine Signaling )( Lab. for Lymphocyte Differentiation )( Lab. for Autoimmune Regulation )Yoriko Saito (left)( Research Unit for Human Disease Model )Masahiro Kitano(Research Unit for Immunodynamics )Mariko Murasawa (right)( Research Unit for Human Disease Model )19

RIKEN Special Postdoctoral Researcher (SPDR) and
Junior Research Associate (JRA)RIKEN Special Postdoctoral Researcher(SPDR) Program

R
01

02

04

IKEN’s program for Special Postdoctoral Researchers wasestablished to provide young and creative scientists theopportunity to be involved in autonomous and independent
research in line with RIKEN objectives and research fields. The
positions are competitive, but if selected, researchers receive
salaries and research budgets (1 million yen) from RIKEN, andthey are able to conduct their research at one of its laboratories.
This year, nine postdoctoral fellows conducted their research at
RCAI through the SPDR program.

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05

06

07

08
10

09
13

Kenichi Asano (Lab. for Innate Cellular Immunity) (01)Kohei Kometani (Lab. for Lymphocyte Differentiation) (02)Yoshitaka Shirasaki (Lab. for Immunogenomics) (03)Nayuta Yakushiji (Lab. for Developmental Genetics) (04)Yuki Horisawa-Takada (Lab. for Developmental Genetics) (05)Mizuho Kajikawa (Lab. for Infectious Immunity) (06)Masahiro Kitano (Research Unit for Immunodynamics) (07)Takashi Kanaya (Lab. for Epithelial Immunobiology) (08)Shinji Fukuda (Lab. for Epithelial Immunobiology) (09)14

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RIKEN’s Junior Research Associate(JRA) Program

T

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12

17

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20

21

22

23

24

25

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

Gaku Nakato ( Lab. for Epithelial Immunobiology) (10)Toshi Yuyama ( Lab. for Epithelial Immunobiology) (11)Masashi Ebisawa ( Lab. for Epithelial Immunobiology) (12)Shintaro Hojo (Lab. for Cytokine Signaling) (13)Beum-Ho Byn (Lab. for Cytokine Signaling) (14)
Yasutaka Motomura ( Lab. for Signal Network) (15)Machiko Sugiyama ( Lab. for Immunogenetics) (16)
Hideaki Takagi ( Lab. for Dendritic Cell Immunobiology) (17)Tomohiro Fukaya ( Lab. for Dendritic Cell Immunobiology) (18)
Izumi Sasaki ( Lab. for Host Defense) (19)
Chihiro Yamazaki ( Lab. for Host Defense) (20)
Saya Moriyama ( Lab. for Lymphocyte Differentiation) (21)Takanori Sawaguchi ( Lab. for Developmental Genetics) (22)Kazunori Kadokura ( Lab. for Epithelial Immunobiology) (23)Daisuke Takahashi ( Lab. for Epithelial Immunobiology) (24)Naomi Hongo ( Lab. for Immune Regulation) (25)2009Laboratory Activities

Group Director

Haruhiko Koseki
Research Scientists : Yixin Dong, Mitsuhiro Endo,Yûichi Fujimura, Kyôichi Isono,
Kyoko Masuda, Jun Shinga,
Daisuke Yamada, Ruken YamanDeveci, Yuki Takada (SPDR),
Nayuta Yakushiji (SPDR)
Technical Staff :Masako Fujita, Hiroshi Kajita,
Iyo Kataoka, Kayoko Katsuyama,
Fuyuko Kezuka, Genta Kitahara,
Yôko Koseki, Tamie Morisawa,
Naoko Ohnaga, Naomi Otsuka,
Rie Suzuki, Momoko Okoshi,
Chieko Tezuka

Visiting Scientists :Mutô Masahiro, Jafar SharifStudent Trainee :Takanori Sawaguchi (JRA),Asami Tsuboi

Administrative Staff : Ryôko Moriizumi

Laboratory for

Developmental
Genetics
T

he Developmental Genetics Research Group fulfills adual role within RCAI. A large portion of the manpower
and financial resources of the group is devoted to the maintenance of a high-standard mouse facility at RCAI. Throughthe Animal Core Facility, the group is also responsible forthe generation of knock-out and transgenic animals for thevarious research laboratories at the center. At the sametime, the laboratory is pursuing a research program to elucidate the molecular mechanisms underlying organ development and stem cell functions、with particular emphasis
on epigenetic regulation mediated by Polycomb group(PcG) genes in development and stem cell functions.
PRC1 suppresses ES cell differentiation programs
through PRC2-dependent and PRC2-independent
mechanisms (Endoh et al., submitted )To clarify the molecular mechanisms linking two distinct
Polycomb complexes, PRC1 and PRC2, and their biological outcomes, we examined the genome-wide distributionsof the PRC1 components Ring1B and Cbx2, and the PRC1catalyzed histone modification, mono-ubiquitinated H2A(H2Aub1), in embryonic stem cells (ESCs). We also integrated data for PRC2 components and the PRC2-catalyzed
modification, trimethylated H3K27 (H3K27me3). H2Aub1
localizes to silent developmental gene promoters, nearly allof which are also enriched for PRC1 and PRC2. H2Aub1
deposition and PRC1 localization were shown to depend on
two parallel pathways. The first pathway is PRC2-dependent and involves recognition of H3K27me3 by Cbx2 component of PRC1. The second is independent of PRC2 andinstead depends on the catalytic activity of Ring1 at key
PRC1 target genes. We propose that these complementarymechanisms combine to repress critical differentiation
genes in ESCs. Such combined mechanisms may be
instrumental in keeping Polycomb-mediated repression at
permissive levels to undergo developmental programs.
Subnuclear architecture involved in mediation oftranscriptional silencing ( Isono et al., in preparation;
Fujimura et al., in preparation )Recent publications
1. Harada M, Murakami H, Okawa A,
Okimoto N, Hiraoka S, Nakahara T,
Akasaka R, Shiraishi Y, Futatsugi
N, Mizutani-Koseki Y, Kuroiwa
A, Shirouzu M, Yokoyama S, Taiji
M, Iseki S, Ornitz DM, Koseki H..
FGF9 monomer-dimer equilibrium
regulates extracellular matrix
affinity and tissue diffusion. Nat
Genet. 41, 289-298 (2009)

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

Figure:Association of Hoxb1, Hoxb7-9 and Ink4a-arf genes with Polycomb bodies in embryonic
fibroblasts (MEFs). Immuno-FISH analysis was performed by using MEFs from Mel18-GFP
knock-in mice. Polycomb bodies represented by distribution of Mel18-GFP fusion protein (Green)
were shown to colocalize with Hoxb1, Hoxb7-9 or Ink4a-arf genes (Red) in the lower panels.

Compartmentalization in eukaryotic nuclei is important for
separation and coordination of various functions that proceed simultaneously within a single nucleus. We therefore
took another approach based on imaging techniques, inparallel with the above genomic approach, to understandthe underlying mechanisms for Polycomb repression. PRC1
components have been shown to exhibit focal accumulations that are designated “Polycomb bodies” in various
tumor cell lines, although the functions of these structures in
Polycomb repression are totally unknown. We have for the
first time identified Polycomb bodies in primary cells andclarified their molecular nature by combining genetic andimaging approaches. We found the role of PRC1 is to condense Hoxb clusters into a single Polycomb body and mediate the repression Fig.). We further identified the role of SAM
domains of polyhometic homologues to mediate Polycomb
body formation by linking multiple PRC1 complexes via
homophilic interactions. Polycomb body formation was further suggested to be a process to enhance recruitment of
PRC1 to polycomb target genes. This study therefore identified an additional layer of Polycomb repression and potentially provides new insight into epigenetic regulation ofcellular differentiation.
In the process of affinity purification of PRC1, we haverepeatedly co-purified α- and β-tubulins, historically known
as the principal components of microtubules. By immuno-

3. Sharif J., Muto M., Takebayashi S.,
Suetake I., Iwamatsu A., Endo T.A.,
Shinga J., Mizutani-Koseki Y., Toyoda
T., Okamura K., Tajima S., Mitsuya K.,
Okano M., Koseki H. The SRA protein
Np95 mediates epigenetic inheritanceby recruiting Dnmt1 to methylatedDNA. Nature 450, 908-912 (2007)

4. Takada Y, Isono K, Shinga J, Turner
JM, Kitamura H, Ohara O, Watanabe
G, Singh PB, Kamijo T, Jenuwein T,
Burgoyne PS, Koseki H. Mammalian
Polycomb Scmh1 mediates exclusionof Polycomb complexes from the XY
body in the pachytene spermatocytes.
Development 134, 579-590 (2007)

fluorescence analysis of ESCs after extraction by detergentsand high salt concentrations, we identified a fraction of
tubulins that remained persistently associated with thenuclear lamina and exhibited a filamentous structure. ChIPchip analysis for tubulins and Lamin-B, a major componentof nuclear lamina, revealed significant co-occupancy of theirtarget genes in ESCs and thus confirmed the interaction of
tubulins with the nuclear lamina. Using Vinblastin to depolymerize tubulins, we found that thelamina-associated tubulins are highly polymerized. Immuno-FISH analysis for several
genes that were bound by both tubulins and Lamin-B,
revealed that they were relocated from the nuclear peripheryto the nuclear interior. This is accompanied by dissociationof these genes from Lamin-B, as shown by ChIP assay.
These observations indicated that the role of tubulins is tolink their targets to the nuclear lamina. Interestingly, tubulins
extensively colocalized with HDACs at the nuclear lamina
and this accumulation of HDACs depends on tubulin polymerization. Our data suggest that tubulins act as a scaffold forthe convergence of chromatin-HDAC-nuclear lamina, andare necessary for maintaining the hypoacetylated status ofgenes that associate with the nuclear lamina.

5. Hiraoka S., Furuichi T., Nishimura G.,
Shibata S., Yanagishita M., Rimoin
D.L., Superti-Furga A., Nikkels P.G.,
Ogawa M., Katsuyama K., Isono
K., Toyoda H., Kinoshita-Toyoda
A., Ishida N., Sanai Y., Cohn
D.H., *Koseki H. *Ikegawa S.
(* Corresponding authors)
Nucleotide-sugar transporter

SLC35D1 is critical to chondroitin
sulfate synthesis in cartilage
and skeletal development inmouse and human. Nat Med.
13, 1363-1367 (2007)

23

Team Leader

Laboratory for

Research Scientists : Tomokatsu Ikawa
Kiyokazu Kakugawa
Kyoko Masuda

Lymphocyte
Development

Hiroshi Kawamoto

Research Associate : Rumi SatohTechnical Staff :Asako Shibano
Yuri Suzuki
Chiho Matsuura

Student Trainee :Johanne Angelina Jacobus Huijskens

Administrative Staff : Yuko Ochi
Visiting Scientists :Yoshimoto KatsuraDuing hematopoiesis, pluripotent hematopoietic stem
cells (HSC) are sequentially restricted to give rise to avariety of lineage- committed progenitors. The major aim of
the Laboratory for Lymphocyte Development is to elucidate
the molecular mechanisms that regulate cell fate decisions
in the process of lineage restriction from HSC to unipotent
progenitors. We have previously established a clonal assay
system that makes it possible to examine the developmental potential of individual progenitor cells toward T, B andmyeloid cell lineages. This work has led to a fundamental
redefinition of lymphoid progenitors and the ontogeny andphylogeny of T- and B-cell development.

Lineage commitment of hematopoietic stem cells
toward unipotent T cell progenitors
T cells are generated from multipotent hematopoietic stem
cells through a series of differentiation steps. Recent studies including ours (Lu et al, J.I., 2002) have indicated thatthe first step in this pathway is the generation of progenitorsthat have lost erythroid/megakaryocyte potential but retain
the capacity to generate other hemopoietic cells including
myeloid, T and B cells. We have recently identified the nextstage beyond these myelo-lymphoid progenitors, which
consists of T cell progenitors that have lost B cell potential
but are still able to generate myeloid cells, DC and NK cells(Wada et al, Nature, 2008). Therefore, the most critical stepfor construction of the T cell lineage is now thought to be at
the point where myeloid potential is terminated.
We have previously shown that the DN2 stage can be
subdivided into two stages based on GFP expression controlled by the proximal lck (plck) promoter. Cells in the earlier, GFP - stage retain non-T lineage potential, including thatfor DC and NK cells, whereas the latter stage GFP+ cells are
determined to the T cell lineage (Masuda et al, J. I. 2007).
We have further shown that DN2 cells still retain myeloid
potential (Wada et al, Nature, 2008). Here we designatethese two stages DN2mt (myeloid-T ) and DN2t ( T-lineage
determined), and term the step between these stages the
DN2-determination step. Such a T cell lineage determination step is thought to be the first critical checkpoint in T cell
development.

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

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

Figure 1: A novel T cell developmental
checkpoint in the midst of the DN2stage is regulated by Bcl11b. (A) Modelof normal T cell lineage determination.
Non-T lineage potential, including thatfor myeloid, DC, and NK lineages, ofT cell progenitors is terminated during
the DN2 stage, subdividing this stage
into DN2mt and DN2t stages. Bcl11b
is required for the DN2mt cells to passthrough this checkpoint. (B) Sustained
IL-7 signaling or Bcl11b deficiency
induces developmental arrest at the
DN2mt stage. The arrested DN2mt cells
acquire self-renewal ability and maintainthe potential to generate macrophages,
NK cells and dendritic cells.

Figure 2: Identification of novel gene essential for the development of
single positive thymocytes. (A) Establishment of ENUinduced mutant line SPOTR that shows reduction in T cellnumbers. CD4 versus CD8 profiles of thymocytes from an 8
week-old wild type and SPOTR mouse. (B) Genetic mappingto narrow down genes responsible for the SPOTR phenotype.
A total of 254 mice were examined by genetic mapping, andthe responsible loci were mapped to the 0.39 centiMorgan(cM) region including D10SNP2 (shown as a red arrow on the
expanded chromosome region). Calculating that 1cM ≈2Mbp,
a 0.78Mbp region was predicted to contain the candidateloci (yellow on chromosome 10). Three genes in this region
in the order Ptprk, E430004N04Rik and 2310057J18Rik
are indicated, with the direction of transcription indicatedby the pointed end of the rectangular bars. (C) A missense
mutation was found in exon 4 of the E430004N04Rik gene,
A1799C, leading to the amino acid substitution T512P. The
coding regions of the E430004N04Rik gene are indicated inlight green and the UTRs are indicated in black. (D) Themis
deficient mice showed a phenotype similar to that of SPOTR
mice. CD4 versus CD8 profiles of thymocytes from a 4
week old wild type and themis deficient mouse are shown.

We recently found that when murine fetal liver progenitors were cultured on immobilized Notch ligand DLL4 in thepresence of a cytokine cocktail including IL-7, the cells werearrested at the DN2mt stage. The arrested DN2 cells did not
die but retained the capacity for self-renewal as well as thepotential to generate myeloid, dendritic, and NK cells. These
self-renewing DN2 cells were induced to T cell lineage
determination by simply reducing the concentration of IL-7,
eventually giving rise to CD4+CD8+ T cells under feeder-free
culture conditions. A similar arrest and self-renewal of progenitors were observed in thymocytes of mice deficient in
the transcription factor Bcl11b. Our study thus identifies theearliest checkpoint of T cell development and demonstrates
that it is Bcl11b-dependent (Ikawa et al, submitted ).
A novel gene essential for the development of single
positive thymocytes
A critical step during intrathymic T cell development is thetransition of CD4+CD8+ double positive (DP) cells to the
MHC I-restricted CD4-CD8+ and MHC II-restricted CD4+CD8single positive (SP) cell stage. In collaboration with the
Immunogenetics Laboratory (Yoshida TL), we identified anovel gene that is essential for this process (Kakugawa et al,

3. Kawamoto, H. and Katsura, Y. A
new paradigm for hematopoietic
cell lineages: revision of theclassical concept of the myeloidlymphoid dichotomy. Trends in
Immunology. 30: 193-200, (2009).

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

MCB, 2009). Through T cell phenotype-based screening of
N-ethyl-N-nitrosourea (ENU)-induced mutant mice, weestablished a mouse line in which numbers of CD4 and CD8
SP thymocytes as well as peripheral CD4 and CD8 T cells
were dramatically reduced. We named this mouse line
SPOTR (Single Positive Thymocyte Reduction). Using linkage analysis and DNA sequencing, we identified a missense
point mutation in a gene, E430004N04Rik, which does notbelong to any known gene family. This orphan gene is specifically expressed in DP and SP thymocytes and peripheral
T cells, whereas in mutant thymocytes, the levels of protein
encoded by this gene were drastically reduced. We generated E430004N04Rik-deficient mice and their phenotype
was virtually identical to that of the ENU mutant mice,
thereby confirming that this gene is essential for the development of SP thymocytes. While we were in the process ofmanuscript submission, we noted that several other groups
have also found this gene and named it “themis”, therefore
we decided to use this name. Since themis is a totally
unknown gene, whose functional domains are so far
unknown, further studies on its role will reveal novel mechanisms in T cell development.

5. Masuda K, Kakugawa K, Nakayama T,
Minato M, Katsura Y, Kawamoto H.
T cell lineage determination
preceedes the initiation of TCRbeta
gene rearrangement. J. Immunol.
179: 3699-3706, (2007)25

Unit Leader

Andrei Rybouchkin
Research scientist : Aleksey Menzorov
Technical Staff :Raul Sakoda

Research Unit for

Lymphocyte
Cloningi

PS cells are considered as one of the most advanced andpromising tools for personalized and regenerative medicine. The potential of these cells will not be realized, however, until development of safe and efficient methods fortheir application to the treatment of diseases. It is generally
accepted that before transplantation into patients, iPS cells
have to be differentiated into the terminally differentiated,functional cells of a particular type, or, alternatively, into
adult stem cells such as mesenchymal stem cells (MSC) or
hematopoietic stem cells (HSC), which, after transplantation would differentiate into the desired cells after homing to
the proper location. Taking this second approach, we areattempting to develop methods to differentiate human andmouse iPS cells into MSC, as MSC may be instrumental information of a supportive environment for survival of transplanted HSC and their function of regeneration of theimmune system. MSC, and their derivatives the stromalcells, are also thought to modulate inflammatory responses
and contribute to regeneration of many tissues by production of multiple growth stimulatory cytokines.
Developing a protocol for differentiation of human
iPS into MSCs or mesenchymal precursors.
In our studies we used human iPS cell lines derived in the
RIKEN BioResource Centre and in the first stage applied
several recently published protocols that reportedly wereeffective in differentiation of human ES cells into MSCs or
their precursors. Two out of four tested protocols resulted in
the robust production of the cell lines with features similar,
but not identical to, donor bone marrow derived MSC (BMMSC). The cell lines have a very similar surface antigen
phenotype and readily differentiated into osteoblasts inosteogenic medium (Alizarin Red staining, Fig.1A), but wereunable to differentiate into adipocytes in the adipogenic
medium (Oil Red O staining, Fig.1B). We concluded that thelines we received are lines of osteoprogenitor (OP) cells. As
reported by others, OP lines can organize hematopoietic
microenvironments (Sacchetti et al., 2007 Cell) and one of
our ideas was to try to test our iPS derived OP lines for thisfunction. In the meantime, we continued our search for a
protocol for differentiation of our human iPS cell lines into

Recent publications
1. A Rybouchkin, Y Kato, Y Tsunoda.
Role of histone acetylation in
reprogramming of somatic nucleus
following nuclear transfer. Biol.
Reprod. 2006, 74: 1083-1089.

2. Watarai H, Rybouchkin A, Hongo
N, Nagata Y, Sakata S, Sekine E,
Dashtsoodol N, Tashiro T, Fujii S,
Shimizu K, Mori K, Masuda K,
Kawamoto H, Koseki H, Taniguchi M.
Generation of functional NKT cells
in vitro from embryonic stem cells
bearing rearranged invariant
Valpha14-Jalpha18 TCRalpha gene.

Osteogenic

Adipogenic
Figure 1: Differentiation of hiPS
derived osteoprogenitors
into (A) osteoblasts, but
not into (B) adipocytes.
(C) Differentiation ofmesenchymal precursors
derived from the same
iPS line by other protocol
into adipocytes.

Figure 2: Percentage of CD140a+ mesenchymal
precursors and total number of thesecells derived by the same differentiation
protocol from R1(129) and B6 ES
cell lines on differentiation day 9.

authentic MSC. Our last month results have finally resulted
in the lines capable of differentiating into adipocytes (Fig.
1C). We hope to continue to fine-tune this protocol evenafter closure of this research unit on March 31, 2010.
Factors affecting differentiation of mouse ES andiPS cells into MSC
We recently derived several mouse iPS cell lines from theC57Bl /6J (B6) mouse embryonic fibroblasts. This strain waschosen because it is one of the most frequently applied
strains for genetic and particularly immunological studies,and a mouse model of iPS to MSC differentiation would
allow us to test properties of iPS derived cells in preclinicalcell transplantation trials. However, there was no reported
protocol for differentiation of B6 ES cells into MSC. Moreover, several published studies have reported significant
difficulties in culture of bone marrow derived adult MSC
recovered from this strain of mice.
We first tested a published ES to MSC precursors (MP)differentiation protocol on the 129 strain-derived ES cellline R1. We obtained robust results in most of the experiments. Cells were positive for MP surface markers (CD140a),
readily expandable, and capable of differentiating into adipocytes and osteoblasts in the appropriate induction media.
The same protocol worked very poorly for the B6 strain ES
cell line. The proportion of CD140a positive cells was similar
to the R1 cell cultures, but the total number of cells andtherefore the number of positive cells was much lower (Fig2). More importantly sorted CD140a positive cells had very

poor expansion potential and less efficient differentiation
potential. Furthermore, results of differentiation experiments
varied greatly from experiment to experiment for no obvious
reasons. We have tried several approaches to improve thisdifferentiation protocol, including varying the concentrationsof serum (10% vs 20%), the differentiation inducing agent(0, 100 and 500nM of retinoic acid), and cells seeded at thestart of differentiation. We also studied the effects of thesurvival stimulating agent dbcAMP the oxygen concentration (5% vs 20%) on cell differentiation and/or expansionof sorted cells. The sorting date of CD140a positive cells
from heterogeneous starting population (day 9 versus day
11 and 13) have also been analyzed. Finally, we have also
tried to identify the reasons for the alarming rate of experiment to experiment variation by testing effects of B6 ES cell
culture media (DMEM based versus IMDM based), feeder
cell density, as well as ES cell confluency level and passage
numbers.
We have come up with a semi-optimized protocol
allowing us to obtain high numbers of B6 ES derived MP
cells in most experiments. Expansion of the resulting cells
has remained, however, a significant problem. Interestingly,
after applying the new protocol to our B6 iPS cell lines, wefound out that MP from these lines are more readily expandable than their B6 ES-derived counterparts. It would be ofinterest to investigate whether better expandability of iPS
derived MP is related to a higher level of leaky expression ofpluripotency inducing transgenes such as c-myc or flk4.

Blood. 2010 Jan 14;115(2):230-7.
Epub 2009 Nov 6.

27

Team Leader

Ichiro Taniuchi
Research Scientists : Hirokazu Tanaka
Taku Naito
Wooseok Seo
Mari Tenno
Technical Staff :Student Trainee :Sawako Muroi
Chizuko Miyamoto
Risa Chihara
Kazuko Kosuge
Sebastian Nieke (IPA)

Administrative Staff : Aiko Iyama

Laboratory forTranscriptional
RegulationO

ne of major questions in developmental biology is
how the fate of progenitor cells differentiating into
opposing lineages is determined. Even as we learn more
about cell fate determination, other questions arise, namely
how genetic programming after lineage specification functions to establish cell identity and then how cell identity,
once established, is maintained in differentiated cells.
Research in my laboratory is directed toward understanding (a) how progenitor cells sense external or intrinsic
stimuli and turn on a genetic program for regulating lineage
choice and (b) how a specific gene expression pattern isestablished during a commitment process as the cellbecomes fully differentiated. We are addressing thesequestions by studying transcription factor networks regulating T lymphocyte development. In particular, we havebeen studying the transcriptional regulation of lineage
choice by CD4+CD8+ double-positive (DP) thymocytes differentiating into either CD4+ helper- or CD8+ cytotoxic-lineage T cells.
Our previous studies revealed that antagonistic interplay between two transcription factors, the Runx transcriptional complexes and ThPOK, plays a central role in thetranscription factor network that governs fate determination by DP thymocytes. While expression of the ThPOK isessential for helper T cell development, in part via antagonizing Runx-mediated gene regulation, repression of ThPOK
expression by Runx complexes via activating the transcriptional silencer in the ThPOK locus is required for cytotoxic
T cell development. We are expanding our findings tounderstand how expression of the ThPOK gene is regulated
and how Runx complexes are involved in immune systemdevelopment.

Mechanism regulating Ztb7b/ThPOK gene
expression
The specificity of TCRs for self MHC molecules correlates
well with the outcome of lineage decision by DP thymocytes. Cells expressing a class I-restricted TCR differentiate
primarily into the CD8+ cytotoxic-lineage, whereas cells
expressing a class II-restricted TCR differentiate into the

Recent publications
1. Setoguchi R., Tachibana M.,
Naoe Y., Muroi S., Akiyama K.,
Tezuka C., Okuda T., Taniuchi I.
Repression of the Transcription
Factor Th-POK by Runx Complexes
in Cytotoxic T Cell Development.
Science 319, 816-819 (2008)

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

TCR signals
reversal of
ThPOK silencer

activation ofenhancers

Runx
distalenhancerA

CbfB+/fl;CreThPOK
silencer

ATG TAA

ThPOK

Ia

CbfBfl/fl;CreIb

B

II III

proximalenhancerC

Figure 1: Cis-regulatory elements regulating
expression of the ThPOK gene. The
expression of ThPOK gene during T cell
development is primarily regulated by the
combined activation and inactivation of thesilencer and the two enhancers. Bindingof the Runx complexes to the silencer isessential, but not sufficient, to activate
the silencer. In order to initiate ThPOK
gene expression upon TCR engagement,
both reversal of the silencer function andactivation of the enhancers are necessary.

10.5

Cbfbfl/fl;Cre
Cbfb+/fl;Cre

Homology

0.0

FoxP3

70%
50%
Foxp3
CNS1 CNS2 CNS3

1kb

Figure 2: Autoimmune disease development following loss of the Runx1/Cbfβ complex in regulatory T cells. (A)Splenomegaly and lymphadenopathy in 8-week-old mice resulting from inactivation of the Cbf β gene in FoxP3+
cells. (B) Reduced expression of FoxP3 protein in Tregs after loss of Cbf β protein. (C) Binding of the Runx1/Cbf β
complex to the regulatory regions, depicted as conserved non-coding sequences (CNS), in the FoxP3 locus.

CD4+ helper-lineage. However, it remains unclear how differences in TCR signaling are sensed and integrated intodevelopmental programming in the cell nucleus of postselection thymocytes. Because expression of ThPOK isboth essential and sufficient to induce helper-lineage fate,
regardless of the MHC specificity of the thymocyte TCR,
understanding of the mechanism that regulates helper-lineage specific expression of the ThPOK gene will be pivotal
to address this issue. Having identified a transcriptionalsilencer (termed as the ThPOK silencer) as an essential cisregulatory element for helper-lineage specific expression of
the ThPOK gene, we are attempting to unravel a molecularmechanism regulating activity of the ThPOK silencer. Our
unpublished results obtained by modifying the activity of theThPOK silencer suggest that reversal of its silencer activity
by an as yet uncharacterized mechanism is necessary forexpression of the ThPOK gene in class II-selected cells. In
order to identify molecules that determine specificity of theThPOK silencer function, we are isolating protein complexes
that bind specifically to the ThPOK silencer.
In addition to the reversal of the ThPOK silencer activity,
activation of positive regulatory elements, such as enhancers and the promoter, is also necessary for expression of theThPOK gene. We have identified two enhancers: a distalenhancer located in the 5’ region proximal to the silencer in
the ThPOK locus, and a proximal enhancer downstream ofexon Ib. While the distal enhancer acts mainly to initiate
ThPOK gene expression in post-selection thymocytes, the

3. Maekawa Y., Minato Y., Ishifune C.,
Kurihara T., Kitamura A., Kojima H.,
Yagita H., Satake-Yanagimoto M.,
Saito T., Taniuchi I., Chiba S., Sone S.,
Yasutomo K. Notch 2 integrates
signaling by the transcription
factors RBP-J and CREB1 topromote T cell cytotoxicity. Nat.
Immunol. 9, 1140-47 (2008)

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

proximal enhancer is necessary to amplify and maintain its
expression at the later developmental stage. Removal ofboth of these enhancers from the ThPOK locus abrogates
expression of the ThPOK gene in CD4+ T cells, but not in B
cells, revealing not only their redundant functions but also
their T lymphocyte specific functions.
Further studies on the molecular control of these regulatory elements in the ThPOK gene will advance our understanding of how cellular signaling initiated by external stimuli
are converted into genetic programs in the cell nucleus.
Role of Runx complexes in regulating function of
FoxP3+ regulatory T cells.
FoxP3+ naturally occurring regulatory T (Treg) cells playessential roles in the maintenance of immunological selftolerance and immune homeostasis by actively suppressing
aberrant or excessive immune responses. By using Treg specific Cre transgenic mice, we show that inactivation of theRunx1 or the Cbfβ gene in FoxP3+ cells results in spontaneous development of autoimmune disease with an attenuated expression of FoxP3. Our chromatin immunoprecipitation
assay revealed that the Runx1/Cbfβ complex binds to regulatory regions in the FoxP3 locus. Thus, the Runx1/Cbfβ
complexes are indispensable for in vivo suppressive function of Treg cells in part via regulating expression of the FoxP3
gene.

5. Kitoh A., Ono M., Naoe Y., OhkuraN., Yamaguchi T., Yaguchi H.,
Kitabayashi I., Tsukada T.,
Nomura T., Miyachi Y., Taniuchi I.,
Sakaguchi S. Indispensable roleof the Runx1-Cbfβ transcription
complex for in vivo-suppressive+function of FoxP3 regulatory T
cells. Immunity. 3, 609-20 (2009)29

Group Director

Takashi Saito
Senior Scientist :Tadashi Yokosuka

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

Administrative Staff : Shihoko Kato, Hiroko Yamaguchi
Visiting Scientists :Hu Zeng, Ikuo Ishige,
Noriko M. Tsuji, Sho Yamasaki

RCAI Central Facility FACS Laboratory :Hanae Fujimoto, Yukiko Hachiman
RCAI Central Facility Confocal Laboratory :Yasutaka Wakabayashi (Leica)
RCAI Central Facility mAb Laboratory :Tomomi Aoyama, Mayuko Matsuda

Laboratory for

Cell SignalingT

he long-term goal of the Cell Signaling group is todetermine the molecular mechanisms of activation,
migration and homeostasis of T cells in order to be able tomodulate T cell activation /function in immunological disorders. Therefore, our group is involved in a range of projects
to analyze the basic mechanisms of antigen recognition,
activation, differentiation, and functional regulation of T cells
from the viewpoint of signal transduction. Particularly, thegroup has been using real-time imaging analysis, which hasprovided new insights into the dynamic regulation of theassembly of the TCR signalsome and the immunologicalsynapse, as well as related downstream signaling pathways
upon antigen recognition. These include regulation ofcytoskeleton and co-stimulation signals. The group also
analyzes regulation at later phases of T cell activation forcell migration and establishment of peripheral antigenspecific effector cell functions.
Dynamic regulation of T cell activation andco-stimulation
We have studied the dynamic movement of signaling molecules in the process of the formation of the immunologicalsynapse and T cell activation upon antigen recognition at
the single-cell level. Using a combination of imaging techniques, we could visualize the behavior of signaling molecules during the dynamic process of T cell activation. Thisvisualization has provided a new insight that microclusters(MC) containing TCR, kinases and adaptors are generated
upon stimulation and function as the minimal signaling unit
responsible for the initial and sustained activation of T cells.
Based upon the discovery of TCR-MCs, we had to revise
the current models for the mechanism of T cell activation
including co-stimulation.
T cell activation upon antigen recognition requires bothantigen recognition signals through theTCR and co-stimulation signals through co-stimulatory receptors. The precise
relationship between these two signals is still unclear. We
have analyzed signal regulation through positive and negative co-stimulation receptors. For the major co-stimulation
signals through CD28, we found that CD28 is accumulated

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

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

Figure :Spatially distinct regulation of T cell co-stimulation : CD28- and CTLA-4-mediated positive/negative
costimulation signals through the “signaling cSMAC”. There are two regions within the cSMAC;
CD3hi and CD3lo. Co-stimulatory receptors CD28 and CTLA-4 are accumulated and compete for theligand binding in the CD3lo area (signaling cSMAC). CD28 recruits and associates with PKCθ to thisregion to mediate costimulation signals while CTLA-4 inhibits CD28-PKCθ accumulation.

in a special co-stimulation regulatory region in the c-SMAC(signaling cSMAC), and recruits and associates with PKCθin this region to induce co-stimulation signals. We also foundthat the negative co-stimulation receptor CTLA-4 is also
accumulated and mediates its inhibitory function by competing for binding of the same ligands as CD28. Thus, wefound the regulation mechanism of co-stimulation signals as“signal 2” through a spatially distinct region of the cSMAC
from that of the TCR-signal mediating MCs. (Fig.)
Furthermore, to analyze mutual activation regulation
between innate and acquired immunity, we have analyzed
the function of TLRs expressed on T cells. We found that
TLR-mediated signals function in T cell co-stimulation. In
these analyses, we found that nucleic acids induce costimulation independently of TLRs, and therefore areattempting to identify the nucleic acid-recognizing sensors
on T cells and the regulation of T cell activation throughthese receptors.

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

4. Yokosuka T., Kobayashi W.,
Sakata-Sogawa K., Takamatsu M.,
Hashimoto-Tane A., Dustin M.L.,
Tokunaga M., Saito T. Spatiotemporal
regulation of T cell costimulation by
TCR-CD28 microclusters throughprotein kinase C θ translocation.
Immunity. 29, 589-601 (2008)

Regulation of T cell dynamics through adhesion
molecules
We have identified CRTAM as a gene that is induced early
during T cell activation, and Necl2 as the CRTAM ligand.
CRTAM is specifically expressed on activated CD8 +T cells
and NK cells whereas Necl2 is expressed on epithelial cells
and DCs. CRTAM binds and functions throughheterotypic interaction with Necl2. Analysis of CRTAMdeficient mice revealed a reduction of effector T cell function
in tumor rejection, DTH response, and virus infection. We
found that CRTAM regulates the retention of activatedT cells upon interaction with DCs within the lymph node
where immune responses are induced. This system thus
regulates maturation and translocation of effector T cells.

5. Yamasaki S., Ishikawa E., Sakuma M.,
Ogata K., Saito, T. Mincle is an
ITAM-coupled activating receptor
that senses damaged cells. Nat.
Immunol. 9,1179-1188 (2008)

6. Hara H., Ishihara C., Takeuchi A.,
Imanishi T., Xue L., Morris SW., Inui
M., Takai T., Shibuya A., Saijo S.,
Iwakura Y., Ohno N., Koseki H.,
Yoshida H., Penninger JM., Saito
T. The adaptor protein CARD9 isessential for the activation of myeloid
cells through ITAM-associatedand Toll-like receptors. Nat.
Immunol. 8, 619-629 (2007)31

Unit Leader

Kumiko Sakata-Sogawa
Technical Staff : Yoshiyuki Sasaki

Research Unit forSingle Molecule
ImagingS

ingle molecule approaches enable us to capture transient intermediates and heterogeneous behavior avoiding ensemble averaging. This ability is powerful in elucidating
mechanisms of cellular functions: we can determine whichmolecule interacts with what, when, where, and how it
works in living cells. Thus fluorescence imaging and quantitative analysis of single molecules are valuable methods tostudy the individual behavior of biological systems.
The main goal of the Research Unit for Single Molecule
Imaging is to elucidate immunological responses and signaling processes with the technique of single molecule
imaging and quantification. To this end we installed a microscope system and we have focused on the development ofnovel types of fluorescence microscopy (e.g., HILO) for use
in single cell/single molecule studies. HILO has an advantage for making observation inside cells due to the highsignal to noise ratio of 7.6. In addition, we are developing
analysis software tools for imaging and quantitative
analysis. We are also applying quantitative approaches tosimulation of cell signaling. Our unit has a tight collaboration
with the Research Unit for Molecular Systems Immunology
for development of new microscopy techniques andanalysis software.

Toward multi-color imaging of cell signaling
For quantitative analysis of the interaction of signaling
proteins, it is important to observe multiple molecules
simultaneously. To this end, we have established methods
to obtain isogenetic cell lines expressing two fluorescencetagged proteins at homogenous and low level. Figure 1
shows examples of isogenetic CHO and Jurkat cell lines.
An expression vector was designed to contain two sets ofpromoter and fluorescence-tagged protein sequences intandem. To optimize the expression level of the fluorescence proteins for single molecule imaging, we selectedsuitable promoter sequences, and inserted insulator
sequences if necessary. This method allows us to analyze
interactions between two different molecules. We are
expanding this technique to allow expression of three ormore fluorescence-tagged proteins.

Recent publications
1. Miletic AV., Graham DB. SakataSogawa K., Hiroshima M., Hamann
MJ., Cemerski S., Kloeppel T.,
Billadeau DD., Kanagawa O.,
Tokunaga M. and Swat, W: Vav
Links the T Cell Antigen Receptor
to the Actin Cytoskelton and T Cell
Activation Independently of Intrinsic
Guanine Nucleotide Exchange Activity.
PLoS One. 4 (8), e6599 (2009)

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

Figure:Control of protein expression levels by careful selection of promoter sequences.
(A) CHO cell line expressing Fucci: fluorescent ubiquitination-based cell cycle
indicator(combination of mKO2-Cdt1) and mAG1-Geminin) in the nucleus.
(B) Jurkat cell line expressing CD28-EGFPA206K and CD3ζ-TagRFP-T. The
cells have been activated with immobilized anti-CD3 antibody. Bars: 10µm.

Dynamics of transcription factors in the nucleus
Transcription factors are known to be involved inregulation of target genes participating in specific signaling
pathways. These protein factors bind to specific shortsequences in DNA and associate with other activator proteins to regulate transcription. Although their association
and regulatory function have been studied by biochemical
approaches, many important features of their dynamics still
remain to be elucidated.
Aiming at visualization of the dynamics of transcription
factor proteins, we constructed Sp1-EGFP, a fusion proteinof EGFP and the transcription factor Sp1, and prepared

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

CHO cell expressing low levels Sp1-EGFP. We confirmedthat the Sp1 fusion protein was still function by testing its
activity in a Luciferase assay. We next observed the cell
nucleus using HILO microscopy, and obtained clear imagesof single Sp1-EGFP molecules. Trajectory analysis showedthat there were several spots that single molecules visit
repeatedly. Quantitative analysis revealed that there are
two peaks in the distribution of Sp1 residence time, suggesting two modes of association, searching for specificsequences and binding. These results further our understanding of the mechanism of association of transcription
factors to specific binding sites on DNA.

4. Yamasaki, S., Sakata-Sogawa,
K., Hasegawa, A., Suzuki, T.,
Kabu, K., Sato, E., Kurosaki, T.,
Yamashita, S., Tokunaga, M.,
Nishida, K., Hirano, T.: Zinc is a novel
intracellular second messenger. J.
Cell Biol. 177, 637-645 (2007)33

Group Director

Laboratory for

Research Scientist : Yuuich Aiba
Hisaaki Shinohara
Kohei Kometani (SPDR)

Lymphocyte
Differentiation

Tomohiro Kurosaki

Visiting Scientist :Technical Staff :Yoshihiro Baba
Yoko Fujii
Megumi Hamadate
Shiori Maeda

Administrative Staff : Mari Kurosaki

S

ignals propagated through B cell receptors (BCRs),
cytokine receptors, and chemokine receptors are crucial for the development of B lymphocytes and their subsequent antigen-triggered differentiation into memory B cells
and antibody secreting plasma cells. The outcomes of thesesignaling events, for example, proliferation, differentiation,or cell migration, are dependent on the developmental stageof the cell and the quality of these signals. The latter is dictated by multiple factors including co-receptors, intracellularsignaling/adapter molecules, and transcription factors. Ourlaboratory has focused on understanding the molecularmechanisms of signaling pathways that lead to crucial cellfate decisions such as memory versus plasma cell differentiation.
Function of Erk kinases in B cell differentiation
B cell maturation can be divided into a series of developmental stages that are characterized by the differential
expression of a variety of marker proteins and changes in
the rearrangement status of the immunoglobulin heavy(Igh) and light (Igl ) chain genes. Igh gene rearrangement isinitiated during the pro-B cell stage, and the resulting µ
heavy chain protein in association with λ5 and VpreB forms
a surface-expressed pre-B cell receptor (pre-BCR). Correct assembly of the pre-BCR triggers a signaling cascadethat induces proliferation, downregulation of the pre-BCR
complex, and differentiation into small pre-B cells. Previously, by using genetic deletion of both Erk1 and Erk2
kinases from early B cell developmental stages, we foundthat these kinases play a crucial role in regulating B cell
development by initiating a transcription regulatory networkand thereby pre-BCR-mediated cell expansion ( Yasuda et.
al; Immunity 2008).
The above genetic strategy hindered the clarificationof Erk’s role in B cell immune responses, because in thissettings peripheral B cells could not develop. Thus, by using
inducible deletion of these kinases during immune
responses, we found that Erk kinases are essential for
generation of plasma cells. Introduction of a dominantnegative mutation in the Erk-mediated phosphorylation

Recent publications
1. Hikida, M., Casola, S., Takahashi, N.,
Kaji, T., Takemori, T., Rajewsky, K.
and Kurosaki, T. PLC-γ 2 is essential
for formation and maintenance ofmemory B cells.
J. Exp. Med. 206, 681-689 (2009).

2. Wang, Y., Deng, X., Zhou, Y., Henderon,E., Mancarella, S., Richie, M.F., Tang,
X.D., Baba, Y., Kurosaki, T., Mori,Y., Soboloff, J. and Gill, D.L. STIM
protein coupling in the activationof Orai channels. Proc. Natl. Acad.
Sci. U.S.A. 106, 7391-7396 (2009).

Figure :Follicular helper T cell-dependent activation of B cells and signaling pathways required for plasma
cell differentiation. B cells expressing a B cell receptor (BCR) that binds antigen are activated, process
the internalized antigen, and then present antigenic peptides in the context of MHC II proteins. Such
activated B cells migrate to the interface between the B cell follicle and the T cell area of the secondary
lymphoid organ in a chemokine-dependent manner. The interaction of antigen-specific B cells with its
cognate follicular helper T cells triggers germinal center formation through the CD40L/CD40 interaction
and the secretion of cytokines. Stimulation of B cells through CD40 and the interleukin-4 (IL-4) receptor
strongly and immediately induces phosphorylation of Erk kinases. Phosphorylated Erk, then, translocates
into the nucleus and promotes transcription of the prdm1 gene, which encodes Blimp-1, through theactivation of the Elk-1 transcription factor. JAK-mediated activation of Stat3 also positively regulates prdm1
transcription. Blimp-1 is a transcriptional repressor that plays a crucial role, together with XBP-1, in theterminal differentiation of germinal center B cells into immunoglobulin-secreting plasma cells. Thus, Erk acts
as a critical signal mediator for plasma cell differentiation through the regulation of Blimp-1 expression.

sites of Elk-1 suppressed IL-4/CD40-mediated Blimp-1
expression. Conversely, enforced expression of Blimp-1 in
Erk-deficient B cells promoted plasma cell differentiation.
Thus, in addition to a critical role of Erk kinases in early B
cell development, these kinases participate in generationof plasma cells by connecting IL-4/CD40 and Blimp-1.
Localization of IgG type memory B cells
Humoral memory is characterized by recall immune
responses that are more rapid than the primary response
and by production of higher serum titers of antigen-specificantibodies, mostly of the IgG isotype. The prevailing view isthat antigen-specific B cells are maintained as a pool ofmemory B cells after clonal expansion during the primary
immune responses. Most memory B cells have been thought
to originate from the germinal center (GC) reaction. In theGC, the combined processes of somatic hypermutation andselection based on the affinity of the BCR for the antigen,are responsible for the generation of high-affinity antibody
variants which ultimately differentiate into long-lived plasma
cells or long-lived memory B cells. In the GC reaction, denovo generated antigen-specific IgG memory B cells are

3. Kurosaki, T. and Hikida, M. Tyrosine
kinases and their substrates in B
lymphocytes. Immunol. Rev.
228, 132-148 (2009).

4. Baba, Y. and Kurosaki, T.
Physiological function and molecularbasis of STIM1-mediated calcium
entry in immune cells. Immunol.
Rev. 231, 174-88 (2009).

thought to acquire intrinsically different traits from their naïve
predecessors, accounting for faster and heightened secondary responses.
Although the importance of IgG memory B cells in longterm humoral memory has been well recognized, the lodging and activation sites of these cells remain elusive, in part,
because of technical difficulties related to in situ detection of
the rare IgG memory B cells specific for a given antigen. By
using painstaking histochemical analysis and genetic
approaches, we have shown that IgG type memory B cells
are mainly located nearby the contracted germinal center(GC)-like structures and that these cells are indeed activatedupon secondary antigen challenge. Based upon theseobservations, together with our evidence that CD4 T cells
exist in close proximity with IgG memory B cells nearby thecontracted GCs, we propose that this close proximity of IgG
memory B cells and memory T cells can explain, at least
partly, the rapid kinetics of memory responses. Unlike in the
primary response, the movement of antigen-specific B cells
and antigen-specific T cells toward the T-B border area andtheir subsequent interaction there would no longer berequired.

5. Kurosaki, T., Shinohara, H. and Baba,
Y. B cell signaling and fate decision.
Ann. Rev. Immunol. (in press).

35

Team Leader

Ji-Yang Wang
Research Scientist : Rika OuchidaStudent Trainee :Chie Kano
Yingqian Li (IPA)

Technical Staff :Hiromi MoriLaboratory for

Immune Diversity

Administrative Staff : Kanae FukuiGerminal center (GC) B cells represent a unique cell
population that is induced during an immune response
against foreign antigens. These rapidly dividing cells undergo
dynamic genetic alterations including somatic hypermutation (SHM) and class switch recombination of the immunoglobulin (Ig) genes and can ultimately differentiate into
antibody-producing plasma cells or memory B cells. Dysregulation of this terminal differentiation pathway can leadto immunodeficiency, autoimmune diseases and B cellmalignancies. The goal of our research is to understandhow the activation-induced cytidine deaminase (AID) can
induce a high frequency of A:T mutations in the Ig genes
and to dissect the molecular mechanisms regulating B cell
activation and terminal differentiation.
SHM is initiated by AID, which catalyzes the deamination of cytosine (C) to uracil (U) and generates a U:G lesion.
One of the intriguing unanswered questions is how AIDtriggered U:G lesions lead to a high proportion of mutations
at non-damaged A:T base pairs. We have shown that GC B
cells, but not other normal tissues/cell types, have an intrinsic property to generate A:T mutations. This property isdependent on the GC B cell environment but is independentof AID expression and the target gene location. We aim toisolate potential GC B-specific factors that are required togenerate A:T mutations.
B cell activation and differentiation into antibody-producing plasma cells or memory B cells is a highly regulated
complex process but the molecular mechanisms still
remain poorly understood. We have identified a numberof uncharacterized genes that are either differentiallyexpressed during B cell activation or highly expressed in GC
B cells, and have been analyzing their physiological roles byusing gene-targeted mice. We have discovered a lysosomal
protein that mediates the degradation of B cell antigen
receptors (BCR) and negatively regulates B cell function.
We have also identified a number of genes that appear to
be involved in B cell activation and antibody production.
Through further analyses of these gene-targeted mice,
we hope to gain additional insights into the molecularmechanisms that control the survival, activation and terminal differentiation of B cells.

Recent publications
1. Ouchida, R., Kurosaki, T. andWang, J.-Y. A role for LAPTM5 in
the negative regulation of surface
BCR levels and B cell activation.
J. Immunol. 185: 294-301 (2010).
(Featured in “In This Issue”)

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

Figure 1: Pathways for A:T mutations in Ig andnon-Ig genes in GC B cells. GC B cells
have an intrinsic property to generate
A:T mutations in Ig and non-Ig genes. In
Ig genes, AID deaminates cytosine (C)to uracil (U) and generates a U:G lesion.
Under normal situations, this U:G lesion
is correctly repaired by the error-free baseexcision repair (BER) pathway. During Ig
gene SHM, however, the U:G lesion canbe resolved by a GC B-specific error-prone
repair pathway, leading to mutations at
non-damaged A:T base pairs. Using alacZ-transgenic system, we found that GC B
cells also efficiently generate A:T mutations
in non-Ig genes in an AID-independent but
POLH-dependent manner, likely utilizing
a similar GC B-specific mechanism.

Figure 2 : LAPTM5 downmodulates cell surface BCR
expression without affecting the levels of
CD19 and CD40. Purified spleen B cells
were stimulated with LPS (10 μg/ml) for
24 h and then transduced with retrovirus
expressing GFP alone or LAPTM5-IRESGFP. The cells were further cultured for 2days and then analyzed by FACS for the cellsurface levels of BCR, CD19 and CD40.

Mechanism of A :T mutations in GC B cells
Using a sensitive lacZ-transgenic ( lacZ-Tg) system to detect
genome-wide mutations, we have found that GC B cells
have an intrinsic propensity to mutate A:T pairs (Fig. 1). Further analysis revealed that the induction of A:T mutations is
independent of AID and the target gene location. Experiments are now in progress to address the following three
non-exclusive hypotheses for the high proportion of A:T
mutations in GC B cells. 1) Rapid cell division of GC B cells
interrupts the normal repair process and causes error-prone
DNA synthesis. 2) GC B cells express a specific factor(s)that function to recruit POLH, a low-fidelity DNA polymerase
essential for the induction of A:T mutations, instead of otherhigh-fidelity polymerases during repair of DNA lesions. 3)
POLH, mismatch repair (MMR) components, or AID itself
undergo specific modifications in GC B cells, resulting in an
atypical repair process not observed in other cell types.

3. Kubagawa, H., Oka, S., Kubagawa,Y., Torii, I., Takayama, E., Kang, D.W.,
Gartland, G.L., Bertoli, L.F., Mori, H.,
Takatsu, H., Kitamura, T., Ohno, H. andWang, J.-Y. Identity of the elusive IgM
Fc receptor (FcµR) in humans.
J. Exp. Med. 206: 2779-2793 (2009).

4. Mori, H., Ouchida, R., Hijikata, A.,
Kitamura, H., Ohara, O., Li, Y., Gao,
X., Yasui, A., Lloyd, R.S., Wang,
J.-Y. Deficiency of the oxidative
damage-specific DNA glycosylase
NEIL1 leads to reduced germinal
center B cell expansion. DNA Repair
8: 1328-1332 (2009). (highlighted
in “This Month in DNA Repair”)

Characterization of genes that are differentially expressed during B cell activation or highly expressedin GC B cells
We have thus far generated mice deficient in each of thesegenes and are now analyzing B cell abnormalities in thesemice. One such gene, LAPTM5, is a lysosomal protein specifically expressed in hematopoietic cells and transiently
down-modulated during B cell activation. We found thatLAPTM5 negatively regulated cell surface BCR levels and B
cell activation. Ectopic expression of LAPTM5 in spleen B
cells downmodulated BCR levels in a dose-dependent fashion, without affecting the levels of CD19 and CD40 (Fig. 2).
We aim to clarify how LAPTM5 specifically targets BCR but
not other cell surface molecules for degradation.

5. Masuda, K., Ouchida, R., Li, Y., Gao,
X., Mori, H. and Wang, J.-Y. A criticalrole for REV1 in regulating theinduction of C:G transitions and A:T
mutations during immunoglobulin
gene hypermutation. J. Immunol.,183: 1846-1850 (2009).

37

Group Director

Toshitada Takemori
Research Scientist : Tomohiro Kaji
Technical staff :Akiko Sugimoto
Jyunko Taka
Tomoko Nishio
Natsumi Yoneda

Visiting Scientists :Yoshimasa Takahashi
Masaki Hikida

Administrative Staff : Satomi Law

Laboratory for

Immunological
MemoryI

mmunological memory is the hallmark of adaptive immunity and is characterized by the prompt and effective
response to antigen upon re-exposure, even a long timeafter initial stimulation. Memory provides a highly selective
mechanism to provide quick protection upon re-exposure
to recurrent pathogens. Therefore, an important question is
how immunological memory is maintained for such long
periods in the absence of repeated antigenic stimulation.
An additional question is how memory and naïve B cells
might differ in their functions, such as their longevity andtheir prompt differentiation into plasma cells that secrete
large amounts of antibodies upon antigen re-exposure.
Finally, another important question is how and when memory B cells develop after initial antigen exposure.
Our goals are to answer these questions, initiallyby utilizing a murine system, which is advantageous forassessing cellular and molecular mechanisms by multiple
approaches. Furthermore, based on the information developed in the mouse, we intend to define the mechanisms
underlying memory B cell maintenance in human.
Class-switched non-mutated memory B cells
develop independently of the GC reaction.
In T-cell dependent response, antigen-activated B cells follow two spatially distinct pathways of differentiation, either
in extrafollicular regions of the secondary lymphoid organs
or in B cell follicles, where antigen-activated B cells form
GCs. While it has been widely accepted that mutated, highaffinity memory B cells originate from the GC response, wesuggested that non-mutated, low affinity memory B
cells develop independently of the GC reaction. Recently,
three different laboratories have confirmed the development of non-mutated memory B cells in the early primary
response, however, little is known of the cell population
dynamics underlying development of GC-independent
memory B cells. To address this issue, we established conditional Bcl6 deficient mice, a strategy that avoids the influence of Bcl6 deletion in immune cells other than B cells.
Our results demonstrated that conditional Bcl6-deletionin B cells impaired the development of NP-specific IgG1+

Recent publications
1. M. Hikida M., Casola. S., TakahashiN., Kaji T., Takemori T., Rajewsky
K., Kurosaki T. PLC-gamma2
is essential for formation andmaintenance of memory B cells.
J.Exp.Med. 206 681-689 (2009)

2. Ato M., Ikebe T., Kawabata
H., Takemori T., Watanabe H.
Incompetence of neutrophils toinvasive group A streptococcus
is attributed to induction ofplural virulence factors by
dysfunction of a regulator. PLoS
One 3[10].e3455 (2008)

Figure:GC B cells in response to NP-CG, whereas IgG1+ nonmutated memory B cells developed independently of GCs
within the first week post-immunization. Thus, it appears
that following T-cell dependent antigen stimulation, classswitched B cells either migrate to extrafollicular sites, where
they differentiate into plasma cells or migrate into follicles,
where they can follow two distinct pathways of differentiation, either towards GC B cells or non-mutated memory B
cells. We previously showed that inhibition of the ICOSICOSL interaction impaired GC B cell development, but not
non-mutated memory B cell development, findings compatible with the notion that development of non-mutatedmemory B cells is helped by a distinct subset of T cells
different from the T follicular helper cells ( TFH ), which play a
crucial role in GC B cell development.
The memory B cell pool sustains its size underhomeostatic regulation and attains functional
maturation after its generation early in the immune
response
The IgG1+ non-mutated memory B cells that develop in theabsence of GCs establish a long-lived IgG1 memory compartment. On the other hand, mutated memory B cells are
generated in the GCs and recruited into the non-mutatedmemory compartment as the immune response progresses.
Because the splenic environment has a limited capacity tosustain memory B cells, the non-mutated cells are graduallyreplaced over time by mutated GC B cell progeny. However,
how such a turnover is regulated remains obscure.
We observed that the early (day 7) memory B cells could
not elicit a significant IgG1 antibody response in the secondary response, however, non-mutated memory cells thatdevelop independently of the GC could elicit a low-affinity
adoptive secondary response at day 40 after immunization,

Memory B cell development early after
immunization. A model for the generation andselection of B cells in the primary immune response.
Following T-cell dependent antigen stimulation, acognate interaction between T and B cells causes
B cell proliferation and isotype class-switchingfrom µ to γ1 at the border of the B-cell/T-cell areasin the spleen and lymph nodes. Subsequently, B
cells either migrate to extrafollicular sites, where
they differentiate into plasma cells, or migrate
into follicles, where they can follow two distinct
pathways of differentiation, either towards GC B
cells under control of T follicular helper cells (TFH)
or toward non-mutated memory B cells under T cell
help by cognate interaction with CD40 and CD40L.

comparable in the number of antibody secreting cells (ASCs)
to wild type day 40 memory B cells. Co-stimulatory molecules needed for T-cell interactions were almost equally
expressed on both day 7 and day 40 memory B cells,
thereby, leading us to speculate that the absence of a
response by day 7 memory B cells may reflect a deficit in acell-intrinsic activity needed for this response. There weredifferences in the expression of certain genes between the
day 7 and day 40 memory B cell populations that develop in
the absence of GCs, leading us to propose that some external signals alter the transcriptional profile of memory B cells,
which promotes their functional maturation. We are attempting to pinpoint the genes responsible for the functional
maturation of memory B cells as the response progresses.
Analysis of memory B cells features in common
variable immunodeficiency (CVID)
Among the humoral primary immunodeficiencies, CVID ischaracterized by antibody deficiencies in childhood or early
to mid adulthood. Because it has been suggested that50 –75% of patients with CVID had reduced numbers ofclass-switched memory B cells in peripheral bloods (PB),
we intended to confirm these findings using multicolor FACS
analysis, enabling us to dissect B cell subsets by new criteria. We observed that all patients tested have a reduction in
the numbers of both class-switched memory B cells andplasma blasts, together with or without an increase in thenumber of transitional B cell fraction 3. These findings may
suggest that CVID is not solely linked to defects in memory
B cell development. We have begun to analyze gene expression profiles in human memory B cells and other subsets ofB cells, information that should provide further insight into
the B cell defects in CVID.

3. Nagawa F, Kishishita N, Shimizu K,
Hirose S, Miyoshi M, Nezu J,
Nishimura T, Nishizumi H, Takahashi Y,
Hashimoto S, Takeuchi M, Miyajima A,
Takemori T, Otsuka AJ, Sakano H.
Antigen-receptor genes of the
agnathan lamprey are assembled
by a process involving copy choice.
Nat. Immunol. 8, 206-213 (2007)

39

Team Leader

Laboratory for

Research Scientists : Koji Hase, Shunsuke Kimura,
Shinji Fukuda (SPDR),
Takashi Kanaya (SPDR)

Epithelial
Immunobiology

Hiroshi Ohno

Technical Staff :Yumi Chiba, Yumiko Fujimura,
Chie Nishigaki, Yoko Ohara,
Masumi Ohmae, Sayuri Sakakibara,
Ayako Yamashita

Student Trainees:

Yasuhiro Date (JRA), Yumiko
Nakanishi (JRA), Gaku Nakato (JRA),
Daisuke Takahashi (JRA), Masashi
Ebisawa (JRA), Toshi Jinnohara(JRA), Kazunori Kadokura (JRA),
Tamotsu Kato (JRA), Masumi
Shinbori, Kaoru Aizawa, Konomi
Ishimaru, Junnosuke Onozato, Ryo
Yamazaki, Chikako Uetake, Hideaki
Shima, Misaho Hanazato

Administrative Staff : Yuuki YamadaThe mucosal epithelium that lines the inner surfaces ofthe body, especially within the intestine, is exposed toa wide range of antigens, including food-borne macromolecules and microorganisms as well as numerous commensal microbiota, and efficient uptake of these antigens is vitalfor maintaining appropriate immune response. Epithelial
cells that overlay the gut-associated lymphoid tissue (GALT ),such as Peyer’s patches (PPs) and isolated lymphoid follicles, are distinct from absorptive epithelial cells of the villi
and are termed follicle-associated epithelium (FAE ). The
FAE contains a specialized subset of epithelial cells, the M
cells, which are thought to play a pivotal role in immune
surveillance by delivering luminal microorganisms to theunderlying lymphoid cells via transcytosis. Despite their significance, the study of M cells has remained in its infancy
until recently, mainly because the low frequency of M cells
and the lack of specific surface markers has made it difficult
to purify the M cells for molecular/biochemical analyses.
Accordingly, one of the primary aims of our laboratory is tounderstand the mechanisms that underlie the differentiationand function of FAE and M cells. Our research team is also
investigating the interaction of commensal microbiota with
the intestinal epithelium and its influence on the mucosaland systemic immune system. These studies may lead tothe development of novel and more efficient mucosal vaccination protocols/drug delivery systems as well as functional foods/preventive medicine based on host-microbiota
interactions.
Uptake by Glycoprotein 2 on M cells of FimH+
bacteria is essential for an efficient mucosal
immune response to these microbes
Toward understanding the molecular mechanisms of M-cellfunction and differentiation, we took advantage of a microarray-based approach for a genome-wide survey of M-cell
specific molecules. We reported last year that glycoprotein-2 (GP-2) was exclusively expressed by Peyer’s patch M
cells, but not by villous epithelial cells, in collaboration with
Dr. Kiyono’s group in the University of Tokyo. We haveextended our finding to show that GP-2 serves as a bacte-

Recent publications
1. Hase K., Kawano K., Nochi T.,
Pontes G. S., Fukuda S., Ebisawa
M., Kadokura K., Tobe T., Fujimura,Y., Kawano S., Yabashi, A., Waguri,S., Nakato G., Kimura S., MurakamiT., Iimura M., Hamura K., Fukuoka
S. I., Lowe A. W., Itoh K, Kiyono H.,
Ohno H. Uptake through Glycoprotein+
2 of FimH bacteria by M cells

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

Figure:rial uptake receptor on M cells. Recombinant GP2 protein
selectively bound a subset of commensal and pathogenic
enterobacteria, including Escherichia coli and Salmonella
enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outermembrane. Consistent with these findings, the same bacteria were colocalized with endogenous GP2 on the apicalplasma membrane as well as within cytoplasmic vesicles ofM cells. Moreover, deficiency of bacterial FimH or host GP2
led to defects in transcytosis of type-I-piliated bacteria
through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer’s patches. GP2 is therefore
a previously unrecognized transcytotic receptor on M cells
for type-I-piliated bacteria and is a prerequisite for the
mucosal immune response to these bacteria. Given that M
cells are considered a promising target for oral vaccination
against various infectious diseases, the GP2-dependenttranscytotic pathway could provide a new target for thedevelopment of M-cell-targeted mucosal vaccines.
M-Sec promotes tunneling nanotube formation inassociation with Ral and the exocyst complex
Cell – cell communication is essential for the development
and homeostasis of multicellular organisms. Recently, a
new type of cell–cell communication was discovered that isbased on the formation of thin membranous nanotubes
between remote cells. These long membrane tethers,

2. Hase K., Kimura S., Takatsu H.,
Ohmae M., Kawano S., Kitamura
H., Ito M., Watarai H., Hazelett C.
C., Yeaman C., Ohno H. M-Sec
promotes membrane nanotube
formation by interacting with Ral
and the exocyst complex. Nature
Cell Biol. 11, 1427-1432 (2009)

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

Schematic representation of
the role of GP2 in mucosal
immune response. FimH+ entericbacteria such as Escherichia coliand Salmonella are captured by
GP2 on the M-cell surface andsubjected to transcytosis to beeventually transferred to dendritic
cells closely associated with M
cells. Subsequently, dendritic
cells process and present bacterial
antigens to T cells to initiate
mucosal immune response
against these bacteria, which
ultimately lead to production ofbacterial antigen-specific IgA.

termed tunneling nanotubes ( TNTs), form an intercellular
conduit and have been shown to enable the transport ofvarious cellular components and signals. However, themolecular basis for TNT formation remains to be elucidated.
We have recently shown that a mammalian protein, M-Sec,
induces de novo formation of numerous membrane protrusions extending from the plasma membrane, some of which
tether onto adjacent cells and subsequently form TNT-likestructures. Depletion of M-Sec by RNA interference (RNAi)
greatly reduced endogenous TNT formation as well asintercellular propagation of a calcium flux in a macrophagecell line. Furthermore, blockage of the interaction of M-Sec
with Ral and the exocyst complex, which serves as a downstream effector of Ral, attenuated the formation of membrane nanotubes. Our results reveal that M-Sec functions as
a key regulator of membrane nanotube formation throughinteraction with the Ral– exocyst pathway. The discovery of
M-Sec as a marker for TNTs and a promoter of TNT formation will help clarify the mechanisms of formation of thesestructures as well as their structural and functional properties. We have recently established M-Sec knockout mice,
which should be a useful model to uncover the physiologicalrole of TNTs. In addition, the identification of M-Sec-targeteddrugs may provide a new strategy for containment of viral
infections such as HIV, given the recent report of the involvement of TNT in intercellular viral propagation and viral pathogenesis.

4. Nakato G., Fukuda S., HaseK., Goitsuka R., Cooper M.D.,
Ohno H. New approach for
M-cell-specific molecules
screening by comprehensive
transcriptome analysis. DNA
Res. 16, 227-235 (2009)

5. Hase K., Takahashi D., Ebisawa
M., Kawano S., Itoh K., Ohno
H. Activation-induced cytidine
deaminase deficiency causesorgan-specific autoimmune disease.
PloS ONE 3, e3033 (2008)41

Team Leader

Sidonia Fagarasan
Research Scientists : Keiichiro Suzuki
Tran Huy Thinh

Laboratory for

Mucosal Immunity

Research Associate : Mikako Maruya
Technical Staff :Yasuko Doi
Yumi Tsutsui

Student Trainee :Shimpei Kawamoto

Administrative Staff : Mari KurosakiOur long-term goal is to understand the cellular andmolecular basis of gut immune regulation, a front lineof body defense, and to apply such knowledge for healthand disease of the mucosal immune system. More specifically, given its safety, low cost and potential efficacy, oralvaccination is considered to be a desirable route for immunization against mucosal as well as systemic infections.
To achieve this goal we have to define and functionallycharacterize the main players in mucosal immunity, namely:
the bacterial community living in our intestinal tract; allimmune cells residing in lymphoid tissues closely associated with bacteria; and the stromal cells that constitute thenatural backbone for the immune cells. Furthermore, acomprehensive view of an immune response in natural
settings requires not only the individual characterizationof these compartments, but also a full understandingof their continuous interactions and cross-talk, which
inevitably lead to dynamic phenotypic and functionalchanges for all players.

T cell-independent and T-cell dependent immunoglobulin A generation in the gut
Immunoglobulin A (IgA) is generated in the gut by both T
cell-dependent and T cell-independent processes. We
recently showed that lymphoid gut structures called isolated
lymphoid follicles (ILF ), which develop in a RORγ t + LTidependent manner after colonization of the intestine withbacteria, serve as sites for IgA synthesis. We found not onlythat ILFs serve as sites where B cell undergo activation,induction of activation-induced cytidine deaminase (AID)
and IgA class switching, but also that this process apparently does not require the help of T cells.
Nevertheless, the number and the quality of IgA plasma
cells present in the intestine of mice having only ILFs differed from those found in mice with Peyer’s patches (PPs)and ILFs. Unlike in the ILFs, the IgAs generated in PPs
require germinal center formation and the presence of CD4+
follicular B helper T ( TFH ) cells. Although TFH cells have been
known for more than a decade, the nature of the TFH cells
present in peripheral lymphoid organs or in the Peyer's
patches has been elusive. We demonstrated that suppressive Foxp3+CD4+ T cells have the potential to differentiate

Recent publications
1. Tran TH, Nakata M, Suzuki K, Begum
NA, Shinkura R, Fagarasan S, Honjo T,
Nagaoka H. B cell-specific andstimulation-responsive enhancers
derepress Aicda by overcoming
the effects of silencers. Nat
Immunol. 2010 Feb;11(2):148-54

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

Figure 1: T cell-dependent induction of IgA+ B cells in the germinal centerof Peyer’s patches. Dendritic cells (DCs) located beneath specialized
M cells take up bacteria, and then migrate to the T-cell zone where
they activate T cells. Upon induction of the transcription factor Bcl-6,
activated CD4+ T cells, express CXCR5 and migrate toward CXCL13
into the FDC network, where they provide help for activated B cells (TFH
cells). Some TFH cells are derived from Foxp3+ T cell precursors that lose
Foxp3-expression in the presence of IL-6 (produced by DCs) and IL-21(produced by T cells). Conventional B-T interactions through MHC-TCR
and CD40-CD40L are critical for activation of B cells, induction of AID
and class switch recombination (CSR)/somatic hypermutation (SHM)
in GCs of PPs. Preferential generation of IgA+ B cells in PPs is due to theabundant production of activated TGF-β1, IL-21 and retinoic acid (RA)in this environment. The IgA+ B cells generated in GC of PPs home to thelamina propria where they further differentiate into IgA plasma cells.

Figure 2: In vivo and in vitro imagesof FDCs. (Left panel) A
horizontal section of Peyer’s
patches showing a B cellfollicle (B220 in green) anda B-cell chemoattractant
produced by FDCs (CXCL13
in red). (Right panel) A
phase-contrast micrograph of
FDCs cultured for two weeks
on a collagen-coated platein the presence of an LT-βR
agonist antibody and TNF-α.

into TFH cells in mouse Peyer's patches. The conversion of
Foxp3+ T cells into TFH cells requires the loss of Foxp3
expression and subsequent interaction with B cells throughCD40-CD40L. Thus, we demonstrated a rather unexpected
plasticity of the CD4+ T cells expressing Foxp3, which undergut environmental conditions (antigens and cytokines) convert to distinct helper T cells such as TFH cells.
The ILF formation/ T-cell independent IgA synthesis
project was in collaboration with Dr. Dan Littman, Skirball
Institute of Biomolecular Medicine, NYU. The T cell-dependent IgA synthesis project was in collaboration with the
Research Unit for Immune Homeostasis led by Dr. Shohei
Hori, the Laboratory for Autoimmune Regulation led by Dr.
Osami Kanagawa, and Professor Tasuku Honjo from Kyoto
University.
Follicular dendritic cells and their role in regulationof mucosal immune responses
Follicular dendritic cells (FDCs) are radio-resistant cells thatwere identified by their ability to retain immune complexes.
More recently it was found that FDCs secrete several cytokines and chemokines involved in the migration and aggregation of lymphocytes, especially B cells into follicles. However,

3. Suzuki K, Fagarasan S. Diverse
regulatory pathways for IgA
synthesis in the gut. Mucosal
Immunol. 2009 Nov;2(6):468-71.

4. Tsuji M, Suzuki K, Kitamura H,
Maruya M, Kinoshita K, Ivaylo II,
Itoh K, Littman DR, Fagarasan S.
Requirement for Lymphoid tissue
inducer cells in isolated follicle
formation and T cell-independent
immunoglobulin A generation in
the gut. Immunity. 2008, Aug, 15.

most of the FDC studies are based on either histologicalcharacterization or generation of cell lines with properties
resembling FDCs. We aimed to characterize FDCs ex vivo inorder to understand their functional compartmentalizationand to determine if FDCs play roles not only for SHM required
for affinity maturation but also for CSR of B cells. Thus, we
set up to evaluate the functional differences between the
FDCs located in organized gut lymphoid tissues (GALT ) andthe FDC from peripheral lymph nodes. Our experiments
revealed that GALT FDCs express surface, cytoplasmic andnuclear receptors that can signal the presence of bacterial
and metabolic products such as RA, the active metaboliteof vitamin A in the gut. Synergistic stimulation of FDCs withbacteria and RA enhances the expression of molecules by
the FDCs, among them many proteins involved in activationof TGF-β1. Our results indicate that gut FDCs, are conditioned by environmental stimuli to express key factors for B
cell migration, survival and preferential generation of IgA in
gut. (Suzuki et al., Immunity, in press.)
This project is in collaboration with the Laboratory for
Immunogenomics led by Dr. Osamu Ohara and with Professor William Agace, from Lund University, Sweden.

5. Suzuki K, Fagarasan S. How
host-bacterial interactions lead to
IgA synthesis in the gut. Trends
Immunol., 2008, 29 (11), 523-531.

43

Team Leader

Tsuneyasu Kaisho
Research Scientist : Katsuaki Hoshino
Technical Staff :Yuri Fukuda
Nana Iwami
Akiko Kato

Student Trainees :Izumi Sasaki
Chihiro Yamazaki
Takeshi Hirashima
Akihiko Iizuka
Eri Hamada

Administrative Staff : Sachiko Haraguchi

Laboratory for

Host DefenseH

ost defense in mammals consists of innate and adaptive immunity. Innate immunity functions as a pathogen sensor and is involved in their eradication. Furthermore,
innate immunity also contributes to the establishment ofadaptive immunity. Dendritic cells (DCs) are antigen presenting cells critically involved in the sequence of these
immune responses. DCs sense various pathogen-derived
molecular components and exert their immunostimulatory
functions by producing inflammatory cytokines or upregulating expression of costimulatory molecules. Those components are termed immune adjuvants based on their ability
to activate DC. Immune adjuvants are recognized by various types of pattern recognition receptors including Toll-likereceptors (TLRs). Identification of new types of immune
adjuvants and characterization of the mechanisms by which
they activate DCs should contribute to the development ofnovel immunoregulatory maneuvers. We are attempting toclarify how DCs are activated through pattern recognition
receptors and to obtain critical information for effectively
manipulating the immune response. Various immune adjuvants, including TLR ligands, and gene targeting mice areimportant tools for our studies.
DC subset-specific mechanisms in TLR7/9-induced
type I IFN gene induction
DCs sense nucleic acid adjuvants and produce type I interferon (IFN) in a subset-dependent manner. Among nucleic
acid sensors, TLR7 and TLR9 are peculiar in that they recognize not only pathogen- but also host-derived nucleic
acids. In fact, accumulating evidence suggests that TLR7/9induced type I IFN production plays important roles in thepathogenesis of autoimmune disorders such as SLE. Therefore, clarifying the TLR7/9 signaling mechanisms should
contribute to the development of therapeutic intervention
for such diseases.
Plasmacytoid DCs (PDC) are a unique DC subset
expressing nucleic acid sensors, TLR7 and TLR9. PDC arefeatured by the ability to produce type I IFN, especially IFNα, in response to TLR7/9. TLR7 and TLR9 are quite similarin their amino acid sequence and can activate similar sig-

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

2. S. K. Pathak, S. Basu, K. K. Basu, A.
Banerjee, S. Pathak, A. Bhattacharyya,
T. Kaisho, M. Kundu, J. Basu.
Direct extracellular interaction
between the early secreted
antigen ESAT-6 of mycobacterium
tuberculosis and TLR2 inhibits TLR
signaling in macrophsages. Nature
Immunol. 8, 610-618 (2007).

Figure: DC subset-specific mechanisms in
TLR7/9-induced type I IFN geneinduction. In response to TLR7/9
signaling, pDC can produce vast
amounts of type I IFNs including IFN-α
and IFN-β, while cDC can produce
only IFN-β. IKKα is required for this
response by both DC subsets. Notably,
the underlying mechanism depends
on the DC subset. IRF-7, which isabundantly expressed in pDC, is criticalfor pDC function. However, it is still
possible that other pDC-specific factors
also contribute to pDC function.

naling pathways, which depend on the cytoplasmic adapter
molecule, MyD88. Downstream of MyD88, signaling pathways bifurcate into NF-κB and IRF-7 activation pathways,
leading to the induction of inflammatory cytokines and type
I IFNs, respectively. We have discovered that a serine threonine kinase, IκB kinase-α (IKKα), is critically involved in thistype I IFN induction pathway in PDC (Hoshino et al. Nature,2006). IKKα associates with and phosphorylates IRF-7,
which is critical for type I IFN gene expression in response to
TLR7/9 signaling. In response to TLR7/9 signaling, conventional DC (cDC) can also produce IFN-β, but not IFN-α. This
IFN-β production requires molecular mechanisms that differ
from those involved in type I IFN production by pDC. However, we have found that IKKα is also required for the IFN-β
production by TLR7/ 9-stimulated cDC. In IKKα-deficient
cDC, IRF-7 activation was not impaired, but activation of
IRF-1 and NF-κB was decreased. These results indicatethat IKKα is involved in TLR7/9-mediated type I IFN induction in a DC subset-specific manner. Thus, IKKα should bea unique target not only for manipulating antiviral immunity
but also for treating autoimmune diseases in which type I
IFN production is elevated. IRF-7 is a critical molecule for
pDC function and is constitutively highly expressed in pDC.
In addition to our studies of the IKKα-IRF-7 axis, we arefurther planning to clarify the underlying molecular mechanisms for pDC function and development.

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

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

Critical roles of TLR3 and TLR3-expressing cDC
Double stranded RNA (dsRNA) is also a nucleic acid adjuvant that can activate DCs. and a synthetic dsRNA, poly (I:C),
is recognized by an endosomal sensor, TLR3, and a cytosolic sensor, MDA5. Each sensor exhibits its own function.
Poly ( I:C) induces IL-12p40 production through TLR3 signaling, while it leads to type I IFN production through MDA5
signaling. We have analyzed the immune adjuvant effects ofanother dsRNA, poly (A:U), and found that it is a ligand for
TLR3 and TLR7, but not for the cytosolic sensor. By using
poly (A:U) and TLR3- and/or TLR7-deficient mice, we havediscovered that TLR3 is involved in mediating the signal
for CD8 T cell responses in vivo, i.e. cross presentation.
Crosspresentation is a unique function of DCs and is criticalfor effective immune responses against tumors or viral infections. cDCs can be further divided into 3 subsets, CD4+8-,
CD4-8+ and CD4-8-. Notably, among these cDC subsets,
CD4-8+ cDCs are characterized by their high crosspresenting activity and are the only DC subset that expresses TLR3
and responds to TLR3 signaling. The other cDCs as well as
pDC fail to respond to TLR3 signaling. We are planning toclarify how CD8+ cDCs are generated and acquire theirfunction and how the function is regulated by TLR3 signaling. For this purpose, we characterize gene expression
profiles of various DC subsets and focus on some CD8+
cDC-specific genes.

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

45

Team Leader

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

Technical Staff :Masami Aoki-Kawasumi
Mari Mito-Yoshida

Administrative Staff : Sachiko Dohi

Laboratory for

Infectious ImmunityWe have demonstrated that the expression level of
MHC class II (MHC II) is regulated through ubiquitination of the MHC II β chain. We also found that MARCH-I,
an E3 ubiquitin ligase, is critical for this process. At present,
however, the physiological importance of MARCH-I-mediated MHC II regulation in vivo is still unknown, and resolving
this issue is the goal of one of our major projects. Given that
MHC II is a critical molecule for initiation of immune
responses, this project will provide new insight into themolecular basis of how antigen presentation regulates
immune responses in vivo.
We are also exploring the molecular mechanism ofubiquitin-regulated trafficking of MHC molecules by using a
new experimental tool, a Tet-on expression system for MIR
E3 ligases. With this new experimental system, we are now
able to analyze the detailed relationship between the statusof ubiquitination and the trafficking of MHC molecules.
Since ubiquitination-mediated regulation of transmembraneproteins (e.g. cytokine receptors) contributes to critical cellsignaling pathways, we will be able to provide important
insight into fields even beyond immunology.
To understand the physiological function of MARCH-I,
we and other groups have examined the regulation of
MARCH-I expression at the transcriptional and post-transcriptional levels. We demonstrated that TLR signals inhibit
the expression of MARCH-I mRNA, and inhibit MHC II ubiquitination in dendritic cells. Also, inhibition of DC maturation
by IL-10 was shown to rescue the down-regulation of
MARCH-I expression and MHC II ubiquitination.
Given that MARCH-I is a physiological E3 for MHC II,
we hypothesize that down-regulation of MARCH-1 mRNA is
an important initiation signal for immune responses. However, the question remains of why constitutive MHC II ubiquitination by MARCH-I is necessary at the steady state invivo. To address this question, we examined T-dependent
immune responses in conventional MARCH-I KO mice.
So far, we have not observed any evidence of enhanced
MHC II function in these mice. Instead, to our surprise, we
observed DC abnormalities in vivo. MARCH-I-deficientsplenic DCs showed high expression of MHC II as expected.

Recent publications
1. Ohmura-Hoshino, M., Matsuki, Y.,
Mito-Yoshida, M., Goto, E.,
Aoki-Kawasumi, M., Nakayama,
M., Ohara, O., and Ishido, S.
Cutting edge: Requirementof MARCH-I-mediated MHC II
ubiquitination for the maintenanceof conventional dendritic cell. J.
Immunol., 2009;183(11):6893-7.

2. Ishido S., Goto, E., Matsuki, Y. andOhmura-Hoshino, M: E3 ubiquitin
ligases for MHC molecules, Curr
Opin Immunol, 2009;21(1):78-83.

Figure:Model for the role of MARCH-I in immune responses. In the steady state, MARCH-I (shownas “E3”) is constitutively expressed in APCs and regulates their expression of MHC II throughubiquitination-mediated lysosomal degradation (left panel). Once APCs are activated andmaturated by infection, MARCH-I expression is down-regulated and pathogen-derived peptides(shown in red) are presented by MHC II stabilized due to loss of ubiquitination (middle panel).
This stabilization results in sustained MHC II expression that eventually delivers as yet to be
identified “inhibitory signals” to APCs, directly or indirectly, halting the immune response.

Interestingly, the co-stimulatory molecule B7-2/CD86 wasalso highly expressed on the KO DCs. Since over-expressionof MARCH-I inhibited B7-2 expression, it seems to beanother physiological MARCH-I substrate. Surprisingly, eventhough the molecules required for antigen presentation werehighly expressed, the ability of splenic DCs to present the
antigens was impaired in the MARCH-I KO, as was LPSinduced cytokine production. Consistent with these observations, T-dependent immune responses were impaired in
the MARCH-I KO mice.
Why are splenic DCs abnormal in MARCH-I KO mice?
Examination of MARCH-I deficient DCs generated in vitro(bone marrow-derived DCs) and the results of transplantation experiments using these cells strongly suggested thatthe DC abnormality is due to an in vivo environmental effect.
Since MHC II and B7-2 are both highly expressed on DCs
and potentially interact with the microenvironment (e.g. T
cells), the contribution of these molecules to the DC abnormality was examined. Each molecule was deleted from
MARCH-I KO mice by crossing them with MHC II or B7-2
KOs. Interestingly, deletion of MHC II completely rescued

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

4. Matsuki, Y., M. Ohmura-Hoshino,
E. Goto, M. Aoki, M. Mito-Yoshida,
M. Uematsu, T. Hasegawa, H. Koseki,
O. Ohara, M. Nakayama, K. Toyooka,
K. Matsuoka, H. Hotta, A. Yamamoto,and S. Ishido. Novel regulation of
MHC class II function in B cells.
EMBO J., 2007 26:846-856.

the abnormality of MARCH-I-deficient DCs, even though
B7-2 was still highly expressed. Consistent with the lack ofa role for B7-2, its deletion had no effect on the abnormality.
These results demonstrated the requirement of MHC II for
the DC abnormality induced by MARCH-I deletion.
It has been demonstrated that stimuli that induce DC
maturation stabilize surface MHC II molecules by inhibiting
MHC II ubiquitination. Furthermore, loss of MHC II ubiquitination is accompanied by the downregulation of MARCH-I
expression. As these events are similar to those that occur
in MARCH-I KO mice, we now hypothesize that matured/activated DCs receive signals that cause the DC abnormalities indirectly, via stabilized MHC II. If this is the case, suchan inhibitory signal may prevent excessive immune reactions, which may prevent detrimental immune responses
such as occur in autoimmune diseases. Consistent with thisidea, several groups have reported on LPS-induced DC
malfunction in vivo. Taken together, our results and those ofothers lead us to propose a new model for the physiologicalrole of MARCH-I-mediated MHC II ubiquitination in the
immune system (see Fig.).

5. Ohmura-Hoshino, M., Y. Matsuki,
M. Aoki, E. Goto, M. Mito, M. Uematsu,
T. Kakiuchi, H. Hotta, and S. Ishido.
Inhibition of MHC class II expression
and immune responses by c-MIR.
J Immunol., 2006 177:341-354.

47

Team Leader

Laboratory for

Research Scientists : Kenichi Asano
Qiu Chunhong
Gen Nishitai

Innate Cellular
Immunity

Masato Tanaka

Technical Staff :Ai Kurita
Ami Nabeyama
Kumiko Ito
Minako Aihara

Administrative Staff : Mizuki ShimuraM

acrophages are key players in the initiation and regulation of inflammation. At the inflammatory site, macrophages infiltrating in response to an acute injury or
infection recognize invading microorganisms or endogenous
adjuvants released by injured cells, such as High MobilityGroup Box Chromosomal Protein 1 (HMGB1) and uric acid,through pattern-recognition receptors. In response to thisrecognition, macrophages produce inflammatory cytokinesand chemokines. Macrophages are also required for the
resolution of inflammation, which is critical for successful
tissue repair. During the late course of inflammation, infiltrated neutrophils and cell debris from tissue injury arecleared by macrophages. A defect of this clearance has
been shown to cause unremitting inflammation in acuteinjury mouse models. In addition to the resolution of inflammation, apoptotic cell clearance by phagocytes also plays acrucial role for maintenance of self tolerance. In the periphery, tissue-resident dendritic cells (DCs) constantly phagocytose apoptotic cells generated during normal tissue
turnover, and migrate to draining lymph nodes where theypresent antigens derived from the apoptotic cells. This presentation of self-antigens leads to deletion or anergy of any
self-reactive T cells, thereby providing another mechanism
to maintain T cell tolerance. Failure of apoptotic cell clearance results in autoimmune disorders. The Laboratory for
Innate Cellular Immunity is investigating the molecularmechanisms for recognition and phagocytosis of dying cellsby phagocytes, and the pathological relevance of impaired
phagocytosis to inflammatory disorders including autoimmune diseases.
Immune regulation by apoptotic cell clearance
Apoptotic cell clearance by phagocytes is essential for the
maintenance of self-tolerance under physiological conditions. Consistent with these findings, the intravenous injection of apoptotic cells can induce specific immunosuppression
or tolerance to the cell associated antigens. We previously
found that this mechanism of tolerance induction requires
the contributions of two populations of cells in the splenicmarginal zone (MZ ). The MZ contains two types of mac-

Recent publications
1. Nabeyama A, Kurita A, Asano K,
Miyake Y, Yasuda T, Miura I, Nishitai
G, Arakawa S, Shimizu S, Wakana S,
Yoshida H, Tanaka M. xCT deficiency
accelerates chemically induced
tumorigenesis. Proc Natl Acad
Sci USA.107,6436-41(2010)

2. Qiu, C. H., Miyake, Y., Kaise, H.,
Kitamura, H., Ohara, O., and Tanaka,
M. Novel subset of CD8α+ dendritic
cells localized in the marginal zone is
responsible for tolerance to cellassociated antigens. J. Immunol.
182, 4127-4136 (2009)

Figure:Brachial lymph node from wild type C57BL/6 was snap frozen in OCT. 5 μm-thickcryosection was stained for CD169 (Green), CD11c (Red) and B220 (Blue) and observed
under confocal microscope. Original magnification; x40 (A), x200 (B).

rophages, marginal metallophilic macrophages (MMM) andmarginal zone macrophages (MZM). These macrophages
contribute to rapid clearance of cellular corpses in the blood
flow. In addition, we found that CD8α+, CD103+ DCs localized in the MZ selectively phagocytose blood-borne deadcells and subsequently present dead cell-associatedantigen to induce antigen-specific immunosuppression or
tolerance.
In contrast to the outcome following intravenous injection, subcutaneously injected apoptotic cells are often
immunogenic and many investigators have taken advantage
of immunogenic tumor corpses for tumor vaccination. Thesefindings suggest that apoptotic cells in the periphery arecleared and processed in a very different way from bloodborne apoptotic cells in spleen. Antigen presentation in
peripheral lymph nodes (LNs) is thought to be a processthat is coordinately performed by migratory DCs fromperipheral tissues and LN-resident APCs. However, little is
known about the role of different APCs in the clearance ofdead cells and the presentation of dead cell-associatedantigens in peripheral tissue or LNs. We are attempthing tounderstand the molecular and cellular mechanisms of antitumor immunity by subcutaneous tumor vaccination. We
previously established transgenic mice in which CD169positive cells could be transiently depleted by diphtheria
toxin (DT ) administration (CD169-DTR mice). We found thatDT treatment selectively depleted sinus macrophages in

3. Tanaka M., Miyake Y., and Asano K.
Maintenance of self-tolerance by
apoptotic cell clearance.
Front. Biosci. 13, 6043-6049 (2008)

4. Miyake Y., Asano K., Kaise, H.,
Uemura, M., Nakayama, M.
and Tanaka, M. Critical role ofmacrophages in the marginalzone in the suppression of
immune responses to apoptotic
cell-associated antigens. J. Clin.
Invest. 117, 2268-2278 (2007)

LNs of these mice. We are now using these mice to examine
the role of sinus macrophages in the presentation of deadcell associated antigens in LNs.
Role of the anti-oxidant system in survival ofactivated macrophages
During the course of inflammation and its resolution, macrophages are exposed to various cytotoxic compounds
including reactive oxygen species (ROS). In the active phaseof inflammation, neutrophils are recruited to the inflammation site, where they produce ROS to kill the invadingorganisms. Macrophages themselves also produce ROS in
response to lipopolysaccaride (LPS). Thus, macrophages
require a protective mechanism against oxidative stress inorder to survive and accomplish the task of resolving
inflammation. We recently established a mutant mouse line
induced by N-ethyl-N-nitrosourea mutagenesis, in which
in vitro generation of macrophages and dendritic cells fromhematopoietic progenitor cells was impaired. Genetic
mapping revealed that the mutation existed in a gene related
to the intracellular anti-oxidant system. We found that thisgene was expressed in infiltrating macrophages and neutrophils at the inflammatory site, and that macrophages fromthe mutant mice showed impaired survival in response to
LPS stimulation. We are investigating the role of the antioxidant system in activated macrophages in vivo by analyzing the mutant mice.

5. Miyake Y., Kaise H., Isono K.,
Koseki H., Kohno K., and Tanaka
M. Protective role of macrophages
in non-inflammatory lung injury
caused by selective ablation ofalveolar epithelial type II cells. J.
Immunol. 178, 5001-5009 (2007)49

Unit Leader

Takashi Tanaka
Technical Staff :Emiri Haga

Administrative Staff : Sachiko Haraguchi

Research Unit for

Inflammatory
RegulationT

here are two sequential phases in the host defense
response. First is the inflammatory response, which is
triggered by inflammatory cytokines produced by dendritic
cells and is critical for eliminating invading pathogens.
Inflammatory responses also direct T-helper (Th) lymphocytes differentiation into distinct subsets, such as Th1, Th2
and Th17, to deal with different varieties of microorganisms.
The second phase is the tissue repair process during which
fibrogenesis and vascularization are induced to repair thedamaged tissues. It is well known that inflammatory
responses can be induced by Toll-like receptor (TLR)-mediated activation of the NF-κB transcription factor, while
TGFβ-mediated activation of the Smad transcription factor
is essential for tissue repair. It is important that these
responses be tightly controlled, otherwise, exaggerated
inflammation can lead to inflammatory diseases, including
rheumatoid arthritis and bronchial asthma. On the other
hand, excess tissue repair activity causes fibrotic disorders,such as pulmonary fibrosis and scleroderma. Our researchgoal is to identify key regulators of signal transduction pathways in inflammatory and tissue repair responses, and toclarify the molecular mechanisms to regulate these
responses. These studies should contribute to the development of new therapeutic tools to control human inflammatory and fibrotic disorders. Our research now focuses on
the role of PDLIM2 (PDZ and LIM-domain containing protein-2) in the negative regulation of these responses.
PDLIM2 is a negative regulator of NF-κB-mediatedsignaling.
PDLIM2, also known as SLIM (STAT-interacting protein),
was originally isolated as a molecule that interacts with the
STAT4 transcription factor. We found that PDLIM2 is a
nuclear ubiquitin E3 ligase that promotes ubiquitination andsubsequent proteasome-mediated degradation of STAT4,
thereby negatively regulating Th1 cell differentiation (Tanaka,
T. et al, Immunity, 22, 729-736, 2005). We have recently
demonstrated that PDLIM2 negatively regulates NF-κB
activity, acting as a nuclear ubiquitin E3 ligase targeting the
p65 subunit of NF-κB ( Tanaka et al, Nat. Immunol. 8, 584-

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

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

Figure : PDLIM2, a nuclear ubiquitin E3 ligase, negatively regulates both inflammatory and tissue repair responses.

591, 2007). PDLIM2 binds to p65 and promotes its polyubiquitination and targeting to discrete intranuclear
compartments, called PML nuclear bodies, where the p65 isdegraded by the proteasome. Consistent with these findings, PDLIM2 deficiency resulted in larger amounts ofnuclear p65, defective p65 ubiquitination and augmented
production of proinflammatory cytokines in response to
innate stimuli. Thus, PDLIM2 can inhibit inflammatory
responses by terminating NF-κB activation through its intranuclear sequestration and subsequent degradation. Thesefindings suggest that upregulating PDLIM2 function can beapplied for treating various inflammatory diseases. We
therefore investigated how the activity of PDLIM2 itself is
controlled. We attempted to identify the molecules thatinteract with PDLIM2 and modify its activity, and haverecently found that heat shock protein 70 (HSP70) binds to
PDLIM2 and promotes the degradation of NF-κB p65
synergistically with PDLIM2.
PDLIM2-mediated regulation of in vivo inflammatory
responses
Several in vivo models are useful for further clarifying the

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

4. Nakahira, M., Tanaka, T., Robson,
B.E., Mizgerd, J.P. and Grusby, M.J.
Regulation of signal transducer andactivator of transcription signalingby the tyrosine phosphatase PTP-BL.
Immunity 26, 163-176 (2007)

biological roles of the PDLIM2-mediated regulatory system,
and for identification of potential PDLIM2-directed therapies.
For example, granuloma formation is a type of host defense
response against intracellular bacteria. We found that bacteria-induced development of granulomatous lesions isenhanced in PDLIM2-deficient mice. Moreover, we recently
demonstrated that PDLIM2 also controls the tissue repair
process. Skin wound healing is a typical model of inflammation-related tissue repair responses. We found that woundhealing is significantly accelerated in PDLIM2-deficient mice.
Moreover, TGFβ–mediated differentiation of myofibroblasts,
the special fibroblasts essential for fibrogenesis and woundhealing, is enhanced in fibroblasts derived from PDLIM2deficient mice, which may account for the accelerated
wound healing observed in these mice. Notably, PDLIM2
bound to Smad2/3, promoting their polyubiquitination anddegradation, thus terminating TGFβ–mediated signaling.
These studies demonstrate that PDLIM2 is essential forthe negative regulation of both inflammatory and tissue
repair responses. PDLIM2 may be a new useful moleculartarget for the treatment of human inflammatory andfibrotic diseases.

5. Hoshino. K., Sugiyama, T., Matsumoto,M., Tanaka, T., Saito, M., Hemmi,
H., Ohara, O., Akira, S., and Kaisho,T. IκB kinase-α is critical forinterferon-α production inducedby Toll-like receptors 7 and 9.
Nature 440, 949-953 (2006)51

Unit Leader

Kanako Shimizu
Technical staff :Miki Asakura

Administrative Staff : Chikako Nishimura

Research Unit for

Therapeutic ModelT

he goal of our laboratory is to develop immunotherapeutic models for cancer. We have been focusing on
the role of in vivo DC maturation as a link between innate
and adaptive immunity. In particular, we have studied thebiological interactions between dendritic cells (DCs) andNKT cells. NKT cells can be stimulated with the glycolipid,
alpha-galactosylceramide (α-GalCer)-loaded on APCs. NKT
cells have unique immunoregulatory features that include
the ability to rapidly produce large quantities of cytokines.
We have been attempting to generate an approach for
inducing adaptive immunity based on the adjuvant effect ofα-GalCer and using in vivo DC maturation, which we havefound to be more effective than ex vivo manipulation of DCs.
Recently, we have developed an immunotherapeuticstrategy using α-GalCer-loaded tumor cells for enhancing
both innate (NKT and NK cells) as well as adaptive immunity( T cells). We further have been elucidating the mechanismof DC functions in detail and continue to analyze memory
T cells. In addition, we developed this approach and established a cellular vector, composed of glycolipidloaded, mRNA-transfected allogeneic fibroblasts to provide
iNKT cell activation, leading to DC maturation and T cell
immunity.
Analyses of critical role of host DCs in immunized
mice with tumor cells loaded with α-GalCer.
We have demonstrated that tumor cells loaded with
α-GalCer (tumor/Gal) are effective APCs for driving innate
and adaptive immune responses, which are able to resist
the establishment of metastases in vivo. With the strategy
of using NKT cell ligand-loaded tumor cells, we initiallyfound that tumor/Gal cells were killed by innate lymphocytes, mainly NKT and NK cells in vivo as above. Subsequently, we have continued to study the mechanisms ofactivation of adaptive immunity through in vivo maturationof DCs and to optimize tumor antigen delivery systems to
DCs. When DCs in situ were analyzed, neighboring DCs
captured dying tumor/Gal cells and then matured by activated NKT cells via CD40-CD40L interactions (Figure).
Importantly, when we analyzed the crucial characteristics

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

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

Figure:Maturing DCs capture tumor cells for cross-presentation in situ. Activated iNKT cells are responsible for DC maturationand cross presentation to T cells in tumor/Gal immunized mice. To understand the mechanism, ex vivo DCs were analyzed
by flow cytometry and confocal microscopy. The maturing DCs captured CFSE-labeled tumor debris for at least 24 h after
tumor/Gal administration. Confocal image of CD8a+DCs that have taken up the injected CFSE-labeled tumor cells.

of DCs in situ in the immunized mice, we have found thatthese cells can play a role in cross presentation of tumor
antigen and glycolipid to T cells and NKT cells, respectively(two types of cross-presentation). After tumor regression
occurred, a variety of T cells responding to a variety of
specific tumor antigens, such as Trp2, Tyrp, Dct and gp100,
persisted as memory T cells.
Antigen mRNA-transfected, allogeneic
fibroblasts loaded with NKT cell ligand confer antitumor immunity.
In the above studies, we demonstrated that the activation ofinvariant (i) NKT cells by administration of tumor/Gal cells
can act as a cellular adjuvant through the maturation of
DCs. In the current study, instead of tumor cells, we usedallogeneic fibroblast cells loaded with α-GalCer and transfected with antigen-encoding mRNA, thus combining the

3. Shimizu K, Fujii S. Adjuvant role ofin situ dendritic cells (DCs) in linking
innate and adaptive immunity. Front
Biosci. 13, 6193-201 (2008)

4. Fujii S, Shimizu K, Hemmi H,
Steinman RM. Innate Vα14+ naturalkiller T cells mature dendritic cells,
leading to strong adaptive immunity.
Immunol Rev. 220, 183-98 (2007)

adjuvant effects of iNKT cell activation with delivery of antigen to DCs in vivo. We found that these cells produce antigen protein and activate NK and iNKT cells. When injected
into MHC mismatched mice, they elicited antigen-specific
T cell responses and provided tumor protection, suggestingthat these immune responses depend on host DCs. Also,
antigen expressing fibroblasts loaded with α-GalCer lead toa more potent T cell response than those expressing NK cell
ligands. Thus, glycolipid-loaded, mRNA-transfected allogeneic fibroblasts act as cellular vectors to promote iNKT cell
activation, leading to DC maturation and T cell immunity.
By harnessing the innate immune system and generating
an adaptive immune response to a variety of antigens,this unique tool could prove clinically beneficial in the development of immunotherapies for malignant and infectious
diseases.

5. Shimizu K, Kurosawa Y, Taniguchi
M, Steinman RM, Fujii S.
Cross-presentation of glycolipid
from tumor cells loaded with
α-galactosylceramide leads topotent and long-lived T cell-mediatedimmunity via dendritic cells.
J. Exp. Med. 204, 2641-53 (2007)

6. Shimizu K, Goto A, Fukui M,
Taniguchi M, Fujii S. Tumor cells
loade with α-galactosylceramide
induced innate NKT and NK
cell-dependent resistance totumor implantation in mice. J
Immunol. 178, 2853-2861 (2007)

53

Group Director

Masaru Taniguchi
Senior Research Scientist : Hiroshi Watarai
Research Scientists :Takuya Tashiro
Michishige Harada

Visiting Scientist :Nyambayar Dashtsoodol

Technical Staff :Sakura Sakata
Etsuko Sekine-Kondo
Yuko Nagata
Sayo Inoue
Tomokuni Shigeura
Natsuki Kobayashi

Student Trainees :Naomi Hongo
Yohei Yoshiga

Administrative Staff :Norie Takeuchi

Laboratory for

Immune Regulation
C

D1d-restricted invariant natural killer T (NKT ) cells
bridge innate and acquired immunity and play animportant role in both protective and regulatory responses.
The nature of the response is dictated by the initial cytokine
environment: interaction with IL-10-producing cells induces
negative regulatory Th2 T cell-type NKT cells, while thatwith IL-12-producing cells results in pro-inflammatory Th1type responses. Particularly in the anti-tumor response,
NKT cells mediate adjuvant activity by their production of
IFN-γ, which in turn activates both innate and acquired
immune systems. Thus, upon activation of NKT cells, both
MHC - and MHC+ tumor cells can be efficiently eliminated.
On the basis of these mechanisms, NKT cell-targeted adjuvant cell therapies have been developed and have shown
great promise in initial clinical trials on cancer patients.

Identification of CD4-CD8- double-negative naturalkiller T cell precursors in the thymus.
It is well known that CD1d-restricted Vα14 NKT cells arederived from cells in the CD4+CD8+ double-positive (DP)population in the thymus. However, the developmental
progression of NKT cells during the earlier stages remains
unclear, and the possible existence of NKT cell presursors
in stages earlier than the DP stage had not been tested. We
were able to demonstrate that NKT cell precursors expressing invariant Vα14-Jα18 transcripts but devoid of surface
expression of the Vα14 receptor are present in the late
CD4-CD8- double-negative (DN) 4 stage and have thepotential to generate mature NKT cells in both in vivo and invitro experimental conditions. Moreover, the DN4 population with NKT cell potential in CD1dKO mice was similar tothat in wild-type C57BL/6 mice, but failed to develop into
NKT cells in vitro. However, these precursors could develop
into NKT cells when co-cultured with normal thymocytes orin an in vivo experimental setting, indicating that functional
NKT cell precursors are present in CD1dKO mice. Together,
these results demonstrate that thymic DN4 fraction contains
NKT cell precursors. Our findings provide new insights into
the early development of NKT cells prior to surface expression of the invariant Vα14 antigen receptor and suggest a

Recent publications
1. Matsuoka N, Itoh T, Watarai H,
Sekine-Kondo E, Nagata N,
Okamoto K, Mera T, Yamamoto H,
Yamada S, Maruyama I,Taniguchi M,
YasunamiY. High-mobility group box 1
is involved in the initial events of earlyloss of transplanted islets in mice. J.
Clin. Invest. 120 (3): 735-743 (2010).

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

Donor islets

Figure:HMGB1 shuttled from nucleus to cytoplasm of islets after transplantation.

possible alternative developmental pathway for NKT cells.
Generation of functional NKT cells in vitro fromembryonic stem cells bearing a rearranged invariant
Vα14-Jα18 TCRα gene.
Establishment of a system for efficient generation of NKT
cells from embryonic stem (ES) cells would enable us to
identify the cells with NKT-cell potential and obtain NKT cellswith desired function. Using cloned ES (NKT-ES) cells generated by the transfer of nuclei from mature NKT cells, wewere able to establish a culture system that preferentiallydeveloped functional NKT cells. We also identified early
NKT progenitors, which first appeared on day 11 as a c-kit+
population in cocultures on OP9 cells expressing Notch
ligand, delta-like1 (OP9/Dll-1). The progenitor cells became
c-kit lo/- on day 14. Interestingly, in the presence of Notch
signals, NKT-ES cells differentiated only into thymic-type
CD44 lo CD24 hi NKT cells producing mainly interleukin-4(IL-4). By contrast, NKT cells resembling CD44 hi CD24 loliver NKT cells producing mainly interferon gamma (IFN-γ )and exhibiting strong adjuvant activity in vivo developed inswitch cultures starting at day 14 in the absence of Notch.
The cloned ES culture system offers a new opportunity forthe elucidation of the molecular events during NKT-celldevelopment and for the establishment of NKT-cell therapy.
High-mobility group box 1 is involved in the initial
events of early loss of transplanted islets in mice.

3. Iwata A, Watanabe N, Oya Y, Owada T,
Ikeda K, Suto A, Kagami S, Hirose K,
Kanari H, Kawashima S, Nakayama T,
Taniguchi M, Iwamoto I, Nakajima H.
Protective roles of B and T
lymphocyte attenuator in NKT
cell-mediated experimental hepatitis.
J. Immunol. 184 (1):127-133 (2010).

Transplanted islets
in the liver

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

Islet transplantation for the treatment of type 1 diabetes
mellitus is limited in its clinical application mainly due toearly loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell–dependent IFN-γ production
by Gr-1+ CD11b+ cells is essential for this loss, but the
upstream events in the process remain undetermined.
We have demonstrated that high-mobility group box 1(HMGB1) plays a crucial role in the initial events of earlyloss of transplanted islets in a mouse model of diabetes.
Pancreatic islets contained abundant HMGB1, which was
released into the circulation soon after islet transplantation
into the liver (Fig.). Treatment with an HMGB1-specificantibody prevented the early islet graft loss and inhibited
IFN-γ production by NKT cells and Gr-1+CD11b+ cells.
Moreover, mice lacking two of the known HMGB1 receptors, TLR2 and receptor for advanced glycation end products (RAGE), but not the third HMGB1 receptor TLR4, failed
to exhibit early islet graft loss. Mechanistically, HMGB1
stimulated hepatic mononuclear cells (MNCs) in vivo and invitro; in particular, it upregulated CD40 expression andenhanced IL-12 production by DCs, leading to NKT cell
activation, and subsequent NKT cell–dependent augmented
IFN-γ production by Gr-1+ CD11b+ cells. Thus, treatment witheither IL-12– or CD40L-specific antibody prevented
the early islet graft loss. These findings indicate that the
HMGB1-mediated pathway eliciting early islet loss is apotential target for intervention to improve the efficiency of
islet transplantation.

5. Burrows PD, Kronenberg M,
Taniguchi M. NKT cells turn ten.
Nat. Immunol. 10 (7):669-671(2009).

55

Team Leader

Katsuaki Sato
Technical Staff :Kaori Sato
Yumiko Sato
Kawori Eizumi

Student Trainee :Tomohiro Fukaya (JRA)
Hideaki Takagi (JRA)Honami Taya

Administrative Staff : Mai Yamamoto
Naomi Uchimura

Laboratory for

Dendritic Cell
Immunobiology
Dendritic cells (DCs) are essential antigen-presenting
cells (APCs) that initiate primary immune responses.
DCs consist of heterogeneous subsets, including conventional DCs (cDCs) and plasmacytoid DCs (pDCs), distinguishable by surface and intracellular phenotypic markers,
immunologic function, and anatomic distribution. ImmatureDCs (iDCs) serve as sentinels, recognizing the presence of
invading pathogens through various pattern-recognition
receptors. Subsequently, they become mature DCs (mDCs)
with up-regulated expression of MHC and costimulatory
molecules under inflammatory conditions. The mDCs thenmove via the afferent lymphatics into the T-cell areas of secondary lymphoid tissues, where they prime rare antigenspecific naive T cells for differentiation into effector T cells(Teff ), including T helper type (TH )1 cells, TH2 cells, and TH17
cells, depending on environmental cues. DCs thereby playa crucial role in the link between innate and adaptive immunity. Conversely, accumulating evidence suggests that systemic iDCs are also crucial for the induction of
immunological tolerance under steady-state conditions.
The tolerogenic mechanisms include recessive tolerance
mediated by clonal deletion and anergy as well as dominant
tolerance involving active immune suppression by
CD4+Foxp3+ regulatory T cells (Tregs ) in the periphery, a function of likely importance in self-tolerance as well as immune
disorders and transplant rejection. However, the precise
functional role of each DC subset in immune responses
remains unclear. Our goal is to characterize the specific DC
subsets involved in immune regulation.
Role of the B7 family of costimulatory molecules
expressed on systemic and mesenteric lymph
node dendritic cells in the differentiation of
Foxp3+regulatory T cells and TH17 cells
In this study, we examined the role of the B7 family of
costimulatory molecules in the function of dendritic cells(DCs) in circulation and in mesenteric lymph nodes (MLNs)
to induce differentiation of antigen-specific naive CD4+Foxp3T cells into CD4+Foxp3+ inducible regulatory T cells (iTregs )and interleukin (IL)-17-producing CD4+T cells ( TH17 cells).

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

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

Figure:Antigen-specific de novo conversion of CD4+Foxp3-T cells into CD4+Foxp3+iTregs in mesentericlymph nodes during oral tolerance. KJ1-26+Foxp3 EGFP-T cells were transferred into BALB/cmice, and the animals were subsequently fed PBS or OVA protein one day later. Cryosections
from mesenteric lymph nodes on day 11 after the adoptive transfer were stained for KJ1-26 (red)and B220 (blue). Foxp3 was detected by green fluorescence from the Foxp3EGFP knock-in allele.

In wild-type (WT) mice, MLN DCs displayed significantly
higher levels of B7-H1 and B7-DC as well as I-A /I-E andCD103 than systemic DCs, whereas they showed similarlevels of CD80, CD86, and B7-H2. MLN DCs markedly
enhanced the generation of CD4+Foxp3+iTregs from
CD4+Foxp3-T cells, whereas, relative to systemic DCs, theysignificantly reduced the conversion of CD4+Foxp3-T cells
into TH17 cells. Analysis of mice deficient in B7 family
costimulatory molecules (B7-/-mice) suggested that B7-H1
and B7-DC expressed on systemic and MLN DCs are
required for the conversion of CD4+Foxp3-T cells into
CD4+Foxp3+iTregs, whereas costimulation with CD80/CD86
through CD28 abrogated this conversion. In contrast, theinduced differentiation of TH17 cells by these DCs wasenhanced in B7h1-/-mice and B7dc-/-mice or decreased in
Cd80/Cd86-/-mice and B7h2-/-mice compared with WT
mice, suggesting that B7-H1 and B7-DC suppress TH17 celldifferentiation, whereas CD80/CD86 and B7-H2 participate
in this development. Collectively, the expression of B7-H1
and B7-DC could be essential for the ability of MLN DCs to
induce the preferential differentiation of iTregs rather than TH17
cells. Thus, our findings suggest that the fine-tuning of iTregs/ TH17 cell equilibrium by MLN DCs through the B7 family is
instrumental in the maintenance of intestinal immune
homeostasis.

and neurotoxicity. Clin. Cancer
Res. 15, 4401-4414 (2009)

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

Essential roles of B7-H1 and B7-DC in theestablishment of oral tolerance mediated throughthe de novo generation of antigen-specificCD4+Foxp3+inducible regulatory T cells
Oral tolerance is a key feature of intestinal immunity, generating systemic tolerance to ingested antigens, however, themolecular mechanisms mediating oral tolerance remain
unclear. In this study, we examined the role of the B7 family
members of costimulatory molecules in the establishment oforal tolerance. Deficiencies of B7-H1 and B7-DC abrogatedoral tolerance; this was accompanied by enhanced antigenspecific CD4+T cell-response and IgG1 production. Furthermore, the antigen-specific conversion of CD4+Foxp3-T cells
into CD4+Foxp3+inducible regulatory T cells (iTregs ) occurredin MLNs to a greater extent than in peripheral organs during
oral tolerance, and this conversion required B7-H1 and
B7-DC, whereas it was severely impaired under inflammatory conditions. Together, B7-H1 and B7-DC appear to benecessary for inducing active immune suppression mediated through the de novo generation of antigen-specificCD4+Foxp3+ iTregs under steady-state conditions, thereby
leading to the establishment of oral tolerance.

4. Fujita S., Sato Y., Sato K., Eizumi K.,
Fukaya T., Kubo M., Yamashita N.,
Sato K. Regulatory dendritic cells
protect against cutaneous chronic
graft-versus-host disease mediated+++through CD4 CD25 Foxp3 T cells.
Blood 110, 3793-3803 (2007)

5. Sato K., Fujita S. Dendritic
Cells-Nature and Classification.
Allergol. Int. 56, 183-191 (2007)

57

Team Leader

Heiichirô Udono
Research Scientists : Shusaku Mizukami
Takashi Imai
Technical Staff :Laboratory for

Immunochaperones

Chiaki Kajiwara
Hidetoshi Sugahara

Administrative Staff : Akiko Imaizumi

C

D8+ T cells recognize naturally processed peptides in
the context of MHC class I molecules. Peptides presented by MHC class I molecules are products of cellular
proteins degraded by the proteasome. Ribosomal de novo
synthesized, misfolded proteins (defective ribosomal products; DRiPs) are recognized by molecular chaperones (heatshock proteins:HSPs), hsc70/hsp40 and hsp90, and are
believed to be polyubiquitinated by the E3 ubiquitin ligase,
CHIP (carboxyl terminus of hsc70 interacting protein).
These ubiquitinated DRiPs are degraded by the proteasomeand have been implicated as the main source of antigen
peptides presented by MHC class I molecules.
During viral infection or malignant transformation, intracellular changes in non-APC (antigen presenting cells) must
be reported to CD8+ T cells, a process that is indispensable
to fighting virus-infected cells and cancer cells. However,the cancer cells cannot act by themselves as APC to prime
T cells, although they express tumor antigens on the cellsurface. Moreover, APC such as DCs (dendritic cells) do not
become infected by every virus, and thus, cannot present
all viral antigens directly to CD8+ T cells. Therefore, DCsmust internalize nearby tumor or infected cells, digest them,and then present antigen peptides to CD8+ T cells in thecontext of MHC class I molecules. This pathway is calledcross-presentation and is believed to be specific to DCs,
not other APC types such as macrophages or B cells.
We hypothesize that molecular chaperones such as
hsp90 also play important roles in this antigen cross-presentation. Our research goal is to identify the mechanismsof proteasome-mediated epitope production and crosspresentation, especially focusing on the role of the molecular chaperone hsp90.
The role of hsp90 in endogenous MHC I antigen
processing
Heat shock protein 90 (hsp90) and the proteasome activator, PA28, stimulate MHC class I antigen processing. However, whether hsp90 influences the proteasome activity to
produce T cell epitopes was unknown, although association of PA28 with the 20S proteasome stimulates its enzyme
activity.

Recent publications
1. Ohkusu-Tsukada K, Toda M, Udono
H, Kawakami Y, Takahashi K. Targeted
inhibition of the p38 MAP kinase of
IL-10-secreting CD25 T regulatory
cells in cancer immunotherapy.
Eur. J. Immunology 2010 in press.

2. Udono H, Ichiyanagi T, Mizukami
S, Imai T. Heat Shock Proteins in
Antigen Trafficking -Implications
on Antigen Presentation to T
cells. International Journal ofHyperthermia 25, 617-625, 2009.

4. exogenous
proteins
2. endogenous proteinstranslocation

Figure:HSPs
endosome

HSPs

HSPs

proteolysis

folding
HSPs

proteolysis

1. newly synthesized
proteins on ribosome

recycling ofamino acids

ER
HSPs

3. degradation
products

We demonstrated that hsp90 is essential for assemblyof the 26S proteasome and, as a result, is involved in epitope
production. Addition of recombinant hsp90α to cell lysates
enhanced chymotrypsin-like activity of the 26S proteasome
in an ATP dependent manner. We pulled down histidinetagged hsp90α-and PA28α-induced, newly assembled 26S
proteasomes from cell extracts for in vitro epitope production assays, and found that these structures are sensitive togeldanamycin, an hsp90 inhibitor. We found that a cleaved
epitope unique to the proteasome was pulled down by bothhsp90α and PA28α, while two different epitopes wereidentified in the hsp90α and PA28α pulldowns, respectively.
Processing of these respective peptides in vivo wasenhanced faithfully by the protein combinations used forthe proteasome pulldowns. Inhibition of hsp90 in vivo bygeldanamycin partly disrupted the 26S proteasome
structure, consistent with downregulated MHC class I
expression. Thus, hsp90 facilitates MHC class I antigen
processing through epitope production in a complex of the
26S proteasome.
The role of hsp90 in antigen cross-presentation
In general, the main functions of HSPs are (i) facilitate protein folding/refolding and prevent aggregation of newly synthesized proteins on the ribosome; (ii) maintain the normal
function of mature proteins; (iii) target misfolded and/or

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

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

HSPs mediate degradationof a few percent of newly
synthesized proteins as well as
mature proteins. In dendritic
cells (DC), HSPs seem tofacilitate the degradation of eveninternalized exogenous proteins
following their translocation to
the cytoplasm. The proteolysis
step depends on the ubiquitinproteasome system to produce
peptides presentable by
MHC class I molecules.

MHC class I

damaged mature proteins for degradation by the proteasome; (iv) transport proteins between distinct microorganisms. All proteins denoted here ultimately undergo
degradation by the proteasome in order to recycle aminoacids and their by-products (degradation products, peptides) are simultaneously utilized as antigen peptides mainlypresented by MHC class I molecules. Thus, the use of proteasomal degradation products as ligands of MHC molecules makes perfect sense in the immune system to alert
the T cell immune system to any antigenic alterations, aswell as life cycle of all proteins with no waste.
Each step towards proteolysis by the proteasome is, atleast in part, regulated by HSPs. To consider how HSPs are
involved in cross-presentation (in terms of translocation ofendosomal antigen to cytosol), there could be two distinct
mechanisms. First is that HSPs direct CHIP mediated polyubiquitinylation of retro-translocated unfolded proteins forproteasomal degradation. Second is that HSPs direct retrotranslocation of proteins from endosome to cytosol, a model
supported by the findings that Bip and cytosolic hsp70/
hsp90 drive polypeptide into the ER and the mitochondria,
respectively. Our recent observations clearly indicate that
HSPs play a pivotal role in antigen trafficking, an activity thatlinks them to antigen cross-presentation as well as endogenous antigen processing.

5, Mizukami S, Kajiwara C, Hiroshi
Ishikawa H, Katayama I, Yui K, and++Udono H. Both CD4 and CD8 T
cell epitopes fused to heat shock
cognate protein 70 (hsc70) canfunction to eradicate tumors. Cancer
Science 99,1008-1015, 2008.

59

Research Unit for

Unit Leader

Shohei Hori
Research Scientists : Masashi Tachibana
Maria Encarnita Mariotti-Ferrandiz

Immune Homeostasis(JSPS Foreign Post-Doctoral Fellow)

Technical Staff :Takahisa Miyao
Nana Shimizu
Erina Furuhata
Guanying WangA

small subpopulation of T lymphocytes known as
regulatory T ( Treg ) cells play a central role in preventing
pathological immune responses including autoimmunity,
inflammation and allergy, and thus ensure dominant tolerance to self and innocuous environmental antigens. Thishas been well illustrated by the findings that the development and function of Treg cells is controlled by the X-linked
transcription factor Foxp3 and that defective generation offunctional Treg cells underlies the catastrophic autoimmune
pathology that develops in Foxp3-mutant scurfy mice andhuman patients with the IPEX syndrome.
The identification of Foxp3 as a central regulator of Treg
cell differentiation and function has provided a key to anumber of outstanding unresolved questions concerning
their role in tolerance and immune regulation, and thephysiology of these cells including their origins, the mechanisms controlling their development and function, and theirantigen specificity. Resolving these issues is the goal of thislaboratory.
Stability and plasticity in regulatory T celldifferentiation
Early studies of organ-specific autoimmunity provoked byneonatal thymectomy, and of transplantation tolerance
induced by previous transplantation of xenogeneic or allogeneic thymic epithelium have suggested that natural
Foxp3+ Treg cells represent a distinct thymus-committed
lineage of T lymphocytes, although it has also become evident that additional Foxp3+ Treg cells can be generated fromperipheral naïve CD4+ T cells upon “tolerogenic” antigen
presentation. Recent findings have challenged, however,this notion of a stable lineage and suggested that Treg cells
may be a flexible population exhibiting developmental plasticity. To reconcile these apparently contradictory views, we
examined the stability of Foxp3 expression and Treg phenotype in natural Foxp3+ T cells isolated from un-manipulated
Foxp3-reporter mice. Our results showed that, whereas the
majority of natural CD4+Foxp3+ T cells maintain stable
Foxp3 expression after adoptive transfer to lymphopenic or
lymphoreplete recipients, a minor fraction enriched within
the CD25- subset actually lose expression. Some of those
Foxp3- T cells adopt effector helper T (Th ) cell functions. Of

Recent publications
1. Kim, J., Lahl, K., Hori, S.,
Loddenkemper, C., Chaudhry A.,
deRoos, P., Rudensky, A., andSparwasser, T. Cutting Edge:+
Depletion of Foxp3 cells leads
to induction of autoimmunity by
specific ablation of regulatory T
cells in genetically targeted mice. J.
Immunol. 183, 7631-7634 (2009)

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

Figure:note, they preferentially differentiated into follicular B
helper T ( TFH ) cells that promoted IgA production in thePeyer’s patches. This minority “plastic” population exhibits
flexible responses to cytokine signals, relying on TGF-β tomaintain Foxp3 expression, and responding to others bydifferentiating into effector Th cells in vitro. In contrast,
CD4+Foxp3+CD25high T cells are resistant to such conversion
to effector Th cells even after many rounds of cell division.
These results demonstrate that natural Foxp3+ T cells are aheterogeneous population consisting of a committed Treg
lineage and an uncommitted subpopulation with developmental plasticity.
To address the origin, nature, and functions of thesecommitted and plastic populations under physiological andpathological conditions, we have recently established amouse model that allows us to track previous history of
Foxp3 expression as well as ongoing Foxp3 transcription.
Our recent analyses revealed that plastic Foxp3+ T cells arecontained predominantly, if not exclusively, within peripherally generated Foxp3+ T cells, whereas thymus-derived
Foxp3+ T cells that are stably incorporated into the peripheral pool exhibit remarkably stable Foxp3 expression (Fig.).
We suggest that the plasticity of Foxp3+ T cells contributes
to the maintenance of a delicate balance between immune
responsiveness and tolerance to non-self antigens in theface of environmental perturbations, whereas the stability of
the Treg lineage ensures robust tolerance to self-antigens
even under inflammatory environments. The molecular andepigenetic basis of Treg lineage commitment in the thymus isone of the next focus areas for our research.
Dissecting the Foxp3-dependent molecular programof Treg cell development and function
In order to understand the molecular mechanisms by which
Foxp3 controls Treg cell differentiation and function, we havebeen addressing whether and how Foxp3 mutations that
had been identified in IPEX patients impair Treg cell develop-

3. Komatsu N., Mariotti-Ferrandiz
M.E., Wang Y., Malissen B.,
Waldmann H., Hori S. Heterogeneity+of natural Foxp3 T cells: acommitted regulatory T celllineage and an uncommitted minor
population retaining plasticity.
PNAS 106, 1903-1908 (2009)

4. Hori, S. Rethinking the moleculardefinition of regulatory T cells.
Eur. J. Immunol. 38, 928-930 (2008)

A model for stability and plasticity in regulatory T
cell differentiation. Immature thymocytes up-regulate
Foxp3 upon recognition of self-antigens presented
by thymic stromal cells. Although some of Foxp3+
thymocytes may fail to stabilize Foxp3 expression andthus undergo abortive Treg differentiation, thymusgenerated Foxp3+ T cells that are stably incorporated
into the peripheral Foxp3+ T cell pool exhibit stable
Foxp3 expression and are thus irreversibly committed,
thereby ensuring tolerance to self-antigens. In contrast,
Foxp3+ T cells generated from peripheral naïve
Foxp3-CD4+ T cells show unstable Foxp3 expression
and can be converted into other effector Th cells uponsuitable environmental stimuli, thereby contributing
a delicate balance between immune responsivenessand tolerance to environmental (non-self ) antigens.

ment and function. We previously showed that all the missense mutations examined were amorphic or hypomorphic
in that, upon retroviral transduction, these mutants fail toconvert conventional T cells into fully functional Treg cells. To
determine the impact of these mutations in vivo, we haveestablished three independent knock-in mouse strains harboring foxp3 mutations that mirror those in human IPEX. We
found that hemizygous mutant males developed a scurfylike lymphoproliferative autoimmune disease, although miceharboring hypomorphic mutations developed a milder disease than mice with scurfy or amorphic mutations. Furthermore, by analyzing healthy heterozygous female mice, inwhich mutant and wild-type Foxp3+ Treg cells co-exist (dueto random X-chromosome inactivation), we found that twoof the three mutations compromised thymic differentiation,
peripheral maintenance, and suppressive effector functionsof Foxp3+ Treg and affected overall transcriptional profiles. In
contrast, Treg cells expressing one of the mutants displayed
normal thymic differentiation, normal in vitro suppressor
function and a transcriptional profile similar to that of wildtype Treg cells. Interestingly, however, the mutant Treg cells
were less competitive than wild-type Treg cells in the periphery and showed altered expression of a small subset of
Foxp3 target genes, suggesting that impaired peripheral
homeostasis of Treg cells underlies the autoimmune pathology in this mutant strain. We are currently investigating
which Foxp3 target gene(s) are responsible for this homeostasis defect and dissecting the molecular mechanisms that
control Treg cell homeostasis.
One of the key questions regarding Treg cells is to understand how Foxp3 controls multiple aspects of Treg cell biology. By learning from experiments in nature, i.e. naturally
occurring foxp3 gene mutations identified in IPEX patients,
we hope that we will be able to dissect the molecular mechanisms by which Foxp3 controls Treg development, function
and homeostasis in vivo.

5. Komatsu, N. and Hori, S.
Full restoration of peripheral+
Foxp3 regulatory T cell poolby radioresistant host cells in
scurfy bone marrow chimeras.
PNAS 104, 8959-8964 (2007)

61

Group Director

Osami Kanagawa
Senior Research Scientist : Michio Tomura
Research Scientists :Naoki Ogawa
Yosiko Mori

Technical Staff :Hidemi Uchiyama
Mitsuyo Takase
Takako Kato
Edith Kanagawa
Gyobai Yu
Saori Misawa

Admimnistrative Staff :Izumi Inoue
Chiyo Takahashi

Laboratory for

Autoimmune
RegulationIn my laboratory, we are developing new technologies forthe analysis of immune responses in vivo and applying
these technologies for the understanding of the autoimmune process. We have established, at present, four new
in vivo imaging technologies: 3D visualization of target organ and cells, monitoring cellular migration/emigration using the photoconvertible fluorescence protein “Kaede”, invivo visualization of cell death with the caspase 3 indicator
SCAT3.1 fluorescence protein, and in vivo visualization oflymphocytes / hematopoietic cell cycle using Fucci fluorescence protein. These technologies can be used to identify
lymphocytes in vivo based on their location, movement andfunctions. Since we have the technology to establish mouselines derived from a single lymphocyte using nuclear transfer, it is now possible to isolate lymphocytes directly fromanimals using these imaging technologies and establish amouse line from a single lymphocyte. These technologies
will be used in all aspects of immunology research. Our
focus is to apply these technologies to understanding thedevelopment of autoimmune type I diabetes in the NOD
mouse model.
Although KAEDE, SCAT3.1 and Fucci imaging systems
are very useful for in vivo imaging, these transgenic mice express fluorescence protein in all cell types due to the use ofviral promoter. This ubiquitous expression of fluorescence
protein often caused a problem with very high background.
Thus, we made knockin (KI) mouse lines in which expression of these proteins can be induced with tissue specific
Cre transgenes. These mouse lines will provide better vivo
imaging systems for cells and tissues of interest.
Monitoring Molecular Movement using Diffracted X-ray
tracking (DXT ) has been undertaken in collaboration with
Drs. Kozono (Tokyo Science Univ.) and Sasaki (Spring8).
DTX can detect sub-micro second level movements of proteins in solution and has been shown to be a useful tool tovisualize molecular movement in physiological conditions.
We used this technology to monitor the movement of the
MHC/peptide complex and found that the movement of thepeptide in the groove is relatively constant among peptides
with different affinity to the MHC. However, movement of

Recent publications
1. Tomura M, Itoh K, Kanagawa O.
Naive CD4+ T Lymphocytes Circulate
through Lymphoid Organs To
Interact with Endogenous Antigens
and Upregulate Their Function. J
Immunol. 184, 4646-53, 2010

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

B Lymphoma

Figure :Comparison of the original
B lymphoma and thelymphoma-derived ES cells.
In vitro cultured cells (left panel)and in vivo tumor/teratoma
formation (right panel)

Lymphoma derived ES cell
MHC differs significantly depending on the peptide in thegroove. This is the first demonstration that peptides boundto the MHC influence the structural stability of the MHC/
peptide complex. Moreover, analysis of the relationshipbetween T cell recognition of MHC/peptide complex andmovement of MHC / peptide complex revealed the following new information. 1) T cell recognition of the complex isgreatly affected, either positively or negatively, by the movement of whole complex. Some T cells prefer a stable complex but others respond exclusively to unstable complexes.
2) comparison of the movement of two low affinity peptides
in the groove revealed distinct differences. This change canalso influence T cell reactivity by more than 100 fold. These
results indicate that the TCR is extremely sensitive to themolecular movement of the MHC/peptide complex. Since
activation of T cells by unstable MHC/peptide is a uniquefeature of diabetes related MHC class II ( I-Ag7 in mouse and
DQ2 and DQ8 in human), our observations here should provide new insights into the TCR/MHC/peptide interactions
that control pathogenic T cell activation for the developmentof autoimmune diabetes.
Pluripotent stem cells (PSCs) provide a promisingsource of cells for the treatment of a wide range of diseases,
particularly in tissue regeneration and repair. To date, PSCs
have been successfully derived from differentiated cells, inthe normal or diseased state, using either a nuclear transfer
or a gene expression protocol. While tumor-derived stem

3. Tomura M, Honda T, Tanizaki H,
Otsuka A, Egawa G, Tokura Y,
Waldmann H, Hori S, Cyster JG,
Watanabe T, Miyachi Y, Kanagawa O,
Kabashima K. Activated regulatory
T cells are the major T cell typeemigrating from the skin during acutaneous immune response in mice.
J Clin Invest. 120 (3):883-93, 2010

4. Tomura M, Mori YS, Watanabe R,
Tanaka M, Miyawaki A, Kanagawa
O. Time-lapse observation ofcellular function with fluorescentprobe reveals novel CTL-target
cell interactions. Int Immunol.
(10) :1145-50, 2009

cells may have significant potential in advancing cancer
research and drug development, there were reported limitations with the reprogramming of various tumor cells to thestem cell state. In collaboration with Dr. Wakayama’s groupat CDB Kobe RIKEN, we demonstrate that PSCs could
indeed be established from a B- and a T-lymphomas at
frequencies comparable to those with normal lymphocytes.
The PSCs generated from a Myc-induced B lymphoma
exhibited gene expression patterns and pluripotency characteristics similar to those of normal ES cells (A comparison
between the original tumor and ES cells can be seen in theFig.). The PSCs can differentiate into tumor cells in vitro thatgrow autonomously and form tumors in vivo. Unexpectedly,
while the generation of the parental B lymphoma cells is
Myc-dependent, the progeny tumor cells have evolved to
be Myc-independent. These results show that the genetic
factors that promote tumor initiation are distinguished fromthose required to maintain tumorigenicity at later stages.
Thus, understanding the mechanism of oncogenesis requires a more comprehensive approach with additional
emphases placed at the later stages of tumor development.
Our study also shows that PSCs derived from tumors could
be a unique and powerful tool for cancer research. We are
currently attempting to establish cancer derived iPS in human system in collaboration with Dr. Yamanaka’s group at
Kyoto University.

5. Hong H, Takahashi K, Ichisaka T,
Aoi T, Kanagawa O, Nakagawa M,
Okita K, Yamanaka S. Suppressionof induced pluripotent stem cell
generation by the p53-p21 pathway.
Nature. 460 (7259):1132-5, 200963

Laboratory forGroup Director

Toshio Hirano
Senior Research Scientist : Toshiyuki Fukada
Keigo Nishida
Research Scientists :Satoru Yamasaki
Wakana Ohashi(Research Associate)

Technical Staff :Ayumi Ito
Masami Kawamura
Mayumi Hara

Student Trainee :Shintaro Hojyo (JRA)Byn Beum-Ho (JRA)Aiko Hasegawa

Administrative Staff :Ryoko Masuda
Mizuki Shimura

Cytokine SignalingD

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

Role of zinc and its transporter in mastcell-mediated allergic reactions
We are investigating the role of Zn of mast cells in allergy
and immune responses. Previously, we have used theZn chelator TPEN to show that Zn is required for Fc
epsilon RI-induced granule translocation to the plasma
membrane, mast cell degranulation, and cytokine
production (Kabu et al., J Immunology. 2006). In addition,
we found that Zn functions as a second messenger capableof converting an external signal into internal events ( Yamasaki et al., J Cell Biol. 2007). When we generated an external signal in mast cells by cross-linking IgE receptors, weobserved an internal wave of free Zn released from thevicinity of the ER within several minutes after stimulation.
These reports suggested that Zn is involved in Fc epsilon
RI-mediated mast cell function. Based on this finding, weare analyzing in role of zinc transporters in mast cell-mediated allergic reactions. We reported that a Zn transporter,
Znt5/Slc30a5 is required for Fc epsilon RI-mediated cytokine
production, but not degranulation. As a result, Znt5-/- mice
had defects in mast cell-mediated delayed-type allergic
reactions but not in the immediate-type reaction. Znt5 was
required for Fc epsilon RI-induced translocation of PKC to
the plasma membrane and the nuclear translocation of NF-

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

2. Sawa Y., Y. Arima, H. Ogura,
C. Kitabayashi, J-J Jiang,
T. Fukushima, D. Kamimura,T. Hirano, and M. Murakami.
Hepatic Interleukin-7 Expression
Regulates T Cell Responses.
Immunity 30, 447-457, (2009)

Figure 1: Role of the zinc transporter, Znt5/Slc30a5
in Fc epsilon RI-mediated signaling pathway
Znt5 is selectively required for Fc epsilon
RI-mediated cytokine production, andis a novel player in PKC signaling.

Figure 2: Significant role of the zinc transporter Slc39a13/Zip13 in connective tissue development.
A. Left: Slc39a13-KO mice show abnormal tooth development and reduction of bone mass. Right:
Slc39a13-KO mice develop kyphosis, and show impairment of cartilage development.
B. Nuclear localization of Smad proteins (green) mediated by BMP4 stimulation is
impaired in Slc39a13-KO osteoblasts. Nuclei (blue), actin (red)C. Involvement of Slc39a13/Zip13 in BMP/TGF-β signaling pathways. Slc39a13 protein, which is expressed in osteoblasts,
odontoblasts, chondrocytes, and fibroblasts, controls nuclear translocation of Smad transcription factors.

kappa B. Thus, Znt5 is selectively required for mast cellmediated delayed-type allergic responses, and is a novel
player in PKC signaling (Fig. 1, Nishida et al., J. Exp. Med.
2009).
Role of Zn and its transporters in immune andnon-immune tissues
We found that the Zn transporter Slc39a6 /Zip6 / Liv1 is a
STAT3 target gene and showed that it has a role in cell
migration during early zebrafish development. ( Yamashita etal., Nature. 2004). We also found that LPS-induced maturation of DCs is partly mediated through lowering the intracellular concentration of free Zn by down regulating Zntransporters, including Slc39a6 (Kitamura et al., Nature
Immunology. 2006). These observations suggest that
intracellular Zn is involved in MHC class II cell surface
expression through regulating endocytosis and membranetrafficking. In order to understand the role and function of Zn
transporters in vivo, we have generated mice deficient in the
Slc39/Zip family of zinc transporters. Recently, we foundthat Slc39a13/Zip13 deficient mice show changes in con-

3. Fukada, T*., N. Civic*, T. Furuichi*,S. Shimoda, K. Mishima, H.
Higashiyama, Y. Idaira, Y. Asada, H.
Kitamura, S. Yamasaki, S. Hojyo, M.
Nakayama, O. Ohara, H. Koseki, H.G.
dos Santos, L. Bonafe, R. Ha-Vinh,
A. Zankl, S. Unger, M.E. Kraenzlin,
J.S. Beckmann, I. Saito, C. Rivolta,
S. Ikegawa, A. Superti-Furga, and

T. Hirano. (*equal contribution). The
Zinc Transporter SLC39A13/ZIP13
is Required for Connective Tissue
Development; Its Involvement in
BMP/TGF-β Signaling Pathways.
PLoS ONE 3 (11), e3642, (2008)

nective tissues reminiscent of the human disease, EhlersDanlos syndrome (EDS). The Slc39a13-KO mice havedefects in the maturation of osteoblasts, chondrocytes,
odontoblasts, and fibroblasts. Impairment of bone morphogenic protein (BMP) and TGF-β signaling was observed in
the corresponding tissues and cells. Homozygosity for a
SLC39A13 loss of function mutation was identified in sibs
affected with a unique variant of EDS that recapitulates thephenotype observed in Slc39a13-KO mice. Hence, ourresults have revealed a crucial role of SLC39A13 in connective tissue development at least in part due to its involvement in BMP/ TGF-β signaling pathways (Fig. 2, Fukada etal, PLoS ONE, 2008). Together these results support theidea that Zn transporters have roles not only in maintaining
Zn homeostasis, but also for mediating intracellular signaling events. We propose the term “Late Zn signaling” for thistype of Zn signaling, in contrast to “Early Zn signaling”. The
former is dependent on changes in the transcription of Zn
transporter genes, whereas the latter is not (Hirano et al,Adv. Immunol., 2008).

4. Hirano, T., M. Murakami, T. Fukada,K. Nishida, S. Yamasaki, and T. Suzuki.
Roles of Zinc and Zinc signaling in
immunity: Zinc is an intracellularsignaling molecule. Advances in
Immunology 97, 149-176, (2008)

5. Ogura, H.*, M. Murakami*, Y.
Okuyama, M. Tsuruoka,
C. Kitabayashi, M. Kanamoto,
M. Nishihara, Y. Iwakura and T. Hirano.
(*equal contribution) Interleukin-17
Promotes Autoimmunity by
Triggering a Positive-Feedback
Loop via Interleukin-6 Induction.
Immunity 29, 628-636, (2008)

65

Team Leader

Masato Kubo
Visiting Scientist :Shin-ichiro Ohno

Student Trainees :Yasutaka Motomura (JRA)Minoru Sawaguchi
Emi Ohkubo
Tatsuki Ogura

Technical Staff :Yoshie Suzuki

Administrative Staff : Eiko Hayashi

Laboratory for

Signal NetworkTcells play a central role in the effector and regulatory
functions in immune surveillance, and aberrations inthese functions can lead to various immunological disorders. T helper 1 ( Th1) cells secrete interleukin-2 (IL-2), IFN-γ
and TNF-α in cellular immune responses against intracellular pathogens and viruses. Th2 cells produce IL-4, IL-5,
IL-6, IL-10 and IL-13, mainly against extracellular pathogens, and account for allergic immune responses. Th17
cells make IL-17 and IL-22 in response to certain bacterial
infections, but are mainly known for their association with
autoimmune tissue inflammation. These cytokines secreted
from effector helper T cells play a critical role in controllingthe outcome of immunological surveillance. The overriding
goal of our laboratory is to understand the molecular basisof helper T cell differentiation.

MINA53, an Il4 co-repressor controlling Th2-bias
Th2-bias, the propensity of naïve CD4 T cells to differentiate
into high-level IL-4 secreting Th2 cells, is a genetic trait
impacting infectious, autoimmune and allergic disease susceptibility. When the immune system encounters a potential
threat, antigen-presenting cells deliver peptide from theinvading pathogen to naïve T cells. These naïve cells
respond by differentiating into mature helper T cells. IL-4
drives Th2 differentiation into high-level IL-4 secreting Th2
cells, resulting in a positive feedback loop that fuels Th2
production while suppressing Th1 production through the
IL4R/STAT6 /GATA3 pathway. The degree of initial IL-4 production resulting in Th2 bias can have serious clinical implications because Th2 bias is tightly associated with thepathology of allergy. It was known that the BALB /c strain ofmice have a strong Th2 bias, producing large quantities of
IL-4 following T cell activation, while B10.D2 mice have astrong Th1 bias. However, a hybrid BALB /c strain that contains chromosome 16 from the B10.D2 strain also exhibitedTh1 bias, suggesting that chromosome 16 includes a gene
pertinent to this phenotype. Closer analysis highlighted
Mina53, a JmjC family member, as a genetic determinant ofTh2-bias. Mina53 specifically binds to and represses the Il4
promoter. Its over-expression in transgenic mice impairs Il4

Recent publications
1. Kubo, M., Notch: filling a hole in
T helper 2 cell differentiation.
Immunity 27, 3-5, 2007

2. Kano, S., Sato, K., Morishita, Y.,
Vollstedt, S., Kim, S., Taki, S.,
Honda, K., Kubo, M., & Taniguchi,T.; Regulation of Th1 vs. Th17
differentiation: Selective contributionof the transcription factor IRF1 tothe IFN-γ-IL-12 axis of signaling+
networks in CD4 T cells. Nature
Immunology 9 (1) :34 - 41, 2008

Low
TH 2 bias

High
TH 2 bias

Mina

Mina

IL - 4

Figure:

AP

AP

Mina

-1
T
N FA

BALB/c-1
T
N FA

C57BL/10

IL - 4

Th2=Allergy

Th1/Th2 bias and Mina expression. Th1 bias strain C57BL/10 (including B10.D2) has high expression of Mina,
while Th2 bias strain BALB/c has low expression of Mina. Mina protein binds to the NFAT binding site on theIl4 promoter and impairs Il4 transcription, leading to repression of Il4 and inhibition of allergic response.

expression, while its knockdown in primary CD4 T cells
leads to Il4 derepression. Together, these findings provide
mechanistic insight into an Il4 regulatory pathway controlling
Th differentiation and genetic variation in Th2-bias.
Natural occurring IL-17 producing T cells regulate
the airway responses.
Effector Th17 cells are a major source of IL-17, a criticalinflammatory cytokine in autoimmune diseases and in hostdefenses during bacterial infections. Recently, splenic lymphoid tissue inducer (LTi )-like cells have been reported to bea source of T cell independent IL-17 ( Takatori, H., J. Exp.
Med. 206:35-41, 2008). Here, we report that the immune
system contains a unique set of naturally occurring IL-17
producing cells, “natural” Th17 (nTh17), which are a memory-like T cell subset. The nTh17 cells can develop in the

3. Lu, L-F., Thai, T-H, Dinis Pedro
Calado, D.P., Chaudhry, A., Kubo,
M., Kentaro Tanaka, K. Gabriel, B.
G., Lee, H., Yoshimura, A., Klaus
Rajewsky, R., Alexander Y. Rudensky
Foxp3-dependent microRNA155
confers competitive fitness toregulatory T cells through targeting
SOCS1. Immunity 30:80-91, 2009.

4. Okamoto, M., Van Stry, M., Chung,L., Koyanagi, M., Sun, X., Suzuki, Y.,
Ohara, O., Kitamura, H., Hijikata, A.,
Kubo, M.*and M., Bix,*; MINA53, an
Il4 co-repressor controlling Th2-bias
Nature Immunology 10, 8, 872-879,
2009.*co-corresponding authors.

absence of the IL-6/STAT3 signaling axis required by inducible Th17 ( iTh17) cells. The nTh17 cell population is distinct
from conventional iTh17 cells, since nTh17 cells express
substantial amounts of IL-17A, but not IL-17F, under thecontrol of the master regulator, RORγ t. The nTh17 cells
simultaneously produce IFN-γ. DO11.10 transgenic ( Tg)mice on a Rag -/- background ( DO11.10 Rag -/- ) lack nTh17
cells, and, following intranasal administration of OVA, IL-17
dependent-neutrophil infiltration occurs in DO11.10 Tg
mice, but not in DO11.10 Rag-/- mice. This impaired neutrophil-dependent airway response is restored by adoptive
transfer of nTh17 cells into the DO11.10 Rag -/- mice. These
results demonstrate that a novel T cell subset, nTh17, facilitates the early phase of antigen-induced airway responses
and host defenses against pathogen invasion prior to theestablishment of acquired immunity.

5. Tanaka, S., Yoshimoto, T., Naka, T.,
Nakae S., Iwakura Y., Cua, D. andKubo, M.; Natural Occurring IL-17
producing T cells regulate theinitial phase of neutrophil mediatedairway responses. J. Immunol.
183(11), 7523-7530, 200967

Team Leader

Hisahiro Yoshida
Research Scientist : Takuwa Yasuda
Technical Staff :Hitomi Fukiage
Ayako Kobayashi
Mikiko Sato
Akimi Banno
Yuko Dokiya

Student Trainee :Machiko Sugiyama

Administrative Staff : Aiko Iyama

Laboratory for

ImmunogeneticsThe main activity of our team is the screening of a large
ethylnitrosourea (ENU) mutant mouse panel and is acollaborative effort with the RIKEN Genomic Sciences Center. An important goal of this project for RCAI is the development of novel mouse models for various immunologicaldisorders, notably, allergic and autoimmune disorders.
Since ENU mutagenesis introduces approximately 3,000
point mutations on a genome, we can expect 100 coding
region mutations in one pedigree. In parallel, we are screening the mutant mice under environmental bias, using a variety of approaches including immunization with allergens
and adjuvants to identify allergic condition modifier genes.
ENU mutant panel study
In order to identify the genetic basis for immune disorders,
we have screened a pool of mutant mice generated by random chemical mutagenesis. ENU induces random singlebase pair changes in genomic DNA at approximately 3,000
sites throughout the entire genome, resulting in approximately 100 sites in protein coding regions per first-generation (G1) mutant mouse.
During the first 4 years of this project we have screened
7,600 recessive inheritances from 80 mouse pedigrees.
For basic phenotypic screening, we examined the levels of
immunoglobulins, cytokines, and autoantibodies, as well as
expression of a panel of cell surface markers on peripheral
blood cells. Using also examined tissue sections from animals at 16 weeks of age. We have thus clarified the mutant
phenotypes by pathological, cytological and molecular biological examinations of affected tissues, lymphoid organs
and blood cells.
A phenotype identified in a mutant candidate line isreexamined at a later development stage, and if the same
phenotype is confirmed, we begin mating the mice for phenotype inheritance tests. The responsible mutant loci aremapped by backcrossing mutant individuals with the C3H /HeJ strain for gene detection by single-nucleotide polymorphism analysis. After approximate mapping, candidate genesequences are compared with wild type sequences in public databases by means of the PosMed research system

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

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

ENU mutant phenotype screen; 80 pedigrees, 4 years

Phenotype 145 lines
Immunoglobulins
Leukocyte number or profiles
Lymphoid organ development
Autoimmune relayed
Leukocyte function

80 lines
45 lines
9 lines
12 lines
10 lines

Allergy related phenotypes(Th2-biased phenotype) 60 lines
IgG1 high
6 lines
IgG1, IgE high15 lines
IgE high
12 lines
Mast cell related
10 lines
Eosinophil related
5 linesAllergic disease
3 linesIgE production to allergen 9 lines

2nd screen byAllergen
Immunization
experimental
allergic conjunctivitisorallergic asthma

Wild type

ENU mutant
With asthmatic
reaction

Figure1: An atopic dermatitis-like mutant mouse was establishedfrom the ENU screen. The scheme indicates the stepwise
progression of AD disease in this mutant animal. The
study of the precise mechanism(s) of the responsible
mutation in this disease is now underway. Photoshows the dermatitis in some of the heterozyogtes.

Figure2: Summary of four years of ENU mutant screening. In total,
approximately 8,000 mutant mice from 80 genomes havebeen screened and 145 phenotypes have been identified inmutant lines. Figures in right bottom show the 3 dimensional
image of lungs after OVA immunization and challenge.

established by RIKEN GSC. Candidate gene mapping has
been done in collaboration with the Phenome Informatic
Team and Mouse Mutation Resource Exploration Team in
GSC, RIKEN

or cure the AD in this animal model.
By ENU recessive mutant screening, we have identified andestablished more than 145 mutant lines of immune or blood
disease models. In keeping with our expectation, more than
50 lines showed phenotypes related to allergic diseases.
From these mutant lines, we are now investigating the
mutant line with experimental allergic asthma, rhinitis or
conjunctivitis by OVA immunization.
We are now backcrossing some of the mice to the C3H/
HeJ strain in order to map the responsible loci. As of
December 2009, we have mapped twenty eight independent mutant-responsible loci to distinct regions, and nine ofthem have been identified to be point mutations of independent genes. Along with allergic disease model mutant
screening, we have identified and characterized a few otherinteresting phenotype mutant line with/without allergic
defects. One of the interesting mutant animal had a defectin thymocyte development resulting in peripheral T cell number reduction. The responsible point mutation was mapped
to a gene coding an unknown molecule participating in single positive thymocyte development in thymus. Although
the mutation of the human orthologue is not reported yet,this gene would be a good candidate for searching thecause of the immunodeficiency condition in humanpatients.

Allergic disease model mutant mouse model
By ENU recessive mutant screening, we identified andestablished a mutant mouse line with phenotypic features
reminiscent of a typical human allergic disease. The ear skinbecame thicker and red and, as the mice aged, they started
to scratch the ear skin or face. The serum IgG1 or IgE levelof these mice gradually increased at 3 weeks after the dermatitis onset. Pathologically, the epidermal layer was hypertrophic and many inflammatory cells were found in thedermis of the lesion. Finally, they developed chronic inflammation in their face and ear skin and not only Th2 serum
immunoglobulin but also Th1 serum immunoglobulin level
increased. These stepwise symptoms and findings are compatible with the criteria for human infantile atopic dermatitis(AD). We have mapped the phenotype responsible point
mutation to a distinct gene and confirmed the phenotype is
induced by the mutation by genetic manipulation of the wild
type animal. Bone marrow transplantation study clarified thedisease is induced by mutant skin tissue but not by mutant
bone marrow. We are now analyzing the mechanism, which
initiates the AD like disease in this mutant animal to prevent

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

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

5. Piao JH, Yoshida H, Yeh WC, Doi T,
Xue X, Yagita H, Okumura K, Nakano
H. TNF receptor-associated factor
2-dependent canonical pathway
is crucial for the developmentof Peyer's patches. J Immunol.
178 (4), 2272-7( 2007)

69

Team Leader

Yasuyuki Ishii
Visiting Scientists : Hidetoshi Akimoto
Satoshi Komaniwa
Omar Duramad
Kenichi Masuda
Technical Staff :Haruka Katagiri
Risa Nozawa

Laboratory for

Vaccine DesignAllergen-specific immunotherapy (ASIT ) for various
pollinosis is globally utilized in the treatment ofallergies. However, in Japan, the only ASIT is subcutaneous
immunotherapy (SCIT ) for Japanese cedar pollinosis and it
is not so popular due to the length of the treatment and an
unclear mechanism of action. We have been studying two
types of vaccine technologies with the goal of clinicalapplication of ASIT for treatment of Japanese cedar pollinosis. The first candidate vaccine is a PEGyalted recombinant fusion protein of Cry j 1 with Cry j 2, which has been
designed to have much less anaphylactic risk than naturalallergen extracts, and could be utilized not only for SCIT but
also for sub-lingual immunotherapy (SLIT ). The second candidate vaccine is a liposomal α-GalCer, a ligand for invariant
natural killer T ( iNKT ) cells, encapsulating the Cry j 1/2
fusion protein. These liposomes showed not only cause
reduction of on-going IgE antibody formation but also suppress tertiary antibody responses. Moreover, oral administration of this vaccine also showed efficacy in suppressing
the IgE antibodies. Experiments have been carried out toelucidate the detailed mechanisms of both vaccines.
PEGylated recombinant Cry j 1/2 fusion protein
Our recombinant Cry j1/2 fusion protein is composed of fulllength mature regions of the Cry j 1 and Cry j 2 proteins
containing all potential human and mouse T-cell epitopes toovercome the problems associated with all the other recombinant polypeptide vaccines so far developed. For the purpose of drug development, we have to develop a method toprepare homogenous samples. However, there are 23 target cysteine residues for PEGylation in the recombinantfusion protein, indicating that PEGylated proteins would bevery heterogeneous. To produce a uniform product, we
generated a mutant fusion protein in which all but one of thecysteine residues was replaced by serine. The fusion protein does not retain any IgE-binding epitopes because thenative tertiary structure of Cry j 1 and Cry j 2 regions aredestroyed by the fusion of these two proteins. In fact, thebinding of IgE antibodies derived from 100-cedar pollinosis
patients to the fusion protein was completely lost, although

Recent publications
1. Yasuda N, Masuda K, Tsukui T, Teng A,
Ishii Y. Identification of canine natural
CD3-positive T cells expressing
an invariant T-cell receptor alphachain. Vet Immunol Immunopathol.
2009 Dec 15;132 (2-4) : 224-231.

2. Fujita H, Teng A, Nozawa R, TakamotoMatsui Y, Katagiri-Matsumura H,
Ikezawa Z, Ishii Y. Production ofboth IL-27 and IFN-gamma after thetreatment with a ligand for invariant
NK T cells is responsible for the
suppression of Th2 response andallergic inflammation in a mouse
experimental asthma model.

Figure:Migration of venus-NKT cells (green) and accumulation of Rho-liposome (red) in vivo

most of the patients were strongly reactive to native Cry j1
protein. This result suggests that PEGylated recombinantfusion protein will not bind to IgE / FcεRI on the surface ofmast cells and basophils, so there should be no risk of anaphylaxis shock. Next, to test whether the recombinant protein could enhance antibody responses against native cedarpollen antigens, mice were immunized with alum-adsorbed
PEGylated recombinant protein and boosted with native Cry
j 1 protein. Anti-native Cry j 1 IgE antibody formation was
not induced by priming with the PEGylated recombinant
protein. On the other hand, anti-native Cry j 1-IgG1 antibody
formation was observed in this situation, although the titerwas remarkably low compared to native Cry j 1-priming.
Although anti-recombinant IgG1 antibody was detected in
the immunized mice, IgE antibody was not. These results
collectively indicate that the PEGylated recombinant fusion
protein is safe because it does not bind to IgE antibodies ofpatients and thee is no induction of cedar antigen-specificIgE antibody formation.
Liposomal α-GalCer - Cry j 1/2 fusion protein
The original idea of the liposome vaccine was born from theobservation that α-GalCer could lead to IFN-γ production by
iNKT cells, Th1 differentiation and the suppression of IgE
antibody responses. However, the injection of liposomal
α−GalCer-recombinant Cry j 1/ 2 fusion protein into native
Cry j-primed mice resulted in the suppression of IgE anti-

body as well as IgG1 and IgG2a antibody responses.
To elucidate the detailed mechanism of liposome vaccine-mediated antigen specific suppression of antibody
responses, the liposome vaccine target cells were first analyzed by using rhodamine-labeled liposomes. The results offlow cytometric analysis indicated that the major target cellsof the liposome vaccine are splenic marginal zone
B220+CD1d high cells but not B220+CD1d low cells. It has beenreported that the chemokine receptor CXCR6 is preferentially expressed on iNKT cells. Flow cytometric analysis
showed that a ligand for CXCR6, CXCL16, was highly
expressed on the surface of rhodamone-positive
B220+CD1d high cells but not on the rhodamine-negative
cells, suggesting that iNKT cells could potentially beattracted to the B220+CD1d high cells in the spleen. Histopathological analysis clearly indicated that iNKT cells colocalized with the cells that had taken up the liposomes(Fig.). The significance of the interaction of iNKT cells with
marginal zone B220+CD1d high cells was supported by the
result that co-administration of neutralizing antibody against
CXCL16 with liposomal α-GalCer -Cry j 1/2 fusion protein in
the primed mice diminished the immunosuppressive effectsof the liposome vaccine. Taken together, these results suggest that the interaction of iNKT cells with marginal zone
B220+CD1d high cells is essential for the immunosuppressive
activity of the liposomal α-GalCer -Cry j 1/ 2 fusion protein.

J Immunol. 2009 Jul
1;183 (1) : 254-260.

71

Unit Leader

Fumihiko Ishikawa
Research Scientists : Yoriko Saito
Yuho Najima
Technical Scientist : Hidetoshi Ozawa
Technical Staff :Nahoko Suzuki
Akiko Sone
Mariko Tomizawa

Student Trainees :Shinsuke Takagi
Yuki Aoki

Administrative Staff : Toshiko Nakamrura

Research Unit for

Human Disease
ModelsC

urrent immunology and biomedical research haslargely been driven by investigations of murine biology.
Although mouse research will continue to provide importantbiological insights in the 21st century, we would also like tobetter understand human immunology and try to overcome
human disorders that cannot be cured with current therapeutics.
To accomplish these goals, we developed an in vivo
model enabling us to investigate normal human hematopoiesis and immunity as well as disorders involving these systems. In the FY2009, we attempted to address the following
three major issues using this in vivo model.
1: Creation of a novel humanized mouse system to evaluate HLA-restricted human immune response
2: Identification of a mechanism underlying human AML
relapse and development of therapeutic interventions
against AML leukemic stem cells
3: Understanding of the developmental pathway fromhuman iPS/ES cells to the hematopoietic lineage
These three issues are considered to be essential totranslate research findings into medicine based on humanimmunology and humanized mouse research. We herereport progress made on two major projects in our laboratory during FY2009.

Creation of a novel humanized mouse with HLArestricted human CTL response
Especially since the IL2rγ-null mutated immune-compromised newborn transplantation model has been established,humanized mice have contributed significantly to humanimmunology research. However, we encounter a major constraint in that human T cells mature and are selected throughtheir interaction with mouse thymic epithelial cells. Therefore human T cells developing in a mouse thymic environment fail to demonstrate HLA-restricted function. To
overcome the lack of HLA-restricted human immune
response, we evaluated human T cell development and its
function in a novel immune-compromised NOD/SCID/
IL2rγ null mouse expressing covalently bound HLA-class I
heavy chain and light chain (NSG-HLA-A2). Transplantation

Recent publications
1. Saito Y, Kitamura H, Hijikata A,
Tomizawa-Murasaza M, Tanaka S,
Takagi S, Uchida N, Suzuki N, Sone A,
Najima Y, Ozawa H, Wake A, Taniguchi
S, Shultz LD, Ohara O, Ishikawa F.
Identification of therapeutic targets
for quiescent chemotherapy-resistanthuman leukemia stem cells. Science
Translational Medicine 2:17ra9, 2010

2. Saito Y, Uchida N, Tanaka S,
Suzuki N, Tomizawa-Murasawa
M, Sone A, Najima Y, Takagi S,
Aoki Y, Wake A, Taniguchi S,
Shultz LD, Ishikawa F. Cell cycle
entry potentiates elimination ofquiescent chemotherapy-resistanthuman AML stem cells. Nature
Biotechnology 28:275-281, 2010

Figure:of purified Lin-CD34+CD38- human hematopoietic stem cells
into NSG-HLA-A2 newborns resulted in the development of
human CD4+ and CD8+ TCRαβ+ T cells and CD4-CD8- andCD8+ TCRγδ + cells in the recipient BM and spleen. Human
CTLs become functionally mature in these mice, as evidenced by the production of granzyme, corresponding to
the phenotypic transition from naïve to effector memory
CTLs. EBV infection in the humanized NSG-HLA-A2 recipients resulted in the formation of lymphoproliferative lesions
consisting mainly of human B cells with scattered human T
cells. Human CTLs developing in the recipients recognized
EBV-derived peptides in an HLA-restricted manner andexerted HLA-restricted cytotoxicity against autologous and
HLA-matched EBV-infected human B cells. The HLAexpressing humanized mouse with functional HLA-restricted
T cells has overcome a major constraint in human immunology research using the humanized mouse model, and willserve as a model for investigation of human immune
responses against pathogens and development of therapeutic strategies against human diseases.
Understanding AML relapse and therapeutic interventions eliminating LSCs
Although the concept of cancer stem cell has yet to becompletely clarified, we previously demonstrated that human
AML consists of heterogenous cell fractions and that theCD34+CD38- cells satisfy all the criteria for leukemic stem
cells proposed by the American Association for Cancer

3. Saito Y and Ishikawa F. The origins
of Blood: Induction of hematopoietic
stem cells from different sources.
Cell Stem Cells 3:8-10, 2008.

4. Ishikawa F, Yoshida S, Saito Y, Hijikata
A, Kitamura H, Tanaka S, Nakamura
R, Tanaka T, Tomiyama H, Saito N,
Fukata M, Miyamoto T, Lyons B,
Ohshima K, Uchida N, Taniguchi S,
Ohara O, Akashi K, Harada M, Shultz L
D. Chemotherapy-resistanthuman AML stem cells home
to and engraft within the bonePreferential location of LSCs in the endosteal
niche. (Upper left) Nuclear staining with DAPI
(Upper right) Staining for Ki67 (Lower left)
Staining for hCD45 (Lower right) Merged image

Research. We further identified the specific location of chemotherapy resistant LSCs as the osteoblastic niche rather
than the perivascular niche. Now we demonstrate that thecells in the central region of the BM are actively cycling,
while the LSCs in the endosteal region are cell cycle quiescent. We further explored the therapeutic strategies foreliminating LSCs to improve patient outcome and to overcome relapse. One of the strategies we have taken is tomodify the cell cycle status of the human AML LSCs. Treatment of AML engrafted mice with G-CSF resulted in the cell
cycle induction of LSCs in the niche. After cell cycle induction, chemotherapy eradicated LSCs to a substantial extent.
The other strategy we take is to compare global gene
expression profiles of normal long-term hematopoietic stem
cells and leukemic stem cells. Both types of stem cells cangenerate all the cells in their respective system and arecharacterized by the CD34+CD38- phenotype. Among various gene categories, we focused on those with characteristics favorable for drug development. We identified specificgenes expressed in LSCs but not in HSCs at the RNA level
and the protein level. Among the candidates, we confirmed
the expression of several genes specifically in AML stem
cells residing in the osteoblastic niche that keep AML LSCs
quiescent and chemotherapy resistant. We are currently
developing an shRNA lentiviral infection system to elucidate
whether down-regulation of identified candidate molecules
results in anti-leukemic activity in vivo.

marrow endosteal region. Nature
Biotechnology 25 (11) :1315-21, 2007.

5. Shultz LD, Ishikawa F, Greiner DL.
Humanized mice in translational
biomedical research. Nature Reviews
Immunol. 7:118-130,2007.

73

Unit Leader

Shin-ichiro Fujii
Technical staff :Yohei Shozaki

Student Trainee :Asako Kikuchi

Administrative Staff : Chikako Nishimura

Research Unit for

Cellular
ImmunotherapyHuman Vα24+NKT cells bearing an invariant Vα24Jα18
antigen receptor are activated by a specific ligand,α-GalCer, in a CD1d-dependent manner. We previously
showed that circulating Vα24 NKT cells present in lungcancer patients were functional. We have been developinga joint clinical study with Chiba University using
α-galactosylceramide (α-GalCer)-pulsed autologous DC
therapy to evaluate the immunological and clinical responses
to NKT cell therapy in advanced non-small cell lung cancer
patients. In the phase I / IIa study we detected an increase in
the number of NKT cells in the high dose DC administrationgroup, and then continued to use that cell number in a subsequent Phase IIa trial. Based on our initial analyses, wehave focused on a careful immunological analysis of lungpre- and post- treatment with this therapy in phase I / IIa
trials (A collaboration with RCAI Director Dr. Taniguchi andDrs. Nakayama and Motohashi in Chiba University).
We have also studied the role of dendritic cell (DC)in situ for tumor immunity by focusing on the link between
innate and adaptive immunity, especially through the interaction between DC maturation and NKT cells. The goalof our laboratory is to establish an antitumor therapeuticstrategy by elucidating the function of effector cells intumor bearing models, leading to the launch of clinical studies, in collaboration with Dr. Shimizu, Therapeutic Model
Research Unit.
Phase I/IIa immunotherapy for advanced
non-small cell lung cancer ( NSCL ) patients( A collaboration with Chiba University and RCAI
Director Dr. Taniguchi )We have been developing a Phase I/ IIa clinical study of theapplication of NKT cell therapy for advanced non-small celllung cancer (NSCLC) patients (stage IIIB, IV or recurrence).
α-GalCer-pulsed APCs (1x109/m2 PBMC-DCs) were intravenously administered four times. Twenty-three patientswere enrolled and 17 patients (73.9%) completed the study.
When we compared the responder group, which had anincreased number of IFN-γ producing NKT cells and thepoor responder group, the MST (median survival time) was

Recent publications
1. Fujii S, Motohashi S, Shimizu K,
Nakayama T, Yoshiga O, Taniguchi
M. Adjuvant activity mediated by
NKT cells. Semin Immunol. (2009)

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

Figure :longer in the responder group (31.9 months) than in thepoor responder group (9.7 months). Thus, the increased
IFN-γ production by NKT cells upon α-GalCer stimulation
was significantly associated with clinical outcome. In theother Phase I/ IIa study with prophylactic therapy by administration of DC/Gal to prevent recurrence in operative lungcancer patients, we have analyzed the NKT cells in thetumor infiltrating lymphocytes ( TILs) in lung where we
detected the higher number of NKT cells capable of producing IFN-γ (Fig.). These phase I/IIa trial results are encouraging and warrant further evaluation of the survival benefit ofthis immunotherapy.
A critical role of host DCs in immunized mice treatedwith α-GalCer-loaded tumor cells (A collaboration
with Dr. Shimizu, Therapeutic Model Research Unit )We showed that tumor cells loaded with α-GalCer are effective APCs for driving innate and adaptive immune responses,

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

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

The IFN-γ producing NKT cells increased in
the lung of NSLC patients treated with DC/
Gal . Prophylactic therapy by administrationof DC/Gal can prevent recurrence in operativelung cancer patients (stage IIB and IIIA) in aphase I/II trial. NKT cells in tumor infiltrating
lymphocytes (TILs) in DC/Gal treated patientswere analyzed at surgery. IFN-γ ELISPOT
assays were performed using mononuclear
cells (MNCs) derived from peripheral bloodor lung after culture in the presence (red) or
absence (blue) of α-GalCer. NKT cells capableof producing IFN-γ persisted in the tumor
region rather than in peripheral blood.

which are able to resist tumor metastases in vivo. With thestrategy of using NKT cell ligand-loaded tumor cells, we
initially found that tumor/Gal cells were killed by innate lymphocytes, mainly NKT and NK cells in vivo. Subsequently,
we have continued to study the mechanisms of activation ofadaptive immunity through in vivo maturation of DCs and to
optimize tumor antigen delivery systems to DCs. When DCs
in situ were analyzed, neighboring DCs captured dying
tumor/Gal cells and were then matured by activated NKT
cells via CD40-CD40L interactions. Importantly, when weanalyzed the crucial characteristics of DCs in situ in thetreated mice, we found that DCs can play a role in crosspresentation of tumor antigen and glycolipid to T cells and
NKT cells, respectively (two types of cross-presentation).
After tumor regression occurred, T cells responding to specific tumor antigens, such as Trp2, Tyrp, Dct and gp100,
persisted as memory T cells.

5. Shimizu K, Kurosawa Y, Taniguchi
M, Steinman RM, Fujii S. Crosspresentation of glycolipids
from tumor cells loaded with
α-galactosylceramide leads topotent and long-lived T cell mediatedimmunity via dendritic cells.
J. Exp. Med. 204, 2641-53 (2007)75

Team Leader

Mariko Okada ( Hatakeyama )Research Scientists : Takeshi Nagashima
Yuichi Shiraishi
Yuko Saeki
Technical Staff :Ryoko Nakayama (until Dec, 2009)

Student Trainee :Yoshimi Naruo

Administrative Staff : Rie Morita

Laboratory for

Cellular System
ModelingG

enome projects have successfully revealed the genetic
features of humans, and enormous amounts of omics
data have accumulated over recent years, yet, it is unclear
how all these parts are connected and function to produce
the desired biological outputs. Understanding of network
structures and regulatory relationship of molecules, extraction of general design principles from this information andits clinical application have been a long time challenge forbiologists. Especially for mammalian cells, such a generalization of design principles was thought to be almost impossible. However, the idea that such universal cellular logic
designs and motifs are distributed in living organisms andcarried over during an evolutionary process has started to
become more widely accepted. Such network structuresoften shift the cellular state to different, irreversible phenotypes in a dynamic cellular reaction process using limited
numbers of genes. The laboratory for Cellular Systems
Modeling has been carrying out research to identify andreconstruct biological networks and basic regulatory principles from quantitative data using computational, theoretical and experimental biology approaches. Particularly ourlaboratory focuses on system analysis of signal transduction networks, which determine cell fate in mammalian cells.
Biological findings, mathematical models and computationalalgorithms developed in our laboratory have been widelyused for prediction of drug effects or identification of biomarker in cancer.
The main topic of our current research is quantitative
analysis of membrane receptor signaling and its transcriptional control in the cell differentiation process. For thispurpose, we take both bottom-up and top-down
approaches. For bottom-up studies, kinetic mathematicalmodeling of pathway and parameter acquisition using computational and experimental approaches are carried out.
This approach is quite useful when the components andtheir interconnections in the reaction network are alreadyknown. However, even in the most well-studied cellular signaling pathways, quantitative reaction kinetics and theireffect on global cellular changes are not well understood.
Our working hypothesis is that small quantitative changes in

Recent publications
1. Nagashima T, Suzuki T, Kondo
S, Kuroki Y, Takahashi K, Ide K,
Hasegawa A, Toyoda T, Kojima T,
Konagaya A, Suzuki H, Hayashizaki
Y, Sakaki Y, Hatakeyama M.
Integrative Genome-wide Expression
Analysis Bears Evidence ofEstrogen Receptor-independent
Transcription in Heregulin-stimulated

MCF-7 Breast Cancer Cells. PLoS
ONE 3(3): e1803. doi:10.1371/
journal.pone.0001803 (2008).
2. Kimura S, Nakayama S, Hatakeyama
M. Genetic network inference asa series of discrimination task.
Bioinformatics 25, 918-925 (2009).

Figure:signaling pathways eventually cause a switch-like responsein the cell decision-making process. The question thenbecomes where, when and what molecules are most
responsible for these events. To answer these questions, wetake time-course quantitative experimental data using cultured cell line (MCF-7 breast cancer cell as a cell model) that
undergoes proliferation and differentiation in response todifferent growth hormones. Based on the data and the literature, we constructed ordinary differential equation (ODE )based kinetic models. For the past few years, we haveworked on the model to explain digital (all-or-none) outputsof c-Fos transcription factor activation (Cell, 2010 ). The
model can explain that a structure with co-existence of asignal-dependent feedforward loop of de novo c-fos transcription and c-Fos stabilization by the same signal (anAND-gate structure) can generate the digital output of
c-Fos. This model is also valid for production of transcription factors in B-cell development and neurite elongation of
PC12 cells, therefore it is most likely that the model capturesgeneral network principles of signal network during cellulardifferentiation process. Once a computational model isestablished, we determine critical kinetic parameters by
experiments for validation and generation of new hypotheses. We collaborate closely with the single molecular imaging laboratory ( Y. Sako, RIKEN ASI ) and obtain association
constants and diffusion constants of proteins in living MCF-7
cells. We believe that membrane receptors may determine
the majority of cellular kinetics, therefore we focus on the

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

4. Birtwistle MR, Hatakeyama M,
Yumoto N, Ogunnaike BA, Hoek JB,
Kholodenko, BN. Ligand-Dependent
Responses of the ErbB Signaling
Network: Experimental and Modeling
Analysis. Mol. Sys. Biol. 3:144
doi:10.1038/msb4100188 (2007).

Cells respond to different growth factors (epidermal
growth factor; EGF and heregulin; HRG) and
induce distinct ERK activation profiles andbinary production of activated Fos proteins. The
binary production of Fos is thought to direct
cells to different phenotypes (EGF; proliferationand HRG; differentiation). Kinetic modelingrevealed an embedded network structure(right) with feedforward, negative feedback
and AND-gate for p-Fos production only in the
differentiation process stimulated by HRG.

receptor and effector proteins with SH2 domains. We also
predict binding kinetics of protein-protein interactions insignaling pathways using molecular dynamics (MD) simulation.
For the top-down approach, we perform transcriptome
and proteome analyses to determine overall systems properties and unknown molecular regulations. Microarray and
ChIP-chip transcriptional analysis have been done for thesame MCF-7 cell platform. We are interested in the signal(context)-dependent activation of transcription factors andrewiring of transcriptional regulation. Therefore we performed phospho-tyrosine and phospho-serine/threonine
proteome analysis using LC mass spectrometry in collaboration with Tokyo Univ. Integrated analysis of transcriptome
and phosphoproteome data generated several hypotheses
that explain drug-resistance mechanisms in breast cancer
cells. We also developed in-house database and statisticalmodels for analysis of these omics data.
Our mathematical model of membrane receptor signaling pathway (Biochem J. 2003 and Mol. Syst. Biol. 2007)
has been referenced and modified for prediction of drug
effects by a group at MIT (Mol Syst Biol 2009) and used fora clinical study to identify biomarkers in drug-resistant breast
cancer by an Edinburgh group (Cancer Res 2009). Since
systems biology studies are not accomplished without close
collaboration with authorities in broad areas of science, wehave established variety of international and domestic
collaborations to resolve our biological problems.

5. Nagashima T, Shimodaira H, Ide
K, Nakakuki T, Tani Y, Takahashi
K, Yumoto N, Hatakeyama M.
Quantitative transcriptional control of
ErbB receptor signaling undergoes
graded to biphasic response
for cell differentiation. J. Biol.
Chem. 282, 4045-4056 (2007).

77

Group Director

Laboratory for

Senior Research Scientist : Hiroshi Kitamura

Immunogenomics

Osamu Ohara
Research Scientist :		 Yayoi Kimura (~May)
Research Associates : Atsushi Hijikata
		 Koichi Oshima (~August),
		 Mai Yamagishi
		 Takuya Kitamoto (~May)
Technical Staff :		 Masatoshi Ito, Miho Izawa,
		 Chisato Kikuguchi (~December),		 Aoi Ozawa
Temporary Employees : Seiko Watanabe, Nobutake Suzuki,
		 Kayoko Nagata (~May) ,		 Atsuo Kobayashi,		 Tomoko Hasegawa,
		 Saori Hayashi, Ryosuke Yashi,
		 Eri Yamamoto, Yoshie Kawase,
		 Kyoko Toyama (October~) ,		 Hideaki Takaku (October~)Students Trainees :		 Nanako Shimura, Noriko Nakagawa
Administrative Staff :		 Kazuyo Nomura

It has been our important and basic mission to function asa “Gateway” to genomics for immunologists. Thus, wehave taken a three pronged approach to our research
activities since our group launched: (1) central support
activities; (2) strategic and collaborative research activities;and (3) exploratory research activities aimed at new technology development. During 2009, as in the past, we havemade a significant effort to enhance strategic and collaborative research activities. In addition, we have initiated a program to provide a cytokine/chemokine profiling service aspart of the central support activities, and our RefDIC database has been enhanced to accommodate these data.
Furthermore, as an important component of the bioinformatic infrastructure for the Primary Immunodeficiency Disease project, we have developed new functions on a newly
developed browser of human mutations. This enables us to
integrate many lines of information about known mutations
in human gene and to detect likely disease-causative
mutation(s) in newly determined human gene sequences.
Besides these activities, we have also made great effort inresearch category (3). As an example, the recent results ofthe macrophages secretome analysis are described below.
Another emphasis in research category (3) is on development of technology for single-cell analyses at the mRNAand protein levels. Based on our experiences so far, we
anticipate that our three-pronged approach will synergistically activate and expedite the development of immunogenomics in RCAI. Described below are two main results ofour research activities in 2009.

Secretome analysis of macrophages
To maintain a dynamic and integrated immune system,
immune cells closely communicate with each other bymeans of humoral factors and cell surface molecules. Thus,
information about membrane and secretory molecules is
especially important for the immunological field. Using conventional transcriptomics, we can obtain a wealth of expression data about membrane/secretory molecules. However,this type of analysis has only provided us with information
about the abundance of transcripts, and this does not necessarily correlate with their actual protein levels. On theother hand, although proteomics is expected to provide

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

RAPID: Resource of Asian Primary
Immunodeficiency Diseases. Nucleic
Acids Res 37, D863-7 (2009)
2. Ohara O. From transcriptome
analysis to immunogenomics:
current status and future direction.
FEBS Lett 583, 1662-1667 (2009)more direct information about the protein profile, there aresome limitations in sensitivity and throughput. Thus, a bridging method between transcriptomics and proteomics would
serve as a key step in immunogenomics. To this end, we
performed DNA microarray analysis of membrane-boundpolysomes, in which membrane/secretory proteins are synthesized. This strategy enables us to obtain genome-wide
expression data at the translation level for membrane/secretory proteins with high sensitivity. In the mouse macrophagelike J774.1 cells, transcripts isolated in this way and detectedby ~3,000 probe sets on a Mouse 430 GeneChip preferentially encode proteins that localize to membrane-boundpolysomes rather than to cytoskeletal-bound and/or freepolysomes. Bacterial LPS affected the expression of 422
transcripts, of which 136 transcripts were not modulated
either in a total RNA pool or cytoskeleton-bound/free polysomes, but in the membrane-bound polysomes. In thegenes thus identified, 25 genes encode functionally uncharacterized membrane/secretory proteins whose expression
changed in peritoneal macrophages as well as in J774.1
cells after LPS stimulation. For example, LAM27 is on thisgene list and is known as an uncharacterized membraneprotein kinase. In agreement with the change in the mRNAlevel in membrane-bound polysomes, LAM27 protein was
temporally decreased in the membrane fraction. Overexpression of this protein attenuated induction of more than
100 proinflammatory genes in macrophages upon LPS
stimulation. This is a nice demonstration that our “SECRETOME” approach can uncover novel regulatory molecules in
activated macrophages.

Expansion of Mutation@A Glance for human genetic
diseases
As we described in last year’s annual report, we have developed a new bioinformatics tool, called Mutation@A Glance,
which enables us to analyze the effects of genetic variations
in genes known to be responsible for primary immunodeficiency diseases (PIDs). Mutation@A Glance has served as a
useful informational platform for DNA diagnosis in PIDs.
However, because of recent progress in the technology toidentify vast numbers of genetic variations, we started toconsider whether this informational platform should be
expanded to cover all the human genes. We have thus
begun to incorporate more comprehensive information
regarding all known human genes into Mutation@A Glance
to serve as a general informational platform for human mutation analyses. As a result, genetic variation data for over
2,400 known disease-associated genes other than PID
genes have been incorporated into this platform and mostprotein-encoding human genes can now be subjected tomutation analysis using this “One Stop” platform.
One of the important issues in mutation analysis is todistinguish bona-fide disease causing non-synonymous (ns)substitutions (one amino acid in a protein replaced byanother due to single nucleotide substitution in the DNA
sequence) from the vast numbers of naturally occurring
non-synonymous single nucleotide polymorphisms (nsSNPs). Although several methods to predict the effect of ns
substitution are already available, none is solely sufficient toaccurately predict the effects of all of the known diseaseassociated mutations. Thus, we felt that inclusion of othertypes of information would improve prediction accuracy.
As a new perspective, we have included a great deal of
information about molecular interactions in Mutation@A
Glance. Using a statistical analysis, we found that thefrequency of disease-associated ns substitutions located at
the sites of molecular interactions is significantly higher thanthat of non-disease associated nsSNPs, which offers arationale for the introduction of this kind of information into
Mutation@A Glance. As a typical example, spatial localization of disease-causing mutations and nsSNPs in STAT3 areshown in Fig. 2.

Figure 1: Enhanced mRNA profile viewer in Ref DIC. (Panel A) Genome-wide
detection of transcripts encoding secretory/membrane proteins l. A
comparison of expression data between membrane-bound polysomes
and cytoskeleton-bound/free polysomes in macrophage-like J774.1
cells is shown. The green oval shows ~3,000 probe sets detecting
transcripts that preferentially localize in membrane-bound polysomes.
(Panel B) Identification of LPS-affected transcripts encoding
secretory/ membrane proteins . Mouse macrophage-like J774.1 cells
were stimulated with LPS or PBS for 4hr. RNA in membrane-boundpolysomes (Mem) , cytoskelton-bound/free polysomes (CSK/Free),and a total RNA pool (total) were subjected to microarray analysis using
a Mouse Genome 430 2.0 GeneChip. Changes in the mRNA level of
136 total genes were detected only in membrane-bound polysomes.

3. Okamoto M, Van Stry M, Chung L,
Koyanagi M, Sun X, Suzuki Y,
Ohara O, Kitamura H, Hijikata A,
Kubo M, Bix M.Mina, an Il4 repressor,
controls T helper type 2 bias.
Nat Immunol 10, 872-879 (2009)

4. Sasuga Y, Iwasawa T, Terada K, Oe Y,
Sorimachi H, Ohara O, Harada Y.
Single-cell chemical lysis method
for analyses of intracellularmolecules using an array of
picoliter-scale microwells. Anal
Chem. 80, 9141-9149 (2008)

Figure 2: Display of molecular interactionsof a PID-causing gene, STAT3.
Two STAT3 subunits (a white spacefilling model and a yellow ribbon modelare depicted) form a homo-dimer andbind to double-stranded DNA (cyan) in
this X-ray crystal structure. The diseasecausing mutations and non diseaseassociated nsSNP sites in STAT3 arecolored magenta and green, respectively.

5. Hijikata A, Kitamura H, Kimura
Y, Yokoyama R, Hori S, Ishii Y.,
Kanagawa O, Kawamoto H, Koseki H,
Kubo M, Kurosaki T, Ohno H, O-Wang
J, Saito H, Saito T, Sato K, Tanaka M,
Taniguchi M, Taniuchi I, Watanabe T,
Aiba Y, Bao Y, Hase K, Kikuchi K,
Nakata M, Oboki K, Okamoto M,
Sakuma M, Sato K, Seino K-I,

Setoguchi R, Suematsu S, Watarai H,
Yamasaki S, Ohara O. Constructionof an open-access databasethat integrates cross-reference
information from the transcriptome
and proteome of immune cells.
Bioinformatics 23, 2934-2941 (2007)79Unit Leader

S. Sujatha Mohan
Research Associate : Suresh Kumar Ramadoss

Research Unit for

Immunoinformatics
An important mission of our research unit is to develop
and maintain an open resource bioinformatics platformand data resources in order to gain insights into Primary
immunodeficiency diseases (PID) through genomic, transcriptomic, proteomic data. Our ultimate goal is to provide
relevant, up-to-date and validated information on PID as
per global community standards in an easily decipherable
and usable format.
RAPID: Resource of Asian Primary Immunodeficiency
Diseases – “An Integrated Informational Platform”
PIDs comprise more than 150 different disorders that affectthe development, function, or both of the immune system.
Patients with these intrinsic defects may have increased
susceptibility to recurrent and persistent infections and, insome cases, elevated susceptibility to autoimmune diseases and cancer. Our main aim is to bring together theclinicians and basic researchers in Asia who are interested
in PID. We have recently developed an open access database on PID designated as “Resource of Asian Primary
Immunodeficiency Diseases – RAPID”, a web-based compendium of molecular alterations and gene expression at
the mRNA and protein levels of all PID genes reported from
PID patients. The database also includes other pertinent
information about protein-protein interactions, mouse studies and microarray gene expression profiles in variousorgans and cells of the immune system and it can beaccessed at http://rapid.rcai.riken.jp / (Fig. 1).
RAPID architecture
RAPID is an object-oriented database. We used Zope (http://
www.zope.org) for the development of RAPID. Zope is aleading open source web application server and is built using
the programming language Python (http://www.python.org).
MySQL is used as a backend data storage system.
RAPID data content
Sequence-variations in PID genes identified in patients aremanually curated from published literature and mapped to
NCBI RefSeq genomic, cDNA and protein sequences as
per the recommendations of the Human Genome Variations
Society (HGVS). Each mutation entry in the database is also
linked to “Mutation@A glance” a web-based graphical user

Recent publications
1. Kandasamy, K.*, Mohan, S.S*, Raju,
R., Keerthikumar, S. et al. NetPath:
A Public Resource of Curated Signal
Transduction Pathways. Genome
Biology (2010) (in press). *Theseauthors contributed equally
2. Kandasamy, K., Keerthikumar, S.,
Rajesh, R., Keshava Prasad, T. S.,

Ramachandra, Y. L., Mohan, S. andPandey, A. PathBuilder – Open Source
Software for Annotating andDeveloping
Pathway Resources. Bioinformatics,
25 (21) : 2860-2 (2009).
3. Shivakumar Keerthikumar, Sahely
Bhadra, Kumaran Kandasamy, Rajeshinterface (GUI) enabled tool which allows users to visualize
the mutation position both at the level of DNA and protein
sequences as well as homology based three-dimensional
structures with various types of information such as SNP,
protein domains and functional sites. RAPID also contains
protein-protein interaction information from the Human Protein Reference Database, expression profiles from Gene
Expression Omnibus and RefDIC, and corresponding mouse
phenotypes from the Mouse genome database (MGI).
RAPID statistics
At present, RAPID comprises a total of 188 PID genes, outof which 171 genes are reported with over 4500 uniquedisease-causing mutation data obtained from more than
1400 citations as of January, 2010. Detailed statistics ofannotated PID mutation data are depicted in Table.
The candidate PID genes prediction program
Currently, much attention has been focused on identifying
novel candidate PID genes through various in-silico
approaches. In this context, with the information available in
RAPID, we have used Support Vector Machine (SVM) based
parameter classification to predict candidate PID genes byscanning genes in the human genome. The genes prioritized in this way will be further tested and the most probablecandidates will be experimentally validated by biomedical
investigators (Published in DNA Research, 2009). Amongthe predicted candidate PID genes, there are 226 signaltransducers, 224 transcriptional regulators, 128 proteins
involved in immune response, 80 cell cycle regulators, 78
apoptotic proteins and 61 inflammatory responsive proteins.
Some of the predicted genes in our catalog have recently

been implicated as PID genes, while others represent attractive candidates to be analyzed in patients where the disease
etiology cannot be ascribed to any of the known PID genes.
RAPID project outlook
Our next focus will be on the analysis of annotated PID datasuch as correlation of mutation types and effects, influenceof mutation occurrence in its functional domains, identification of hotspot mutations, and disease-causing mutation
distribution and frequency with reference to ethnic groups.
Subsequent interpretation of all this analyzed information
should lead to a greater PID knowledgebase.
The semantic web, machine-interpretable descriptions,will be initiated in RAPID for all annotated entries using
Resource Description Framework (RDF ) and Web Ontology
Language (OWL ) file formats. The salient feature of thistechnology is being exploited in automation of web-based
information and also facilitates data exchange and sharingof PID data among other interested research groups from allover the world.
Collaborations & Funding
The PID project has been initiated in collaboration with theInstitute of Bioinformatics (IOB, Bangalore, India), the Immunogenomics research group at RIKEN RCAI, Japan and theKazusa DNA Research Institute (KDRI ), Japan.
This research unit is supported by “The Asia S&T Strategic
Cooperation Promotion Program, Special Coordination
Funds for Promoting Science and Technology by the Ministry of Education, Culture, Sports, Science and Technology(MEXT ), Japan”.

Table: Highlighted list of annotated
PIDgenes mutation data

Missense
Frameshift
Nonsense
Splice anomalies
Amino acid deletion

No. ofgenes

No. ofentries

124
114
102
79
46

2703
1219
1094
778
132

155
114
48
43

4476
1164
241
162

162
79
4
3
3

5320
707
19
22
3

Mutation type
Substitution
Deletion
Duplication
Insertion

Figure: Home page of
RAPID showing
variousmenus, data
content andorganization,
PID network,news andcollaboration

Sequence features
Exon
Intron5 Prime UTR
Promoter
3 Prime UTR

Raju, Ramachandra Y.L, Chiranjib
Bhattacharyya, Osamu Ohara, Sujatha
Mohan and Akhilesh Pandey. Predictionof candidate primary immunodeficiency
disease genes using a support vector
machine learning approach. DNA
Research, 16(6): 345-51 (2009).
4. Keerthikumar S, Raju R, Kandasamy K,
Hijikata A, Ramabadran S, Balakrishnan

L, Ahmed M, Rani S, Selvan LD,
Somanathan DS, Ray S, Bhattacharjee
M, Gollapudi S, Ramachandra YL,
Bhadra S, Bhattacharyya C, Imai K,
Nonoyama S, Kanegane H, Miyawaki T,
Pandey A, Ohara O, Mohan S. RAPID:
Primary Immunodeficiency disease
database. Nucleic Acids Research,
37 (Database issue): D863-7 (2009).

5. Prasad, T. S. K., Goel, R., Kandasamy,
K., Keerthikumar, S., Kumar, S.,
Mathivanan, S., Telikicherla, D.,
Raju, R., Shafreen, B., Venugopal,
A., Balakrishnan, L., Marimuthu, A.,
Banerjee, S., Somanathan, D. S.,
Sebastian, A., Rani, S., Ray, S., Kishore,
C. J. H., Kanth, S., Ahmed, M., Kashyap,M., Mohmood, R., Ramachandra, Y. L.,
Krishna, V., Rahiman, A. B., Mohan, S.,

Ranganathan, P., Ramabadran, S.,
Chaerkady, R. and Pandey, A. Human
Protein Reference Database - 2009
update. Nucleic Acids Research, 37(Database issue): D767-72 (2009).

81Unit Leader

Takaharu Okada
Research Scientists : Masahiro Kitano (SPDR)
Yoshikazau Ando
Technical Staff :Research Unit for

Immunodynamics

Noriko Takahashi

Administrative Staff : Mari KurosakiThe focus of our research is to understand the mechanisms regulating dynamic cellular interactions in thetissues that shape immune responses. For this purpose weuse real time imaging, in particular multi-photon microscopy, to analyze in vivo cellular migration and interactions.
Multi-photon microscopy, which was introduced only
recently to the field of immunology, has been revealing striking dynamics of immune cells in the lymphoid organs,underscoring the importance of this approach for resolving
the complexity of the immune system. Utilizing this technique, along with other methods, we are currently working
on two projects. The first one is to understand the regulation of B cell–helper T cell interactions that determine thefate of B cells in the antibody response. Antigen-engaged B
cells undergo proliferation, selection, and multidirectional
differentiation to achieve both immediate and long-term
humoral immunity. T cell help plays a pivotal role in most ofthese processes, but it is not known how these processes
are differentially regulated by B cell–helper T cell interactions. We tackle this problem by tracking individual B cellinteractions with helper T cells and their proliferation anddifferentiation. This lab also studies the interactions between
immune cells and tumor cells in the tumor-draining lymph
node. We aim to mechanistically understand the impact oflymph node tumor metastasis on the activation and differentiation of tumor specific T cells.
Roles for stable conjugate formation between
B cells and T cells in B cell proliferation anddifferentiation.
Our previous studies showed that cognate antigen-engaged
B and T cells form stable conjugates early after immunization. Stable B-T conjugates are motile, with the B cells
migrating ahead of conjugated T cells, possibly allowing theexchange of partners among conjugates to generate theoptimal responses. We also recently found that B-T conjugates migrate along the stromal cell network, which has
been reported to serve as a migration scaffold for naïve B
and T cells ( Fig. 1). To study the role of the conjugation in B
cell proliferation in the T-dependent antibody response, we

Recent publications
1. Ebisuno Y., Katagiri K., Katakai T.,
Ueda Y., Nemoto T., Inada H.,
Nabekura J., Okada T., Kannagi R.,
Tanaka T., Miyasaka M., Hogg N.,
Kinashi T. Rap1 controls lymphocyte
adhesion cascades and interstitialmigration within lymph nodes in
RAPL-dependent and -independent
manners. Blood. Epub ahead of print.

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

Figure :developed the imaging system to visualize the cell cycle ofantigen-engaged B cells in vivo in collaboration with Dr.
Miyawaki’s group (Laboratory for Cell Function Dynamics,
BSI, RIKEN) and Dr. Kurosaki’s group (Laboratory for Lymphocyte Differentiation, RCAI, RIKEN). We are also collaborating with Dr. Midorikawa’s group (Laser Technology
Laboratory, ASI, RIKEN) to optimize the two-photon excitation for multi-probe cell cycle imaging. Our initial imaging
results have begun to reveal the relationship between B-T
conjugation and cell cycle progression at various phases of
the immune response. After several cycles of proliferation,
antigen-engaged B cells form germinal centers, which areimportant for long term humoral immunity. As evidence suggests that prolonged B-T conjugation is critical for germinal
center formation, we are attempting to develop imaging systems to track differentiation of germinal center B cells inparallel with B-T interactions.
Interactions between tumor cells and tumor-specific
T cells in the lymph node
Cytotoxic T cells have the capacity to induce tumor regression. Although several studies have analyzed dynamics oftumor-infiltrating CTLs and their interaction with tumor cells,
these studies used exogenous model antigens to target

Migration of a B cell-T cell
conjugate on stromal cells in
the lymph node. The time-lapse
images show dynamics of cognate
antigen-specific B cells (green)and helper T cells (red). In
cyan are primarily irradiationresistant stromal cells. The
mouse lymph node was imagedby two-photon laser microscopy
36 hr after immunization withthe cognate antigen. Arrowheads
indicate the same B-T conjugate.
Elapsed time is shown as
minutes:seconds. Scale bar, 30µm. The images are presented
as 60 µm z-projections.

CTLs to tumor cells. On the other hand, most authentic
tumor-associated antigens are self antigens. Thus, thedynamics of CTLs reactive to ‘self’ tumor-associated antigens in the tumor environment is not known. In addition,interactions of CTLs with tumor cells that have metastasized
into the lymph node have not been dynamically analyzed.
We investigated the dynamic behavior of melanocyte antigen-reactive CD8 T cells and their interactions with melanoma cells in the lymph node in collaboration with Dr.
Shimizu ( Research Unit for Therapeutic Model) and Dr. Fujii(Research Unit for Cellular Immunotherapy). We found thatafter vaccination with specific peptides these self-reactive
CD8 T cells became sessile, most likely due to stable conjugation with DCs, proliferated robustly, and produced
interferon-γ just like exogenous antigen-engaged CD8 T
cells in the lymph node. However, these self-reactive CTLs
were unable to form stable conjugates with melanoma cells
that had metastasized into the lymph node parenchyma.
This is in contrast to the efficient conjugation observed
between model antigen-specific CTLs and tumor cells exogenously expressing the model antigen. Our further study ofthis immune ignorance will help understand the mechanismof tumor escape from the immune system.

3. Grigorova I.L., Schwab S.R., Phan
T.G., Pham T.H., Okada T., Cyster J.G.
Cortical sinus probing, S1P1dependent entry and flow-based
capture of egressing T cells. Nat.
Immunol. 10, 58-65 (2009)83Unit Leader

Research Unit forStudent Trainees : Akira Okada
Yuma Itou
Jun Takimoto

Molecular Systems
Immunology

Makio TokunagaOur laboratory has developed technologies that allow
immunological responses and signaling processes tobe visualized at the single-molecule level. Single molecule
imaging coupled with the ability to simultaneously visualize
several different proteins in cells has enabled the quantification of molecular dynamics, interactions, and kinetics.
Based on these three-dimensional and temporal parameters, we examine numerical modeling and computer simulations of cell functions. Using the combination of single
molecule quantification and "in silico" modeling, we aim toopen up new frontiers for understanding immune cells as
molecular systems.
Single Molecule Imaging and Molecular
Quantification in Cells
We have demonstrated that clear visualization of single
molecules in cells enables their molecular quantification.
Clear single-molecule visualization was achieved using
TIRF and HILO microscopy. We have been involved in
the development of total internal reflection fluorescence(TIRF) microscopy, a light-microscopic technique. TIRF has
become a widespread technique for single-molecule imaging at surfaces, but cannot be used for molecular imaging
inside cells.
To overcome this limitation, we have devised an
approach, called highly inclined and laminated optical sheet(HILO) microscopy for single molecule imaging inside cells(Fig. 1a). The main technical challenge of single-molecule
fluorescence imaging is increasing the signal / backgroundratio. We have achieved notable success in this by inclining
the illumination beam and by minimizing the illumination
area. The incident laser beam is highly inclined by a large
refraction, and is laminated as a thin optical sheet at the
specimen side. In HILO microscopy, this thin optical sheet
is used for illumination.
To evaluate the HILO microscopy technique, we reconstructed three-dimensional images of the nuclear porecomplexes (NPCs) in cells from z-scanned serial images(Fig. 1b). Clear, point-like images of NPCs were obtained
without the need for deconvolution to remove out-of-focus

Recent publications
1. Fukagawa, A., Hiroshima, M.,
Sakane, I., Tokunaga M.: Stochastic
emergence of multiple intermediates
detected by single-molecule
quasi-static mechanical unfolding ofprotein. BIOPHYSICS, 5, 25-35 (2009).

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

abcargo through nuclear pores in cells as bright spots on the
nuclear envelope. Molecular interactions with the assembled
NPC were quantified by single molecule analysis. Retention
times, the number of associated molecules, the dissociation
constant, and stoichiometry of import were all determined.

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

haze. The fluorescence intensity of the point images of
NPCs followed the theory of image formation quite well.
Further, there was much less photobleaching than in conventional confocal microscopy because of the lower intensity and non-focused nature of the illumination.
We examined the thickness of the illumination beam
and obtained illumination intensity profiles in the z-direction.
We decreased the illumination thickness by the reduction of
the illumination diameter. Notably, the full width at half maximum of the profile, i.e., the illumination thickness was lessthan 7mm at the diameter below 20mm.
We further evaluated the signal/background ratioof images in HILO. Inclination of the illumination beamincreases intensities of the fluorescence images up to 2.8fold compared with epi-illumination. This 2.8-fold increase isin excellent agreement with the theory. In contrast, thebackground intensity is substantially decreased by illumination inclination. As the background is composed of out-offocus images, the decrease is explained by the reduction of
the illuminated range. As a result, illumination inclination
increased the ratio of image to background (signal/background) up to 3.1-3.5-fold. Reduction of the illumination
diameter further decreased the background intensity. Consequently, reduction of the diameter increased the ratio of
signal to background up to 2.2-2.9-fold. The end result isthat HILO illumination microscopy increased the signal/
background ratio up to 7.6-fold.
To explore potential new uses of this technology, we
performed quantitative analysis on nuclear import to demonstrate its application to kinetic studies. We could visualize
single molecules of GFP-importin β mediating the import of

3. Fukagawa, A., Hiroshima, M., Sakane,
I., Tokunaga, M.: Direct Observationof Multiple and Stochastic Transition
States by a Feedback-controlled
Single-molecule Force Measurement.
Anal. Sci. 25 (1), 5-7 (2009).

4. Tokunaga, M., Imamoto, N., SakataSogawa, K.: Highly inclined thin
illumination enables clear singlemolecule imaging in cells. Nature
Methods 5 (2), 159-161 (2008).

“In silico” Modeling and Simulation
As shown above, molecular interactions with the assembled
NPC were quantified by single molecule analysis. In order tounderstand the molecular mechanism of nuclear import, anumerical import model was constructed using these kinetic
parameters. Computer simulation was carried out based on
the model with two types of binding sites. The simulation fitvery well with both the results of single-molecule experiments and the molecular kinetic features in cells.
We are now expanding the simulation studies into
whole-cell simulation of single lymphocytes based on single
molecule imaging and quantification ( Fig. 2 ). Direct comparison with molecular imaging is indispensable for thesimulation, since the values of parameters have a huge
number of degrees of freedom. We demonstrated that clear
visualization of single molecules in cells enabled the quantification. The combination of single molecule quantification
and “in silico” reconstructions of cell functions opens new
approaches for developing molecular systems biology.

ab

Figure 2: The combination of single molecule quantification and “in silico”
modeling opens new approaches for developing molecular systems
biology. (a) Aiming at understanding immune cells as molecularsystems, we are going to construct “in silico” cell models basedon single-molecule quantification. Bidirectional research isessential to reconstruct cell functions in silico; research frommolecules to systems by single molecule analysis, and feedback
research from systems to molecules. (b) Two-dimensional'topographic' energy landscape of single-molecule protein
unfolding by MD simulations. Three representative trajectories
are shown as lines. Multiple transition states were detected, andindividual trajectories took highly heterogeneous pathways. Thus,
single-molecule measurement has achieved notable success indetecting stochastic features of protein conformations. We arenow expanding this type of study from molecules into cells.

5. Yokosuka, T., Kobayashi, W.,
Sakata-Sogawa, K., Takamatsu,
M., Hashimoto-Tane, A., Dustin,
M. L., Tokunaga, M., Saito, T.:
Spatiotemporal regulation of
T cell co-stimulation by TCRCD28 microclusters through
PKCq translocation. Immunity
29 (4), 589-601 (2008).

85Unit Leader

Willem van Ewijk
Research Scientist : Katrin Ishii-Schrade

Research unit for

ThymicMicroenvironmentsIn the thymic environment, lympho-stromal interaction
forms the basis for both lymphoid and stromal cell development (Fig.1). Thus, on the one hand, thymic epithelial
cells ( TECs) promote maturation of T lymphocytes, while inturn, developing thymocytes induce maturation and functional stability of the thymic stroma, a phenomenon earlier
designated as “thymic crosstalk” (1-3).
Notably, in the absence of developing thymocytes, thethymic environment is perturbed and unexpected epithelial
cells appear such as ciliated cells, goblet cells and absorptive cells, forming cysts (Fig. 2). Importantly, this abnormal
stromal thymic phenotype can be quickly repaired, once
developing thymocytes are added back to the thymic environment, highlighting both the impressive plasticity of thethymic microenvironment, and the instructive role of developing T lymphocytes in TEC development (3,4).
The immunodeficient thymus
In 2009, we have studied the development of the thymic
environment in the absence of developing thymocytes, specifically focusing on the origin and generation of the aberrant epithelial cell types localized in thymic cysts (Fig.2). It
can be argued that these cells may have an extra-thymic
origin, and in that case these cells should not express thymic epithelial specific transcription factors. However, in
studies using FoxN1 reporter mice, we have shown that thecyst lining epithelial cells have passed through a Foxn1dependant developmental pathway, univocally demonstrating that they have originated from normal TECs. We also
found that cysts are not caused by downregulation of Foxn1
at the protein level. Also in normal TEC’s FoxN1 is downregulated with prolonged age, questioning the role of Foxn1
in the maintenance of thymic environments in general.
We then analyzed the thymic origin of cyst lining epithelial cells in an in vitro experimental system, where corticaland medullary TECs were tested for their potential to create
thymic cysts under the influence of deoxyguanosine, apotent blocking agent for T cell development. We showedthat medullary, rather than cortical TECs had the capacity toform cysts.
In a separate study, we have analyzed the molecularnature of factors involved in thymic crosstalk. We showed

References
1. Ewijk W van, Shores EW, Singer
A. Crosstalk in the mouse thymus.
Immunol Today. 1994, 15: 214-217.

2. Holländer GA, Wang B,
Nichogiannopoulou A, Platenburg
PP, Ewijk W van, Burakoff SJ,
Gutierrez-Ramos J-C,Terhorst C.
Developmental control point in
induction of thymic cortex regulated
by a subpopulation of prothymocytes.
Nature 1995, 373:350-353.

Figure 1: Foxn-1 (green)
expression in
subpopulations ofprofessional thymic
epithelial cells in 23 wk
old Foxn-1 EGFP mice,stained by antibodies
directed to cytokeratin
5 (red; medullaryepithelial cells) andcytokeratin 8 (blue;
cortical epithelial cells)

Figure 2: Scanning electron
micrograph of athymic cyst.

that Notch-1 activation on TECs by DLL-1 is important in
the generation of the professional 3-D phenotype of TEC’s(5). Thus, the Notch-1-DLL-1 interaction is a prime molecular example of thymic crosstalk, because both types of molecules are reciprocally expressed on TECs and developing T
lymphocytes, and this molecular interaction drives both
TEC development as well as T cell development. This latteraspect is now being employed to generate an artificial
thymic environment.
The artificial thymus
Importantly, Notch signaling in developing hemato-progenitor cells induces restriction of these cells along the T-lineage(5). We have set-up an in vitro system to generate T lymphocytes, based on Notch signaling. By infecting the fibroblast
cell line TsT with a DLL-1 containing retrovirus, hematopoietic progenitor cells added to the monolayer culture arecommitted into the T cell lineage, generating CD4+8+ thymocytes. However, CD4 and CD8 T cells did not develop. To
promote the final steps in T cell development we have added
MHC expressing cells to the TsT4/DLL1 culture. Thus, bonemarrow dendritic cells (BMDCs) were generated in vitro fromRag 1-/- mice, using GM-CSF and LPS. CD11c+ MHCII+BMDCs were sorted by FACS and added with T cell precursors to TsT4/DLL1 monolayers. Such isolated BMDC’s
nicely develop as MHC II expressing cells within the TsT4/
DLL1 monolayer, but so far these cells have been unable toinduce the final steps in T cell maturation. We then questioned whether viral expression of the cortical TEC-specificproteosomal subunit β5T on BMDC’s could promote positive selection of CD8 T cells. So far, the results of this study
are not yet conclusive. We are now analyzing the interaction

3. Ewijk W van, Holländer G, Terhorst
C, Wang B. Stepwise developmentof thymic microenvironments invivo is regulated by thymic subsets.
Development 2000, 127: 1583-1591.

4. Germeraad, W.T.V., Kawamoto, H.,
Itoi, M.,Jiang, Y., Amagai, T., Katsura,Y., van Ewijk, W. Developmentof thymic microenvironments invitro is Oxygen dependent andrequires permanent presence ofT cell progenitors. J. Histochem.
Cytochem. 2003, 51:1225-1235.

of ß5T-pulsed DC’s and pre-selection DP thymocytes, in amore precise way (where we reduce cell heterogeneity).
An alternative approach to generate an artificial thymus
includes studies on the trans-differentiation capacity of nonthymic epithelial cells into thymic epithelial cells. Using our invivo approach we have shown that intra-thymic transfer ofepithelial cells derived from the intestine results in integration of these extra-thymic cells into the thymic environment.
Interestingly, these cells develop morphologically into 3-D
organized TEC’s, expressing keratin (Fig. 3). A minor subpopulation of these newly formed TEC’s also expresses
MHCII, making these cells candidate “teachers” in T cellselection.
Using an in vitro approach, where we have implanted
tissue fragments or cell suspensions of other organs into
fetal thymi, we have confirmed the induction capacity of thethymic environment. We found that epithelial cells derived
both from the fetal intestine and lung can trans-differentiate
into TEC’s. In these domains, host-derived thymocytes integrate into the new epithelium (Fig. 4), supporting the notionthat these newly formed environments support T cell development.
The construction of an artificial thymic environment
generating a balanced T cell repertoire is not a simple task.
We have established the first steps on the way to define
minimal requirements for the development of T lymphocytes
outside the body. In our in vitro approach we need to search
for molecules and cell types completing the final steps in T
cell maturation. Our in vivo approach will concentrate bothon the plasticity of thymus-induced epithelial cell differentiation as well as the complete functional maturation of intrathymic implanted epithelial cells.
Figure 3: Keratin expression in
3-D GFP+ intestinederived epithelial cells, 4
weeks after intra-thymic
injection in the mouse
thymus. GFP:green;
DAPI:blue; keratin:red.

Figure 4: CD 45+ thymocytes (red)reside amongst GFP+ intestinederived epithelial cells (green)in fetal thymic organ culture.

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

6. Vroegindeweij, E, Itoi M, Satoh R,
Zuklys S, Crobach S, Germeraad WTV.
Cornelissen JJ, Cupedo T, Holländer
G, Kawamoto H, van Ewijk W. Thymic
cysts originate from Foxn1 positive
thymic medullary epithelium. Mol.
Immunol. 2010, 5: 1106-1113.

87

Reseaerch Unit forLaboratory Head

Miguel Vidal
Research Scientists : Kaori Hisada
Technical staff :Asako Shibano

Immunoepigenetics
Epigenetic regulation by the Polycomb group (PcG) ofgenes silences gene expression through mechanisms
not fully understood. PcG activity is essential for self-renewalof adult stem cells and the maintenance of their developmental potential, as well as for the stable repression ofgenes in imprinted domains or in the inactive X-chromosome. Distinctive PcG complexes bear either histone H3K27
methyltransferase or histone H2A monoubiquitin ligase
activities, that sometimes act coordinately. PcG targets arecell type and differentiation context dependent. Recruitingof PcG complexes to their targets is thought to occur
through DNA binding proteins and/or non-coding RNAs.
Some time ago, we identified a protein that interacts with
the Polycomb E3 monoubiquitin ligases Ring1A and Ring1B,
and also with the DNA binding protein YY1. The propertiesof the so- called RYBP protein (Ring1 and YY1 binding protein) made it a candidate for PcG recruiting and, indeed,
recently it has been shown that it contributes to PcGdependent repression of regions of the HoxD cluster with
YY1 binding sites. In addition, RYBP is an essential geneduring embryonic development and it has a function in adultB-cell homeostasis (our unpublished observations).
Genetic analysis of RYBP function in an embryonicstem cell model
Biochemical analysis by other groups has identified RYBP
as a member of the pluripotency network of transcriptional
regulators of embryonic stem (ES) cells. To study RYBP
function we are using a conditional loss-of-function modelin embryonic stem (ES) cells. These ES cells carry both afloxed RYBP allele and a Cre-ER fusion protein which isactivated in the presence of hydroxytamoxifen. RYBP gene
inactivation, as with that of other PcG products, has little
effect on ES cell self-renewal. In contrast, genes
derepressed in RYBP-deficient ES cells are rather unrelated
to the set of developmentally relevant genes that are
maintained repressed by PcG products. However, many ofthese PcG targets are bound by RYBP in ES cells, suggesting that either the paralog Yaf2 substitutes functionally forthe absence of RYBP in mutant cells, or that its contribution

Recent publications
1. Calés, C., Román-Trufero, M., Pavón,
L., Serrano, I., Melgar, T., Endoh, M.,
Pérez, C., Koseki, H. and Vidal, M.
(2008) Inactivation of the Polycomb
group protein Ring1B unveils an
antiproliferative role in hematopoietic
cell expansion and cooperation withtumorigenesis associated to Ink4a
deletion. Mol. Cell. Biol. 28:1018-28

2. Román-Trufero, M., Méndez-Gómez,H., Pérez, C., Hijikata, A., Fujimura, Y.,
Endo, T., Koseki, H., Vicario-Abejón,
C. and Vidal, M. (2009) Maintenanceof undifferentiated state andself-renewal of embryonic neuralstem cells by Polycomb protein
Ring1B. Stem Cells 27: 1559-1570

Figure:RYBP gene control in ES cells. (Top Panel) Gene Ontology (GO) analysis ofprobes derepressed in ES cells carrying a floxed RYBP four days after additionof 4-hydroxytamoxifen. (Bottom Panel) two examples of targets bound by RYBP:
Mae1 (right) is derepressed in mutant ES cells, whereas expression of Gata6(left) is unaffected by RYBP inactivation. The panels show the different binding
patterns of Ring1B, a Polycomb component that interacts with RYBP.

to the repression of PcG targets in ES cells is not essential.
Interestingly, though, among derepressed probes in RYBP
mutant cells we found some corresponding to a subset ofgermline specific genes and other genes that are also
derepressed in ES cells lacking components of the DNA
methylation machinery. We are currently exploring the

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

possibility that among RYBP functions in early embryogenesis, one is in the stable silencing (involving DNA methylation) of specific genes in somatic cells. Also, to evaluate
functional redundancy, we are generating mice carrying
conditional alleles of RYBP and its only paralog Yaf2.

4. Hirabayashi and, Y., Suzki, N., Tsuboi,
M., Endo, T.A., Toyoda, T., Shinga, J.,
Koseki, H., Vidal, M. Gotoh, Y. (2009)
Polycomb limits the neurogenic
competence of neural precursor
cells to promote astrogenic fatetransition. Neuron 63: 600-1389Core Members :Haruhiko Koseki (Group Director )iPS Project TeamKyoko Masuda ( Research Scientist )Momoko Okoshi-Sato ( Technical Staff )Chieko Tezuka ( Technical Staff )Adjunct Members :Hiroshi Kawamoto ( Team Leader )Fumihiko Ishikawa ( Unit Leader )Osamu Ohara ( Group Director )Hiroshi Watarai ( Senior Research Scientist )Daisuke Yamada ( Research Scientist )Genta Kitahara ( Technical Staff )Induced pluripotent stem (iPS) cells possess tremendous
therapeutic potential not only in the field of regenerative
medicine but also for immune therapy. We have begun anactivity to apply iPS technology for mouse and humanimmunology research and therapeutic development. The
core facility for iPS research is engaged in developing efficient protocols to reprogram various lymphocytes andinduce differentiation of iPS cells into lymphoid lineage on acollaborative basis with individual researchers in RCAI. This
activity is partly supported by CREST from JST.

Masako Fujita ( Technical Staff )Sakura Sakata ( Technical Staff )Yuko Nagata ( Technical Staff )Mariko Tomizawa ( Technical Staff )Yuri Suzuki ( Technical staff )Identification of genes that resist against
reprogramming (Yamada et al., in progress)
Reprogramming to generate iPS cells ( iPSCs) is a less efficient process when using mature lymphocytes comparedto fibroblasts. We hypothesized the presence of genes thatare difficult to fully reprogram in lymphocytes. To test this,we established 20 and 5 independent iPSC clones frommature B- and NKT cells, respectively. We have investigated gene expression profiles of 6 B cell-derived and 2
NKT cell-derived NKT cells and compared them with thoseof ESCs and identified a group of genes which reproducibly
failed to be reprogrammed in lymphoid cells.

Reprogramming of natural killer T (NKT )cells to iPS cells and their development intofunctional NKT cells in vitro. (Watarai et al.,submitted)
Although iPSCs hold tremendous potential for cellreplacement therapy, the efficacy of cells with desired functions differentiated in vitro from iPSCs has not been
rigorously assessed. We established iPSCs derived fromnatural killer T (NKT ) cell cloned mice (NKT mice), whichharbor functional NKT cell-specific rearranged T cell receptor loci in the germline. These iPSCs could be efficiently
differentiated into NKT cells in vitro. Moreover, adoptive
transfer of iPSC-derived NKT cells into NKT cell-deficient

Recent publications
1. Schwermann J, Rathinam C,
Schubert M, Schumacher S,
Noyan F, Koseki H, Kotlyarov A,
Klein C, Gaestel M. MAPKAP kinase
MK2 maintains self-renewal capacityof haematopoietic stem cells.
EMBO J. 28:1392-1406. (2009).

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

Figure :mice recapitulated the known adjuvant effects of natural
NKT cells and suppressed tumor growth in vivo. Our study
shows for the first time that fully functional immune cells canbe generated from iPSCs and also demonstrates the feasibility of expanding functionally competent NKT cells via an
iPSC phase in a mouse model, thereby, illustrating the clinical potential of this approach for NKT cell-targeted adjuvant
therapy in humans.

Development of human iPS-derived
hematopoietic stem & progenitor cells( Ishikawa et al., in progress )We are attempting to dissect the developmental programand pathway from human ES/ iPS cells to the humanhematopoietic lineage. When human iPS cells were cultured
with SCF/TPO/FL / TGFβ1 under feeder-free conditions for
7-14 days, the expression level of SSEA4 was decreased
and there was reciprocal up-regulation of SSEA1 expression. The cultured cells did not retain the morphologicalfeatures characteristic of iPS colonies, and formed embryoid
bodies. However, the hematopoietic induction was not sufficient, as demonstrated by the lack of expression of the
pan-leukocyte antigen, CD34, or Flk-1 on the surface of thecultured human iPS-derived cells. We then set up co-cultures of human iPS cells and OP9 cells. The stromal support with or without Notch signal via delta-like ligand 1(DLL1) successfully induced the development of the mesodermal lineage, as evidenced by the appearance of hCD45hCD34+ cells and subsequent development of hCD45+hCD34+
cells in vitro. We are currently investigating the differentiation potential of these candidate mesodermal or hematopoietic progenitor cells in vivo and in vitro.

3. Watarai H, Rybouchkin A, Hongo
N, Nagata Y, Sakata S, Sekine E,
Dashtsoodol N, Tashiro T, Fujii
SI, Shimizu K, Mori K, Masuda K,
Kawamoto H, Koseki H, and Taniguchi
M. Generation of functional NKT
cells in vitro from embryonic stem
cells bearing rearranged invariant
Vα14-Jα18 TCRα gene. Blood,
14, 115 (2) 230-237 (2010)

Chimeric mice generated
from NKT cell-derived
iPSCs are shown in the left
panel. Black pups represent
germline transmission of NKT
cell-derived iPSCs (right).

Functional differences between ES and iPS
cells revealed by B cell induction in an in vitro
culture system ( Kawamoto et al., in progress )We investigated whether iPS cells have the same features
as ES cells. We examined ES and iPS cells in terms of theirdevelopmental potential to produce hematopoietic cells.
We tested a total of 20 iPS clones including those derived
from mature B cells, from NKT cells and from mouse embryonic fibroblasts (MEF), in comparison with several ES lines.
All ES cell clones produced all types of hematopoietic cells
in the in vitro culture systems, including erythrocytes, myeloid lineage cells, T cells and B cells. In contrast, eight out ofnine iPS cell clones did not produce B cells, although theother types of hematopoietic cells were generated. This can
not be due to insufficient reprogramming, because the sameobservations were seen in several iPS cell clones derived
from mature B cells. These results clearly show that iPS
cells are not identical to ES cells in terms of their ability to
produce B cells.

A novel technology for cloning of human mAb
using by using iPS cells ( Kawamoto et al., inprogress )We are using iPS cell technology to develop a new method
to make human antibody for therapeutic use. We plan to i )immunize humanized mice with NP-antigen, ii ) make iPS
cells from antigen-specific human B cells, iii) collect mAb
from B cells induced from iPS cells. To establish the above
system, we performed a pilot study. Antigen-specific B cells
were isolated from immunized normal mice, and we successfully induced iPS cells from these B cells. From these
iPS cells, we produced chimeric mice, and confirmed that
antigen-specific B cells were generated.

4. Saito Y, Uchida N, Tanaka S,
Suzuki N, Tomizawa-Murasawa
M, Sone A, Najima Y, Takagi S,
Aoki Y, Wake A, Taniguchi S, Shultz
L.D, Ishikawa F. Cell cycle entry
potentiates elimination of quiescentchemotherapy-resistant humanleukemia stem cells. Nature
Biotechnology 28 (3), 275-280 (2010)91

Central Facilities
C

entral Facilities in RCAI provide all researchers in the Center with access to the most advanced equipment
and technologies. The Central Facilities consist of five sections; the Genomics Laboratory managed by
Osamu Ohara (Lab. for Immunogenomics), the Animal Facility managed by Haruhiko Koseki (Lab. for Developmental
Genetics), and the Confocal, Monoclonal Antibody, and FACS Laboratories managed by Takashi Saito (Lab. for
Cell Signaling) .

Genomics Lab.
Senior Research Scientist: Hiroshi Kitamura
Research Scientist: Yayoi Kimura
Research Associate: Mai Yamagishi, Atsushi Hijikata
Technical Staff: Tomoko Hasegawa, Saori Hayashi, Ryosuke Yashi, Nobutake Suzuki, Akio Kobayashi

The Genomics Lab provides various services to the members of the Center: proteomics analysis, in vitro translation,
multiplex suspension array, DNA microarray, DNA sequencing, and cDNA/Genomic clone distribution (Table). In 2009, westarted a new program to distribute primer/labeled probe sets for qRT-PCR analysis of immune cells.
The lab organized a RIKEN Technical Support Workshop on Oct. 8-9, 2009, and exchanged views with 70 staff
members from other RIKEN Centers, Omics Science Center, Brain Science Institute, Center for Developmental Biology,
Bioinformatics And Systems Engineering Division, and Advanced Science Institute. The goal was to improve the technologies of the Central Facilities by sharing information with other RIKEN’s Centers. As part of this process, a new website
in RIKEN SciNes was constructed by the Genomics Lab. (Fig.).

Table: Services provided by the Genomics Lab in 2009
Proteomics# of samples
Two-dimensional
15
electrophoresis
Mass Spectrometry Analysis
393
In vitro translation#ofsamples( recombinant protein synthesis )3
Multiplex suspension array# of samples
2,723
Affymetrix GeneChip# of arrays( Exon array, Gene array )
Human
223
Mouse
376
Total
599
DNA sequencing# of samples
36cm capillary
15,56350cm capillary
12,507
Total
28,070cDNA clone delivery# of clones
58# of teams
1
1# of teams
1# of teams
11# of teams
4
21
23# of teams
14
11
17# of teams
8

Figure : A top page of a new website for information exchange among
RIKEN technical assistance groups on RIKEN SciNes

Animal Facility
Senior Technical Scientist: Takanori Hasegawa
Technical Scientist: Shinobu Mochizuki
Technical Staff: Masashi Matsuda, Tomoyuki Ishikura, Isamu Hisanaga,Yusuke Iizuka, Hiromi Tatsumi,
Yurie Kawamoto
Admnistrative Staff: Hiroko Iwamoto
Animal care takers (outsourced ) : 22 people
The animal facility at RCAI provides a high-standard facility for animal experiments for RIKEN Yokohama Institute. The facility
also supports the generation of knock-out, transgenic and humanized mice (See Research Highlights section)

92

1.

Maintenance, generation and cryopreservation of genetic resources
In 2009, over 51,200 mice and 5 rats were maintained in the SPF area and 500 mice in an isolated area. 502 mouselines were introduced into the SPF area by a combination of in vitro fertilization (IVF ) and embryo transfer. Cryostocksof 424 lines were also generated. The facility also maintained relatively large colonies of several commonly usedstrains such as NOD/SCID/Cγ KO, Rag1KO and various Cre recombinase deleters and provided them to users on
demand. The facility provided technical assistance to generate chimeras (169 lines) and transgenic mice (14lines) andto establish and maintain ES cells. In addition, they generated a new internally available database for genetic
resources.

2.

Introduction of human BAC clones into NOD/SCID/Cγ KO mice
In collaboration with the Lab. for Immunogenomics, the Lab. for Developmental Genetics and the Res. Unit for Human
Disease Model, the facility has launched an activity to improve the efficacy of transplantation of human hematopoietic
stem cells into NOD/SCID/Cγ KO mice by “humanizing” the host strain. For this purpose, we have introduced large
genomic fragments containing human genes encoding MHC, cytokines, adhesion molecules, virus receptors andothers into NOD/SCID/Cγ KO mice. To date, they have established 6 BAC transgenics and confirmed the expressionof human genes on a C57BL6 background and begun back-crossing these mice onto the NOD/SCID/Cγ KO background using the speed-congenic method.

Photo :

Animal Facility won
RCAI Outstanding
Contribution of theYear 2009. (Fromleft, Chie Yoshida,
Hiroko Iwamoto,
Tomoyuki Ishikura,
Takanori Hasegawa,
Shinobu Mochizuki,
Isamu Hisanaga,
Yusuke Iizuka andHiromi Tatsumi)

Confocal Lab.
Collaborative Researcher: Yasutaka Wakabayashi ( Leica Microsystems K.K.)

hours

The Confocal Lab provides imaging equipment and technical support in collaboration with Leica Microsystems. Currently,
six confocal microscope systems are available:
1. Inverted system with visual and multiphoton MP laser that is suitable for time-lapse imaging of living cells andorgans.
2. Inverted system with a 405 nm laser that is suitable for time-lapse imaging of living cells in a controlled environment(CO2, temperature, and humidity).
3. Inverted system with visual and UV lasers that can be used for calcium detection.
4. Upright system with visual and UV lasers that is suitable for standard fixed specimen observation.
5. Intravital upright system with visual and MP lasers thatcan be used for in vivo imaging of various tissues such300
as lymph nodes.
250
6. Intravital upright system with a single visual laser,
double MP lasers and a high speed scanner that can
200
be used for in vivo imaging and for other specialized
applications such as light stimulation.
150
During 2009, the Lab. provided a training course for 21
investigators. The total running time of the microscopes
100
was more than 2302 hours (Fig.).
50

0
Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Dec

Figure : Running time of the microscopes in 200993

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

The Monoclonal Antibody (mAb) Lab restarted its activity in April 2009 after a period of inactivity during 2008. This newactivity is partially supported by Becton Dickinson, which will continue its support in 2010. Two technical staff members,
Tomomi Aoyama and Mayuko Matsuda, are primarily engaged in the generation of mAbs at the request of individual RCAI
investigators. The mAb Lab has produced mAbs against 20 different antigens, which were requested by 7 laboratories,
including mAb against a Zn transporter and a surface marker on regulatory dendritic cells. The lab will continue to produce
mAbs that meet the needs of RCAI researchers, and will also focus on development of mAb directed to strategic proteinssuch as allergy related molecules and several G protein-coupled receptors, which are important but difficult targets.

FACS Laboratory
Technical Scientist: Hanae Fujimoto
Technical Staff: Yukiko Hachiman

The FACS Lab provides a range of support for flow cytometry and cell sorting, procedures that are essential for nearly all
immunological experiments. In 2009, two new FACS machines, Aria II and Canto II were installed ( Table). In addition, thecapacity to sort single cells onto glass microscope slides was added (Photo). The laboratory’s activities are divided into
three main parts: technical support and training for FACS users, cell sorting operation services, and management/maintenance of the FACS equipment.
Table: FACS instruments in the central facilities

Analyzer

Sorter•FACS

no of machines

Calibur

6

LSR

1

Canto II

1

Aria I

2

Aria II

1

Vantage

2

Diva (digital vantage)

1

Photo: Sorting single cells onto glass microscope slides

Technical support and training
In 2009, the facility offered 15 technical courses (10 for cell sorting and 5 for cell analysis). Courses were held at 7
different levels, Calibur basic, Calibur option, Canto II, Vantage basic, Vantage option, Aria basic and Aria option. A
total of 52 RCAI researchers participated in these courses.

•

Cell sorting operation service
The FACS Lab provides a cell sorting operation service. Our experienced operators perform the actual cell sorting after
consultation on experimental design with RCAI researchers. In 2009, 343 sorting experiments were performed by our
FACS operators.

•

Management / maintenance of FACS equipment
FACS instruments are available for registered users 24 hours a day and reservations are accepted up to one month inadvance through an internal website. All the necessary information including instructions, reservations and user fees
can be accessed via the intranet. In addition to the in house FACS Lab staff, engineers from Becton Dickinson visit
once a week to provide maintenance and technical support.

94

2009

Collaborative Networks

RCAI and Torii Collaborate for the Allergy Vaccine

I

In December, 2009, RIKEN and Torii Pharmaceuticaln 2009, RCAI established a program for bridging researchCo., Ltd. agreed to collaborate for the development of cedar(translational research) platform. To fill the critical gap
pollen allergy vaccine under this program.
between basic research and later stage drug development,
Cedar pollen allergy afflicts an estimated more thanthe program offers an ‘exchange zone’ where RCAI, univer20 % of the Japanese population. The new vaccine involves
sities, hospitals and pharmaceutical companies work
polyethylene glycol bound to a recombinant fusion protein
together for the drug development.
of two cedar (cryptomeria japonica) pollen antigens (Cry j1 /The process from the discovery of a new molecule to its
Cry j2 ). Through animal trials, RCAI has found the vaccine
marketing is long, expensive and very uncertain. Developing
effective and it reduces the likelihood of anaphylactic shock.
a drug intended to treat a rare disease does not allow theUsing Torii's technology for making chemical compounds,
recovery of the capital invested for its research. Thus, sponthey will develop a vaccine for human use.
sors are reluctant to develop these ‘orphan drugs’ and to
take the high-risks for early stage research for innovative
drug discovery.
The new program aims to support the development of orphan drugs or innovative drugs thatprivate companies would hesitate to develop bythemselves. The program is comprised of two
steps; 1 ) RIKEN, in collaboration with universities,
will analyze the mechanisms, pharmacologicaleffects, efficacy and safety. 2) Based on the results,
companies will develop the drug and conductclinical trials. (Fig.)“This is like an exchange zone in athletic relays.
The sprinters run together in the exchange zone to
give/receive the baton firmly and smoothly. The
key is how smoothly the baton is taken over fromone runner to the next. RCAI creates the baton( innovation) and passes firmly to the next runner,”said Director Taniguchi.
Figure: Two-step scheme for the bridging research

Open Laboratory for Allergy ResearchR

CAI launched an open laboratory for allergy researchwhere researchers from external institutes, universities
or hospitals can have access to the Center’s resources
and conduct collaborative projects on allergy research. In
FY2009, three projects were launched under this program.
Dr. Kenji Matsumoto (National Center for Child Healthand Development ) and Dr. Naoki Shimojo (Chiba Univ.)
started an allergy cohort study combined with the generationof humanized allergy mouse model. Dr. Noriko Tsuji ( Advanced
Industrial Science and Technology ) will analyze the mechanisms of oral tolerance and its application for food allergy. Dr.
Masato Kubo ( Tokyo Univ. of Science ) will analyze IL-4 andthe class switch of B cells to IgE producing plasma cells.

96

Table: Members of Open Laboratory for Allergy Research
Kenji Matsumoto

National Center for Child Health and Development

Naoki Shimojo

Chiba University

Noriko Tsuji

Advanced Industrial Science and Technology

Masato Kubo

Tokyo University of Science

Multidisciplinary Research ProjectsW

ith the aim of developing new paradigms in immunology, RCAI launched a program to support a limited
number of multidisciplinary collaborative projects leadby RCAI researchers. The program provides 5-10 million

JPY/year to each collaborative research projects for up to
five years.
In FY2009, five projects were selected for the support.

Table: Awardees of RCAI Multidisciplinary Research Projects 2009

RCAI Project Leader

Project Title

Collaborators

Hiroshi Ohno

Genomic, transcriptomic and metabolomic
analysis of the human ulcerous colitis

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

Satoshi Ishido

Modeling of antigen presentation

Yuji Sugita (RIKEN Advanced Science Institute)

Ichiro Taniuchi

Regulation of gene expressions for thymic
development

Atsushi Mochizuki (RIKEN Advanced Science Institute)

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

Creation of the artificial immune cells

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

Hisaaki Shinohara

Modeling of NF-κB activation signals

Mariko Hatakeyama (RIKEN Advanced Science Institute)

RCAI and the Univ. of Palermo Signa Cooperative AgreementOn September 11, 2009, RCAI and the Univ. of Palermo
agreed to cooperate for scientific and educational
exchanges. Dr. Taniguchi, Director of RCAI, and Professor
Lagalla, the rector of the Univ. of Palermo, signed the agreement at the ceremony held at the Univ. of Palermo. The
agreement includes a) the exchange of professors andresearchers for conferences or seminars, b) the exchange ofscientific informative materials, and c) the co-organization ofadvanced study courses, seminars and research activities.
Palermo Univ. is located in north-western Sicily. The
university was officially founded in 1806, but its earliest
history dates back to the 15th century. A number of important scientific personalities did their work at Palermo University, including the astronomer Giuseppe Piazzi, the chemist
Stanislao Cannizzaro, the architect Giuseppe Venanzio
Marvuglia, and the physicist Emilio Segre.
Photo: Agreement and a medal given by the Univ. of Palermo

97

Harvard Summer School Program at RCAIR

CAI provides an undergraduate student internship program in collaboration with Harvard
University’s Summer School. From this year, the participants earn university credits in the Harvard
Summer School Program at RCAI.
In 2009, RCAI accepted three students, Ms. Denise Ye, Ms. Kelly Brock and Mr. Andrew Hardiganfrom June 8 to August 16. They conducted a research project at Lab. for Lymphocyte Development,
Lab. for Immunogenomics, and Lab. for Cell Signaling respectively. They also participated in a seriesof basic immunology lectures, RCAI’s International Summer Program ( RISP ), the RCAI-JSI International
Symposium on Immunology and basic Japanese classes. Besides these studies, they enjoyed thecultural exchange with students of Yokohama Science Frontier High School (photos),and excursions to Kamakura and the Nasu mountains.

Photo: Japanese culture visit at
Yokohama Science FrontierHigh School. Harvard Summer
School students learned Kendo(left) and Sado (right) fromJapanese high school students.

RIKEN Joint Graduate School Program
International Program Associate

R

CAI accepted three international students as RIKEN International Program Associates (IPA).
Under this IPA program, RCAI lab heads host students from collaborating overseas graduate
schools and supervise their Ph.D. program as Joint Supervisors. The students receive a daily living
allowance and housing costs for up to a maximum of three years.
The IPA students studying at RCAI in 2009 were
Li Yingqian (Nanjing University, China) studied at Lab. for Immune Diversity (01)Sebastian Nieke (Tübingen Univ, Germany) studied at Lab. for Immune Transcription (02)Li Shuyin (China Agriculture University, China) studied at Lab. for Immune Diversity (03)

01

98

02

03

Kyoto T cell Conference ( KTCC ) June 1- 4, 2009K

TCC 2009, the 5th International Workshop of Kyoto
T Cell Conference, was held at Shiran Kaikan, Kyoto
University on June 1-4, 2009. This was the time of theworldwide Swine Flue scare, and although the organizers
were worried about possible cancellations of the trip toJapan by guests and participants, the meeting was held
successfully with about 300 attendees including 93 scientists from 12 overseas countries. These included the Czech
Republic, Estonia, France, Germany, Sweden, Switzerland,
the Netherlands, United Kingdom, Canada, Australia, Korea,
and USA.
KTCC 2009 is the 5th meeting in a series of linked international meetings, named the “Global Thymus Network”(GTN), and dedicated to the study of T cells and the thymus.
The GTN is composed of four individual thymus meetings,
Rolduc in Europe, ThymOz in Australia, ThymUS in USA,and KTCC in Japan. Each year, one of these domestic
meetings is designated as an international conference.
KTCC has its annual domestic meeting on T cell development and function in Kyoto. As president, Dr. Takashi Saito(01), RIKEN, worked to develop this international meeting,and the organizing committee for the meeting was composed of representative members of the GTN. The meeting
was co-organized with The 3rd Symposium for the MEXT
Priority Research on “Immunological Self –Recognition and
Its Disorders–” which had a similar scope of research, andwas partially supported by RIKEN RCAI. Since KTCC has
always been organized and managed without any funding,the international KTCC was similarly organized; namely theworkshop did not support travel expenses for any speakers
from the GTN, but instead did provide travel awards to 20
foreign young investigators.
At the meeting, Dr. Michael Bevan (Univ. Washington,
Seattle) (02) presented his keynote lecture entitled “Theimportance of TCR affinity in selection and response”, andduring the next three days there were 78 presentations. The
topics discussed covered thymic microenvironments, lymphoid progenitors and lineage commitment, selection anddifferentiation, recognition and activation, immune response
and memory, immune regulation and diseases. There wereparticularly hot discussions on the discovery of a new genethemis, which is critical for positive selection (M.S. Partickand K. Kakugawa(03)), on the mechanism of lineage commitment (H. Rodewald (04) and H. Kawamoto (05)) and on
the function of the aire gene in thymocyte development.
Very active poster sessions were held in the evenings:
94 posters were equitably evaluated by members of the
GTN. Four posters were recognized for their research excellence and three of the authors received prizes of a one-year
subscription to eminent journals, such as Immunity, Nature
Immunology and Trends in Immunology, and one author
received a monetary prize from KTCC 2009 to encourage
their studies.
The 2010 international meeting will be held next as ThymOz in Australia, whereas the 20th domestic KTCC will bein Kyoto in that year.

The 5th International Workshop of

Kyoto T Cell Conference
June 1-4, 2009
Shiran-Kaikan, Kyoto University, Kyoto, Japan

Tentative Topics:
Thymus microenvironments
Lymphoid progenitors and lineage commitment
Selection and differentiation
Recognition and activation
Immune response and memory
Immune regulation and diseases
Keynote Speaker
Michael Bevan
Speakers (tentative)
Graham Anderson
Toshinori Nakayama
Mark Anderson
Ed Palmer
Avinash Bhandoola
Warren Pear
Thomas Boehm
P! rt Peterson
Daniel Campbell
Freddy Radtke
Daniel Gray
Ellen Rothenberg
Adrian Hayday
Hans-Reimer Rodewald
Fumihiko Ishikawa
Takashi Saito
Manami Itoi-Watanabe Shimon Sakaguchi
Stephen Jameson
Stephen Schoenberger
Hiroshi Kawamoto
Mari Shinohara
Ludger Klein
Alfred Singer
Shigeo Koyasu
Jonathan Sprent
Charles SuhrBruno Kyewski
Yousuke Takahama
Charles Mackay
Bernard Malissen
Ichiro Taniuchi
Masahide Tone
Nancy Manley
Mitsuru Matsumoto
Juan C Zúñiga-Pﬂücker
Nagahiro Minato
KTCC2009 Organizers
Manami Itoi-Watanabe Toshinori Nakayama
Takashi Saito (chair)
Hiroshi Kawamoto
Shimon Sakaguchi
Shigeo Koyasu
Yousuke Takahama
Nagahiro Minato
Global Thymus Network Organizers
KTCC

ThymOzManami Itoi-Watanabe Richard Boyd
Hiroshi Kawamoto
Ann Chidgey
Takashi Saito
Yousuke Takahama

Rolduc meeting

ThymUS

Graham Anderson
Georg H! llander
Naomi Taylor
Rose Zamoyska

Janko Nikolich-!ugich
Howard Petrie

Abstract deadline: March 31, 2009

Selection for oral and poster presentation will be made by the organizers.

Travel bursary of 100K JPY (approx 1000 USD) per abstract application is available
for 20 postdocs and graduate students from outside of Japan.
Registration fee: 5000 JPY

For more information, visit our website: http://ktcc.umin.jp
Co-organized with The 3rd Symposium for the Mext Priority Research on

Immunological Self

-recognition and its disordersPartially supported by
RIKEN Research Center for Allergy and Immunology

01

03

02

Contact:
saito@rcai.riken.jp
takahama@genome.tokushima-u.ac.jp

04

05

99

The 2 nd Symposium for PID in AsiaThe second Symposium for Primary Immunodeficiency
Diseases (PID) in Asia was held on February 4-5 at the
Kazusa Academia Hall, Chiba, Japan. This event was organized by Dr. Michio Oishi (01), Director, Kazusa DNA
Research Institute and Dr. Toshio Hara (Leader, PID Study
Group in Japan, Ministry of Health, Labour and Welfare). It
was well attended by many prominent PID physicians not
only from Japan but also from other parts of the world
including USA, France, Finland, China, India, Hong Kong,
Korea, Thailand, and Vietnam.
PID are a genetically heterogeneous group of disordersthat mainly affect distinct components of the innate andadaptive immune systems, such as neutrophils, macrophages, dendritic cells, natural killer cells, and T and B
lymphocytes. The major focus of this meeting was to discuss the prevailing issues and challenges encountered in
PID diagnosis and treatment. In addition to establishing anactive Asian PID network among interested groups, it was
hoped that this type of interaction would facilitate mutual
exchange of relevant information required for timely PID
diagnosis and therapeutic intervention to save many precious innocent lives.
The meeting proceedings began with a “PID network”
session, chaired by Dr. Shigeru Tsuchiya, in which the status
of PID in Japan was discussed by Dr. Toshiro Hara and subsequently, Dr. Kosuke Imai highlighted significant developments in the Primary Immunodeficiency Database in Japan(PIDJ) since its inception a couple of years ago. Next there
was a presentation by Dr. Capucine Picard, a well-known
PID expert from the French national PID reference center,
Necker Hospital, Paris. She vividly explained her clinicalexperiences in identifying PID patients who have been susceptible to both invasive and non-invasive bacterial infections along with early/severe / transient clinical phenotypes.
These PIDs affect key signaling molecules, IRAK-4 and
MyD88 and Dr. Picard also delineated the functional role in100

IL1-beta and Toll-like receptor ( TLR) dependent pathways.
This session was followed by talks describing recent developments in basic and clinical PID research, including effective introduction of gene therapy protocols for the treatmentof various PIDs – Wiskott Aldrich syndrome ( WAS) andchronic granulomatous disease (CGD). This later topic was
lucidly presented by two speakers, the first Dr. Anne Galy(02), INSERM Research Director at Genethon, a French notfor profit organization for biotherapies, who has been a pioneer in establishing clinical gene therapy programmes forvarious human diseases particularly for WAS, and now has
a phase I/II gene therapy clinical trial underway, using theex-vivo gene transfer approach. The second speaker on this
topic was Dr. Joong Gon Kim, Seoul National University,
Korea, who has been involved in designing gene therapy
protocols using retroviral vectors for treating X-CGD patients.
Dr. John Ziegler, a PID physician from Sydney Children’s
Hospital, Australia then discussed the working network system of ASCIA - Primary Immunodeficiency Diseases Register of Australia and New Zealand - and explained the current
status of veno-occlusive disease in these regions.
The second day of the symposium commenced with
topics related to pathophysiology of PIDs and the challenges in establishing PID diagnosis. Dr. Mei W. Baker (03),
Science Advisor, Newborn Screening Program, University of
Wisconsin-Madison, USA, described the workflow of newborn screening for SCID, in which two screening systems
are used: T cell receptor excision circles (TRECs) by a realtime PCR system or flowcytometric analyses of lymphocyte subpopulations. She further described patients who
have been successfully treated by hematopoietic stem
cell transplantation. Next, Dr. Yu-lung Lau (04), a well known
PID physician from the Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, China,
explained how PID care can be successfully establishedamong PID physicians in Asia using an e-consultation

approach thereby ensuring an efficient working group for
PID diagnosis. This approach would save a great deal oftime and energy, and also promote free genetic diagnosis,
as has already been clearly demonstrated by the e-consultation system that is now being practiced under his supervision. Dr. Mauno Vihinen (05), from the Institute of Medical
Technology, University of Tampere, Finland, next presented
an overview of novel systematic classifications of PID
based on clinical, pathological and laboratory parameters
using advanced computational tools for clustering andnetwork analysis. This method should help users to understand the intricacies of genotype and phenotype correlations that could be further applied to any systems
biology-based human disease classification. During thissession, all participants had a nice opportunity to observe
the high through-put sequencing facility that has been set
up at the Department of Human Genome Research, Kazusa
DNA Research Institute (KDRI), Japan under the directorship of Dr. Osamu Ohara. The final session dealt with thecurrent status of PID clinical experiences observed amongdeveloping countries in South East Asia and included discussions about available economical therapeutic modalities
to control PIDs.
At its conclusion, all of the participants agreed that thiskind of meeting would provide the right ambience for initiating global collaborations through strategic research programs, thereby sharing PID information, infrastructure andlaboratory facilities among the PID community.

Table: Program
4-Feb
1. PID network
Toshiro Hara (Kyushu University, JAPAN)

PID in Japan : Past, present and future

Kosuke Imai ( National Defense Medical College Hospital, Japan )PIDJ ( primary immunodeficiency database in Japan), 2008 - 2009

Capucine Picard ( Necker Hospital, INSERM, Paris Descartes University, France)

Clinical features and outcome of patients with IRAK- 4 and MyD88 deficiency

2. Recent Developments in Clinical PID Researches
Xiaochuan Wang ( Children’s Hospital of Fudan University, China )Promoting the recognition of primary immunodeficiency in clinical practiceJohn Ziegler ( Sydney Children's Hospital, Australia )Veno-occlusive disease and immune deficiency : an update

Anne Galy ( INSERM, France )Gene therapy for Wiskott Aldrich syndrome

JoongGon Kim ( Seoul National University, Korea )Gene therapy for CGD : Korean experience

3. Recent Developments in Basic PID Researches
Yoji Sasahara ( Tohoku University, Japan )The role of WIP in Wiskott-Aldrich syndrome

Yoshiyuki Minegishi ( Tokyo Medical and Dental University, Japan )Molecular origins and mechanisms of hyper-IgE syndrome

Fumihiko Ishikawa ( RIKEN RCAI, Japan )Humanized PID mouse model for basic and translational immunology

Osamu Ohara ( RIKEN RCAI ; Kazusa DNA Research Institute, Japan )Exploration of PID causative mutations: An Japan model and the future direction

5-Feb
1. Pathogenesis/pathophysiology of PID
Taizo Wada ( Kanazawa University, Japan )Revertant somatic mosaicism in primary immunodeficiency

Satoshi Okada ( Hiroshima University, Japan )Severe congenital neutropenia in Japan

Toshio Heike ( Kyoto University, Japan )Pitfall for diagnosis of autoinflammatory disorders

2. Diagnosis: Present and Future
Tomohiro Morio ( Tokyo Medical and Dental University, Japan )Common variable immunodeficiency (CVID): Molecular basis of immune
dysfunction

Mei W. Baker ( University of Wisconsin-Madison, USA )Newborn screening for SCID: What we've learned so far

Yu-lung Lau ( The University of Hong Kong, PR China )Promoting PID care in Asia through e-consultation and free genetic diagnosis

3. Bioinformatic/Genomic Researches for PID
Mauno Vihinen ( University of Tampere, Finland )Novel systematic classification of PIDs

Sujatha Mohan ( RIKEN RCAI, Japan )Resource of Asian Primary Immunodeficiency Diseases : RAPID : A road ahead

01

02

03

04

05

101

International Research Collaboration AwardT

he RCAI International Research Collaboration Award is aand Drs. Osato, Ito and Taniuchi’s work on Runx1 deficiency

unique program supporting researchers outside of Japan

resulted in successful publication ( Jacob et al. Blood 115,in setting up semi-independent research units within the labora-

1610-1620 (2010)). Drs. Ellmeier and Taniuchi’s paper ontory of their collaboration partner at the Center. The programtranscription factor network that control CD4/CD8 cell fateprovides up to 10 million JPY/year to each collaborative researchdecision of DP thymocytes is now in press (Sakaguchi et al,

projects for up to three years.

Nature Immunology, in press). Two collaborative projects took

Since the program started in 2004, 14 projects have beenplace in FY2009, and contributed not only to scientificfunded (Table). In FY2009, Drs. Bix and Kubo published theirwork on Mina, an Il4 repressor, that controls T helper type 2

discoveries but also for international exchange of people andinternational visibility.

bias (Okamoto et al. Nature Immunology, 10, 872-879 (2009))

Table: Awardees of RCAI International Collaboration Award Program

102

Year

Awardee

Title of Research

Host Lab.

2004-2006

Michael Dustin
New York University School of Medicine

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

Takashi Saito
Lab for Cell Signaling

2004-2006

Willem van Ewijk
Leiden University Medical Center

Regulatory role of lymphoid progenitors during
development of thymic microenvironments

Hiroshi Kawamoto
Lab for Lymphocyte Development

2004-2006

Miguel Vidal
Centro de Investigaciones Biologicas, CSIC

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

Haruhiko Koseki
Lab for Developmental Genetics

2004-2005

Steven F. Ziegler
Benaroya Research Institute at Virginia
Mason Medical Center

Role of NKT cells in TSLP-mediated allergic inflammation

Masaru Taniguchi
Lab for Immune Regulation

2004-2006

Peter D. Burrows
University of Alabama at Birmingham

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

Jiyang O-Wang
Lab for Antigen Receptor Diversity

2005-2007

Wilfried Ellemeier
Institute of Immunology, Medical University Vienna
Hilde Cheroute
La Jolla Institute for Allergy and Immunology

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

Ichiro Taniuchi
Lab for Transcriptional Regulation

2005-2007

Mark Bix
University of Washington, Seattle, Washington

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

Masato Kubo
Lab for Signal Network

2005-2006

Yun-Cai Liu
La Jolla Institute for Allergy and Immunology

Gene-array analysis and proteomics of Th2 tolerance

Yasuyuki Ishii
Lab for Vaccine Design

2005-2007

Kenneth M. Murphy
Howard Hughes Medical InstituteWashington University School of Medicine

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

Osami Kanagawa
Lab for Autoimmune Regulation

2005-2007

Facundo Damian Batista
Cancer Research UK London

Role of signaling molecules in B cell synapse formationand its maintenance

Tomohiro Kurosaki
Lab for Lymphocyte Differentiation

2006-2008

Sunhwa Kim and Michael Karin
Department of Pharmacology, Univ. of California,
San Diego

Identification of novel necrotic molecules from necrotic
hepatocytes and examination of its effect on the
inflammatory response

Masato Tanaka
Lab for Innate Cellular Immunity

2007

Andrea Brendolan
Cornell University Medical Center, Department of
Cell and Developmental Biology

A study on the spleen and lymph nodes mesenchymal
Takeshi Watanabe
cells that participate in the assembly of artificial secondary
Research Unit for Immune Surveilance
lymphoid organs

2008-

Stefano Casola
IFOM-The FIRC Institute of Molecular Oncology
Foundation

Nuclear reprogramming of terminally differentiated plasma
Sidonia Fagarasancells to study the specific role of IgA in mucosal andLaboratory for Mucosal Immunity
systemic immunity and B cell development

2008-

Yoshiaki Ito and Dr. Motomi Osato
Institute of Molecular and Cell, National Universityof Singapore

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

Ichiro Taniuchi
Laboratory for Transcriptional Regulation

2009

EventsRCAI International Summer Program
2009T

he fourth RCAI International Summer Program (RISP 2009) was held in Yokohama July 3-10. Fortytwo graduate students and postdoctoral fellows from sixteen countries participated in RISP 2009.
The first part of the program was held at RCAI and featured poster and oral presentations by the participants and a series of lectures by invited speakers from RCAI, Japanese universities, and abroad. The
second part of the program was the joint RCAI/Japan Society of Immunology meeting. "Cellular andGenetic View on Autoimmunity" held at the Pacifico Conference Center in Yokohama. Four of the RISP
participants also stayed on for a month-long laboratory internship at RCAI.
The focus of the lectures at RCAI was on regulation of immune responses, in particular by regulatory T cells and TH17 cells, but other topics included lymphocyte development/differentiation, immunity
to infection, mucosal immunity, and innate immunity. These lectures provided a broad overview of theimmune system, with approaches ranging from biochemical to whole animal analyses and human clinical trials. The invited speakers incorporated introductory material as well as recent highlights from theirown research into their talks. The research interests of the participants were similarly varied, making fora unique opportunity for cross-fertilization among immunology subdisciplines during the oral and poster
presentations as well as in more informal settings. The question periods following talks by the invitedlecturers and participants were spirited and stimulating.
Several of the invited lecturers also gave talks at the RCAI/JSI meeting, where they described further new developments in their research programs. There were also many new speakers and topics,
thus further increasing the breadth of the immunology coverage for the RISP participants. Awards forbest RISP posters were presented at a reception during the RCAI/JSI meeting. All of the participants
gathered to express their appreciation to the organizers for inviting them to RISP 2009, an exceptional
experience from both scientific and cultural perspectives. In a survey completed after the RISP, theparticipants all agreed that they would recommend the program to colleagues, and more than halfindicated that they would consider a postdoctoral position at RIKEN. The success of this unique program is due to the efforts of the Organizing Committee, chaired by Dr. Tanaka and Dr. Ohno, and the
RISP Secretariat, Ms. Aki, who kept the entire operation running smoothly, as well as to the efforts of
the outstanding participants. Planning is already underway for RISP 2010, scheduled for August, justprior to the 14 th International Congress of Immunology in Kobe.

104

Table 1 : Lectures
Lecturer

Title

Hiroshi Kawamoto, RIKEN RCAI, Japan

Lineage Restriction Pathway in Hematopoiesis: revision of the classical concept of
myeloid-lymphoid dichotomy

Clifford Lowell, University of California, USA

The STIM1 calcium sensor is required for activation of the phagocyte oxidase during
inflammation and host defense

Hiroshi Nakajima, Chiba University, JapanTh2 cells, Th17 cells, and allergic airway inflammation

Takashi Tokuhisa, Chiba University, Japan

Role for Bcl6 in development and maintenance of germinal center B cells

Sidonia Fagarasan, RIKEN RCAI, JapanT cell-independent and T cell-dependent f IgA synthesis in gut

Ellen Rothenberg, California Institute of Technology, USA

Gene regulatory guidance mechanisms for early lymphocyte development

Masaru Taniguchi, RIKEN RCAI, JapanNKT cell-mediated adjuvant cell therapy for cancer patients

Diane Mathis, Harvard Medical School, USA

Central tolerance

Alexander Rudensky, Memorial-Sloan Kettering Cancer Center,
USA

Class struggle and regulatory T-cells

Thomas Tedder, Duke University Medical Center, USA

Regulatory B cells and B10 cells during immune responses, inflammation,
autoimmunity and cancer

Vijay Kuchroo, Harvard Medical School, USA

Differentiation of Th17 cells

Yong-Jun Liu, M.D. Anderson Cancer Center, USA

Negative feedback regulation of type 1 IFN response by pDC specific receptor ILT7
and ligand

Takaharu Okada, RIKEN RCAI, Japan

Lymphocyte migration and interactions during the antibody response

Christian Münz, University Hospital of Zürich, Switzerland

Macroautophagy in innate and adaptive immunity

Table 2 : ParticipantsName (country)
John Altin (Australia)

Susan Johnson (Australia)

Jing Pan (China)

Thomas Tiller (Germany)

Carrie Arnold (USA)Marko Knoll (Germany)

Hemanth Ramaprakash (USA)

Damon Tumes (Australia)

Rosa Maria Barreira da Silva (Portugal) Meghan Koch (USA)Andrea Reboldi (Italy)

Marian Turner (Australia)

Niklas Björkström (Sweden)

Andreia Lino (Portugal)

Joseph Reynolds (USA)Aaron Tyznik (USA)Joaquim Carreras (Spain)

Cindy Ma (Australia)

Dipayan Rudra (India)

Yvonne Vercoulen (Netherlands)

Aranzazu Cruz-Adalia (Spain)

Ramon Mayoral (Spain)

James Scott-Browne (USA)Gabriel Victora (Brazil)

Priya Dedhia (USA)

Jaclyn McAlees (USA)

Doo-Hee Shim (Korea)

Jennifer Walker (UK)

Jessy Deshane (USA)Matthew Meredith (USA)Sunita Singh (India)

Ricardo Weinlich (Brazil)

Swantje Hammerschmidt (Germany)

Jessica Moffat (Australia)

Heidi Snider (USA)

Hakim Yadi (UK)Mark Headley (USA)

Yingting Mok (Singapore)

Todd Suscovich (USA)

Juandy Jo (India)

Diego Mourao Sa (Brazil)

Duncan Sutherland ( Australia)

105

RCAI- JSI International Symposium on
Immunology 2009
Cellular and Genetic View on AutoimmunityT

he fifth RCAI-JSI International Symposium on Immunology, hosted by the RIKEN Research Center
for Allergy and Immunology (RCAI) in conjunction with the Japanese Society for Immunology (JSI),
was held on July 9-10 in Yokohama. Organized jointly by RCAI Director Masaru Taniguchi and JSI President Kayo Inaba, the symposium is part of a series of annual conferences aimed at providing a forum fordiscussions of cutting-edge immunological research. This year’s event proved a great success, drawing
more than 350 participants from around the world with 21 internationally recognized speakers describing
their research on the topic of “Cellular and Genetic View on Autoimmunity”.
The symposium consisted of four sections focusing on different aspects of autoimmunity: “Negative
Regulation of Autoimmunity”, “Cellular and Molecular Basis of Autoimmunity”, “Genetics in Autoimmunity ”, and “Inflammation and Autoimmunity”.
In the session on Cellular and Molecular Basis of Autoimmunity, Professor Kiyoshi Takeda of Osaka
University described his research on lamina propria dendritic cells (DC). Findings from his group indicatethat ATP, the molecular unit of currency in intracellular energy transfer, also acts as a bacterial mediatorin the development of TH17 cells, a helper T cell subset thought to play a key role both in defending
against certain pathogens and, conversely, in driving autoimmune diseases. This discovery is an
important example of the interplay between intestinal microbiota and the host immune system.
In a later section on Inflammation and Autoimmunity, Dr. Toshio Hirano of Osaka University and RCAI
described an “IL-6 amplifier ” which appears to be involved in the etiology of autoimmune and inflammatory diseases. Dr. Josef Penninger of the Austrian Academy of Sciences outlined his group’s research on
the RANK /RANK ligand (RANKL), which plays an important role in bone metabolism. In experiments
using a knockout strategy, Dr. Penninger found that mutant mice without RANKL can no longer mount a
febrile response to a variety of stimuli, and that humans with RANK deficiency, like the mice, also sufferfrom both life-threatening osteopetrosis and absence of the physiologically important febrile response.
In bringing together cutting- edge immunological researchers, this year’s RCAI-JSI symposiumprovided participants a unique opportunity to share ideas and learn about the latest discoveries. Next
year’s symposium promises further advances in this rapidly evolving field.

106

Table : Program
Session I : Negative regulation of autoimmunity
Shimon Sakaguchi, Kyoto University, JapanT cell signaling, regulatory T cells, and autoimmunity

Alexander Rudensky, Memorial Sloan-Kettering Cancer Center and
HHMI, USA

Plasticity of regulatory T cell suppression program

Christopher A. Hunter, University of Pennsylvania, USA

Role of IL-27 in regulation of inflammation

Vijay K. Kuchroo, Harvard Medical School, USA

Differentiation of Th17 cells

Christophe Benoist, Harvard Medical School, USA

Genomics of tolerance

Session II : Cellular and molecular basis of autoimmunity
Thomas F. Tedder, Duke University Medical Center, USA

Regulatory B cells and B10 cells during immune responses, inflammation,
autoimmunity and cancer

Diane Mathis, Harvard Medical School, USA

Initiation of arthritis by commensal microbes

Jun-ichiro Inoue, The University of Tokyo, Japan

TRAF6 in controlling autoimmunity

Hiroki Yoshida, Saga University, Japan

Interleukin 27 signaling pathways in regulation of immune and autoimmune
responses

Fiona Powrie, University of Oxford, UK

Intestinal homeostasis: a balancing act between effector and regulatory T
cells

Kiyoshi Takeda, Osaka University, Japan

Commensal bacteria-derived ATP mediates development of intestinal Th17
cells

Session III : Genetics in autoimmunity
Henri-Jean Garchon, Cochin Institute and Inserm U567, France

Shaping the genetic risk for autoimmunity at the major histocompatibility
complex

Jacek Majewski, McGill University, Canada

Genome-wide analysis of transcript isoform variation in humans

Kazuhiko Yamamoto, The University of Tokyo and RIKEN CGM, Japan

Genome-wide association studies of rheumatoid arthritis and the ethnic
differences

Session IV : Inflammation and autoimmunity
Carola G. Vinuesa, The Australian National University, Australia

Autoimmunity as a consequence of aberrant positive selection of germinal
center B cells by TFH cells

David A. Hafler, Harvard Medical School, USA

Natural human CD4+CD25 high Tregs and Th17 effector cells

Yoichiro Iwakura, The University of Tokyo, JapanThe role of DCIR in the development of autoimmune arthritis

Toshio Hirano, Osaka University and RIKEN RCAI, Japan“IL-6 amplifier”: a key player in autoimmune and inflammatory diseases

Josef M. Penninger, Austrian Academy of Sciences, Austria

Learning from SARS – common injury pathways in lung infections?

Luigina Romani, University of Perugia, Italy

IOD as a key to balancing inflammation and tolerance to microbiota

Michel Gilliet, The University of Texas, USA

Antimicrobial peptides in plasmacytoid dendritic cell-driven autoimmunity

107

US-Japan International
Cancer Systems Biology MeetingT

he US-Japan International Cancer Systems Biology Meeting was held October 28-29 at the
RIKEN Yokohama Institute. The event was hosted by the US National Cancer Institute, The Systems Biology Institute, RIKEN RCAI, and the Japanese Foundation for Cancer Research. The goalsof the meeting were threefold: 1) to bring together leading experts in systems biology and cancer toexchange current state-of-the-art research, 2) to identify challenges and barriers that may be inhibiting progress in the field, in particular in terms of bringing current knowledge into the clinic, and 3) to
identify opportunities for interactions and collaborations at the individual and institutional levels.
Cancer is an ideal, albeit difficult, target for the systems biology approach. It has long been
known that cancer is a complex disease and it is becoming more appreciated that there is considerable heterogeneity even in an individual patient, both within the tumor and over time. The
talks at the meeting were very diverse, but there were common themes and issues. Much of
the deep analysis of cancer as a system uses cell lines rather than primary tumor cells. The potential pitfalls of this approach have long been recognized by reductionist biologists andare appreciated by systems biologists as well. Nonetheless, cell lines are invaluable as astarting point; knowledge gained from cell line studies can provide the foundation for subsequent ex vivo analyses of primary cells. A point made by several speakers is the importanceof having a common repository of cancer cell lines so that all researchers in the field are applying systems biology tools to as uniform a starting population as possible. Such cell line panels are currently maintained in the US by the NCI (NCI60) and in Japan by the JapaneseFoundation for Cancer Research (JFCR39). However, the participants were in agreement thathaving one central source of single-cell-derived, well-characterized, clonal cell lines would be highly
desirable.
One of the goals of systems biology as it relates to cancer is to develop personalized cancer
therapy. It is now possible to screen patients for mutations/amplifications in known oncogenes andtumor suppressor genes, and this information can sometimes provide an initial rational approach toappropriate therapy. However, as pointed out by several speakers, typically only a fraction of patients
categorized in this way will respond to a given therapy. The genetic complexity of individual tumors
and the existence of multiple different additional mutations clearly play a role in treatment failures,and ferreting out this type of higher order information is within the realm of the systems biologist.
Although not yet possible, the goal is to be able to provide patients with a profile of their cancer that willallow for effective therapy. The development of tools to accomplish this is a major challenge for systems biology.
One presentation that generated considerable discussion was by Stephen Friend of Sage Bionetworks. He predicted that as the tools now in use develop, there will be a ten-fold increase in data
generation, and that the systems biology community is unprepared to handle this data explosion. He
pointed out that taking individual network approaches, e.g. transcriptome, metabolome, etc., even ascomplex as these each are, is insufficient for making progress against cancer and other diseases, andthat a mechanism is needed to link all data sets together. Although there seemed to be general agreement on the value of this approach, many issues still remain, for example patient privacy regulations
and the lack of electronic medical records in many hospitals, making any “omics” data incomplete
and sometimes useless if it cannot be tracked back to clinical outcomes. Other concerns include
intellectual property rights, which might preclude some investigators, often at the insistence of their
institutional guidelines, from submitting data to such a repository, as well as a more general problem,
allocation of credit to individuals in an area that is of necessity big Team Science.
Clearly systems biology has great potential yet is experiencing some growing pains. The Yokohama participants agreed that meetings such as this one were very fruitful, as scientists wrestle withcomplex and sometimes thorny issues.

108

Table : Program
Oct. 28
Hiroaki Kitano (Sony CSL, Inc., The Systems Biology Institute,Japan)

Cancer systems biology: a road ahead

Roger Brent (Fred Hutchinson Cancer Research Center, USA)For cell signaling systems-- what is the signal and what information does it carry?

Alexander Hoffman (University of California, San Diego, USA)

Signaling dynamics and the cellular response

Takao Yamori (Japanese Foundation for Cancer Research, Japan)

Drug discovery of molecular targeted drugs by an information-rich cancer cell line
panel JFCR39

Edison Liu (Genome Institute of Singapore, Singapore)

Transcriptional regulation on a genomic scale

Ju-Seog Lee (M. D. Anderson Cancer Center, USA)

Decoding cancer signatures: systems biology approaches

Harukazu Suzuki (RIKEN Omics Science Center, Japan)

The transcriptional network in a human acute myeloid leukemia cell line

Mariko Okada-Hatakeyama (RIKEN RCAI, Japan)

Network design of cell differentiation

Kevin White (University of Chicago, USA)

Defining genomic networks in breast cancer

Samik Ghosh (The Systems Biology Institute, Japan)

A systems biology approach towards targeted cancer therapeutics: a study in the
MAPK pathway

Oct. 29
Yosef Yarden (The Weizmann Institute, Israel)

Defective feedback regulation of signal transduction networks in tumors

Kohei Miyazono (University of Tokyo, Japan)

Regulation of cancer progression by TGF-beta signaling

Stephen Friend (Sage Bionetworks, USA)

Sage: Together building probabilistic causal disease models via an open access
contributor network

Osamu Ohara (RIKEN RCAI, Kazusa DNA Research Institute,Japan)

From transcriptome analysis to immunogenomics

Joe Nevins (Duke University Medical Center, USA)

Dissecting the complexity and heterogeneity of human cancer

Bonghee Lee (Gachon University of Medicine and Science, Korea)

A proteome-wide map of protein translocation identifies an essential protein
complex for human glioma progression

Michiyuki Matsuda (Kyoto University, Japan)

The exhaustive modeling approach: Collection of missing parameters of EGF-RasERK signaling cascade

Gordon Mills (M. D. Anderson Cancer Center, USA)

Systems approach to personalized medicine

109Briefing session for the
RCAI adjunctgraduate school programs
May 23, 2009R

CAI accepts graduate students from various
universities through the adjunct professorshipmechanism. There were several new adjunctappointments in 2009. Dr. Tsuneyasu Kaisho (Team
Leader, Laboratory for Host Defense) became anew visiting professor at Yokohama City University.
Furthermore, the Research Institute of Biological
Sciences at Tokyo University of Science agreed tocollaborate with RCAI for research education. Dr.
Osamu Ohara (Group Director, Lab. for Immunogenomics) and Dr. Shohei Hori (Unit Leader, Res.
Unit for Immune Homeostasis) were appointed as
visiting professors, and Dr. Tadashi Yokosuka(Senior Researcher, Lab. for Cell Signaling) became
a visiting associate professor of the university. RCAI
will start accepting graduate students from Tokyo
University of Science in 2011. The total number ofadjunct /associate professors in RCAI is now 22,
and the Center collaborates with six university graduate schools ( Table).

On May 23, RCAI held a briefing session on
adjunct graduate school programs. The aim of thissession was to introduce this RCAI program to students who are interested in studying immunology.
Twenty participants gathered from Aomori, Tottori,
Nagano, Kyoto, Hyogo, Saitama, Kanagawa andTokyo. Dr. Saito, Deputy Director, first briefly summarized the adjunct graduate school programs, andthen representatives from seven laboratories introduced their research topics. After the oral session,
twelve laboratories had poster presentations andthen each of the participants had the opportunity tovisit the laboratories of his /her interest.
The session provided an opportunity for students to visit and talk directly with lab leaders, andto consider their future directions. “This briefing
was very fruitful for me, because I could talk directlywith researchers,” a participant commented.

Table : RCAI adjunct graduate school programs
Graduate School of Frontier Bioscience,
Osaka University

Tomohiro Kurosaki ( Visiting professor)
Ichiro Taniuchi ( Visiting professor )Keigo Nishida (Visiting associate professor)

Graduate School of Medicine,
Osaka University

Osami Kanagawa (Visiting professor)
Tsuneyasu Kaisho (Visiting professor)
Toshiyuki Fukada (Visiting associate professor)

Department of Immunology,
School of Medicine,
Chiba University

Takashi Saito (Visiting professor)
Haruhiko Koseki (Visiting professor)
Hiroshi Ohno (Visiting professor)
Shin-ichiro Fujii (Visiting associate professor)
Yasuyuki Ishii (Visiting associate professor)
Fumihiko Ishikawa (Visiting associate professor)

School of Biomedical Science,
Tokyo Medical and Dental University

Takashi Saito (Visiting professor)
Tomohiro Kurosaki (Visiting professor)
Masato Kubo (Visiting professor)
Sidonia Fagarasan ( Visiting associate professor)

International Graduate School of Arts and Sciences,
Yokohama City University

Hiroshi Ohno ( Visiting professor)
Tsuneyasu Kaisho ( Visiting professor)
Satoshi Ishido (Visiting associate professor)

Research Institute of Biological Sciences,
Tokyo University of Science

Osamu Ohara (Visiting professor)
Shohei Hori (Visiting professor)
Tadashi Yokosuka (Visiting associate professor)

110

RIKEN Yokohama
Open Campus
July 4, 2009T

he RIKEN Yokohama Institute Open Campus
was held on July 4, 2009. The number of visitors to this event increased for the fifth consecutive
year, this time numbering 2,614, including 841 children and 1,773 adults, a 26% increase compared
to last year. Twenty-one RCAI laboratories participated in this event and set up exhibits together inthe recreation hall of the main building of the Yokohama Institute (photo above). Using posters, movies or experimental samples, the RCAI scientists
explained their research topics to the visitors in a
way easy to understand. One of the new RCAI
exhibits this year was a cell culture of cardiac myocytes differentiated from murine iPS cells. Visitors
were surprised to see the synchronized regularbeats of the cultured cells. Another new exhibit wasconstructed in part to honor Dr. Osamu Shimomura,
who received the 2008 Nobel Prize "for the discov-

ery and development of the green fluorescent protein, GFP". This fluorescent protein is now used in awide variety of biological research fields. A tissue
sample from a GFP transgenic mouse enthralled
the visitors, who also saw how the fluorescence
could be excited by ultraviolet light (photo below).
Many children at the Open Campus were especiallyattracted to exhibits of experimental animals suchas xenopus, planaria and zebrafish. The visitors,
ranging in age from kindergarten to retirees, enthusiastically asked questions of the researchers andshared their common interests in science.

RCAI Retreat
Aug. 31- Sep. 1, 2009T

he aim of the RCAI Retreat is to improve theresearch activities of the Center by gathering
the members at a quiet place with few distractions.
This setting provides the RCAI scientists with theopportunity for in depth discussion and critiques ofongoing research projects, and this open communication promotes the development of new interactions and collaborations. At the 2009 RCAI Retreat,
all in attendance including researchers and staff
members (technical, and assistant) were encour-

aged to exchange information and initiate new collaborations. This year, Dr. John F. Foley, the editor of
Science Signaling, attended the meeting at the
Nihon Aerobics Center in Chiba Prefecture together
with 124 RCAI members.
On August 31, two poster presentation sessions were held. There were 90 posters in total, andboth new unpublished data and ongoing projects
were introduced to the retreat participants. The top
nine posters were selected by vote of the laboratory

111

The dedicated efforts of the organizers contributed to the success of the retreat. Drs. Sato andWang were in charge of the general organization of
the meeting. Ms. Yamamoto, Ms. Uchimura and
Ms. Fukui cooperated to coordinate travel, foodand accommodation, and ensured that the program
went very smoothly. Several researchers commented that the quality of the retreat continues torise every year.

heads and these received the Excellent Poster
Awards from the Director (See Research Highlights).
Oral presentations were held on September 1.
Dr. Foley introduced science related journals, Science Signaling and Science Translational Medicine,which at that time was planned for almost immediate launch. Ten selected young postdoctoral fellows
then introduced their projects, which stimulated
active discussions from the floor.

Super Science
High School
July 31, 2009

“S

uper Science High Schools” are schools
designated by the Ministry of Education,
Culture, Sports, Science and Technology tostrengthen and improve scientific education. RCAI
holds a workshop every year for students from theSuper Science High Schools in Kanagawa prefecture. This year, 22 students and 6 teachers from
Hakuyoh, Seisho, Yokosuka and Kanagawa Sohgoh High Schools were invited. After special lectures on “Immunological Organs” and “AIDS Virusand Immunity” by Drs. Hisahiro Yoshida and Tomohiro Kurosaki, the students had the opportunity for
informal discussions with the lecturers over lunch.
In the afternoon, the students got hands-on experience with mouse dissection and histological staining of the immunological organs. In the final session,
students were divided into groups for discussion
and presentations. This year’s theme for discussion
was “Development of a specific antibody for the112

new influenza and the possible problems”. “ Thetopic was timely but difficult. I learned how hard it is
to develop logical ideas and then present them well.
I’ll have to be tough,” one student said. “I was worried about the dissection, but now I am interested inanatomy,” another said. Through this experience,the students had a chance to look ahead to their
future career paths.
In addition to this workshop, RCAI invited
students from Jisyukann Junior High School inAugust and Takasaki High School in September. Dr.
Yasuyuki Ishii explained to them the basics
of allergy and answered their questions about
asthma and hay fever.

2009

Data and Statistics

Publications ( Apr. 2009-Mar. 2010 )Table : RCAI Publidations (Apr. 2009-Mar. 2010)
Journal Title

IF (2008)

FY 2009

Nature31.4

2Nature Genetics

30.3

1

Nature Immunology

25.1

5

Nature Biotechnology

22.3

1

Immunity

20.6

2

Nature Cell Biology

17.8

1

Journal of Clinical Investigation

16.6

3

Journal of Experimental Medicine

15.5

4

Neuron14.2

1

Immunol Rev

11.8

2

Current Opinion in Immunology

10.5

1

Blood

10.4

6

Trends in Immunology

9.93

J Allergy Clin Immun

9.8

1

Am J Respir Crit Care Med

9.8

1

PNAS

9.4

1

Journal of Cell Biology

9.1

1

EMBO Journal

8.3

2

Stem Cells

7.7

2

Clin Cancer Res

6.5

1

J Cell Sci

6.2

3

Genome Biol

6.2

1

Journal of Immunology

6.0

13

Molecular and Cellular Biology

5.92

J Med Genet

5.7

1

Am J Pathol

5.7

1

Journal of Biological Chemistry

5.5

1

Curr Top Microbiol Immunol

5.1

1

DNA Repair

5.1

1International Immunology

3.2

8

Other Journals

31

Total

104

1.

An, W. W., Kanazawa, Y., Ozawa, M., Nakaya, K., Saito, T., Tanaka, A. &Bradley, W. G. Dendritic cell differentiation and tumor cell apoptosis induced by components of a poly-phenylpropanoid polysaccharide complex.
Anticancer Res Feb. 2010, 30; 613-622.

2.

Angkasekwinai, P., Chang, S. H., Thapa, M., Watarai, H. & Dong, C. Regulation of IL-9 expression by IL-25 signaling. Nat Immunol Mar. 2010, 11;
250-256.

3.

Baba, Y. & Kurosaki, T. Physiological function and molecular basis of
STIM1-mediated calcium entry in immune cells. Immunol Rev Sep. 2009,
231; 174-188.

4.

Bruno, L., Mazzarella, L., Hoogenkamp, M., Hertweck, A., Cobb, B. S.,
Sauer, S., Hadjur, S., Leleu, M., Naoe, Y., Telfer, J. C., Bonifer, C., Taniuchi,
I., Fisher, A. G. & Merkenschlager, M. Runx proteins regulate Foxp3 expression. J Exp Med Oct. 2009, 206; 2329-2337.

5.

114

Burrows, P. D., Kronenberg, M. & Taniguchi, M. NKT cells turn ten. Nat
Immunol Jul. 2009, 10; 669-671.

6.

Candolfi, M., Yagiz, K., Foulad, D., Alzadeh, G. E., Tesarfreund, M., Muhammad, A. K., Puntel, M., Kroeger, K. M., Liu, C., Lee, S., Curtin, J. F., King,G. D., Lerner, J., Sato, K., Mineharu, Y., Xiong, W., Lowenstein, P. R. &Castro, M. G. Release of HMGB1 in response to proapoptotic glioma killing strategies: efficacy and neurotoxicity. Clin Cancer Res Jul. 2009, 15;4401-4414.

7.

Delevoye, C., Hurbain, I., Tenza, D., Sibarita, J. B., Uzan-Gafsou, S., Ohno,
H., Geerts, W. J., Verkleij, A. J., Salamero, J., Marks, M. S. & Raposo, G.
AP-1 and KIF13A coordinate endosomal sorting and positioning during
melanosome biogenesis. J Cell Biol Oct. 2009, 187; 247-264.

8.

Ebisuno, Y., Katagiri, K., Katakai, T., Ueda, Y., Nemoto, T., Inada, H.,
Nabekura, J., Okada, T., Kannagi, R., Tanaka, T., Miyasaka, M., Hogg, N.
& Kinashi, T. Rap1 controls lymphocyte adhesion cascade and interstitialmigration within lymph nodes in RAPL-dependent and -independent manners. Blood Jan. 2010, 115; 804-814.

9.

Fujita, H., Teng, A., Nozawa, R., Takamoto-Matsui, Y., Katagiri-Matsumura,
H., Ikezawa, Z. & Ishii, Y. Production of both IL-27 and IFN-gamma after
the treatment with a ligand for invariant NK T cells is responsible forthe suppression of Th2 response and allergic inflammation in a mouse
experimental asthma model. J Immunol Jul. 2009, 183; 254-260.

10. Fukuyama, S., Nakano, T., Matsumoto, T., Oliver, B. G., Burgess, J. K.,
Moriwaki, A., Tanaka, K., Kubo, M., Hoshino, T., Tanaka, H., McKenzie, A.
N., Matsumoto, K., Aizawa, H., Nakanishi, Y., Yoshimura, A., Black, J. L. &Inoue, H. Pulmonary suppressor of cytokine signaling-1 induced by IL-13
regulates allergic asthma phenotype. Am J Respir Crit Care Med Jun 1.
2009, 179; 992-998.
11. Furuichi, T., Kayserili, H., Hiraoka, S., Nishimura, G., Ohashi, H., Alanay,
Y., Lerena, J. C., Aslanger, A. D., Koseki, H., Cohn, D. H., Superti-Furga,
A., Unger, S. & Ikegawa, S. Identification of loss-of-function mutations of
SLC35D1 in patients with Schneckenbecken dysplasia, but not with othersevere spondylodysplastic dysplasias group diseases. J Med Genet Aug.
2009, 46; 562-568.
12. Fushimi, S., Ogino, T., Hara, J., Takahata, T., Wakabayashi, H., Watanabe,
H., Arashima, Y., Kubo, M. & Matsukawa, A. Forced expression of suppressor of cytokine signaling 3 in T cells protects the development of
concanavalin A-induced hepatitis in mice. Clin Immunol Dec. 2009, 133;
437-446.
13. Hara, H. & Saito, T. CARD9 versus CARMA1 in innate and adaptive immunity. Trends Immunol May. 2009, 30; 234-242.
14. Hara, S., Nakaseko, C., Yamasaki, S., Hattori, M., Bos, J. L., Saito, Y., Minato, N. & Saito, T. Involvement of Rap-1 activation and early terminationof immune synapse in CTLA-4-mediated negative signal. Hematology Jun.
2009, 14; 150-158.
15. Hase, K., Kawano, K., Nochi, T., Pontes, G. S., Fukuda, S., Ebisawa, M.,
Kadokura, K., Tobe, T., Fujimura, Y., Kawano, S., Yabashi, A., Waguri, S.,
Nakato, G., Kimura, S., Murakami, T., Iimura, M., Hamura, K., Fukuoka, S.,
Lowe, A. W., Itoh, K., Kiyono, H. & Ohno, H. Uptake through glycoprotein 2of FimH (+) bacteria by M cells initiates mucosal immune response. Nature
Nov. 2009, 462; 226-230.
16. Hase, K., Kimura, S., Takatsu, H., Ohmae, M., Kawano, S., Kitamura, H., Ito,
M., Watarai, H., Hazelett, C. C., Yeaman, C. & Ohno, H. M-Sec promotes
membrane nanotube formation by interacting with Ral and the exocyst
complex. Nat Cell Biol Dec. 2009, 11; 1427-1432.
17. Hashimoto, T., Tashiro, T., Kitahara, T., Mori, K., Sasaki, M. & Takikawa, H.
First synthesis of (+/- ) -basidifferquinone C, an inducer for fruiting-bodyformation in Polyporus arcularius. Biosci Biotechnol Biochem Oct. 2009,73; 2299-2302.
18. He, F., Saito, K., Kobayashi, N., Harada, T., Watanabe, S., Kigawa, T., Guntert, P., Ohara, O., Tanaka, A., Unzai, S., Muto, Y. & Yokoyama, S. Structuraland functional characterization of the NHR1 domain of the Drosophila
neuralized E3 ligase in the notch signaling pathway. J Mol Biol Oct. 2009,393; 478-495.

19. Higashi, A. Y., Ikawa, T., Muramatsu, M., Economides, A. N., Niwa, A.,
Okuda, T., Murphy, A. J., Rojas, J., Heike, T., Nakahata, T., Kawamoto, H.,
Kita, T. & Yanagita, M. Direct hematological toxicity and illegitimate chromosomal recombination caused by the systemic activation of CreERT2. J
Immunol May. 2009, 182; 5633-5640.
20. Hirabayashi, Y., Suzki, N., Tsuboi, M., Endo, T. A., Toyoda, T., Shinga, J., Koseki, H., Vidal, M. & Gotoh, Y. Polycomb limits the neurogenic competenceof neural precursor cells to promote astrogenic fate transition. Neuron
Sep. 2009, 63; 600-613.
21. Hori, S. c-Rel: a pioneer in directing regulatory T-cell lineage commitment?
Eur J Immunol Mar. 2010, 40; 664-667.
22. Imada, M., Masuda, K., Satoh, R., Ito, Y., Goto, Y., Matsuoka, T., Endo, S.,
Nakamura, A., Kawamoto, H. & Takai, T. Ectopically expressed PIR-B on T
cells constitutively binds to MHC class I and attenuates T helper type 1
responses. Int Immunol Oct. 2009, 21; 1151-1161.
23. Inoue, T., Okino, N., Kakuta, Y., Hijikata, A., Okano, H., Goda, H. M., Tani,
M., Sueyoshi, N., Kambayashi, K., Matsumura, H., Kai, Y. & Ito, M. Mechanistic insights into the hydrolysis and synthesis of ceramide by neutral
ceramidase. J Biol Chem Apr. 2009, 284; 9566-9577.
24. Ishido, S., Matsuki, Y., Goto, E., Kajikawa, M. & Ohmura-Hoshino, M.
MARCH-I: a new regulator of dendritic cell function. Mol Cells Mar. 2010,29; 229-232.
25. Iwata, A., Watanabe, N., Oya, Y., Owada, T., Ikeda, K., Suto, A., Kagami,S., Hirose, K., Kanari, H., Kawashima, S., Nakayama, T., Taniguchi, M.,
Iwamoto, I. & Nakajima, H. Protective roles of B and T lymphocyte attenuator in NKT cell-mediated experimental hepatitis. J Immunol Jan. 2010,
184; 127-133.
26. Jacob, B., Osato, M., Yamashita, N., Wang, C. Q., Taniuchi, I., Littman,
D. R., Asou, N. & Ito, Y. Stem cell exhaustion due to Runx1 deficiency isprevented by Evi5 activation in leukemogenesis. Blood Feb. 2010, 115;
1610-1620.

34. Keerthikumar, S., Bhadra, S., Kandasamy, K., Raju, R., Ramachandra, Y. L.,
Bhattacharyya, C., Imai, K., Ohara, O., Mohan, S. & Pandey, A. Predictionof candidate primary immunodeficiency disease genes using a support
vector machine learning approach. DNA Res Dec. 2009, 16; 345-351.
35. Kim, J., Lahl, K., Hori, S., Loddenkemper, C., Chaudhry, A., deRoos, P.,
Rudensky, A. & Sparwasser, T. Cutting edge: depletion of Foxp3+ cells
leads to induction of autoimmunity by specific ablation of regulatory T cells
in genetically targeted mice. J Immunol Dec. 2009, 183; 7631-7634.
36. Kimura, S., Nakayama, S. & Hatakeyama, M. Genetic network inference asa series of discrimination tasks. Bioinformatics Apr. 2009, 25; 918-925.
37. Kimura, S., Shiraishi, Y. & Okada-Hatakeyama, M. Inference of genetic
networks using linear programming machines. IEEE Press, 2009.
38. Kitajima, M., Iwamura, C., Miki-Hosokawa, T., Shinoda, K., Endo, Y.,
Watanabe, Y., Shinnakasu, R., Hosokawa, H., Hashimoto, K., Motohashi,S., Koseki, H., Ohara, O., Yamashita, M. & Nakayama, T. Enhanced Th2cell differentiation and allergen-induced airway inflammation in Zfp35deficient mice. J Immunol Oct. 2009, 183; 5388-5396.
39. Kitoh, A., Ono, M., Naoe, Y., Ohkura, N., Yamaguchi, T., Yaguchi, H., Kitabayashi, I., Tsukada, T., Nomura, T., Miyachi, Y., Taniuchi, I. & Sakaguchi,
S. Indispensable role of the Runx1-Cbfbeta transcription complex for invivo-suppressive function of FoxP3+ regulatory T cells. Immunity Oct.
2009, 31; 609-620.
40. Kochi, Y., Myouzen, K., Yamada, R., Suzuki, A., Kurosaki, T., Nakamura, Y.
& Yamamoto, K. FCRL3, an autoimmune susceptibility gene, has inhibitory
potential on B-cell receptor-mediated signaling. J Immunol Nov. 2009,
183; 5502-5510.
41. Kohu, K., Ohmori, H., Wong, W. F., Onda, D., Wakoh, T., Kon, S., Yamashita,M., Nakayama, T., Kubo, M. & Satake, M. The Runx3 transcription factor
augments Th1 and down-modulates Th2 phenotypes by interacting withand attenuating GATA3. J Immunol Dec 15. 2009, 183; 7817-7824.

27. Kaisho, T. & Takeda, K. The study of innate immunity in Japan: a historicalperspective. Int Immunol Apr. 2009, 21; 313-316.

42. Koso, M., Tashiro, T., Sasaki, M. & Takikawa, H. First synthesis and absoluteconfiguration of a beta- farnesene-trimethoxystyrene conjugate isolated
from Pachypodanthium confine. Tetrahedron Nov. 2009, 65; 9142-9145.

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

43. Kubagawa, H., Oka, S., Kubagawa, Y., Torii, I., Takayama, E., Kang, D. W.,
Gartland, G. L., Bertoli, L. F., Mori, H., Takatsu, H., Kitamura, T., Ohno, H. &Wang, J. Y. Identity of the elusive IgM Fc receptor ( FcmuR) in humans. J
Exp Med Nov. 2009, 206; 2779-2793.

29. Kandasamy, K., Keerthikumar, S., Raju, R., Keshava Prasad, T. S.,
Ramachandra, Y. L., Mohan, S. & Pandey, A. PathBuilder--open source
software for annotating and developing pathway resources. Bioinformatics Nov. 2009, 25; 2860-2862.

44. Kunii, N., Horiguchi, S., Motohashi, S., Yamamoto, H., Ueno, N., Yamamoto,S., Sakurai, D., Taniguchi, M., Nakayama, T. & Okamoto, Y. Combination
therapy of in vitro-expanded natural killer T cells and alpha-galactosylceramide-pulsed antigen-presenting cells in patients with recurrent head andneck carcinoma. Cancer Sci Jun. 2009, 100; 1092-1098.

30. Kandasamy, K., Mohan, S. S., Raju, R., Keerthikumar, S., Kumar, G. S.,
Venugopal, A. K., Telikicherla, D., Navarro, J. D., Mathivanan, S., Pecquet,C., Gollapudi, S. K., Tattikota, S. G., Mohan, S., Padhukasahasram, H.,
Subbannayya, Y., Goel, R., Jacob, H. K., Zhong, J., Sekhar, R., Nanjappa,
V., Balakrishnan, L., Subbaiah, R., Ramachandra, Y. L., Rahiman, B. A.,
Prasad, T. S., Lin, J. X., Houtman, J. C., Desiderio, S., Renauld, J. C.,
Constantinescu, S. N., Ohara, O., Hirano, T., Kubo, M., Singh, S., Khatri, P.,
Draghici, S., Bader, G. D., Sander, C., Leonard, W. J. & Pandey, A. NetPath:a public resource of curated signal transduction pathways. Genome Biol
Jan. 2010, 11; R3.
31. Katagiri, K., Katakai, T., Ebisuno, Y., Ueda, Y., Okada, T. & Kinashi, T. Mst1
controls lymphocyte trafficking and interstitial motility within lymph nodes.
Embo J May. 2009, 28; 1319-1331.
32. Kawamoto, H. & Katsura, Y. A new paradigm for hematopoietic cell lineages: revision of the classical concept of the myeloid-lymphoid dichotomy.
Trends Immunol May. 2009, 30; 193-200.
33. Kawamoto, H., Wada, H. & Katsura, Y. A revised scheme for developmental
pathways of hematopoietic cells: the myeloid-based model. Int Immunol
Feb. 2010, 22; 65-70.

45. Masuda, K., Germeraad, W. T., Satoh, R., Itoi, M., Ikawa, T., Minato, N.,
Katsura, Y., van Ewijk, W. & Kawamoto, H. Notch activation in thymic
epithelial cells induces development of thymic microenvironments. Mol
Immunol May. 2009, 46; 1756-1767.
46. Masuda, K., Ouchida, R., Li, Y., Gao, X., Mori, H. & Wang, J. Y. A critical rolefor REV1 in regulating the induction of C:G transitions and A:T mutations
during Ig gene hypermutation. J Immunol Aug. 2009, 183; 1846-1850.
47. Matsuda, T., Kido, Y., Asahara, S., Kaisho, T., Tanaka, T., Hashimoto, N.,
Shigeyama, Y., Takeda, A., Inoue, T., Shibutani, Y., Koyanagi, M., Hosooka,T., Matsumoto, M., Inoue, H., Uchida, T., Koike, M., Uchiyama, Y., Akira, S.
& Kasuga, M. Ablation of C/ EBPbeta alleviates ER stress and pancreatic
beta cell failure through the GRP78 chaperone in mice. J Clin Invest Jan.
2010, 120; 115-126.
48. Matsuoka, N., Itoh, T., Watarai, H., Sekine-Kondo, E., Nagata, N., Okamoto,K., Mera, T., Yamamoto, H., Yamada, S., Maruyama, I., Taniguchi, M. &Yasunami, Y. High-mobility group box 1 is involved in the initial events ofearly loss of transplanted islets in mice. J Clin Invest Mar. 2010, 120;735-743.

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49. Meek, B., Cloosen, S., Borsotti, C., Van Elssen, C. H., Vanderlocht, J.,
Schnijderberg, M. C., van der Poel, M. W., Leewis, B., Hesselink, R., Manz,
M. G., Katsura, Y., Kawamoto, H., Germeraad, W. T. & Bos, G. M. In vitrodifferentiated T/natural killer-cell progenitors derived from human CD34+
cells mature in the thymus. Blood Jan. 2010, 115; 261-264.
50. Miletic, A. V., Graham, D. B., Sakata-Sogawa, K., Hiroshima, M., Hamann,
M. J., Cemerski, S., Kloeppel, T., Billadeau, D. D., Kanagawa, O., Tokunaga,
M. & Swat, W. Vav links the T cell antigen receptor to the actin cytoskeleton and T cell activation independently of intrinsic Guanine nucleotide
exchange activity. PLoS One Aug. 2009, 4; e6599.
51. Mori, H., Ouchida, R., Hijikata, A., Kitamura, H., Ohara, O., Li, Y., Gao, X.,
Yasui, A., Lloyd, R. S. & Wang, J. Y. Deficiency of the oxidative damagespecific DNA glycosylase NEIL1 leads to reduced germinal center B cell
expansion. DNA Repair (Amst ) Nov. 2009, 8; 1328-1332.
52. Mori, K. & Tashiro, T. Synthesis of all the four stereoisomers of (1' S)1-ethyl-2-methylpropyl 3, 13-dimethylpentadecanoate, the major sex
pheromone component of Paulownia bagworm, Clania Variegate. Tetrahedron Lett Oct. 2009, 50; 3266-3269.
53. Morita, T., Tanimura, A., Baba, Y., Kurosaki, T. & Tojyo, Y. A Stim1-dependent,
noncapacitative Ca2+-entry pathway is activated by B-cell-receptor stimulation and depletion of Ca2+. J Cell Sci Apr. 2009, 122; 1220-1228.
54. Moro, K., Yamada, T., Tanabe, M., Takeuchi, T., Ikawa, T., Kawamoto, H.,
Furusawa, J., Ohtani, M., Fujii, H. & Koyasu, S. Innate production of T( H ) 2
cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells.
Nature Jan. 2010, 463; 540-544.
55. Nagashima, T., Ushikoshi-Nakayama, R., Suenaga, A., Ide, K., Yumoto, N.,
Naruo, Y., Takahashi, K., Saeki, Y., Taiji, M., Tanaka, H., Tsai, S. F. & Hatakeyama, M. Mutation of epidermal growth factor receptor is associatedwith MIG6 expression. Febs J Sep. 2009, 276; 5239-5251.
56. Nagatake, T., Fukuyama, S., Kim, D. Y., Goda, K., Igarashi, O., Sato, S.,
Nochi, T., Sagara, H., Yokota, Y., Jetten, A. M., Kaisho, T., Akira, S., Mimuro,
H., Sasakawa, C., Fukui, Y., Fujihashi, K., Akiyama, T., Inoue, J., Penninger,
J. M., Kunisawa, J. & Kiyono, H. Id2-, RORgammat-, and LTbetaRindependent initiation of lymphoid organogenesis in ocular immunity. J
Exp Med Oct. 2009, 206; 2351-2364.
57. Nakagawa, T., Tsuruoka, M., Ogura, H., Okuyama, Y., Arima, Y., Hirano, T.
& Murakami, M. IL-6 positively regulates Foxp3+CD8+ T cells in vivo. Int
Immunol Feb. 2010, 22; 129-139.
58. Nakata, M., Takada, Y., Hishiki, T., Saito, T., Terui, K., Sato, Y., Koseki, H.
& Yoshida, H. Induction of Wnt5a-expressing mesenchymal cells adjacentto the cloacal plate is an essential process for its proximodistal elongation and subsequent anorectal development. Pediatr Res Aug. 2009, 66;
149-154.
59. Nakato, G., Fukuda, S., Hase, K., Goitsuka, R., Cooper, M. D. & Ohno, H.
New approach for m-cell-specific molecules screening by comprehensive
transcriptome analysis. DNA Res Aug. 2009, 16; 227-235.
60. Nishida, K., Hasegawa, A., Nakae, S., Oboki, K., Saito, H., Yamasaki,
S. & Hirano, T. Zinc transporter Znt5 / Slc30a5 is required for the mastcell-mediated delayed-type allergic reaction but not the immediate-type
reaction. J Exp Med Jun. 2009, 206; 1351-1364.
61. Numaga, T., Nishida, M., Kiyonaka, S., Kato, K., Katano, M., Mori, E.,
Kurosaki, T., Inoue, R., Hikida, M., Putney, J. W., Jr. & Mori, Y. Ca2+ influxand protein scaffolding via TRPC3 sustain PKCbeta and ERK activation in
B cells. J Cell Sci Mar. 2010, 123; 927-938.
62. Ohara, O. ORFeome cloning. Methods Mol Biol Aug. 2009, 577; 3-9.
63. Ohara, O. From transcriptome analysis to immunogenomics: current status
and future direction. FEBS Lett Jun. 2009, 583; 1662-1667.
64. Ohmura-Hoshino, M., Matsuki, Y., Mito-Yoshida, M., Goto, E., Aoki-Kawasumi, M., Nakayama, M., Ohara, O. & Ishido, S. Cutting edge: requirementof MARCH-I-mediated MHC II ubiquitination for the maintenance of
conventional dendritic cells. J Immunol Dec. 2009, 183; 6893-6897.

116

65. Okamoto, M., Van Stry, M., Chung, L., Koyanagi, M., Sun, X., Suzuki, Y.,
Ohara, O., Kitamura, H., Hijikata, A., Kubo, M. & Bix, M. Mina, an Il4 repressor, controls T helper type 2 bias. Nat Immunol Aug. 2009, 10; 872-879.
66. Park, J. H., Adoro, S., Guinter, T., Erman, B., Alag, A. S., Catalfamo, M.,
Kimura, M. Y., Cui, Y., Lucas, P. J., Gress, R. E., Kubo, M., Hennighausen,
L., Feigenbaum, L. & Singer, A. Signaling by intrathymic cytokines, not T
cell antigen receptors, specifies CD8 lineage choice and promotes thedifferentiation of cytotoxic-lineage T cells. Nat Immunol Mar. 2010, 11;257-264.
67. Qiu, C. H., Miyake, Y., Kaise, H., Kitamura, H., Ohara, O. & Tanaka, M.
Novel subset of CD8 { alpha } + dendritic cells localized in the marginal zone
is responsible for tolerance to cell-associated antigens. J Immunol Apr.
2009, 182; 4127-4136.
68. Roman-Trufero, M., Mendez-Gomez, H. R., Perez, C., Hijikata, A., Fujimura,Y., Endo, T., Koseki, H., Vicario-Abejon, C. & Vidal, M. Maintenance of
undifferentiated state and self-renewal of embryonic neural stem cells by
Polycomb protein Ring1B. Stem Cells Jul. 2009, 27; 1559-1570.
69. Saito, Y., Kitamura, H., Hijikata, A., Tomizawa-Murasawa, M., Tanaka, S.,
Takagi, S., Uchida, N., Suzuki, N., Sone, A., Najima, Y., Ozawa, H., Wake,
A., Taniguchi, S., Shultz, L. D., Ohara, O. & Ishikawa, F. Identification oftherapeutic targets for quiescent, chemotherapy-resistant human leukemia stem cells. Sci Transl Med Feb. 2010, 2; 17ra19.
70. Saito, Y., Uchida, N., Tanaka, S., Suzuki, N., Tomizawa-Murasawa, M., Sone,
A., Najima, Y., Takagi, S., Aoki, Y., Wake, A., Taniguchi, S., Shultz, L. D. &Ishikawa, F. Induction of cell cycle entry eliminates human leukemia stem
cells in a mouse model of AML. Nat Biotechnol Mar. 2010, 28; 275-280.
71. Sasaki, Y. & Kurosaki, T. Immobile BCRs: the safety on the signal trigger.
Immunity Feb. 2010, 32; 143-144.
72. Sato, I., Obata, Y., Kasahara, K., Nakayama, Y., Fukumoto, Y., Yamasaki, T.,
Yokoyama, K. K., Saito, T. & Yamaguchi, N. Differential trafficking of Src,
Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4
domain. J Cell Sci Apr. 2009, 122; 965-975.
73. Sato, K., Eizumi, K., Fukaya, T., Fujita, S., Sato, Y., Takagi, H., Yamamoto,M., Yamashita, N., Hijikata, A., Kitamura, H., Ohara, O., Yamasaki, S., Saito,
T. & Sato, K. Naturally occurring regulatory dendritic cells regulate murine
cutaneous chronic graft-versus-host disease. Blood May. 2009, 113;
4780-4789.
74. Schwermann, J., Rathinam, C., Schubert, M., Schumacher, S., Noyan, F.,
Koseki, H., Kotlyarov, A., Klein, C. & Gaestel, M. MAPKAP kinase MK2maintains self-renewal capacity of haematopoietic stem cells. Embo J
May. 2009, 28; 1392-1406.
75. Seo, J. H., Suenaga, A., Hatakeyama, M., Taiji, M. & Imamoto, A. Structuraland functional basis of a role for CRKL in a fibroblast growth factor 8-induced feed-forward loop. Mol Cell Biol Jun. 2009, 29; 3076-3087.
76. Setoguchi, R., Taniuchi, I. & Bevan, M. J. ThPOK derepression is required
for robust CD8 T cell responses to viral infection. J Immunol Oct. 2009,
183; 4467-4474.
77. Shima, H., Takatsu, H., Fukuda, S., Ohmae, M., Hase, K., Kubagawa, H.,
Wang, J. Y. & Ohno, H. Identification of TOSO / FAIM3 as an Fc receptor for
IgM. Int Immunol Mar. 2010, 22; 149-156.
78. Shinohara, H. & Kurosaki, T. Comprehending the complex connection
between PKCbeta, TAK1, and IKK in BCR signaling. Immunol Rev Nov.
2009, 232; 300-318.
79. Shinohara, H., Yamasaki, S., Maeda, S., Saito, T. & Kurosaki, T. Regulationof NF-kappaB-dependent T cell activation and development by MEKK3. Int
Immunol Apr. 2009, 21; 393-401.
80. Suzuki, H., Forrest, A. R., van Nimwegen, E., Daub, C. O., Balwierz, P.
J., Irvine, K. M., Lassmann, T., Ravasi, T., Hasegawa, Y., de Hoon, M. J.,
Katayama, S., Schroder, K., Carninci, P., Tomaru, Y., Kanamori-Katayama,M., Kubosaki, A., Akalin, A., Ando, Y., Arner, E., Asada, M., Asahara, H.,
Bailey, T., Bajic, V. B., Bauer, D., Beckhouse, A. G., Bertin, N., Bjorkegren,

J., Brombacher, F., Bulger, E., Chalk, A. M., Chiba, J., Cloonan, N., Dawe,
A., Dostie, J., Engstrom, P. G., Essack, M., Faulkner, G. J., Fink, J. L.,
Fredman, D., Fujimori, K., Furuno, M., Gojobori, T., Gough, J., Grimmond,
S. M., Gustafsson, M., Hashimoto, M., Hashimoto, T., Hatakeyama, M.,
Heinzel, S., Hide, W., Hofmann, O., Hornquist, M., Huminiecki, L., Ikeo, K.,
Imamoto, N., Inoue, S., Inoue, Y., Ishihara, R., Iwayanagi, T., Jacobsen, A.,
Kaur, M., Kawaji, H., Kerr, M. C., Kimura, R., Kimura, S., Kimura, Y., Kitano,
H., Koga, H., Kojima, T., Kondo, S., Konno, T., Krogh, A., Kruger, A., Kumar,A., Lenhard, B., Lennartsson, A., Lindow, M., Lizio, M., Macpherson, C.,
Maeda, N., Maher, C. A., Maqungo, M., Mar, J., Matigian, N. A., Matsuda,
H., Mattick, J. S., Meier, S., Miyamoto, S., Miyamoto-Sato, E., Nakabayashi,K., Nakachi, Y., Nakano, M., Nygaard, S., Okayama, T., Okazaki, Y., OkudaYabukami, H., Orlando, V., Otomo, J., Pachkov, M., Petrovsky, N., Plessy,C., Quackenbush, J., Radovanovic, A., Rehli, M., Saito, R., Sandelin, A.,
Schmeier, S., Schonbach, C., Schwartz, A. S., Semple, C. A., Sera, M.,
Severin, J., Shirahige, K., Simons, C., St Laurent, G., Suzuki, M., Suzuki,T., Sweet, M. J., Taft, R. J., Takeda, S., Takenaka, Y., Tan, K., Taylor, M.
S., Teasdale, R. D., Tegner, J., Teichmann, S., Valen, E., Wahlestedt, C.,
Waki, K., Waterhouse, A., Wells, C. A., Winther, O., Wu, L., Yamaguchi, K.,
Yanagawa, H., Yasuda, J., Zavolan, M., Hume, D. A., Arakawa, T., Fukuda,S., Imamura, K., Kai, C., Kaiho, A., Kawashima, T., Kawazu, C., Kitazume,Y., Kojima, M., Miura, H., Murakami, K., Murata, M., Ninomiya, N., Nishiyori, H., Noma, S., Ogawa, C., Sano, T., Simon, C., Tagami, M., Takahashi,Y., Kawai, J. & Hayashizaki, Y. The transcriptional network that controls
growth arrest and differentiation in a human myeloid leukemia cell line.
Nat Genet May. 2009, 41; 553-562.
81. Takahama, Y., Saito, T., Kawamoto, H., Itoi, M., Boyd, R. L., Chidgey, A.,
Zamoyska, R., Hollander, G. A., Anderson, G., Taylor, N., Petrie, H. T. &Nikolich-Zugich, J. The Global Thymus Network: past, present and future.
Trends Immunol May. 2009, 30; 191-192.
82. Takahashi, A., Obata, Y., Fukumoto, Y., Nakayama, Y., Kasahara, K., Kuga,
T., Higashiyama, Y., Saito, T., Yokoyama, K. K. & Yamaguchi, N. Nuclear
localization of Src-family tyrosine kinases is required for growth factorinduced euchromatinization. Exp Cell Res Apr. 2009, 315; 1117-1141.
83. Takashima, K., Matsunaga, N., Yoshimatsu, M., Hazeki, K., Kaisho, T.,
Uekata, M., Hazeki, O., Akira, S., Iizawa, Y. & Ii, M. Analysis of binding site
for the novel small-molecule TLR4 signal transduction inhibitor TAK-242
and its therapeutic effect on mouse sepsis model. Br J Pharmacol Aug.
2009, 157; 1250-1262.
84. Takeuchi, A., Itoh, Y., Takumi, A., Ishihara, C., Arase, N., Yokosuka, T.,
Koseki, H., Yamasaki, S., Takai, Y., Miyoshi, J., Ogasawara, K. & Saito, T.
CRTAM confers late-stage activation of CD8+ T cells to regulate retention
within lymph node. J Immunol Oct. 2009, 183; 4220-4228.
85. Tanaka, S., Yoshimoto, T., Naka, T., Nakae, S., Iwakura, Y., Cua, D. & Kubo,
M. Natural occurring IL-17 producing T cells regulate the initial phaseof neutrophil mediated airway responses. J Immunol Dec 1. 2009, 183;7523-7530.
86. Taniguchi, M., Tashiro, T., Dashtsoodol, N., Hongo, N. & Watarai, H. The
specialized iNKT cell system recognizes glycolipid antigens and bridges
the innate and acquired immune systems with potential applications forcancer therapy. Int Immunol Jan. 2010, 22; 1-6.
87. Taniuchi, I. Transcriptional regulation in helper versus cytotoxic-lineage
decision. Curr Opin Immunol Apr. 2009, 21; 127-132.
88. Tashiro, T., Nakagawa, R., Hirokawa, T., Inoue, S., Watarai, H., Taniguchi,
M. & Mori, K. RCAI-37, 56, 59, 60, 92, 101, and 102, cyclitol and carbasugar analogs of KRN7000 : their synthesis and bioactivity for mouse
lymphocytes to produce Th1-biased cytokines. Bioorg Med Chem Sep.
2009, 17; 6360-6373.
89. Tomura, M., Honda, T., Tanizaki, H., Otsuka, A., Egawa, G., Tokura, Y.,
Waldmann, H., Hori, S., Cyster, J. G., Watanabe, T., Miyachi, Y., Kanagawa,O. & Kabashima, K. Activated regulatory T cells are the major T cell typeemigrating from the skin during a cutaneous immune response in mice. J
Clin Invest Mar. 2010, 120; 883-893.

90. Tomura, M., Mori, Y. S., Watanabe, R., Tanaka, M., Miyawaki, A. & Kanagawa, O. Time-lapse observation of cellular function with fluorescentprobe reveals novel CTL-target cell interactions. Int Immunol Oct. 2009,
21; 1145-1150.
91. Tong, H., Miyazaki, Y., Yamazaki, M., Hara, H., Waldmann, H., Hori, S. &Yoshida, H. Exacerbation of delayed-type hypersensitivity responses in
EBV-induced gene-3 ( EBI-3 )-deficient mice. Immunol Lett Feb. 2010,
128; 108-115.
92. Tran, T. H., Nakata, M., Suzuki, K., Begum, N. A., Shinkura, R., Fagarasan,
S., Honjo, T. & Nagaoka, H. B cell-specific and stimulation-responsive
enhancers derepress Aicda by overcoming the effects of silencers. Nat
Immunol Feb. 2010, 11; 148-154.
93. Tsukamoto, Y., Nagai, Y., Kariyone, A., Shibata, T., Kaisho, T., Akira, S.,
Miyake, K. & Takatsu, K. Toll-like receptor 7 cooperates with IL-4 inactivated B cells through antigen receptor or CD38 and induces classswitch recombination and IgG1 production. Mol Immunol Apr. 2009, 46;
1278-1288.
94. Udono, H., Ichiyanagi, T., Mizukami, S. & Imai, T. Heat shock proteins inantigen trafficking--implications on antigen presentation to T cells. Int J
Hyperthermia Dec. 2009, 25; 617-625.
95. Wada, H., Yasuda, T., Miura, I., Watabe, K., Sawa, C., Kamijuku, H., Kojo,
S., Taniguchi, M., Nishino, I., Wakana, S., Yoshida, H. & Seino, K. Establishment of an improved mouse model for infantile neuroaxonal dystrophy thatshows early disease onset and bears a point mutation in Pla2g6. Am J
Pathol Dec. 2009, 175; 2257-2263.
96. Wang, Y., Deng, X., Zhou, Y., Hendron, E., Mancarella, S., Ritchie, M. F.,
Tang, X. D., Baba, Y., Kurosaki, T., Mori, Y., Soboloff, J. & Gill, D. L. STIM
protein coupling in the activation of Orai channels. Proc Natl Acad Sci U S
A May. 2009, 106; 7391-7396.
97. Watarai, H., Rybouchkin, A., Hongo, N., Nagata, Y., Sakata, S., Sekine, E.,
Dashtsoodol, N., Tashiro, T., Fujii, S., Shimizu, K., Mori, K., Masuda, K.,
Kawamoto, H., Koseki, H. & Taniguchi, M. Generation of functional NKT
cells in vitro from embryonic stem cells bearing rearranged invariant
Valpha14-Jalpha18 TCRalpha gene. Blood Jan. 2010, 115; 230-237.
98. Xue, X., Kimura, W., Wang, B., Hikosaka, K., Itakura, T., Uezato, T., Matsuda, M., Koseki, H. & Miura, N. A unique expression pattern of Tbx10 in
the hindbrain as revealed by Tbx10 ( lacZ ) allele. Genesis Feb. 2010.
99. Yamaguchi, K., Inoue, S., Ohara, O. & Nagase, T. Pulse-chase experiment
for the analysis of protein stability in cultured mammalian cells by covalent
fluorescent labeling of fusion proteins. Methods Mol Biol Aug. 2009, 577;
121-131.
100. Yan, P., Fu, J., Qu, Z., Li, S., Tanaka, T., Grusby, M. J. & Xiao, G. PDLIM2
suppresses human T-cell leukemia virus type I Tax-mediated tumorigenesis by targeting Tax into the nuclear matrix for proteasomal degradation.
Blood Apr. 2009, 113; 4370-4380.
101. Yasuda, N., Masuda, K., Tsukui, T., Teng, A. & Ishii, Y. Identification ofcanine natural CD3-positive T cells expressing an invariant T-cell receptoralpha chain. Vet Immunol Immunopathol Dec 15. 2009, 132; 224-231.
102. Yokosuka, T. & Saito, T. Dynamic regulation of T-cell costimulation through
TCR-CD28 microclusters. Immunol Rev May. 2009, 229; 27- 40.
103. Yokosuka, T. & Saito, T. The immunological synapse, TCR microclusters, andT cell activation. Curr Top Microbiol Immunol Jan. 2010, 340; 81-107.
104. Yuri, S., Fujimura, S., Nimura, K., Takeda, N., Toyooka, Y., Fujimura, Y.,
Aburatani, H., Ura, K., Koseki, H., Niwa, H. & Nishinakamura, R. Sall4 isessential for stabilization, but not for pluripotency, of embryonic stem cellsby repressing aberrant trophectoderm gene expression. Stem Cells Apr.
2009, 27; 796-805.

117

Invited Presentations
Speaker

Title

MeetingMonthYear

Fagarasan, S.

Dynamic interactions between bacteria and B cells in GALT

Tolerance and Development, Fondation des Treilles, France

Apr- 09

Tokunaga, M.

Single molecule imaging in living cells

4th Global COE International Symposium, Tokyo, Japan

May - 09

Tokunaga, M.

Clear visualization of single molecules living cells

Extreme Photonics Symposium of RIKEN, Wako, Japan

May - 09

Mohan, S.

RAPID: Resource of Asian Primary Immunodeficiency Diseases - An Integrated
Informational Platform

Keystone Symposia Conference, Human Immunology and
Immunodeficiencies, Beijing, China

May - 09

Hirano, T.

“Interleukin 6 Amplifier”, a key player in inflammatory diseases ---How extensive aview from the top of the mountain is!

Symposium of The Crafoord Prize, Stockholm, Sweden

May - 09

Hatakeyama, M.

Binary response of ErbB signaling to determine cell fate

The 2nd RIKEN - University of Edinburgh Joint Workshop for
Computational and Systems Biology, Tokyo, Japan

May - 09

Saito, T.

Spatiotemporal regulation of lymphocyte activation

The 2nd RIKEN - University of Edinburgh Joint Workshop for
Computational and Systems Biology, Tokyo, Japan

May - 09

Fukada, T.

A model mouse with mutated zinc transporter

The 2nd Tokyo Anti-aging Academy, Tokyo, Japan

May - 09

Fukada, T.

Zinc and zinc transporter: involvement in cellular functions and inherited
pathogenesis

The 9th Scientific Meeting of The Japanese Society of Anti-Aging
Medicine, Tokyo, Japan

May - 09

Kurosaki, T.

Erk signaling in B lymphocytes

FASEB Summer Research Conferences, Snowmass Village, U.S.A.

Jun - 09

Saito, T.

Sensing self and non-self danger by ITAM-coupled innate receptor Mincle

FASEB Summer Research Conferences, Snowmass Village, U.S.A.

Jun - 09

Tanaka, M.

Mechanism of tolerance induction to cell-associated antigens by apoptotic cell
clearance

Gordon Research Conferences, Apoptotic Cell Recognition &Clearance, New London, USA

Jun - 09

Kubo, M

Control of skin inflammation by cytokine signaling

Immuno-Cardiology Conference, Tokyo, JapanJun - 09

Kurosaki, T.

PLC g/calcium signaling in immune cells

Protein Coupling in Cellular Networks, Freiburg, Germany

Jun - 09

Taniuchi, I.

Transcription factors network that regulates thymocyte differentiation

Seminar at Chiba University, Chiba, JapanJun - 09

Kurosaki, T.

Erk signaling in B lymphocytes

Singapore-Osaka, 1st Joint SIgN-IFReC Meeting, Singapore

Jun - 09

Saito, T.

Visualization of T cell activation and co-stimulation

Singapore-Osaka, 1st Joint SIgN-IFReC Meeting, Singapore

Jun - 09

Hirano, T.

“IL-6 amplifier”: a key player in autoimmune and inflammatory diseases

Singapore-Osaka, 1st Joint SIgN-IFReC Meeting, Singapore

Jun - 09

Ishii, Y.

Immunotherapy for cedar pollinosis by new vaccine technologies

The 21st Spring Meeting of Japanese Society of Allergology, Gifu,Japan

Jun - 09

Kaisho, T.

Molecular mechanisms on dendritic cell activation by nucleic acid adjuvants

The 21st Spring Meeting of Japanese Society of Allergology, Gifu,Japan

Jun - 09

Ishikawa, F.

Reconstitution of human immune system in mice

The 5th International Workshop of Kyoto T Cell Conference 2009,
Kyoto, JapanJun - 09

Kawamoto, H.

Retention and termination of myeloid potential in T cell progenitors

The 5th International Workshop of Kyoto T Cell Conference 2009,
Kyoto, JapanJun - 09

Kubo, M

Runx/CBF-β is a critical gatekeeper that directs the Th1 and Th17 differentiation

The 5th International Workshop of Kyoto T Cell Conference 2009,
Kyoto, JapanJun - 09

Taniuchi, I.

Transcriptional control of CD4/CD8 lineage choice

The 5th International Workshop of Kyoto T Cell Conference 2009,
Kyoto, JapanJun - 09

van Ewijk, W.

The thymus….follow me!

The 5th International Workshop of Kyoto T Cell Conference 2009,
Kyoto, JapanJun - 09

Saito, T.

Dynamic assembly of signaling complex for positive and negative costimulationof T cell activation

The 5th International Workshop of Kyoto T Cell Conference 2009,
Kyoto, JapanJun - 09

Kurosaki, T.

Erk signaling in B lymphocytes

The 5th Leukocyte Signal Transduction Workshop, Heraklion, Greek

Jun - 09

Hirano, T.

Interleukin 6 hunting

The 74th Japanese Society of Interferon and Cytokine Research,
Kyoto, JapanJun - 09

Fujii, S.

NKT cell-dendritic cell (DC) cross-talk.

The 74th Japanese Society of Interferon and Cytokine Research,
Kyoto, JapanJun - 09

Fukuda, S.

Multiple Omics Identified Microbial Metabolites to Protect Host fromEnterohemorrhagic Escherichia coli O157:H7 Lethal Infection

The 8th International Workshop on Advanced Genomics, Tokyo,
Japan

Jun - 09

Kawamoto, H.

Making iPS cells from lymphocyte

Japanese Association for Animal Cell Technology 2009, Tsukuba,Japan

Jul - 09

Hirano, T.

“IL-6 amplifier”: a key player in autoimmune and inflammatory diseases.

RCAI-JSI International Symposium on Immunology 2009, Kanagawa,Japan

Jul - 09

Taniuchi, I.

Transcriptional control of helper versus cytotoxic lineage choice

Seminar at MD Anderson Cancer Center, Houston, USA

Jul - 09

Fukada, T.

The role of zinc transporter Slc39a13/Zip13 in connective tissue development ofmouse and humanThe 20th Annual Scientific Meeting of the Japanese Society for
Biomedical Research on Trace Element, Tokyo, Japan

Jul - 09

Ishikawa, F.

The role of AML stem cell niche

The 27th Annual Meeting of the Japanese Society for Bone andMineral Research, Osaka, Japan

Jul - 09

Hirano, T.

Is Dysregulation of IL-6 Amplifier a Key Event of Rheumatoid Arthritis?

The 9th World Congress on Inflammation, Industry Sponsored
Symposium 2, Tokyo, Japan

Jul - 09

Saito, T.

Dynamic regulation of T cell recognition and activation.

The JSI Summer School, Hyogo, Japan

Jul - 09

Hirano, T.

“A view from the summit”-- Lessons from mountain climbing-- three decades with
Interleukin 6

The JSI Summer School, Hyogo, Japan

Jul - 09

Tanaka, M.

Strategy of tolerance induction by apoptotic cell clearance

4th Medical Biotech Forum, Dalian, China

Aug - 09

Sakata-Sogawa,K. & Tokunaga, M.

Single molecule imaging and quantitative analysis of T cell signaling initiation

CREST Symposium, Tokyo, Japan

Aug - 09

Taniuchi, I.

Essential requirement of Cbfβ2 variant for Smad4-mediated TGFβ signals and fordevelopment and homeostasis of immune system

EMBO Workshop on RUNX Transcription Factors, Oxford, UK

Aug - 09

Kawamoto, H.

Insight into the phylogenic origin of lymphocytes

Japanese Association for Developmental and Comparative
Immunology, Annual Meeting, Fujisawa, Japan

Aug - 09

Saito, T.

Spatiotemporal regulation of T cell activation

Meeting for the MEXT Priority Research on the Molecular
Mechanisms and function of Transportsome, Aso, Japan

Aug - 09

Sakata-Sogawa,K. & Tokunaga, M.

Single molecule imaging analysis of T cell signaling initiation

Scientific Research of Priority Areas Symposium, Tokyo, Japan

Aug - 09

Fukuda, S.

Construction of Gut Environment Assessment System Based on Multiple Omics

19th Annual Meeting of the Japanese Society for Mathematical
Biology, Tokyo, Japan

Sep - 09

Taniguchi, M.

A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to
IL-25 and contributes to airway hyperreactivity

2nd European Congress of Immunology, Berlin, Germany

Sep - 09

Ohno, H.

Molecular mechanism for the formation of tunneling nanotube connecting distantcells

Bioscience Seminar, Chiba University, School of Medicine, Chiba,Japan

Sep - 09118

Wang, J-Y.

How can AID induce a high frequency of A:T mutations in the germinal center B
cells?

College of Biological Sciences, China Agricultural University, Beijing,
China

Sep - 09

Kurosaki, T.

Role of PLCg2 in humoral memory

EMBO Conference Signaling in the Immune System, Siena, Italy

Sep - 09

Kawamoto, H.

How are cell fate decisions made during hematopoiesis?

Japanese Society of Mathematical Biology, Annual Meeting, Tokyo,
Japan

Sep - 09

Yamada,D.
Koseki, H.

Application of iPS technology for NKT cell-mediated adjuvant therapy in mice

Joint Cold Spring Harbor Laboratory/ Wellcome Trust Conference,
Hinxton, UK

Sep - 09

Taniuchi, I.

Transcription factors network that governs cell fate determination by thymocyte

JSMB 2009, Tokyo, Japan

Sep - 09

Kawamoto, H.

Induction of self-renewal program in developing hematopoietic progenitors by
inhibiting their differentiation

JST-CIRM Workshop, Kyoto, Japan

Sep - 09

Fagarasan, S.

T-dependent and T-independent IgA responses in the gut

Microanatomy of the Immune Responses in Health and Disease,
University of Birmingham, UK

Sep - 09

van Ewijk, W.

The thymus, classrooms, teachers and students

Southwestern Medical University, Dallas, USA

Sep - 09

Udono, H.

A role of immunochaperones in host-defense mechanism

Symposium of Immunity on mammals and plants, Tokyo, Japan

Sep - 09

Kaisho, T.

Vaccination strategy based on plasmacytoid dendritic cell biology

The 13th annual meeting of The Japanese Society for Vaccinology,
Hokkaido, Japan

Sep - 09

Tokunaga, M. &Sakata-Sogawa,
K.

Single molecule imaging in living cells

The 58th Discussion Meeting of Japan Polymer Science Society,Kumamoto, Japan

Sep - 09

Sato, K.

Control of immune response by regulatory dendritic cells

The 7th Seminar for New Animal Breeding & Genetics, JapaneseSociety of Animal Breeding and Genetic, Tsukuba, Japan

Sep - 09

Fukada, T.

The zinc transporter Zip13 is required for mammalian tooth development; its
implication in dental physiology and pathogenesis

The Satellite Symposium of the 51st Annual Meeting of theJapanese Society for Oral Biology, Niigata, Japan

Sep - 09

Taniguchi, M.

NKT cells bridging innate and acquired immunity

University of Palermo, Palermo, Italy

Sep - 09

Taniguchi, M.

IL-17RB+ NKT subset responsible for development of AHR

University of Palermo, Palermo, Italy

Sep - 09

Taniguchi, M.

Clinical application of NKT cell-targeted therapy and its future direction

University of Palermo, Palermo, Italy

Sep - 09

Ohno, H.

GP-2-mediated bacterial uptake by M cells and intestinal immune response

52nd Frontier Bioscience Colloquium, Osaka University, Japan

Oct - 09

Ishido, S.

E3 ubiquitin ligase for MHC molecules

82nd annual meeting of the Japanese biochemical society, Kobe,Japan

Oct - 09

van Ewijk, W.

The thymus, classrooms and students

City of Hope, Pasadena, USA

Oct - 09

Kawamoto, H.

The myeloid-based model: revised scheme for hematopoiesis

Kanazawa University, special seminar, Kanazawa, Japan

Oct - 09

Sato, K.

Immunotherapy with regulatory dendritic cells against allergic asthma

The 121th Annual Meeting of Kanto Area of JapanesePharmacological Society, Tokyo, Japan

Oct - 09

Hirano, T.

Zinc, immunity, allergy and inflammation: Zinc is a signaling molecule.

The 16th Hiroshi Kobayashi Memorial Lecture at The 415th Osaka
Dermatological Association, Osaka, Japan

Oct - 09

Udono, H.

Unique features of HSP-Ag fusion protein as a cancer vaccine

The 4th International Congress on Stress Response in Biology andMedicine, Sapporo, Japan

Oct - 09

Nishida, K

Role of zinc/zinc transporter in mast cell-mediated allergic reaction

The 59th Annual Meeting of Japanese Society of Allergology, Akita,Japan

Oct - 09

Sato, K.

Immunotherapy with regulatory dendritic cells

The 59th Annual Meeting of Japanese Society of Allergology, Akita,Japan

Oct - 09

Koseki, H.

Application of iPS technology for NKT cell-mediated adjuvant therapy in mice

The 71st Annual Meeting of the Japanese Society of Hematology,
Kyoto, Japan

Oct -09

Fukada, T.

The role of zinc transporter Slc39a13/Zip13 in mammalian health and disease

The 82nd Annual Meeting of the Japanese Biochemical Society,
Kobe, Japan

Oct - 09

Kubo, MRUNX is a critical gatekeeper that directs the Th1 and Th17 differentiation

The 82nd Annual Meeting of the Japanese Biochemical Society,
Kobe, Japan

Oct - 09

Nishida, K.

Role of zinc in mast cell-mediated allergic reaction

The 82nd Annual Meeting of the Japanese Biochemical Society,
Kobe, Japan

Oct - 09

Sakata-Sogawa,K. & Tokunaga, M.

Single molecule imaging and quantitative analysis of T cell signaling initiation

The 82nd Annual Meeting of the Japanese Biochemical Society,
Kobe, Japan

Oct - 09

Ohara, O.

From transcriptome analysis to immunogenomics

US-Japan International Cancer Systems Biology Meeting,Yokohama, Japan

Oct - 09

OkadaHatakeyama, M.

Network design of cell differentiation

US-Japan International Cancer Systems Biology Meeting,Yokohama, Japan

Oct - 09

Hirano, T.

What does 'self' mean for the immune system? The plasticity of self.

2009 Tekijuku Memorial Lectures, Osaka, Japan

Nov - 09

Saito, T.

CARD9 and innate immunity

Center for the Study of Inflammatory Bowel Disease 19th Annual
Workshop, Boston, USA

Nov - 09

Kawamoto, H.

The myeloid-based model: insight into original stage of mixed type leukemia

Japanese Society of Pediatric Hematology, Annual Meeting, Chiba,Japan

Nov - 09

Wang, J-Y.

Learning from the immune system

Kwansei Gakuin University, Hyogo, Japan

Nov - 09

Tokunaga, M.

Clear visualization of single molecules living cells

Seminar of Graduate School of Life Science, Hokkaido University,
Sapporo, Japan

Nov - 09

Ishikawa, F.

In vivo model for Human Leukemia

The 16th RIKEN President’s Discretionary Fund workshop, Gifu,Japan

Nov - 09

Fukada, T.

The zinc transporter Zip13: its roles in skeletogenesis and inherited connective
tissue disorders

The 1st Orthopedic Research Club, Kisarazu, Japan

Nov - 09

Tanaka, T.

PDLIM2, a nuclear ubiquitin E3 ligase, negatively regulates inflammatory
responses

The 2009 Fall Conference of The Korean Association of
Immunologists, Seoul, Republic of Korea

Nov - 09

Fukada, T.

The zinc transporter SLC39A13/ZIP13 is required for bone, tooth and connective
tissue development in mouse and human; its involvement in BMP/TGF-β
Signaling Pathways.

The 26th Naito Conference of Osteobiology, Hyogo, Japan

Nov - 09

Ishikawa, F.

In vivo investigation of human diseases using humanized mice

The 37th Annual Meeting of the Japanese Society of Clinical
Immunology, Tokyo, Japan

Nov - 09

Ishikawa, F.

Translational medicine using humanized mouse

The 39th Annual Meeting of the Japanese Society for Immunology,
Osaka, Japan

Nov - 09

Ishikawa, F.

Translational medicine using humanized mouse

The 39th annual meeting of The Japanese Society for Immunology,
Osaka, Japan

Nov - 09

Ishikawa, F.

Humanized mouse for biomedical research

The 3rd Chiba Global COE Symposium, Chiba, Japan

Nov - 09

Naito, T.

Ikaros-mediated epigenetic regulation of Notch signaling in T cell
development and leukemeogenesis

The 3rd Chiba Global COE Symposium, Chiba, Japan

Nov - 09

Suzuki, K.

T-independent IgA responses in the gut

The First CSI/JSI/KAI Joint Symposium on Immunology, Shanghai,
China

Nov - 09119

Ishikawa, F.

Humanized mouse for biomedical research

The First CSI/KAI Joint Symposium on Immunology, Shanghai, China

Nov - 09

Fukada, T.

The zinc transporter ZIP13 is required for bone, tooth and connective tissue
development in mouse and human; its involvement in BMP/TGF-β signaling
pathways2009 International Society for Zinc Biology Meeting, Jerusalem,
IsraelDec -09

Fagarasan, S.

T-dependent and T-independent IgA responses in the gut

Annual Meeting of the Japanese Society for Immunology, Osaka,Japan

Dec - 09

Kawamoto, H.

Insight into original stage of mixed type leukemia based on the
myeloid-based model

Japanese Society of Hematology, Kinki-area Meeting, Kyoto, Japan

Dec - 09

Kawamoto, H.

T cell lineage determination: termination of myeloid potential in T cell progenitors

Japanese Society of Immunology, Annual Meeting, Symposium,
Osaka, Japan

Dec - 09

Taniuchi, I.

Transcriptional control of helper versus cytotoxic lineage choice in thymus

Seminar at MPI, Berlin, Germany

Dec - 09

Okada, T.

Real-time imaging of cell-cell interactions during acquired immune responses

The 32th Annual meeting of Japan Molecular Biology Society,
Yokohama, Japan

Dec - 09

Sakata-Sogawa,K. & Tokunaga, M.

Single molecule imaging and quantitative analysis of living cells

The 32th Annual meeting of Japan Molecular Biology Society,
Yokohama, Japan

Dec - 09

Tokunaga, M. &Sakata-Sogawa,
K.

Future of Quantitative Biology: Single molecule imaging and quantification of livingcells

The 32th Annual meeting of Japan Molecular Biology Society,
Yokohama, Japan

Dec - 09

Ishido, S.

Novel immune regulation by ubiquitination

The 32th Annual meeting of Japan Molecular Biology Society,
Yokohama, Japan

Dec - 09

Yokosuka, T.

Dynamic regulation of T-cell costimulation by TCR microclusters and the c-SMAC

The 38th Annual Meeting of the Japanese Society for Immunology,
Osaka, Japan

Dec - 09

Sato, K.

Crucial roles of B7-H1 and B7-DC expressed on mesenteric dendritic cells in oral
tolerance

The 38th Annual Meeting of the Japanese Society for Immunology,
Osaka, Japan

Dec - 09

Aiba, Y.

Preferential localization of IgG memory B cells adjacent to waned germinal
centers

The 38th Annual Meeting of the Japanese Society for Immunology,
Osaka, Japan

Dec - 09

Baba, Y.

Store-operated calcium entry in B cells

The 38th Annual Meeting of the Japanese Society for Immunology,
Osaka, Japan

Dec - 09

Hori, S.

Robustness and plasticity in regulatory T cell differentiation

The 38th Annual Meeting of the Japanese Society for Immunology,
Osaka, Japan

Dec - 09

Kaisho, T.

Lecture for The Japanese Society for Immunology AwardThe 38th Annual Meeting of the Japanese Society for Immunology,
Osaka, Japan

Dec - 09

Haruhiko Koseki,

HP1g links histone H3K9 methylation to homologus chromosome pairing duringmelotic prophase

The Epigenetics 2009 Australian Scientific Conference at
Epigenetics and Development, Melbourne, Australia

Dec - 09

Taniuchi, I.

Roles of Runx proteins during T cell development

TWINCORE Seminar, Hanover, Germany

Dec - 09

Kubo, M

Immuno-epigenetics of allergic diseases

XXI World Allergy Congress (WAC) 2009, Buenos Aires, Argentine

Dec - 09

Ohara, O.

Functional genomics focused on immune systems

XXI World Allergy Congress (WAC) 2009, Buenos Aires, Argentine

Dec - 09

Ohno, H.

Uptake via Glycoprotein 2 by M cells is important for mucosal immunosurveillance+of FimH Bacteria

Adaptive and Innate Immune Responses to Neglected Tropical
Diseases Conference, US-Japan Cooperative Medical Science
Program, San Diego, CA, USA

Jan -10

Taniuchi, I.

Transcriptional regulations of T lymphocyte development

Seminar at Kumamoto Univ., JapanJan -10

Sakata-Sogawa,K. & Tokunaga, M.

Single molecule microscopy for immunogical cell signaling

Sweden-Japan joint colloquium, Stockholm, Sweden

Jan -10

Yokosuka, T.

Dynamic regulation of T-cell costimulation by TCR microclusters and the c-SMAC

The 13th International Membrane Research Forum / The 6th iCeMS
International Symposium, Featuring Membrane Meso Mechanisms,
Kyoto, JapanJan -10

Hase, K.

GP2-dependent antigen transcytosis by M cells initiates mucosal immune
response.

The 4th Stage Surface Barrier Immunology Study Group (SBARIS).
2nd Meeting, Tokyo, JapanJan -10

Saito, T.

Dual specificity receptor for damaged cells and pathogen

US-Japan Joint Immunology Board & Parasitic Disease Panel
Conference, San Diego, USA

Jan -10

Kawamoto, H.

The myeloid-based model: revised scheme for hematopoiesis

Yamaguchi University, Immunology Seminar, Kyoto, JapanJan -10Ohno, H

Glycoprotein 2 and M-Sec, genes specifically expressed in M cells, specialized
intestinal epithelial cells

21st International Training Program Seminar, Okayama University,Japan

Feb -10

Kawamoto, H.

Lineage restriction pathway in hematopoiesis:Revision of the classical concept of
myeloid-lymphoid dichotomy

Harvard Medical School, Wednesday Immunology Seminar Series,
Boston, USA

Feb -10

Saito, T.

Molecular dynamics for T cell activation and costimulation

Keystone Symposia, Lymphocyte Activation and Gene Expression,
Brekenbridge, USA

Feb -10

Hori, S.

Stability and plasticity in regulatory T cell differentiation

Keystone Symposia, Tolerance and Autoimmunity, Taos, New
Mexico, USA

Feb -10

Ohara, O.

Exploration of PID-causative mutations: Japan model and the future direction

The 2nd Symposium of PID in Asia, Chiba, Japan

Feb -10

Fukada, T.

Zinc and zinc transporter: their roles in osteogenesis and bone disease

The 4th Bone Research Seminar, Tokyo, Japan

Feb -10

Tokunaga, M.

TIRF principle and applications with a focus on biological samples

DKFZ/EMBL Practical Course, Heidelberg, Germany

Mar-10Ohno, H.

Comprehensive analysis of the protective mechanism of enterohaemorrhagic
Escherichia coli O157:H7 infection by probiotic Bifidobacteria via multi-omics
approach

Symposium of the Azabu Academic Frontier Project–Matching Fund
Subsidy for Private Universities, Tokyo, Japan

Mar-10

Fukada, T.

Involvement of zinc transporter Slc39a13/Zip13 in hereditary connective tissue
diseaseThe 130th Annual Meeting of the Pharmaceutical Society of Japan,
Okayama, Japan

Mar-10Hirano, T.

Zinc signaling in immunity, allergy and inflammation

The 130th Annual Meeting of the Pharmaceutical Society of Japan,
Okayama, Japan

Mar-10

Nishida, K.

Role of zinc transporter in allergic reaction

The 130th Annual Meeting of the Pharmaceutical Society of Japan,
Okayama, Japan

Mar-10

Fukuda, S.

Elucidation of the effectiveness of probiotics against lethal infection ofEnterohemorrhagic Escherichia coli O157:H7

The 83rd Annual Meeting of Japanese Society for
Bacteriology, Yokohama, Japan

Mar-10

Fukada, T.

Zinc transporter Zip13: its involvement in dental development anddisorders

The 8th Oral Medical Science Frontier, Fukuoka, Japan

Mar-10

Fukada, T.

The Zinc transporter Zip13: its involvement in skeletogenesis and pathogenesis inmouse and human

The Satellite Symposium on Bone of the 14th International Congressof Endocrinology, Osaka, Japan

Mar-10120

RCAI Seminars
Date

Title

Lecturer

11-May- 09

Expression dynamics of endogenous antisense /non-coding RNA in mammals: Expression analysis bygenomic science approach

Dr. Hidenori Kiyosawa (RIKEN BRC)

26-May-09

Mutant mice analysis reveals roles of Kif7 in Hedgehog
pathway and Pericentrin in interneuron migration

Dr. Setsu Endoh-Yamagami (Genentech, Inc. / Universityof California, San Francisco, USA)

1-Jun - 09

An ENU screen for modifiers of epigenetic
reprogramming

Dr. Emma Whitelaw (NHMRC Australia Fellow,
Queensland Institute of Medical Research, Australia)

2-Jun - 09

Anti-IgE therapy for severe asthma and allergy:
rationale and drug actions

Dr. Tse Wen Chang (Distinguished Research Fellow,
Genomics Research Center Academia Sinica, Taipei,
Taiwan)8 - Jun - 09

Subversion of PKCalpha: impact on during B
cell lineage commitment, development andleukaemogenesis

Dr. Alison Michie (Senior Lecturer of Molecular
Lymphopoiesis, University of Glasgow, UK)

16 - Jun - 09

The mammalian transcriptome

Dr. Piero Carninci (RIKEN OSC)

30- Jun -09

Reprograming the cardiac cell fate and remodeling theheart formation

Dr. Jun K. Takeuchi (Cardiovascular Research, Global
Edge Institute, Tokyo Institute of Technology)

2 - Jul - 09

Host defence system of larval asteroid

Dr. Ryouhei Furukawa (University of Keio)

23 - Jul - 09

Interactive T-like and B-like lymphocytes inlampreys:Evolutionary implications

Dr. Masayuki Hirano ( Emory Vaccine Center and
Department of Pathology and Laboratory Medicine, Emory
University , USA)30 - Jul - 09

A voyage of hematopoetic stem cell and her pilot

Dr. Yoshio Katayama (Kobe University Graduate Schoolof Medicine)30 - Jul - 09

Nuclear export factor hCAS/CSE1L associates withchromatin and regulates expression of selective p53
target genes

Dr. Tomoaki Tanaka (Chiba University Hospital,
Department of Clinical Cell Biology,)

19 - Aug - 09

Identification of a human CD4+T-cell clone thatrecognizes and kills renal cell carcinoma cells throughalpha/beta TCR-mediated recognition of the soluble
TRAIL /DR4 complex

Dr. Ken-ichi Hanada (National Cancer Institute, NIH,
USA)

14 - Oct - 09

Epigenetic regulators supporting nuclear receptor
function in gene regulations

Dr. Shigeaki Kato (Institute of Molecular and Cellular
Biosciences, The University of Tokyo)

121

122

28 -Oct - 09

A novel function for RANKL: Switching on M celldifferentiation in small intestinal epithelial cells

Dr. Ifor Williams (Department of Pathology Emory
University School of Medicine, USA)2 - Nov - 09

Unc93B1 biases Toll-like receptor responses to nucleic
acid in dendritic cells towards DNA- but against
RNA-sensing

Dr. Kensuke Miyake (Division of Infectious Genetics,The
Institute of Medical Science, The University of Tokyo)

5 - Nov - 09

Polycomb function in B-cell development and adaptive
immunity

Dr. Stefano Casola (IFOM, the FIRC Institute of Molecular
Oncology Foundataion, Milano, Italy)

19 - Nov - 09

Kinetic Monte Carlo simulations of EGFR clustering inheterogeneous cell membranes

Dr. Michail Stamatakis (Department of Chemical
Engineering, University of Delaware, USA)30 - Nov - 09

Gut homing T cells, retinoic acid and intestinal DCs

Dr. William Agace (Lund University, Sweden)

8 - Dec - 09

The selective pressures on becoming a T cell

Dr. Eric Huseby (University of Massachusetts Medical
School, USA)8 - Dec - 09

Ubiquitin-mediated pathways that regulate T cell
tolerance and effector function

Dr. Paula Oliver (University of Pennsylvania, USA)8 - Dec - 09

Summary of XFEL

Dr. Tetsuya Ishikawa (RIKEN RSC)

9 - Dec - 09

The transcriptional interactome; Spatial clustering of
co-regulated genes at transcription factories

Dr. Peter Fraser (The Babraham Institute, Babraham
Research Campus, Cambridge, UK)

14 - Dec - 09

Live cell imaging of Xic homologous pairing inmouse ES cells: implications for the initiation of X
chromosome inactivation

Dr. Osamu Masui (Mammalian Developmental Epigenetics
Group, Curie Institute, France)

10 - Jan -10

bZIP transcription factor Batf regulates germinal
center formation and class switch recombination

Dr. Wataru Ise (Washington University School of
Medicine, USA)

1- Feb - 10

Role of the composition of the commensal microbiota
in Th17 cell differentiation and function

Dr. Ivaylo I. Ivanov (New York University School of
Medicine, USA)22- Feb -10

Visualizing antigen-specific helper T cell immunity:
how, where, and when

Dr. James McLachlan (Tulane University School of
Medicine, USA)16 - Mar -10

Control of immunity by SAP family molecules

Dr. Andre Veillete (Clinical Research Institute of Montreal,
Canada)Budget, Personnel and Patents

RCAI Budget (JPY 100 Million )RCAI Budget FY2001-2009(JPY 100 Million)

70

100 million yen

60

Note: Budgets for FY2001-2003 include
construction expenses for RCAI facility

50

RCAI Budget (JPY 100 Million )

40
30
20
10
0
2002

2000

2004

2006

2008

2010

Fiscal Year

2001

41.74

2002

54.23

2003
2004
2005

60.48
40.10
39.02

2006

35.90

2007

34.56

2008

32.61

2009

31.86

RCAI Staff Composition(as of Mar. 2010)
Category

NumberDirector

1

Senior Advisor

1

Group Director

7300Team Leader

14

250

Unit Leader

12

200

Coordinator

1

Senior Scientist

8

Research Scientist

40

Postdoctoral Researcher

18

RCAI Personnel
400

RCAI Personnel

350

150
100
50

Research Associate

70Student Trainee

51

Intern1

Research Consultant

2

Research Fellow

3

Visiting Scientist

63

Senior Technical Scientist

1

2002

2000

2004

2006

Fiscal Year

RCAI Personnel
2001

84

2002

156

2003

191

2004

328

2008

2010

Technical Scientist

7

Technical Staff I

26

Technical Staff II

53
20

2005

363

Assistant

2006

366

Part-time Staff

2

2007

339

2008
2009

326
380

Temporary Employment

42

Total

380

RCAI Patents FY 2009 (as of Mar. 2010)
There were 28 patents filed from April
2009- March 2010.

123Original Photos of the Cover and Front Pages

Front cover
The formation of tunneling nanotubes (TNTs). The formation of TNTs
connecting mouse macrophage cells (left) is blocked when M-Sec is depleted byknockdown of gene expression (right). (See Research Highlights section)
Image courtesy of Laboratory for Epithelial Immunology

Front page of Research Highlights section
Control of protein expression levels by judicious selection of promoter
sequences. CHO cells expressing a Fucci: fluorescent ubiquitination-based cell cycle
indicator (combination of mKO2-Cdt1 and mAG1-Geminin) in the nucleus.
For quantitative analysis of the interaction of signaling proteins, it is important toobserve multiple molecules simultaneously. To this end, the Research Unit for Single
Molecule Imaging and the Research Unit for Molecular Systems Biology establishedmethods to obtain isogenetic cell lines expressing two fluorescence tagged proteinsat a homogenous and low, but detectable level. An expression vector was designed
to contain two cassettes of promoter and fluorescence-tagged protein sequences intandem. To optimize the expression level of the fluorescence proteins for single
molecule imaging, the investigators selected suitable promoter sequences, and inserted
insulator sequences if necessary. This method allows to the precise analysis ofinteractions between two different molecules. (See Laboratory Activities section)
Image courtesy of the Research Unit for Single Molecule Imaging and the Research Unit for
Molecular Systems Biology

Front page of Laboratory Activities section
In vitro images of follicular dendritic cells (FDCs). A phase-contrast micrographof FDCs cultured for two weeks on a collagen coated plate in the presence of an
LT-bR agonist antibody and TNF-a.
Follicular dendritic cells (FDCs) are radio-resistant cells that were identified bytheir ability to retain immune complexes. More recently it was found that FDCs secrete
several cytokines and chemokines involved in the migration and aggregation oflymphocytes, especially B cells into follicles. The Laboratory for Mucosal Immunity
aims to characterize FDCs ex vivo in order to understand their functional compartmentalization and to determine if they play roles not only in somatic hypermutation
required for affinity maturation but also for class switch recombination in B cells. (SeeLaboratory Activities section)
Image courtesy of Laboratory for Mucosal Immunity

124Front page of Collaborative Network section
Localization of CD169-positive sinus macrophages in lymph nodes. A cryosection of lymph nodes was stained for CD169(Green), CD11c (Red ) and B220 (Blue) and observed by confocal microscopy. Original magnification; x40 (A), x200 (B).
CD169-positive sinus macrophages are localized in the sinus of the brachial lymph nodes, CD11c-positive dendritic
cells are localized in the T cell area, and B220-positive B cells are localized in follicles. The Laboratory for Innate Cellular
Immunity has previously established transgenic mice in which CD169-positive cells can be transiently depleted by diphtheria
toxin (DT) administration (CD169-DTR mice). Using this mouse, they study the role of sinus macrophages in the presentationof dead cell-associated antigens (See Laboratory Activities section)
Image courtesy of Laboratory for Innate Cellular Immunity

Front page of Events section
Scanning electron micrograph of a thymic cyst.
The Research Unit for Thymic Microenvironments studies development of the thymic environment, a.o. specifically focusing on the origin and generation of the aberrant epithelial cell
types localized in thymic cysts. In the absence of developing thymocytes, the thymic environment is perturbed and unusual epithelial cells appear, such as ciliated cells, goblet cells andabsorptive cells, forming cystic structures. Importantly, this abnormal stromal thymic phenotype can be quickly repaired, once developing thymocytes are added back to the thymic
environment, highlighting both the impressive plasticity of the thymic microenvironment, andthe instructive role of developing T lymphocytes in creating intact functional thymic environments. (See Laboratory Activities section)
Image courtesy of Research unit for Thymic Microenvironments

Front page of Data and Statistics section
Antigen-specific de novo conversion of CD4+Foxp3 - T cells into CD4+Foxp3+ iTregs in mesenteric lymph nodes during oral
tolerance. KJ1-26+Foxp3 EGFP- T cells were transferred into BALB/c mice, and the animals were subsequently fed PBS or OVA
protein one day later. Cryosections from mesenteric lymph nodes on day 11 after the adoptive transfer were stained for KJ1-26(red) and B220 (blue). Foxp3 was detected by green fluorescence from the Foxp3EGFP knock-in allele.
Oral tolerance is a key feature of intestinal immunity, generating systemic tolerance to ingested antigens. Antigen-specificconversion of CD4+ T cells into CD4+Foxp3+ inducible regulatory T cells ( iTregs) occurred in mesenteric lymph nodes (MLNs) during
oral tolerance induction. The Laboratory for Dendritic Cell Immunobiology studies the role of the B7 family of costimulatory moleculesin this conversion. (See Laboratory Activities section)
Image courtesy of Laboratory for Dendritic Cell Immunobiology