WPI Immunology Frontier Research Center FY 2016

World Premier International
Research Center

Osaka University

Annual Reportof IFReC
FY 2016

Edit : Research Planning & Management Office, IFReC
Published in June, 2017

Osaka University

Copyright : Immunology Frontier Research Center (IFReC ), Osaka University

Immunology
Frontier
ResearchCenterMessage from the Director

Contents

As the Director of the Immunology Frontier Re-

In FY2016, IFReC organized the “International

search Center (WPI-IFReC) at Osaka University, I

Symposium on Advanced Immunology” to cele-

am very pleased to present the IFReC annual re-brate IFReC’s first decade and Professor Tadamitsu

port for fiscal 2016.

Kishimoto’s 77th birthday. In the symposium,

Since its inception in 2007, IFReC has establishedthe researchers of IFReC recognized past achieve-

itself as a Visible International Research Center ofments, and shared the challenges in diverse re-

Immunology with the support of many people

search fields for the future.

Message from the Director ・
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Outreach Activities

Looking back on IFReC’s activities over the years ・
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Science Cafe ・
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including the WPI Program Director and the Pro-

Super Science High School Students Fair / Students Visit ・
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gram Officer.

to scientific advances through research and edu-

WPI 10 years Memorial Symposium ・
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IFReC was created as part of the WPI program, acation and evolvement into a world top immu-

AAAS 2017 Annual Meeting ・
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national project led by the Japanese Ministry of

nology research center.

Organization
Organization Chart ・
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Committee and Advisory Board for IFReC ・
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Administrative Office of IFReC ・
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Education, Culture, Sports, Science and TechnolResearch Projects
Support Programs for Fusion Researches ・
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Laboratories

Young Scientist Support Program for Research Abroad ・
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ogy. However, from FY2017, IFReC will mark a
new stage in its history with a novel agreement
for academic-industry partnership. This gover-Immunology Groups ・
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Imaging Groups ・
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Data

nance system is an ambitious program without

Informatics Groups ・
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Common Facilities ・
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Units for Combined Research Fields ・
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Kishimoto Foundation Fellowship ・
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Although the governing structure will change,

IFReC's New PIs ・
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Major Awards ・
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our most important mission “Constructing a

Chugai Lab・
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Composition ・
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Finance・
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Symposia & Seminars
International Symposium on Advanced Immunology ・
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Selected Articles ・
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Publications ・
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& the 77 anniversary og Kishimoto Tadamitsu's birth-

precedent.

world-top immunology research center” remains
the same. We will make unceasing efforts to develop immunology research to ensure translation
to medical science.

-Commemorating the 10 th anniversary of IFReCth

We are committed to continuing contributions

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IFReC Seminars ・
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IFReC Colloquia ・
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EventsThe 6 th NIF Winter School on Advanced Immunology ・
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Japanese Lessons ・
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Shizuo Akira, MD/PhD
Director
WPI Immunology Frontier Research Center

1

Looking back on IFReC’s activities over the years

Table 2. Performance Comparison between IFReC and Other Institutions

Jun Sakanoue ( Research Planning and Management Office, IFReC )Staf f (approx.)

200

250

1,000

619

715

1,247

Citation Number

36,32522,72040,447

Citation Impact

58.7

31.8

32.4

Papers in

IFReC’s Research Achievements over the past decade

Immunology,
Biochemistry &Molecular Biology,
Cell Biology

FY2017. However, the conclusion of contracts between Osaka
University and several pharmaceutical companies offered abright outlook and people began to look toward the futureagain for the next generation of IFReC. For this reason, thenumber of researchers at IFReC increased again in 2016, andthe number of papers recovered.

Since the establishment of IFReC in 2007, there have been
1270 scientific articles published by IFReC researchers. Over
10% of these articles have appeared in high impact journals
such as Science, Nature, Cell and their affiliates.
The productivity of scientific articles by IFReC researchers isshown in Figure 1. In 2015, the number of articles dipped whichis thought to be due to the end of the WPI grant by MEXT fromh-index

77

71

94

Documents in Top
1%

6.6%

4.7%

5.4%

Documents in Top
10%

29.8%

23.4%

27.3%

Web of Science™ © 2017 Clarivate Analytics/Citation number and h-index as of March 10, 2017

Number of Papers

Number of Citation
12000

200

10000
150
8000
100

ably with the other two institutes, allowing us to conclude that
IFReC researchers have produced high-quality papers for the
past ten years.

Since IFReC has a lower number of researchers than LIAI or
WEHI, the total citation number or h-index of IFReC did notmatch these institutes. However, IFReC’s figures for Citation Impact, and Documents in Top 1% and Top 10% compared favor-

6000
4000

50
2000
0

Internationalization and Interdisciplinary Researches02008 2009 2010 2011 2012 2013 2014 2015 20162008 2009 2010 2011 2012 2013 2014 2015 2016

Web of Science ™ © 2017 Clarivate Analytics as of March 10, 2017

Figure 1. Research Output in 2008 -2016

researchers in the immunology field are around 25% (data notshown). IFReC had been internationalized to some extent at an
early stage of the WPI program, and generally continued to beflat thereafter. The ratio of international collaborative papers of
LIAI and WEHI were 52% and 48%, respectively (2014-2016).
The ratio of papers in interdisciplinary researches areas in
IFReC was 21% (2014-2016), and is still increasing.

Promotion of internationalization and interdisciplinary research is an important mission of the WPI program and WPI
institutions including IFReC.
The ratio of international co-authored papers is considered
to be one of the indexes for internationalization of the institute. The ratio for IFReC remained around 41 to 43% after 2010
(Figure 2), whereas the average values by all Osaka University

Table 1 shows the extracted parameters for institution evaluation in all the research fields of IFReC.

41.7

Number of Papers

1,270

Number of Citations

54,840Citation Impact

43.2

h-index

96

41.3

35.6

Table 1. Number of Published Papers and their Citations in
All the Research Fields

International Co-authored (%)
Interdisciplinary Research (%)

21.0
14.7

13.1
Figure 2. International Co-authored &Interdisciplinary Research2008 -2010

2011-20132014 -2016

Web of Science ™ © 2017 Clarivate Analytics as of March 10, 2017

IFReC, which has created a large number of quality sciencepapers, and promoted internationalization and interdisciplinary
Table 2 shows the performance comparison between IFReC
and two world top research institutions in the life science field,
La Jolla Institute for Allergy and Immunology (LIAI), and Walterand Eliza Hall Institute of Medical Research (WEHI), Australia.

Since each of the three institutes has a different composition ofresearch fields, to be fair, the parameters for comparison were
calculated only in the three research areas of Immunology, Biochemistry & Molecular Biology, and Cell Biology.

research, is surely commensurable with institutes honored withWorld Premier Status.

References
・ Annual Reports of IFReC FY2010-2015
・ Web of ScienceTM © 2017 CLARIVATE ANALYTICS・ IncitesTM © 2017 CLARIVATE ANALYTICS

2

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Organization

As of March, 2017

Organization Chart

Immunology GroupMEXT
WPI Initiative
Program Committee

JSPS

WPI Working GroupInternational
Scientific
Advisory Board

Director

Management
Committee

Board ofRepresentatives

Administrative
Director
General Affairs Section

Imaging Group

Research Groups

Deputy Directors

Host Defense ............................................................................................. Shizuo Akira
Immunoglycobiology ............................................................................. Taroh Kinoshita
Immunopathology .................................................................................. Atsushi Kumanogoh
Immunochemistry ................................................................................... Hisashi Arase
Immune Regulation ................................................................................ Tadamitsu Kishimoto
Mucosal Immunology ............................................................................ Kiyoshi Takeda
Immune Regulation ................................................................................ Hitoshi Kikutani
Experimental Immunology .................................................................. Shimon Sakaguchi
Cell Signaling ............................................................................................ Takashi Saito
Lymphocyte Differentiation ................................................................ Tomohiro Kurosaki
Lymphocyte Development ................................................................. Fritz Melchers
Malaria Immunology .............................................................................. Cevayir Coban
Vaccine Science ....................................................................................... Ken J. Ishii
Immunoparasitology ............................................................................. Masahiro Yamamoto
Biochemistry and Immunology ......................................................... Shigekazu Nagata

Single Molecule Imaging ..................................................................... Toshio Yanagida
Biofunctional Imaging ........................................................................... Yoshichika Yoshioka
Immunology and Cell Biology ............................................................. Masaru Ishii
Nuclear Medicine ..................................................................................... Jun Hatazawa
Chemical Imaging Techniques ........................................................... Kazuya Kikuchi
Biophotonics ............................................................................................. Nicholas Isaac Smith
Immune Response Dynamics ............................................................. Kazuhiro Suzuki
Brain-Immune Interaction .................................................................... Ben Seymour

Informatics Group
Information Systems .............................................................................. Yutaka Hata
Systems Immunology ............................................................................. Daron M Standley

Accounting Section

Units for Combined Research Fields

Administrative Department

Quantitative Immunology .................................................................... Yutaro Kumagai
Diego Diez
Immuno-Genomics ................................................................................. Alexis Vandenbon
Hiromasa Morikawa

Research PlanningandManagement Office

Common Facilities

Core Instrumentation Facility
Animal Resource Center for Infectious Diseases
Network Administration OfficeOsaka University
Institute for Frontier Medical Sciences, Kyoto University

President

Administrative Bureau

Domestic RIKEN Center for Integrative Medical Sciences
National Institute of Biomedical Innovation, Health and Nutrition

Cooperative
Institutions
Overseas

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Convergent Research Consortium for Immunologic Disease,
Seoul, St Mary's Hospital, Catholic University of Korea
Indian Institute of Science Education and Research, India

7

Committee and Advisory Board for IFReC

International Scientific Advisory Board

As one of the nine centers selected for the World Premier International Research Center Initiative (WPI), IFReC has been the subjectof evaluations including site visits and follow-ups by the WPI Program Committee.
In close cooperation with the Program Directors, the Program Officer and working group members, the WPI Program Committee
conducts follow-up activities on progress being made by the WPI institutes including IFReC, with an eye to developing them into“highly visible research centers.”

The International Scientific Advisory Board conducts evaluations on scientific achievements of IFReC PIs by reviewing their reports ordirect interviews.
Jeffrey Ravetch

The Rockefeller University, USA

Immunology

Christopher Goodnow

Australian National University

Immunology

Richard Locksley

University of California, San Francisco, USA

Immunology

Lewis L. Lanier

University of California, San Francisco, USA

Immunology

Anne O'Garra

The Francis Crick Institute, UK

Immunology

Kiyoshi Takatsu

Toyama Prefectural Institute for Pharmaceutical Research, Japan

Immunology

Kazuo Sugamura

Miyagi Cancer Center Research Institute, Japan

Immunology

Yale Goldman

University of Pennsylvania, USA

Imaging

Program Committee Members

Yasuyoshi Watanabe

Center for Life Science Technologies, RIKEN, Japan

Imaging

Toshiaki Ikoma

Special Adviser, Canon Inc., Japan

Masamitsu Iino

Nihon University School of Medicine, Japan

Imaging

Hiroto Ishida

President Emeritus, Kanazawa Gakuin University, Japan

Akinori Kidera

Yokohama City University, Japan

Informatics

Shinichiro Ohgaki

President, Japan Water Research Center, Japan

Maki Kawai

Director General, Institute for Molecular Science, National Institutes of Natural Sciences, Japan

Kiyoshi Kurokawa

Adjunct Professor, National Graduate Institute for Policy Studies, Japan

Makoto Kobayashi

Honorary Professor Emeritus, High Energy Accelerator Research Organization, Japan
Nobel Laureate in Physics (2008)

Norihiko Suzuki

Chair of the Board/President, Akita International University

Ryozo Nagai

President, Jichi Medical University, Japan

Michiharu Nakamura

Counselor to the President, Japan Science and Technology Agency (JST), Japan[Chairperson]
Ryoji Noyori

Director-General, Center for Research and Development Strategy, JST, Japan
Nobel Laureate in Chemistry (2001)

Michinari Hamaguchi

President, JST, Japan

Hiroshi Matsumoto

President, RIKEN, Japan

Rita Colwell

Distinguished Professor, University of Maryland, USA

Richard Dasher

Consulting Professor, Stanford University, USA

Victor Joseph Dzau

President, National Academy of Medicine, USA

Klaus von Klitzing

Director, Max Planck Institute for Solid State Research, Germany Nobel laureate in Physics (1985)

1. Level of achievement

Chuan Poh Lim

Chairman, Agency for Science, Technology and Research, Singapore

2. Scientific/technical merit

Harriet Wallberg

Former president, Karolinska Institutet, Sweden

3. Research output and outcomes (research achievements, patents, invited lectures, etc.)

Jean Zinn-Justin

Scientific adviser, IRFU/CEA, France

4. Promoting interdisciplinary research

WPI Program Committee
Program Director
Toshio Kuroki

as of FY2016

Senior Advisor, Research Center for Science Systems, Japan Society for the Promotion of Science (JSPS), Japan

Deputy Program Director
Akira Ukawa

Deputy Director, RIKEN Advanced Institute for Computational Science, Japan

Working Group Leader and Assigned Members

8

as of FY2016

All researches performed at IFReC are required to be reviewed by external scientists. Therefore an in-depth evaluation of research
activities at IFReC was performed in a peer-review style by the International Scientific Advisory Board (ISAB), which consists of ten
highly qualified scientists. Since this is the final year of WPI program, the entire research activities since joining IFReC of each PI were
evaluated.
The evaluation was conducted through both document and interview. For each PI, one board member was assigned for document
evaluation and two board members were assigned for the interview evaluation. Ten ISAB members gathered at IFReC on February 2nd
and 3 rd, 2017 for the interviews.
The document evaluation was based on a submitted research report including future prospectus, publication list, obtained patents,
awards, invited lectures and list of external funding. In addition, the PIs attached copies of the five most important publications as anappendix to their progress report.
The board members evaluated the following items and provided the score and the comments.

Evaluation Items

as of FY2016

5. Future prospects
Score 5 : Excellent, 4 : Good, 3 : Average, 2 : Fair, 1 : Poor

[Program Officer ]Takehiko Sasazuki

University Professor, Institute for Advanced Study, Kyushu University, Japan

Hiroshi Kiyono

Professor, Institute of Medical Science, The University of Tokyo, Japan

Nagahiro Minato

Executive Vice-President for Research, Planning, and Hospital Administration, Kyoto University, Japan

Kazuhiko Yamamoto

Professor and Chairman, Department of Allergy and Reumatology, Graduate school of Medicine, The Universityof Tokyo, Japan

Günter J. Hämmerling

Professor, Tumorimmunology Program, German Cancer Research Center DKFZ, Germany

Hisataka Kobayashi

Associate (chief ) scientist, Molecular Imaging Program, National Cancer Institute, National Institutes of Health,USA

Philippe Kourilsky

Professor, Collège de France/Honorary Director-General, The Institute of Pasteur, France

After summarizing the evaluation results, anonymous comments and scores made by board members were conveyed individually to
PIs. The evaluations and advice received are invaluable to each PI and will surely contribute to their research in the future. Also, IFReC
will make use of the comments and suggestions to open up new paths over the next ten years.

9Administrative Office of IFReC

General Affairs Section■ Employment /acceptance of researchers and staff procedures■ Social insurance (part-time)/ employment insurance■ Management of work hours■ Procedures related to patents■ Issuing various certificates■ Procedures related to international students■ Support for international researchers

Accounting Section■ Budget drafting / implementation / management■ Purchasing procedures■ Acceptance and implementation of third-party funding■ Payment of payroll, travel expense and honorarium■ Health insurance procedures■ Management of buildings and assets

Laboratories■ RI (Radio Isotope) procedures

Research Planning & Management Office■ Research Promotion & Support( Consultation for grants & patents, Fusion research program, etc. )■ Establishing Research Environments( Facility & Safety management, Research agreement, etc.)■ Fostering Young Scientists( Winter School etc.)■ Organizing Scientific Events( Symposia, Colloquia, Seminars, etc.)■ Public Relations( Publishing, Website, Outreach to citizens, etc.)■ WPI evaluation issues( Progress report, Advisory Board meeting, etc.)

As of March, 201710

Shizuo Akira, MD/PhD

Professor

Shizuo Akira

Associate Professor

Kazuhiko Maeda

Assistant Professor

Takashi Satohendogenous RNase that destabilizes a set of mRNAs throughcleavage of their 3' UTRs such as IL-6 and IL-12p40 in macrophages and c-Rel, Ox40, and IL-2 in CD4+ T cells. Regnase-1 itself
is cleaved by Malt1 protease after T cell receptor (TCR) stimulation and enhances T cell activation. Consistently, the proteaseactivity of Malt1 is involved in control of the mRNA stability of T
cell effector genes. This indicates that dynamic control of Regnase-1 expression is critical for modulation of T cell activation.

Based on these findings, we promote understanding of the
precious role of Regnase-1 in immune cells by using Regnase-1
mutant mice and tissue-specific Regnase-1 deficient mice.

Immunology

Host Defense

We are studying to achieve the goal of a comprehensive understanding of the innate immune system and to develop an effective treatment for immune-related inflammatory diseases.

Kenta Maruyama
Hiroki Tanaka

We are focused on the innate immune system, which is an evolutionally conserved host defense mechanism against various
pathogens. Innate immune responses are initiated by patternrecognition receptors (PRRs), which recognize specific structuresof microorganisms. Toll-like receptors (TLRs) are capable of sensing organisms ranging from bacteria to fungi, protozoa and viruses, and play a major role in innate immunity. Individual TLRs
recognize different microbial components, and give rise to different patterns in gene expression.

Identification of an atypical monocyte and committedprogenitor involved in fibrosis
Macrophages consist of at least two subgroups, M1 and M2.
M1 macrophages are pro-inflammatory and have a central role inhost defense. Whereas, M2 macrophages are associated with responses to anti-inflammatory reactions, and tissue remodeling.
Previously we reported that Trib1 is critical for the differentiationof tissue-resident macrophages—that share characteristics withM2 macrophages (which we term M2-like macrophages). Trib1
deficiency results in a severe reduction of M2-like macrophagesin various organs, including bone marrow, spleen and adipose
tissues. Trib1 is critical for adipose tissue maintenance and suppression of metabolic disorders by controlling the differentiationof tissue-resident M2-like macrophages.
Monocytes and macrophages comprise a variety of subsetswith diverse functions. It is thought that these cells play a crucialrole in homeostasis of peripheral organs, key immunological processes and development of various diseases. Among these dis12

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eases, fibrosis is a life-threatening disease of unknown aetiology.
Its pathogenesis is poorly understood, and there are few effective therapies. The development of fibrosis is associatedwith activation of monocytes and macrophages. However,the specific subtypes of monocytes and macrophages thatare involved in fibrosis have not yet been identified. We showedthat Ceacam1+Msr1+Ly6C-F4/80-Mac1+ monocytes, which wetermed segregated-nucleus-containing atypical monocytes (SatM),
share granulocyte characteristics, are regulated by CCAAT/enhancer binding protein beta (C/EBPβ), and are critical for fibrosis.
Cebpb-deficiency results in a complete lack of SatM. Furthermore, the development of bleomycin-induced fibrosis, but not
inflammation, was prevented in chimeric mice with Cebpb-/haematopoietic cells. Adoptive transfer of SatM into Cebpb-/mice resulted in fibrosis. Notably, SatM are derived from Ly6CFcεRI+ granulocyte/macrophage progenitors, and a newly
identified SatM progenitor downstream of Ly6C-FcεRI+ granulocyte/macrophage progenitors, but not from macrophage/dendritic-cell progenitors. Our results show that SatM are critical for
fibrosis and that C/EBPβ licenses differentiation of SatM fromtheir committed progenitor.

Figure 1.
The morphology of SatM

Recent Publications■ Satoh T, et al. Identification of an atypical monocyte and committed pro-

Regulation of mRNA stability by CCCH-type zinc-finger
protein Regnase-1 in immune system
Regnase-1, also known as Zc3h12a, is a member of the CCCHtype zinc finger domain containing proteins. Regnase-1 knockout
mice developed spontaneous autoimmune diseases accompanied by splenomegaly and lymphadenopathy. Regnase-1 is an

Figure 2.
Concept of disorder specific macrophages

genitor involved in fibrosis. Nature 541, 96-101 (2017).
■ Maeda K & Akira S. Regulation of mRNA stability by CCCH-type zinc-finger

proteins in immune cells. Int. Immunol. (2017) in press.

■ Satoh T & Akira S. Toll-Like Receptor Signaling and Its Inducible Proteins.

Microbiol. Spectr. 4 (6), doi:10. 1128/microbiolspec. MCHD-0040 (2016).
■ Maeda K & Akira S. TLR7 Structure: Cut in Z-Loop. Immunity 45, 705-707(2016).

■ Kozaki T, et al. Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose) polymerase-mediated antiviral response. Proc. Natl. Acad. Sci. USA
114, 2681-2686 (2017).

13

Immunology

Immunoglycobiology
Taroh Kinoshita, PhD

Professor

Taroh Kinoshita

Associate Professor

Yusuke Maeda
Yoshiko Murakami

Assistant Professor

Yuko Tashima
Noriyuki Kanzawa

More than 150 different proteins with various functions, suchas receptors, adhesion molecules, enzymes, enzyme inhibitorsand complement regulatory proteins, are anchored to the cellsurface via glycosylphosphatidylinositol (GPI) that is amidebonded to the carboxyl-terminus. Our laboratory has been working to clarify the mechanism of biogenesis, biological functions
and medical significance of GPI anchors and GPI-anchored proteins (GPI-APs). In 2016 we made several discoveries.
Characterization of new pathogenic mutations responsible forinherited GPI deficiencies (IGDs):We reported the first cases of inherited GPI deficiency caused by mutations in PIGC and PIGP
genes in collaborations with research groups from Canada andIsrael (Edvardson, S. et al., J. Med. Genet., 54:196-201, 2016; Johnstone, D. L. et al, Hum. Mol. Genet., 2017). Three affected individuals from two families having global developmental delay, severe
intellectual disability and seizures had homozygous or compound heterozygous loss-of-function mutations in the PIGC
gene. Two siblings with biallelic loss-of-function PIGP mutations
showed early-onset seizures, hypotonia and global developmental delay. Both PIGC and PIGP are components of the enzyme thatmediates the initial step in GPI biosynthesis, transfer of N-acetylglucosamine to phosphatidylinositol (PI) (Figure). To date, pathogenic mutations that cause IGDs were identified in 16 of 27 genes
involved in biosynthesis, transfer to proteins and maturation of
GPI anchors.

14

Postdoctoral Fellow

2

Research Assistant

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Visiting Scientist

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Demonstration of new function of PIGN: PIGN is an enzymethat transfers ethanolaminephosphate side-chain to the firstmannose in step 8 in GPI biosynthesis (Figure). In collaboration
with Dr. Ihara’s group in the National Institute of Genetics, weshow PIGN is required for quality control in mammalian cells(Ihara, S. et al, J Cell Sci., 130:602-613, 2017). In C. elegans pign-1
mutants, several proteins fail to be secreted and instead form abnormal aggregation in the endoplasmic reticulum (ER). PIGNknockout HEK293 cells also showed similar protein aggregation.
Certain mutation in C. elegans pign-1 caused protein aggregationin the ER without affecting GPI-anchor biosynthesis. These results
show that PIGN-1/ PIGN has a conserved and non-canonical function to prevent deleterious protein aggregation in the ER independently of the GPI-anchor biosynthesis. A defect in the noncanonical function of PIGN may contribute to clinical symptoms
of individuals affected by IGD caused by PIGN mutations.
In collaboration with Dr. Yamaguchi’s group in RIKEN, we havebeen characterizing p24γ2 complex, a cargo receptor for efficienttransport of GPI-anchored proteins from the ER (Figure). In 2016,
we reported crystal structures of GOLD domains of p24β1, p24γ2
and p24δ1 component of the cargo receptor (Nagae, M. et al, J.
Mol. Biol., 428:4087-4099, 2016; Proteins, in press). The results support the idea that the p24 cargo receptor is an octamer consistingof two each of four components.

Figure.
Biosynthesis and transport of GPI-anchored proteins. PIGC and PIGP are components ofN-acetylglucosaminine transferase that mediates the initial step in GPI biosynthetic pathway.
We reported individuals with inherited GPI deficiencies caused by pathogenic mutations
in PIGC and PIGP. PIGN is involved in step 8 in GPI biosynthesis. We reported that PIGN isrequired for protection of protein aggregation in addition to GPI biosynthesis. GPI-anchoredproteins are packaged into transport vesicles by a cargo receptor consisting of four p24
proteins, α2, β1, γ2 and δ1. We report evidence for an octameric complex including two eachof four components.

Recent Publications■ Lee GH, Fujita M, Takaoka K, Murakami Y, Fujihara Y, Kanzawa N, Murakami K,■ Hirata T, Fujita M, Nakamura S, Gotoh K, Motooka D, Murakami Y, Maeda Y

Kajikawa E, Takada Y, Saito K, Ikawa M, Hamada H, Maeda Y and Kinoshita
T. A GPI processing phospholipase A2, PGAP6, modulates Nodal signaling
in embryos by shedding CRIPTO. J. Cell Biol. 215, 705-718 (2016).

and Kinoshita T. Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport. Mol. Biol. Cell 26, 30713084 (2015).

■ Makrythanasis P, Kato M, Zaki M, Saitsu H, Nakamura K, Santoni F,

Miyatake S, Nakashima M, Issa MY, Guipponi M, Letourneau A, Logan C,
Roberts N, Parry DA, Johnson CA, Matsumoto N, Hamamy H, Sheridan E,
Kinoshita T, Antonarakis SE and Murakami Y. Pathogenic variants in PIGG
cause intellectual disability with seizures and hypotonia. Am. J. Hum.
Genet. 98, 615-626 (2016).

■ Theiler R, Fujita M, Nagae M, Yamaguchi Y, Maeda Y and Kinoshita T.

Thealpha helical region in p24γ2 subunit of p24 cargo receptor is pivotal forthe recognition and transport of glycosylphosphatidylinositol-anchoredproteins. J. Biol. Chem. 289, 16835-16843 (2014).

■ Nagae M, Hirata T, Morita-Matsumoto K, Theiler R, Fujita M, Kinoshita T

and Yamaguchi Y. 3D structure and interaction of p24β and p24δ Golgi
dynamics domains: implication for p24 complex formation and cargotransport. J. Mol. Biol. 428, 4087-4099 (2016).

15

Immunology

Immunopathology
Atsushi Kumanogoh, MD/PhD

Our research team is involved in two approaches; basic andclinical immunology. As basic aspects of our projects, our proposed study is the regulation of immune cell motility and migratory behavior in vivo by soluble and membrane-bound ‘immune
guidance molecules’ such as semaphorins and their receptors.
Semaphorins were originally identified as axon-guidance molecules that function during neuronal development. However, cumulative evidence indicates that semaphorins also participate inimmune responses, both physiological and pathological, andthey are now considered to be potential diagnostic and/or therapeutic targets for a range of diseases. Beyond such basic implications, we are trying to apply the findings from this proposed
study into diagnosis/therapy for human immunological disorders, such as autoimmunity, allergy, immune deficiency, cancer/
metastasis, and neurodegenerative diseases. We focus on thefunctions of Semaphorin 4D (SEMA4D) as an immune check point
for neutrophils and its pathological involvement in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).
We found that serum levels of soluble SEMA4D were elevatedin AAV patients and were correlated with clinical disease scores.
In neutrophils from AAV patients, cell surface expression of SEMA4D was significantly down-regulated, a consequence of ADAM17-mediated neutrophils-specific proteolytic shedding of SEMA4D. Soluble SEMA4D exerted pro-inflammatory effects onendothelial cells. Moreover, we found that neutrophil membrane-bound SEMA4D interacted with plexin B2 on endothelial
cells, and this binding was required for the suppression of reactive oxygen species (ROS) production and resultant neutrophil
16

Professor

Atsushi Kumanogoh

Assistant Professor

Shohei Koyama

Support Staff

5

Figure. Graphical scheme of this study.
Under healthy conditions, the interaction between endothelial plexin B2 ligand andneutrophil cells surface SEMA4D receptor inhibits Rac1 activation and negatively regulates
the generation of ROS and NET formation. In contrast, in AAV patients, neutrophil surface
SEMA4D is shed by ADAM17. Soluble SEMA4D had pro-inflammatory functions on endothelial
cells. In addition, alteration of the SEMA4D–plexin B2 interaction results in aberrant activationof neutrophils, and this dichotomous effect is involved in the pathogenesis of AAV.

extracellular trap (NET) formation. Treating neutrophils with recombinant plexin B2 led to the suppression of Rac1 activation
through SEMA4D’s intracellular domain, and resulted in the inhibition of the pathogen or ANCA IgG-induced neutrophil oxidative burst. Collectively, neutrophil surface SEMA4D functions as anegative regulator of neutrophil activation. Proteolytic cleavageof SEMA4D as observed in AAV patients may amplify neutrophilmediated inflammatory responses. SEMA4D is a promising biomarker and potential therapeutic target for AAV.

Recent Publications■ Nishide M, Nojima S, Ito D, Takamatsu H, Koyama S, Kang S, Kimura T,■ Nojima S, Toyofuku T, Kamao H, Ishigami C, Kaneko J, Okuno T, Takamatsu H,

Morimoto K, Hosokawa T, Hayama Y, Kinehara Y, Kato Y, Nakatani T,
Nakanishi Y, Tsuda T, Park JH, Hirano T, Shima Y, Narazaki M, Kumanogoh A.
Semaphorin 4D inhibits neutrophil activation and is involved in thepathogenesis of neutrophil-mediated autoimmune vasculitis. Ann.
Rheum. Dis. (2017) in press.

Ito D, Kang S, Kimura T, Yoshida Y, Morimoto K, Maeda Y, Ogata A, Ikawa M,
Morii E, Aozasa K, Takagi J, Takahashi M, Kumanogoh A. A point mutationin Semaphorin 4A associates with defective endosomal sorting andcauses retinal degeneration. Nature Commun. 4, 1406 (2013).
■ Kumanogoh A, Kikutani H. Immunological functions of the neuropilins■ Kimura T, Nada S, Takegahara N, Okuno T, Nojima S, Kang Sujin, Ito D,

Morimoto K, Hosokawa T, Hayama Y, Mitsui Y, Sakurai N, Sarashina-Kida H,
Nishide M, Maeda Y, Takamatsu H, Okuzaki D, Yamada M, Okada M and
Kumanogoh A. Polarization of M2 macrophages requires Lamtor1 that
integrates cytokine and amino-acid signals. Nature Commun. 8, 14711(2017).

and plexins as receptors for semaphorins. Nat. Rev. Immunol. 13, 802-14(2013).

■ Yoshida Y, Ogata A, Kang S, Ebina K, Shi K, Nojima S, Kimura T, Ito D,

Morimoto K, Nishide M, Hosokawa T, Hirano T, Shima Y, Narazaki M, Tsuboi
H, Saeki Y, Tomita T, Tanaka T, Kumanogoh A. Semaphorin 4D contributes
to rheumatoid arthritis by inducing inflammatory cytokine production:
Pathogenic and therapeutic implications. Arthritis Rheumatol. 67, 1481
(2015).
17

Hisashi Arase, MD/PhD

Professor

Hisashi Arase

Associate Professor

Tadahiro Suenaga

Assistant Professor

Masako Kohyama

Misfolded proteins complexed with MHC class II molecules are targets for autoimmune diseases
MHC class II allelic polymorphisms are associated with susceptibility to many autoimmune diseases. However, it has remained
unclear how MHC class II molecules are involved in autoimmune
disease susceptibility. We found that cellular misfolded autoantigens are rescued from protein degradation by MHC class II molecules (Jiang et al. Int. Immunol. 2013). Furthermore, we foundthat misfolded proteins complexed with MHC class II molecules
are targets for autoantibodies in autoimmune disease patients(Jin et al. Proc. Natl. Acad. Sci. USA. 2014; Tanimura et al. Blood.
2015). In addition, we could detect autoantibodies against β2GPI/HLA class II complex in the patients with refractory cutaneous ulcers (Arase et a. Br. J. Dermatol. 2017). Autoantibody binding to
misfolded proteins transported to the cell surface by MHC class II
molecules was strongly correlated with susceptibility to autoimmune disease. This suggested that misfolded proteins, which
normally would not be exposed to the immune system, can betargets for autoantibodies as ‘neo self’ antigens, which are involved in the pathogenicity of autoimmune diseases (Figure 2).

Immunology

Immunochemistry

Kouyuki Hirayasu

We have been working the interactions between pathogens
and various paired receptors. In addition, we have found thatMHC class II molecules function as molecular chaperons to transport cellular misfolded proteins to the cell surface. Analyses of
misfolded proteins transported to the cell surface revealed thatthese proteins are involved in autoimmune diseases as a target
for autoantibodies.

Interaction between immune receptors and herpesviruses
PILRα is one of the immune inhibitory receptors that are expressed on various immune cells. We have previously found thatboth PILRα and PILRβ recognize CD99 as a host ligand (Shiratori
et al. J. Exp. Med. 2004). In addition, we have identified PANP as a
new ligand for PILR (Kogure et al. Biochem. Biophys. Res. Commun. 2011). PILRα is a unique receptor that that has binding sites
for both sugar chains and protein structures (Wang et al. J. Immunol. 2008; Kuroki et al. Proc. Natl. Acad. Sci. USA. 2014). We
found that PILRα associates with glycoprotein B (gB), an envelope
protein of herpes simplex virus-1 (HSV-1), and the interaction between PILRα and gB is involved in membrane fusion during HSV-1
infection (Satoh et al. Cell 2008; Wang et al. J. Virol. 2009). We further analyzed host cell molecules that associate with HSV-1 gB
and found that non-muscle myosin heavy chain (NMHC-IIA) associates with gB and is involved in HSV-1 infection (Arii et al. Nature 2010). We also found that Siglec-4 (MAG, myelin associatedglycoprotein) associates with VZV gB and mediates VZV infection.
Because Siglec-4 is specifically expressed in neural tissues, Si18

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glec-4 seemed to be involved in neurotropic characteristic of VZV(Suenaga et al. Proc. Natl. Acad. Sci. USA. 2010; Suenaga et al. J.
Biol. Chem. 2015).

PILRα plays an important role in regulation of inflammation
We analyzed function of PILRα in immune response using
PILRα-knockout mice. PILRα-deficient mice were susceptible to
LPS-induced endotoxin shock. Further analyses revealed that infiltration of neutrophils in liver and lung was significantly increased in PILRα-deficient mice. (Wang et al. Nat. Immunol. 2012).
Furthermore, PILRα-deficient mice showed severe DSS-induced
colitis (Kishida et al. Int. Immunol. 2015) as well as increased tissue fibrilization (Kohyama et al. Eur. J. Immunol. 2016). Thesefindings indicated that PILRα plays an important role in the regulation of inflammation by regulating integrin function.

Identification of new sensing system for immune abnormalities
We found that LILRA2 (also called ILT1, LIR-7 and CD85H), an
orphan activating receptor expressed on human myeloid cells,
recognizes abnormal immunoglobulins cleaved by microbial
proteases but not normal immunoglobulins. Because immunoglobulins are quite important molecules in host defense, degradation of immunoglobulins is a very dangerous situation for immunity. Therefore, LILRA2 is a sensor to detect immunoglobulin
abnormalities in microbial infection (Figure 1, Hirayasu et al. Nat.
Microbiol. 2016).

Figure 1. Microbially cleaved immunoglobulins are sensed by the innate
immune receptor LILRA2
Microbial pathogens produce various proteases and degrade immunoglobulins
to evade immune system. However, immune system has acquired a novel
receptor, LILRA2, which specifically recognizes degraded immunoglobulins todetect immune abnormalities caused by microbial pathogens. The recognitionof degraded immunoglobulins by LILRA2 plays an important role in hostdefense against pathogens (Hirayasu et al. Nat. Microbiol. 2016).

Figure 2. Misfolded proteins transported to the cell surface by MHC class
II molecules are targets for autoantibodies
Cellular misfolded proteins are generally degraded in the cells and are nottransported to outside the cells. Therefore, misfolded proteins transported
to the cell surface by MHC class II molecules may be recognized as ‘neoself’ antigens by immune system, which might initiate aberrant immune
response to self-antigens (Jiang et al. Int. Immunol. 2013; Jin et al. Proc.
Natl. Acad. Sci. USA. 2014; Tanimura et al. Blood 2015, Arase Adv. Immunol.
2016; Arase et al. Br. J. Dermatol. 2017, Hiwa et al. Arthritis Rheumatol.
2017).

Recent Publications■ Hirayasu K, Saito F, Suenaga T, Shida K, Arase N, Oikawa K, Yamaoka T, Murota H,■ Wang J, Shiratori I, Uehori J, Ikawa M, Arase H. Neutrophil infiltration during

Chibana H, Nagai H, Nakamura Y, Katayama I, Colonna M, Arase H. LILRA2 isan innate immune sensor for microbially cleaved immunoglobulins. Nat. Microbiol. 1, 1-7 (2016).

inflammation is regulated by PILRα via modulation of integrin activation. Nat.
Immunol. 14, 34-40 (2012).
■ Suenaga T, Satoh T, Somboonthum P, Kawaguchi Y, Mori Y and Arase H. My-■ Tanimura K, Jin H, Suenaga T, Morikami S, Arase N, Kishida K, Hirayasu K,

Kohyama M, Ebina Y, Yasuda S, Horita T, Takasugi K, Ohmura K, Yamamoto K,
Katayama I, Sasazuki T, Lanier L L, Atsumi T, Yamada H and Arase H β2glycoprotein I / HLA class II complexes are novel autoantigens in antiphospholipid syndrome. Blood. 125, 2835-2844 (2015).

elin-associated glycoprotein mediates membrane fusion and entry of neurotropic herpesviruses. Proc. Natl. Acad. Sci. USA 107, 866-871 (2010).

■ Jin H, Arase N, Hirayasu K, Kohyama M, Suenaga T, Saito F, Tanimura K,

Matsuoka S, Ebina K, Shi K, Toyama-Sorimachi N, Yasuda S, Horita T, Hiwa R,
Takasugi K, Ohmura K, Yoshikawa H, Saito T, Atsumi T, Sasazuki T, Katayama I,
Lanier LL and Arase H. Autoantibodies to IgG/HLA-DR complexes are associated with rheumatoid arthritis susceptibility. Proc. Natl. Acad. Sci. USA. 111,
3787-3792 (2014).
19

Tadamitsu Kishimoto, MD/PhD

Professor

Tadamitsu Kishimoto

Assistant Professor

Kazuya Masuda

Postdoctoral Fellow

4Support Staff2function with high mortality. We examined whether Arid5a is involved in the pathogenesis of acute lung inflammatory diseases.
Interestingly, we observed that Arid5a deficient mice are resistant to bleomycin-induced lung injury-mediated mortality. Arid5a deficiency suppressed not only for IL-6 production, but also
reactive oxygen species (ROS) expression in lung. Moreover, immunohistological data of lung tissue suggests that Arid5a deficiency could protect mice from bleomycin-induced lung fibrosis
which indicates the important role of Arid5a in lung tissue fibrosis. Therefore, the control of Arid5a expression should be a potential therapeutic target for treatment of acute lung inflammatory
disorders.

Rabex-5 is a lenalidomide target molecule that negatively regulates TLR-induced type 1 IFN production.
(Published)
A recent study identified Cereblon as the receptor for immunomodulatory drugs (IMiDs) compounds responsible for thalidomide teratogenicity. To understand the mechanism underlying
the anti-inflammatory properties of IMiDs we generated Cere-

Introduction
Interleukin-6 (IL-6) is in important cytokine in early phase of
acute immune responses, by which lymphocytes, hematopoietic
cells and vascular endothelial cells are activated to protect the
body against invasion of pathogens. In contrast, persistent production of IL-6 leads to development of various chronic diseases
such as rheumatoid arthritis (RA) and juvenile idiopathic arthritis(JIA). Regarding the significance of IL-6 in the pathogenesis ofchronic diseases, tocilizumab, a humanized anti-IL-6 receptor antibody successfully proved its outstanding efficacy against thosechronic immune disorders. However, high levels of IL-6 expression have also been revealed in the sera of patients from systemic
inflammatory responses syndrome (SIRS) and cytokine release
syndrome (CRS).
To pursue the molecular mechanisms of overproduction of IL6, we focused on IL-6 gene expression in activated macrophages
under TLR signaling. IL-6 mRNA is tightly regulated by RNA binding proteins, in which dysfunction led to sustain IL-6 production.
Previously, we identified an RNA-binding protein, AT-rich interactive domain 5a (Arid5a), which binds to the 3'UTR of IL-6 mRNA.
Arid5a has been shown to stabilize IL-6 mRNA (Figure 1). In fact,
Arid5a deficient mice are resistant to development of endotoxinshock and EAE mice model. In addition, we found a T cell-intrinsicrole of Arid5a through stabilization of STAT3 mRNA. Reduction of
STAT3 level in Arid5a deficient T cells led to the imbalance of
STATs (STAT1, 3, 5) activation under Th17 polarizing conditions,which contributed to the alteration of the character of inflammatory T cells into that of anti-inflammatory T cells. Importantly, we
20

blon deficient mice using the CRISPR/Cas9 system. Importantly,
thalidomide was effective in inhibiting TLR4 induced TNFα production even in the absence of Cereblon. Thus, we concluded
that the inhibitory effect of thalidomide on TLR induced cytokine
production occurs independently of Cereblon. The significanceof this study is that certain therapeutic properties of IMiDs can beseparated from pathways leading to teratogenicity. However, theanti-inflammatory properties of IMiDs are unlikely to be entirely
Cereblon-independent. To identify IMiD target molecules thatmight contribute to the anti-inflammatory properties of IMiDs wesearched for Cereblon interacting proteins affected by IMiD binding. We subsequently identified Rabex-5 as a Cereblon interacting protein. Signficantly, IMiD binding prevented the associationof Cereblon with Rabex-5. Knockdown of Rabex-5 in a humanmacrophage cell line upregulated TLR induced type-1 interferon
production via a MYD88 dependent STAT1 activating pathway.
We have proposed that disruption of the Cereblon-Rabex-5 complex releases Rabex-5 to suppress TLR induced STAT1 activation,
contributing to the efficacy of these compounds in autoimmune
disorders (Figure 2).

Immunology

Immune Regulation

revealed that Arid5a regulates naïve T cells-fate through selective
stabilization of STAT3 mRNA, suggesting that Arid5a is requiredfor RNA stabilization for IL-6 and STAT3 mRNAs.

Arid5a exacerbates IFN-γ-mediated septic shock bystabilizing T-bet mRNA. (Published)
Arid5a deficient mice are shown to be completely resistant tolipopolysaccharide (LPS) mediated shock. Histological analysis by
H&E staining of lung, liver and spleen tissue indicated that Arid5a
deficient mice are protected from tissue injury after LPS induced
shock mice model. Recently, our studies have shown that Arid5a
regulates IFNγ production through Tbx21 mRNA stabilization,
which encodes T-bet, known as master regulator of Th1 cell differentiation. Consistent with the data, serum level of IFNγ wassignificantly reduced in LPS-induced Arid5a deficient mice. Consequently, reduction of IFNγ production contributed to the recovery of mortality in endotoxin shock. Additionally, Arid5a-deficient mice in Propionibacterium acnes-primed endotoxin shockmodel, which elicits Th1 responses, showed lower levels of IFNγ,
IL-6 and TNFα, with a higher survival rate. These results strongly
suggest that Arid5a controls the stabilization of many inflammatory mRNAs, which in turn, contributes to the pathogenesis of
inflammatory and autoimmune disease (Figure 1).

Figure 1.
Amplification of IL-6 level by Arid5a.

Figure 2.
Lenalidomide disrupts a Celeblon-Rabex-5 complex to suppress TLR
induced type-1 interferon production.

Recent Publications■ Dubey PK, et al. Arid5a-deficient mice are highly resistant to bleomycin-

induced lung injury. Int. Immunol. 29(2), 79-85 (2017).

■ Millrine D, et al. Rabex-5 is a lenalidomide target molecule that negatively

regulates TLR-induced type 1 IFN production. Proc. Natl. Acad. Sci. USA.
113(38), 10625-30 (2016).

■ Tanaka T, et al. Regulation of IL-6 in Immunity and Diseases. Adv. Exp.

Arid5a-deficient mice are highly resistant to bleomycin-induced lung injury (Published)
Acute lung injury (ALI) and acute respiratory distress syndrome(ARDS) are clinical conditions involving acute respiratory dys-Med. Biol. 941, 79-88 (2016).

■ Masuda K, et al. Arid5a regulates naive CD4+ T cell fate through selective

stabilization of Stat3 mRNA. J. Exp. Med. 213(4), 605-19 (2016).
■ Zaman MM, et al. Arid5a exacerbates IFN-γ-mediated septic shock by sta-bilizing T-bet mRNA. Proc. Natl. Acad. Sci. USA. 113(41), 11543-11548(2016).

21

Kiyoshi Takeda, MD/PhD

Professor

Kiyoshi Takeda

Associate Professor

Eiji Umemoto

Assistant Professor

Hisako Kayama
Ryu Okumura
Mari Murkami

Our laboratory is analyzing the mechanisms how intestinal homeostasis is maintained to reveal the pathogenesis of inflammatory bowel diseases (IBD). Several recent evidences have indicatedthat both intestinal environmental factors including microbiota
and intestinal immunity (genetic predisposition) contribute to
the development of IBD. However, there exists a single layer ofintestinal epithelial cells between intestinal environmental factors and intestinal immunity. We thought that intestinal epithelial cells possess some barrier functions that separate intestinal
environmental factors and intestinal immunity, and analyzed thebarrier function of intestinal epithelial cells.

Barrier function of intestinal epithelial layers
Genome-wide association studies have identified several susceptible loci for IBD including ulcerative colitis (UC). Amonggenes that are located within these loci, we tried to find a genethat is highly expressed in colonic epithelial cells. A gene encoding RING finger protein 186 (RNF186), which was located within
UC-susceptible loci, was found to be highly expressed in epithelial cells in the colon. Therefore, we analyzed the role of RNF186 in
the regulation of intestinal homeostasis. RNF186, which harbored
RING finger domain, acted as an E3 ligase mediating polyubiquitination of its substrates. Comparison of protein expression levels
using liquid chromatography-tandem mass spectrometry showedthat expression of several proteins was increased in colonic epithelial cells of Rnf186−/− mice. These included occludin. RNF186
was found to associate with these proteins and induce polyubiquitination. The polyubiquitination induced by RNF186 was K4822

Research Assistant

6

Support Staff

3

mediated, which leads to protein degradation. These findings indicate that RNF186 regulates protein expression the colonicepithelial cells by inducing polyubiquitinaton. The disturbed protein homeostasis in Rnf186−/− mice correlated with enhanced endoplasmic reticulum (ER) stress in colonic epithelia and increased
sensitivity to intestinal inflammation after dextran sulfate sodium(DSS) treatment. A previous deep sequencing study of the UCsusceptible loci had found a single nucleotide polymorphism(SNP) in a coding region of the RNF186 gene. Introduction of an
UC-associated Rnf186 SNP led to impaired E3 ligase activity andincreased sensitivity to DSS-induced intestinal inflammation in
mice. Taken together, these findings demonstrate that RNF186 is
responsible for the maintenance of gut homeostasis by controlling ER stress in colonic epithelia (Figure 1).

Dysbiosis triggers arthritis development
We also analyzed the involvement of microbiota in several disorders other than IBD. Several previous studies using mousemodels of arthritis indicated that the intestinal microbiota is involved in arthritis pathogenesis. Therefore, we analyzed whether
microbiota is involved in the pathogenesis of rheumatoid arthritis (RA). We first compared the composition of fecal microbiota
between healthy volunteers and RA patients. Altered microbiota
composition, called dysbiosis, was found in some RA patients.
These RA patients harbored intestinal microbiota dominated by
Prevotella copri. We then analyzed how dysbiosis contributes toarthritis development using arthritis-prone SKG mice harboring
human microbiota. SKG mice reared in a specific pathogen-freefacility developed arthritis upon fungal component injection;
however, elimination of intestinal microbiota by oral administration of antibiotics protected these mice from joint inflammation.
Then, germ-free SKG mice were generated and these mice werecolonized with human fecal microbiota from healthy volunteers
and Prevotella-dominated RA patients. SKG mice harboring boththe microbiota of healthy controls and the Prevotella-dominatedmicrobiota from RA patients (RA-SKG) were successfully produced. RA-SKG mice developed severe arthritis after fungal component injection. Lymphocytes in regional lymph nodes and colon, but not spleen, of RA-SKG mice showed enhanced IL-17
responses to the arthritis-related autoantigen, RPL23A. The
RPL23A-induced T cell response was attenuated in germ-free SKG

mice. Naïve SKG T cells, co-cultured with P. copri-stimulated dendritic cells, responded to RPL23A and rapidly induced arthritis.
SKG mice monocolonized with P. copri developed severe arthritis.
Thus, SKG T cells are activated to vigorously react to autoantigens
by dysbiotic microbiota in the intestine, causing joint inflammation (Figure 2). These findings indicate that dysbiosis is an environmental factor that triggers arthritis development in genetically susceptible mice.

Figure 1.
RNF186 is responsible for the maintenance of protein homeostasis in thecolonic epithelial cell. In the absence of RNF186, expression of RNF186targeted protein was increased, causing high susceptibility to stressinduced cell death, which leads to the high sensitivity to intestinal
inflammation.

Figure 2.
A subpopulation of rheumatoid arthritis (RA) patients showed dysbiotic
microbiota dominated by Prevotella copri. SKG mice harboring Prevotellacopri -dominated microbiota from RA patients (RA-SKG mice) showed
severe inflammatory arthritis as well as increased number of Th17 cells in
the regional lymph nodes and large intestine. Moreover, lymphocytes inregional lymph nodes and large intestine of these mice showed enhanced
IL-17 responses to arthritis-related autoantigen (RPL23A). Thus, dysbiosis
triggers arthritis development via activation of autoreactive T cells in theintestine.

Immunology

Mucosal Immunology

Recent Publications■ Kitada S, Kayama H, Okuzaki D, Koga R, Kobayashi M, Arima Y, Kumanogoh A,■ Okumura R, Kurakawa T, Nakano T, Kayama H, Kinoshita M, Motoka D,

Murakami M, Ikawa M& Takeda K BATF2 inhibits immunopathologicalTh17 responses by suppressing Il23a expression during Trypanosoma
cruzi infection. J. Exp. Med. (2017) in press.

Gotoh K, Kimura T, Kamiyama N, Kusu T, Ueda Y, Wu H, Iijima H, Barman S,
Osawwa H, Matsuno H, Nishimura J, Ohba Y, Nakamura S, Iida T,
Yamamoto M, Umemoto E, Sano K, & Takeda K. Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia. Nature 532, 117121 (2016).

■ Fujimoto K, Kinoshita M, Tamura A, Tanaka H, Okuzaki D, Shimada Y,

Kayama H, Okumura R, Furuta Y, Narazaki M, Hatakeyama S, Ikawa M,
Tsuchiya K, Watanabe M, Kumanogoh A, Tsukita S, & Takeda K. Regulationof intestinal homeostasis by the ulcerative colitis-associated gene
RNF186. Mucosal Immunol. 10, 446-459 (2017).
■ Maeda Y, Kurakawa T, Umemoto E, Motooka D, Ito Y, Gotoh K, Hirota K,■ Tsai SH, Kinoshita M, Kusu T, KayamaH, Okumura R, Ikeda K, Shimada Y,

Takeda A, Yoshikawa S, Kurashima Y, Sato S, Umemoto E, Kiyono H,
Karasuyama H & Takeda K. Ectoenzyme E-NPP3 (CD203c) negatively regulates ATP-dependent chronic allergic responses by basophils and mast
cells. Immunity 42, 279-293 (2015).

Matsushita M, Furuta Y, Narazaki M, Sakaguchi N, Kayama H, Nakamura S,
Iida T, Saeki Y, Kumanogoh A, Sakaguchi S, & Takeda K. Dysbiosis contributes to arthritis development via activation of autoreactive T cells in theintestine. Arthritis Rheum. 68, 2646-2661 (2016).
23

Immunology

Immune Regulation
Hitoshi Kikutani, MD/PhD

Professor

Hitoshi Kikutani

Assistant Professor

Shuhei Sakakibara

Postdoctoral Fellow

1

Support Staff

1

Figure 1.
X-ray crystallography of anti-DNA antibody 71F12.
The Fab binds to thymines through a stacking interaction (W101 of the heavy chain) andseveral hydrogen bonds. It should be noted that mutated F33 of the heavy chain locates at the
antigen recognition site.

Structural basis for antigen recognition of anti-DNA antibody in systemic lupus erythematosus (SLE)

Tracking of allergy-related IgE in chronic rhinosinusitis
with nasal polyposis (CRSwNP)

Anti-DNA antibody is the established criterion for SLE diagnosis and its contribution to clinical symptoms in SLE has been
widely accepted. It is still unclear how anti-DNA antibodies exert
pathogenicity. This may be due to a lack of precise structural
studies of anti-DNA antibodies.
In the current study, we isolated high-affinity anti-DNA monoclonal antibodies (mAbs) from acute SLE subjects. Unlike previously reported anti-DNA mAbs obtained from SLE model mice,
they exhibited nano-molar KD in surface plasmon resonance(SPR). In collaboration with Prof. Junichi Takagi (Institute of Protein Research, Osaka Univ), the crystallography of ligand-boundFab of one mAb clone (designated as 71F12) was solved (Figure 1).
The structural analysis clearly demonstrated a contribution of somatic hypermutation (SHM) to the antigen recognition, in which
nucleobases are rigidly grabbed by the Fab through the characteristic stacking interaction. Our result provides a new structural
insight into antigen recognition by anti-DNA antibodies in SLE.

CRSwNP is characterized by eosinophilic inflammation and nasal polyposis. Nasal polyps (NPs) of CRSwNP patients contain ahigh concentration of IgE presumably originated from infiltrating
B cells. We sought to understand the development pathway of
IgE-producing B cells in NPs.
We first determined antigens of IgE produced in NPs by using
antibody cloning and expression of monoclonal IgE antibodies.
The majority of isolated mAbs appeared to be specific to bacteria
which normally inhabit in sinonasal cavity, such as Streptococcus
pyogenes. Next, to comprehend the development pathway ofsuch antigen-specific IgE, deep sequencing of NP-associated BCR
repertoires was performed (Figure 2). It showed that major clonal
lineages of IgE were often found in IgG or IgA1 clonal lineages,
indicative of sequential class switch to IgE in NPs. Taken together,
CRSwNP is derived from protective immune response against nasal bacteria, in which unnecessary class switching to IgE takes
place concomitantly. Immune cells involved in IgE production
remains to be elucidated.

Figure 2.
Clonal connectivity between isotypes in NP-associated immunoglobulins.
Deep sequencing analysis of expressed BCR repertoires unraveled the clone distribution
across immunoglobulin isotypes. Large IgE clones were strongly connected with IgG and IgAlineages. This analysis was performed by Dr. Shota Nakamura, "Dr. Kazuo Yamashita" and Prof.
Daron M. Standley in RIMD, Osaka Univ.

24

25

Immunology

Experimental Immunology
Shimon Sakaguchi, MD/PhD

Professor

Shimon Sakaguchi

Assistant Professor

Wing James Badger
Norihisa Mikami
Noriko Sakaguchi
Kenji Ichiyama

This laboratory studies: (i) the cellular and molecular basis of
immunologic self-tolerance, in particular the roles of regulatory T
cells; (ii) the strategy for eliciting effective immune responses to
autologous tumor cells, or inducing immunologic tolerance toorgan transplants, by manipulating the mechanism of immunologic self-tolerance; and (iii) the cause and pathogenetic mechanism of systemic autoimmune diseases, such as rheumatoid arthritis, by utilizing an animal model established in our laboratory.
Treg cells, which specifically express the transcription factor
Foxp3, are actively engaged in the maintenance of immunological self-tolerance and homeostasis. The majority of them develop
in the thymus as a functionally distinct and mature T-cell subpopulation, with their stable Foxp3 expression chiefly maintained by
Treg-specific DNA demethylation (Ohkura et al., Immunity 2012,2013; Morikawa et al., PNAS 2014). It is poorly understood, however, how Treg-specific transcriptional and epigenetic changes
are initiated and coordinated to determine the Treg cell lineage
in the thymus. This year, with recently demonstrated associationsof super-enhancers with cell type-specific gene regulation andlineage determination in various cell types, we identified Treg
cell-specific super-enhancers (Treg-SEs), many of which were associated with the Treg signature genes, such as Foxp3, Ctla4 andIl2ra (Kitagawa et al., Nat. Immunol. 2017). The establishment ofTreg-SEs developmentally began in Treg progenitor cells before
Foxp3 transcription and Treg-specific DNA demethylation, facilitating early induction of the associated genes. It required the genome organizer Satb1, which bound to Treg-SEs before their activation and extended its binding sites within the SEs along Treg
26

Postdoctoral Fellow

3

Research Assistant4

Support Staff

6cell differentiation. T cell-specific deletion of Satb1 impaired
Treg-SE formation in Treg precursor cells, hindering both Tregspecific DNA demethylation and the transcription of Treg-SE-associated genes including Foxp3. The consequent arrest of Treg
cell differentiation at the precursor stage resulted in spontaneousdevelopment of severe autoimmunity and IgE hyperproduction.
Our results thus demonstrate how Satb1-dependent Treg-SE establishment and subsequent transcriptional and epigenetic
changes control Treg cell linage specification in the thymus, andhow molecular anomaly in this process causes autoimmune andother immunological diseases via affecting Treg cell development.
Based on the findings described above with mice, we are now
studying how Treg-specific transcriptional and epigenetic alterations control the function, development, and functional stabilityof Treg cells in humans, and how genetic variations of Treg-specific functional genes contribute to the development of autoimmune and other immunological diseases.

Figure. Establishment of Treg-specific super-enhancers for Treg cell development
A model of super-enhancer (SE) establishment and subsequent transcriptional
regulation during thymic Treg cell development. Treg-SE regions are poised at leastfrom the CD4/CD8 double-positive (DP) stage and bound by Satb1. Upon receiving
the signal to direct Treg cell differentiation via the immature CD4 single-positive(SP) stage, Treg-SE regions undergo Satb1-dependent activation, likely through therecruitment of epigenetic modifying enzymes and become Treg lineage-committed
precursor cells. As Treg cell development proceeds, enhancer-promoter looping
facilitates the expression of associated Treg cell signature genes, including Foxp3,
as well as other epigenetic modifications such as Treg-specific DNA demethylation,
histone modification of promoter regions and chromatin loosening of enhancer
and promoter regions. Once Treg cell development is complete, Foxp3 amplifies
pre-established molecular features and Satb1 transcription is repressed by Foxp3;
however, mediator, cohesin, and various transcription factors, including Foxp3,occupy the sites where Satb1 initially bound, maintaining the local chromatin
structure.

Recent Publications■ Kitagawa Y, Ohkura N, Kidani Y, Vandenbon A, Hirota K, Kawakami R,■ Ito Y, Hashimoto M, Hirota K, Ohkura N, Morikawa H, Nishikawa H, Tanaka A,

Yasuda K, Motooka D, Nakamura S, Kondo M, Taniuchi I, Kohwi-Shigematsu T,
Sakaguchi S. Guidance of regulatory T cell development by Satb1-dependent super-enhancer establishment. Nature Immunol. 18, 173-183,(2017).

Furu M, Ito H, Fujii T, Nomura T, Yamazaki S, Morita A, Vignali DA, Kappler JW,
Matsuda S, Mimori T, Sakaguchi N, and Sakaguchi S. Detection of T-cell
responses to a ubiquitous cellular protein in autoimmune disease. Science. 346, 363-368 (2014).

■ Saito T, Nishikawa H, Wada H, Nagano Y, Sugiyama D, Atarashi K, Maeda Y,■ Wing JB, Ise W, Kurosaki T, and Sakaguchi S. Regulatory T-cells control an-

Hamaguchi M, Ohkura N, Sato E, Nagase H, Nishimura J, Yamamoto H,
Takiguchi S, Tanoue T, Suda W, Morita H, Hattori M, Honda K, Mori M, Doki Y
and Sakaguchi S. Two FOXP3+CD4+ T-cell subpopulations distinctly control the prognosis of colorectal cancers. Nature Med. 22, 679-684 (2016).

tigen-specific expansion of Tfh cell number and humoral immune responses via the coreceptor CTLA-4. Immunity. 41, 1013-1025 (2014).

■ Maeda Y, Nishikawa H, Sugiyama D, Ha D, Hamaguchi M, Saito T, Nishioka M,

Wing JB, Adeegbe D, Katayama I, Sakaguchi S. Detection of self-reactive
CD8+ T cells with an anergic phenotype in healthy individuals. Science.
346, 1536-1540 (2014).

27

Takashi Saito, PhD

Professor

T cells play central roles in initiating and regulating immune
responses. They start immune response, induce activation, andgenerate various effector T cells, which function for protection
against infection and tumorigenesis. Aberrancy in T cell function
results in infectious and autoimmune diseases. Our group aims todetermine the molecular mechanism of T cell activation, differentiation and homeostasis, particularly from the perspective of signaling regulation.
T cells are activated upon antigen recognition by association
with antigen-presenting cells such as dendritic cells (DC). Association between T cells and DC generates immune synapse at theinterface. We have found that activation is induced through TCRmicroclusters (MC), the signaling clusters to induce T cell activation, prior to immune synapse formation. TCR-MCs are generated
by recruiting TCR and proximal signaling molecules such as kinases, adaptors, and effector molecules. We found that each TCRMC is surrounded by an adhesion-ring composed of integrin andfocal adhesion molecules, such as LFA-1, paxillin, Pyk2 and vinculin, at the initial stage of activation. The structure resembles immune synapse in micro-scale, thus termed microsynapse (Figure).
TCR-MCs, ZAP70, SLP76 and F-actin were detected at the center,and LFA1, Paxillin, Pyk2 are in the outside ring of microsynapse.
Microsynapse supports integrin outside-in signal and cell adhesion, which help the formation of MCs and cSMAC, and consequently T cell activation. Microsynapse is particularly importantin activation of T cells under weak stimulation conditions because
weak stimulation induces long-lasting microsynapse whereas
28

Takashi Saito

function of STING activation in T cells is analyzed. Surprisingly,
STING stimulation resulted in inhibition of T cell proliferation andinduces anti-viral and anti-tumor responses including type I-IFN
secretion. Therefore, STING ligands possibly serve as specificmodulators of activated effector T cells.
The ultimate aim of our approaches to analyze T cell signaling
and function is to elucidate the mechanism of inducing autoim-

mune diseases through aberrant T cell signaling and function
and to be able to modulate signaling and function in order to inhibit/prevent autoimmunity and allergic inflammation. We are
analyzing function and regulation by phosphatase (PTPN22,
PTPN2) whose mutations and deficiency are related to inductionof autoimmune diseases. Then the onset of autoimmunity will beanalyzed in mouse models.

Immunology

Cell Signalingstrong stimulation merely induces transiently. Thus, microsynapse and MCs seem to be the major structures to induce activation signal in T cells. Under weak stimulation, T cells do not induce the formation of cSMAC, however, TCR-MCs function to
induce activation with the support of microsynapse for cell adhesion.
We continued to analyze spatio- and temporal-regulation of T
cell activation, particularly by analyzing individual critical activation pathways. Since activation of the Ras-MAPK pathway is spatially regulated, dynamics of two Ras GEFs, Ras-GRP1 and Sos,
were investigated. Whereas Sos is initially co-localized with TCRMC, Ras-GRP1 is accumulated in a unique mesh-like cytoskeletal
structure upon stimulation. Since RasGRP1 dominantly functions
as GEF in T cells, we concentrated on analyzing the dynamics ofRasGRP1. The membrane localization was mediated through Cterminus region of RasGRP1. Mass analysis identified Plectin, aknown cytoskeletal linker, as a binding molecule to this region of
RasGRP1. Plectin-deficient T cells failed to generate the meshstructure of RasGRP1 and consequently reduced IL-2 production
upon TCR stimulation. Thus, Ras activation is mediated by spatial
regulation of RasGRP1 through linking cytoskeleton.

Figure. Microsynapse composed of micro-adhesion ring surrounding TCR-microcluster isessential for T cell activation(A) Focal adhesion molecule Pyk2 (green) forms micro-adhesion ring around TCRmicroclusters (red) upon T cell stimulation.
(B) Microsynapse composed of micro-adhesion ring around TCR-microcluster is formedin early activation and help formation of microcluster and cSMAC and T cell activation,
particularly under weak stimulation.
(C) Adhesion-ring in microsynapse serves inducing outside-in signals through LFA1/focal
adhesion molecules helps inducing activation signals through TCR-microclusters.

Recent Publications+
CTL, a Cytotoxic Subset of CD4+ T cells, Their
Differentiation and Function. Front. Immunol. 8, 194 (2017).

■ Takeuchi A, et al. CRTAM determines the CD4+ cytotoxic T lymphocyte+
thymocytes through phosphorylation of SHP-1. Nat. Commun. 7, 12756(2016).

■ Matsumoto T, et al. Overexpression of CTLA-4 prevents atherosclerosis in■ Takeuchi A and Saito T. CD4■ Ishikawa E, et al. Protein kinase D regulates positive selection of CD4

We have also investigated the regulation of T cell activationand function by innate signals. We have already analyzed thefunctions and signaling of TLRs, and nucleic acid recognition in T
cells. STING is now a major intracellular DNA sensor in innate
cells. Since we found that STING is highly expressed in T cells, thelineage. J. Exp. Med. 213, 123-138 (2016).

mice. Arterioscl. Throm. Vas. 36, 1141-1151 (2016).

■ Hashimoto-Tane A, et al. The Micro adhesion-ring surrounding each TCR

microclusters forms synapse-like structure essential for T cell activation. J.
Exp. Med. 213, 1609-1625 (2016).

29

Tomohiro Kurosaki, MD/PhD

Professor

Tomohiro Kurosaki

Associate Professor

Yoshihiro Baba

cence as previously thought. Mechanistically, upon provision ofadequate T cell help, Foxo1-/- GC LZ B cells could not induce transcription factors, c-Myc ant BATF, both of which are essential for
GC B cell proliferation. We confirmed that BATF was transiently
induced in LZ B cells in a Foxo1-dependent manner and that de-pletion of BATF similarly led to GC disruption. Thus, our results
support a model in which the switch from the LZ to DZ is triggered after receipt of T cell help, and suggest that Foxo1-mediated BATF up-regulation plays an important role in this process(Fig.2).

Immunology

Lymphocyte Differentiation

Wataru Ise
Assistant Professor

Takeshi Inoue
Ryo Shinnakasu
Shinya Tanaka

Research Assistant2

Support Staff

4

Figure 1. Strong T cell help confers the differentiation potential toward plasma cells
GC-B cells with high affinity BCR receive strong T cell help via CD40, which leads to upregulation of adhesion molecules such as ICAM-1 or SLAM. The consequence of strong
interaction with TFH is the induction of IRF4+ Bcl6lo GC B cells which favor plasma celldifferentiation rather than GC re-cycling.

The immune system acts in a trade-off manner between enhanced immune response and an increased risk of autoimmunity.
For example, the PTPN22R620W polymorphism provides increased
protection against tuberculosis and is associated with increased
susceptibility to several autoimmune diseases.
Involvement of DNA demethylases, Tet2 and Tet3 in B cell
tolerance
Genome-wide association studies (GWAS) have identified
hundreds of gene polymorphisms that are associated with an increased risk of developing autoimmunity. Among them, Tet3 was
identified to be associated with SLE; Tet3 is one of the recently
identified Ten-eleven translocation (Tet) dioxygenase family proteins (Tet1, Tet2, and Tet3). They act as DNA demethylase by catalyzing the conversion of 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC) and other intermediate products inthe DNA demethylation pathway. We showed that deficiency of
Tet DNA demethylases, Tet2 and Tet3, in B cells led to accumulation of effector T cells, autoantibody formation, and nephritis. By
using this mouse model, we found that Tet2- and Tet3-deficient B
cells manifested hyper-responsive to B cell receptor, thereby inducing co-stimulatory molecules CD80/86 at the initiating phasein lupus. At the promoting phase, treatment of anti-CD4 antibodies ameliorated aberrant activation of B cells and autoantibody
titers. Thus, our results suggest that dysregulation of CD80/86 on
B cells is one of the key drivers for initiating the positive loop between autoreactive B and T cells, resulting in full-blown autoimmunity.
30

Selection of high-affinity antibodies during germinal center
reactions
The hallmark of antibody-mediated adaptive immunity is the
progressive increase in the ability of serum antibodies to bind antigen and efficiently provide immune protection. This phenomenon, known as affinity maturation, takes place majorly in the germinal center (GC), where antigen-specific GC B cells diversify their
BCRs by somatic hyper-mutation. We found that the IRF4+Bcl6lo
light zone (LZ) GC subset with high affinity BCRs favored the plasma cell fate over GC cycling. Cells in this plasma-prone fraction
was increased or decreased by stronger T cell help or haplo-insufficiency of CD40, respectively. ICAM and SAP on GC B cells wereupregulated by strong CD40 signals, thereby affording stronger
interaction between IRF4+Bcl6lo cells and TFH cells. Together, ourdata suggest that high CD40 signal induces stronger T-B interaction, thereby conferring differentiation potential towards plasma
cells concomitantly with losing GC potential (Fig.1).
Regulation of germinal center B cell proliferation by Foxo1
Foxo1, forkhead box protein O1, is a transcriptional regulator
that localizes to the nucleus and controls gene expression. As GCderived lymphomas frequently carry mutations in Foxo1, Foxo1
might play a critical role in controlling gene expression within GC
B cells. To address Foxo1 function in GC biology, we conducted
an experimental system in which Foxo1 is inducibly deleted specifically in GC B cells. We found that ablation of Foxo1 after GC
development led to the loss of the GC DZ B cells, suggesting that
Foxo1 exerts a proliferative role, rather than promoting quies-

Figure 2. Regulation of GC B cell proliferation by Foxo1(Left) Without Foxo1, germinal center structure is disorganized and mice could not produce
high affinity antibodies against foreign antigens. (Right) DZ is lost and the GC is collapsed
upon Foxo1 ablation. Foxo1 has also important roles in the LZ B cells, including antigen
presentation to TFH cells and expression of DZ program genes, including BATF.

Recent Publications■ Shinnakasu R, Kurosaki T. Regulation of memory B and plasma cell differ-entiation. Curr. Opin. Immunol. 45, 126-131 (2017) Epub ahead of print.

■ Shinnakasu R, Inoue T, Kometani K, Moriyama S, Adachi Y, Nakayama M,■ Inoue T, Shinnakasu R, Ise W, Kawai C, Egawa T, Kurosaki T. The transcrip-

Takahashi Y, Fukuyama H, Okada T, Kurosaki T. Regulated selection of germinal center cells into the memory B cell compartment. Nat. Immunol. 17(7), 861-9 (2016).

tion factor Foxo1 controls germinal center B cell proliferation in response
to T cell help. J. Exp. Med. 214 (4), 1181-1198 (2017).

■ Kometani K, Kurosaki T. Differentiation and maintenance of long-lived

plasma cells. Curr. Opin. Immunol. 33, 64-9 (2015).
■ Kuwahara M, Ise W, Ochi M, Suzuki J, Kometani K, Maruyama S, Izumoto M,

Matsumoto A, Takemori N, Takemori A, Shinoda K, Nakayama T, Ohara O,
Yasukawa M, Sawasaki T, Kurosaki T, Yamashita M. Bach2-Batf interactions
control Th2-type immune response by regulating the IL-4 amplification
loop. Nat. Commun. 7, 12596 (2016).

31

Cevayir Coban, MD

Malaria continues to be a global burden to the world that kills
millions every year due to a lack of a fully successful vaccine orpotent drugs for the complete elimination of Plasmodium parasites. Therefore, research in our laboratory focuses on the elucidation of host-pathogen interactions in the context of malaria tounderstand better how these parasites interact with the host. Our
broad and final aim is to investigate host-parasite interactions
both in animal models and humans, and translate our understanding into safe intervention such as vaccines and drugs.
During blood-stage life cycle of Plasmodium parasites, infection can cause disease manifestations such as fever, vomiting,muscle pain and anemia and severe complications including cerebral malaria and, respiratory distress, suggesting that the interaction between infected erythrocyte and tissue environment
could be different. Therefore, our studies have mainly focused on
the understanding of tissue-specific immunopathology caused
by malaria in different tissues/organs. Our final goal is to create
new interventions from this understanding as adjunct therapy toanti-malarials (Figure 1).
We have taken advantage of using imaging technologies suchas MRI, multiphoton live imaging and micro-computed tomography (μCT) readily available at IFReC facilities in accordance with
IFreC/WPI program’s aims.
One of the topics we have investigated is the pathology of cerebral malaria, an acute and deadly outcome caused by Plasmo32

Professor

Cevayir Coban

Postdoctoral Fellow

2

Research Assistant2

Support Staff

2

we recently showed that infection causes significant and longterm bone loss in adult mice, and growth retardation in young
mice. Acute malaria infection severely suppresses bone homeostasis, but sustained accumulation of Plasmodium products in thebone marrow niche induces MyD88-dependent inflammatory
responses in osteoclast and osteoblast precursors, leading to increased RANKL expression and over-stimulation of osteoclastogenesis favoring bone resorption. Infection with a mutant parasite with impaired hemoglobin digestion that produces little hemozoin, a major Plasmodium by-product, did not cause boneloss. Importantly, supplementation of alfacalcidol, a vitamin D3
analog, could prevent bone loss. These results highlight the riskof bone loss in malaria-infected patients and the potential benefits of coupling bone therapy with anti-malarial treatment (Lee etdium falciparum infections in humans and P. bergheiANKA parasites in mice. We have recently reported a new understanding of
cerebral malaria pathogenesis using ultra-high field MRI duringexperimental cerebral malaria. Deep investigation of the brain by
ultra-high field MRI showed that the olfactory bulb is physicallyand functionally damaged by Plasmodium parasites. Live multiphoton imaging of the olfactory bulb confirmed that the complex trabecular small capillaries comprising the olfactory bulb
show parasite accumulation and cell occlusion followed by micro-bleeding, events associated with high fever and cytokine
storm. With these findings, we have provided evidence that olfactory functional impairment (loss of smell) could be a valuable
early diagnosis marker for cerebral malaria (Cell Host Microbe,2014).
On the other hand, despite severe complications, the majorityof patients recover from malaria. However, there is evidence thatmalaria survivors experience long-term ‘hidden’ pathologies thatare as yet poorly defined. For instance, chronic exposure to Plasmodium parasites promotes prolonged immune activation andenhances the risk of lymphoma formation. Growing evidencessuggest that physical growth retardation in young children in Africa is associated with infectious diseases, with a high prevalence
among malaria-infected children regardless of nutritional status.
An increased incidence of porous bone lesions has also been reported in malaria endemic regions, suggesting infection may
compromise bone integrity. Using well-established mouse models mimicking various aspects of human Plasmodium infection,al., Science Immunology, 2017, in press).
The final goal of our research is the translation of our understanding of host-pathogen interactions into vaccines or drugs totreat disease(s). Given that there is still more to do for the development of potent vaccines against malaria, we focus on the adjuvants, because if rationally designed, adjuvants improve vaccine
efficacy. We have developed a new adjuvant called synthetic hemozoin, a synthetic analog of Plasmodium-produced hemozoin,
and completed its preliminary GLP non-clinical safety and toxicology studies in several animals and infection models (Lee et al.,Vaccine, 2016). These studies were performed according to GLPprocedures in rats and concluded that synthetic hemozoin is asafe adjuvant that displays very low autoimmune properties.

Immunology

Malaria Immunology

Figure.
Blood stage Plasmodium infection causes deadly complications. However, most people
develop partial immunity and suffer from mild symptoms. Recent evidences suggest thatthe incomplete recovery from infection causes chronic illnesses such as bone loss or growth
retardation. High prevalence of drug resistance and look of successful vaccines are drawbacks
in the efforts to eliminate malaria. Therefore additional treatment modalities should be
investigated to prevent and treat acute and chronic complications caused by malaria. Our
approach to this problem is to use imaging technologies to understand immunopathology
caused by Plasmodium parasites.

Recent Publications■ Lee MSJ, Maruyama K, Fujita Y, Konishi A, Lelliott PM, Itagaki S, Horii T, Lin JW,■ Zhao H et al. Lipocalin 2 bolsters innate and adaptive immune responses

Khan SM, Kuroda E, Akira S, Ishii KJ, Coban C. Plasmodium products persist
in the bone marrow and promote chronic bone loss. Sci. Immunol. (2017)
in press.

to blood-stage malaria infection by reinforcing host iron metabolism. Cell
Host Microbe 12(5), 705-16 (2012).
■ Coban C et al. Immunogenicity of Whole Parasite Vaccines Against Plas-■ Lee MSJ, Igari Y, Tsukui T, Ishii KJ, Coban C. Current status of synthetic he-mozoin adjuvant: A preliminary safety evaluation. Vaccine 34(18), 205561 (2016).

modium falciparum Involves Malarial Hemozoin and Host TLR9. Cell Hostand Microbe 7(1), 50-61 (2010).

■ Zhao H et al. Olfactory Plays a Key Role in Spatiotemporal Pathogenesis of

Cerebral Malaria. Cell Host Microbe 15(5), 551-63 (2014).
33

Ken J. Ishii, MD/PhD

Professor

Ken J. Ishii

Associate Professor

Etsushi Kuroda
Norifumi Iijima
Takuya Yamamoto

THE primary goal of our laboratory is to understand the immunological mechanisms of the intra- and inter-cellular signaling
pathways that mediate the immunogenicity of successful vaccines, as well as biological responses to adjuvants. Such knowledge will enable us to develop novel concepts, modalities andnext generation immuno-preventive and/or therapeutic agentsagainst infectious diseases, cancer and allergy as well as othernon-communicable diseases.
Our recent study revealed another face of aluminum nano- tomicro-particle as a part of PM2.5 (Kuroda E et al, Immunity 2016 inpress).
<Basic and translational vaccine science>

Old, but newly evolving adjuvant research
It is known that particle pollutions such as PM2.5 are involved
in allergic inflammation. Many papers have shown that these particulates have a strong adjuvant activity and induce Type-2 immune responses. However, the mechanisms by which particulates trigger allergic responses are unclear. In FY2016-2017, wehave shown that particulates induce alveolar macrophage deathand release IL-1α as a DAMP (damage-associated molecular pattern). Released IL-1α contributes to IgE production and inducible
bronchus-associated lymphoid tissue (iBALT) formation in thelungs. We suggest that iBALT formation plays an important role
for local IgE responses induced by particulate in the lungs. In thisstudy, we examined the detailed mechanisms of iBALT formationin the lungs (Kuroda E et al Immunity 2016).
We used alum as inflammatory particulate. Alum was adminis34

Assistant Professor

Natsuko Kishishita

Postdoctoral Fellow

4

Research Assistant

4

Visiting Scientist

6

Support Staff

1

tered by intratracheal (i.t.) instillation and then mice were exposed to OVA aerosol. Previously, we found that iBALT formation
and IgE responses were regulated by IL-1R signaling. In addition,
we observed the migration of DCs into the lungs are also controlled by IL-1R signaling. Using CD11c-DTR mice, we examined
the role of migrated DCs for iBALT formation and found that depletion of CD11c+ cells displayed reduced levels of IgE responses
and iBALT formation. Next, we examined the role of Tfh cells in
the lungs using Cd4-cre Bcl6flox/flox mice, as a model of Tfh-deficient mouse. The same as requirement of DCs, Tfh cells werealso required for iBALT formation, in addition to IgE responses.
These results indicate that DCs and Tfh cells play a pivotal rolefor particulate-induced lung inflammation, and might be a goodtherapeutic target for treatment of this type of inflammation(Figure 1).

actions (Manuscript submitted). Adding another data of AS04 adjuvant demonstrated that current data work as a reliable andstandardized core for new adjuvant characterization. We believe
this approach will be an important platform for adjuvant biology
and immunology, and contribute new adjuvant development in
the future.
Cohort as well as retrospective analysis of human samples obtained from volunteers of vaccine clinical trials and patients ofrelevant immunological disorders are being conducted by fourgroups including our lab in IFReC and those in National Instituteof Biomedical Innovation, Health and Nutrition (NIBIOHN). Preliminary results suggest serum miRNA may provide useful biomarkers to predict safety and immunogenicity of adjuvanted vaccines (manuscript in preparation).

Future prospect

Figure 1.
Lung-specific allergic inflammation by PM2.5 and other allergen via alveolar
macrophage and IL-1.

Figure 2.
Adjuvant data base project to evaluate adjuvant efficacy as well as safety bycomprehensive transcriptome analysis.

The Forefront Vaccine Development: Development of nextgeneration vaccines is verging on practical possibility based onmolecular designing. Toward such an end, outcomes from studies in my lab at IFReC are also expected to make considerable
contributions. These include searching of modality of antigen
delivery system and adjuvants. Vaccine target diseases are now
not only restricted to a framework of infectious diseases but include a broad range of diseases such as cancer, allergy, Alzheimer's disease, and many other lifestyle-related diseases. We willcontinue innovative research and development vaccines against
these diseases together with National Institute of Biomedical Innovation, Health and Nutrition (NIBIOHN) accompanying active
exchanges of researchers.

Immunology

Vaccine Science<Human immunology, clinical development of novel adjuvants and their biomarkers>

Clinical studies on seeking bio-marker(s) for safety as
well as efficacy of adjuvanted vaccines
In 2012 we launched a national project called Adjuvant Data
Base project supported by Ministry of Health, Labor and Welfare(Figure 2). We conducted a whole organ transcriptome as well as
miRNA analysis after the most representative 20-25 adjuvants,
some of which are approved in the market or are being developed, injected in mice and rats. Our datasets provided a detailed
biological feature of each adjuvant in vivo, unbiasedly classified
those adjuvants into 6 groups with associated mechanisms of the

Recent Publications■ Kanuma T, et al. CD63-mediated antigen delivery into extracellular vesi-■ Kobiyama K, et al. Species-dependent role of type I IFNs and IL-12 in thecles via DNA vaccination results in robust CD8+ T cell responses J. Immunol.
(2017) in press.

CTL response induced by humanized CpG complexed with β-glucan. Eur.
J. Immunol. 46(5), 1142-51 (2016).

■ Kuroda E, et al. Inhaled Fine Particles Induce Alveolar Macrophage Death■ Temizoz B, et al. Vaccine adjuvants as potential cancer immunotherapeu-

and Interleukin-1α Release to Promote Inducible Bronchus-AssociatedLymphoid Tissue Formation. Immunity. 45(6), 1299-1310 (2016).

tics. Int. Immunol. 28(7), 329-38 (2016).

■ Kitahata Y, et al. Circulating nano-particulate TLR9 agonist scouts out tu-

mor microenvironment to release immunogenic dead tumor cells. Oncotarget. 7(31), 48860-48869 (2016).
35

Immunology

Immunoparasitology
Masahiro Yamamoto, PhD

Professor

Masahiro Yamamoto

Assistant Professor

Miwa Sasai
Jisu Ma
Youngae Lee

Support Staff

The host immune system produces a proinflammatory cytokine, interferon γ (IFN-γ), against infection of the obligatory intracellular protozoan pathogen Toxoplasma gondii. IFN-γ stimulates
the cell-autonomous innate immune response, causing the disruption of the membranes of parasitophorous vacuole (PV),which T. gondii forms inside infected cells, by IFN-γ-inducible GTPases, such as immunity-related GTPases (IRGs) and guanylatebinding proteins (GBPs). GBPs and effector IRGs (called GKS-IRGs),
such as Irga6 and Irgb6, are recruited to the T. gondii PV membranes and are thought to cooperate in PV disruption. The regulatory IRGs (called GMS-IRGs), such as Irgm1 and Irgm3 (Irgm1/m3), and some essential autophagy components, such as Atg3,
Atg5, Atg7, and Atg16L1, positively control the recruitment of
IRGs and GBPs to the T. gondii PVs.
When a healthy host is infected with T. gondii, it develops acquired immunity involving a parasite-specific CD8+ T cell response, which is important for both acute and chronic resistanceto T. gondii and protective immunity against infection. Endogenous parasite-derived proteins, including GRA6, or a model protein such as ovalbumin (OVA) that is secreted into the PVs, hasbeen shown to efficiently elicit strong parasite-specific CD8+ T
cells. Professional hematopoietic antigen-presenting cells (APCs),
such as dendritic cells (DCs) and macrophages, as well as nonprofessional nonhematopoietic cells, such as fibroblasts and astrocytes, process the vacuolar proteins and present the antigens on
major histocompatibility complex class I (MHCI) molecules in a
TAP1-dependent manner. Furthermore, the direct interaction between the PV and the host endoplasmic reticulum (ER) provides a36

1route of entry for the MHCI-restricted antigen presented on DCs.
The priming of macrophages by the IFN-γ and tumor necrosis factor alpha (TNF-α) further enhances the activation of antigen-specific CD8+ T cells in an Irgm3-dependent manner, suggesting a
potential role for IFN-γ-inducible GTPases in antigen presentation. However, the molecular link between IFN-γ-induced cellautonomous innate immunity and vacuolar-antigen-specificCD8+ T cell-dependent acquired immunity remains unclear.
Here, we show that p62/Sqstm1, a protein with a ubiquitinbinding domain and that is involved in the selective autophagic
elimination of intracellular pathogens, is recruited together withubiquitin to the T. gondii PVs, following IFN-γ stimulation, andplays a specific role in vacuolar-antigen-specific CD8+ T cell activation after PV disruption by IFN-γ-inducible GTPases.

Figure.
p62 Plays a Specific Role in Interferon-γ-Induced Presentation of a
Toxoplasma Vacuolar Antigen

Recent Publications■ Man SM, Karki R, Sasai M, Place DE, Kesavardhana S, Temirov J, Frase S, Zhu Q,■ Lee Y, Sasai M, Ma J, Sakaguchi N, Ohshima J, Bando H, Saitoh T, Akira S,

Malireddi RK, Kuriakose T, Peters JL, Neale G, Brown SA, Yamamoto M,
Kanneganti TD. IRGB10 Liberates Bacterial Ligands for Sensing by the
AIM2 and Caspase-11-NLRP3 Inflammasomes. Cell. 167, 382-396 (2016).

Yamamoto M (Corresponding author). p62 plays a specific role ininterferon-γ-induced presentation of a Toxoplasma vacuolar antigen. Cell
Reports 13, 223-233 (2015).

37

Shigekazu Nagata, PhD

Phospholipids in plasma membranes are asymmetrically distributed between inner and outer leaflets. Phosphatidylserine(PtdSer), one of most abundant phospholipids in eukaryotic plasma membranes, is exclusively localized in the inner leaflet. Thisasymmetrical distribution of phospholipids is maintained by an
ATP-dependent phospholipid flippase, that translocates PtdSer
and phosphatidylethanolamine from outer to inner leaflets.
When cells undergo apoptosis, or platelets are activated, theasymmetrical distribution of phospholipids is disrupted by ascramblase(s) that non-specifically scrambles phospholipids between the inner and outer leaflets of plasma membranes, leading
to PtdSer-exposure. The PtdSer, thus exposed to the cell surface,works as an “eat me” signal of apoptotic cells, and as scaffolds on
the activated platelets for blood clotting factor. The PtdSer-exposure is also observed in activated lymphocytes, capacitatedsperm, aged erythrocytes, exosomes, and enveloped virus.
We recently identified three P4-type ATPases (ATP8A2, ATP11A
and ATP11C) and their subunit CDC50A as flippases that activelytranslocate PtdSer from outer to inner leaflets of the plasma
membrane. Among these three flippases, ATP8A2 is specifically
expressed in brains, while ATP11A and ATP11C are ubiquitously
expressed in various cells including lymphocytes and hepatocytes. ATP11A and ATP11C contain two or three caspase-recognition sites in the middle of molecules, and their flippase activity is
destroyed during apoptosis. A high concentration of Ca2+ inhibits
their flippase activity, too.
There are two families of membrane proteins that support
non-specific scrambling of phospholipids at plasma membranes.
38

Professor

Shigekazu Nagata

Associate Professor

Katsumori Segawa

Postdoctoral Fellow

4

Research Assistant

3

Visiting Scientist

1

Support Staff

1

Five members (TMEM16C, 16D, 16F, 16G and 16J) of the TMEM16
family, that contain 10 transmembrane segments, function as
Ca2+-dependent scramblases by forming a dimer. Thus, when cells
are activated, the high intracellular concentration of Ca2+ reversibly inactivates the flippases, and reversibly activates TMEM16F
scramblase to expose PtdSer to the cell surface (Figure 1).
When the intracellular Ca2+ level returns to normal, the flippase
re-establishes the asymmetrical distribution of phospholipids.
Among these five members that function as scramblases,
TMEM16F is ubiquitously expressed in various cells, while others
are tissue-specific; they are specifically expressed in the brains or
intestines. Human and mouse platelets express only TMEM16F asa Ca2+-dependent scramblase. The TMEM16F-/- activated platelets
cannot expose PtdSer, leading to the reduced ability to produce
thrombin for blood clotting. In fact, human patients of Scott syndrome, a rare congenital bleeding disorder, were found to carry aloss of function mutation in TMEM16F.
Three members (Xkr4, Xkr8 and Xkr9) of the Xkr family that aresupposed to carry 10 transmembrane segments, support scrambling phospholipids during apoptosis. These members contain a
caspase-recognition site in the C-terminal tail region and arecleaved by caspase during apoptosis. Thus, when cells undergo
apoptosis, caspase cleaves and irreversibly inactivates ATP11A
and ATP11C flippases, and cleaves and activates Xkr4, Xkr6, andXkr9 scramblases, to quickly and irreversibly expose PtdSer (Figure 2).
The PtdSer, thus on the dead cell’s surface, is recognized bymacrophages for engulfment. Xkr4 and Xkr9 are rather tissuespecifically expressed in the brains and intestines, respectively.

Whereas, Xkr8 is ubiquitously expressed in various tissues. When
Xkr8-/- mouse embryonal fibroblasts (MEF) and fetal thymocytes
undergo apoptosis, they do not expose PtdSer and can not be
engulfed by macrophage. The unengulfed dead cells are believed to undergo secondary necrosis, and intracellular materials
are released from dead cells. This may activate the immune sys-tem, leading to systemic lupus erythematosus-type autoimmune
disease.
We are studying the molecular mechanism of how flippases
and scramblases flip and scramble phospholipids at plasma
membranes, and also the physiological and pathological roles ofthe flippases and scramblases.

Figure 1. The molecular mechanism for PtdSer exposure in cells with highCa2+-concentration.
The flippase comprised of P4-ATPase (ATP11A or ATP11C) and CDC50A,
and a Ca2+-dependent scramblase (TMEM16F) are schematically shown.
In activated platelets, the intracellular Ca2+ concentration increases andactivates TMEM16F to scramble phospholipids, while it inactivates P4ATPases and reduces their flipping activity. When the Ca2+ concentration
returns to normal level, TMEM16F stops scrambling phospholipids, while
P4-ATPases resume flipping PtdSer and PtdEtn. Thus, PtdSer is onlytransiently exposed to the cell surface in this process, and likely depends
on the intracellular concentration of ATP and Ca2+. The constant flippingof PtdSer prevents the PtdSer-exposing cells from being engulfed bymacrophages.

Figure 2. The PtdSer-exposure in apoptotic cells.
A caspase-dependent phospholipid scramblase of Xkr8, and flippase(ATP11A/ATP11C associated with CDC50A) are schematically shown.
When cells undergo apoptosis, caspase 3 or caspase 7 in the downstreamof the caspase-cascade cleaves Xkr8 to activate its scramblase activity,
while the same caspases cleave and inactivate ATP11A and ATP11C. Thisis an irreversible process, and the PtdSer exposed on the cell surface isrecognized by macrophages for engulfment.

Immunology

Biochemistry and Immunology

Recent Publications■ Nagata S, Tanaka M. Programmed cell death and the immune system. Nat.

Rev. Immunol. 17, 333-340 (2017).

■ Fujii T, Sakata A, Nishimura S, Eto K, Nagata S. TMEM16F is required forphosphatidylserine exposure and microparticle release in activatedmouse platelets. Proc. Natl. Acad. Sci. USA 112, 12800-12805 (2015).

■ Suzuki J, Imanishi E, Nagata S. The Xkr8 phospholipid scrambling complex

in apoptotic phosphatidylserine exposure. Proc. Natl. Acad. Sci. USA 113,
9509-9514 (2016).

■ Segawa K, et al.

Caspase-mediated cleavage of phospholipid flippase for
apoptotic phosphatidylserine exposure. Science 344, 1164-1168 (2014).

■ Gyobu S, et al. A role of TMEM16E carrying a scrambling domain in sperm

motility. Mol. Cell Biol. 36, 645-659 (2016).
39

Toshio Yanagida, PhD

Professor

Toshio Yanagida

Associate Professor

Hideaki Fujitaspite the non-deterministic manner of T cells, we further investigated the effect of Treg cells (Fig. 2), and found that in the presence of Treg cells, activation of T cells was suppressed in theabsence of antigen peptide, whereas activation probability did
not change in the presence of antigen peptide. This observation
show that Treg cells act as a faucet for a leaky immune system.

Imaging

Single Molecule Imaging

Tomoyuki Yamaguchi
Yutaka Komai
Postdoctoral Fellow

1

Visiting Scientist

2

Support Staff

1

Figure 1.
Multicellular model of T cell response. Schematic illustration of the model (left), response of
T cell population with respect to antigen presentation (middle), and average number of Treg
cells as a function of the fraction of the target antigen (right).

Use of Morin hydrate for manganese mapping
Recently, manganese (Mn) became an important paramagnetic contrast agent in magnetic resonance imaging. Mn2+ can enter
neurons through voltage dependent Ca2+ channels, and thus acts
as an indirect marker of calcium channel activity. Morin is a chemical isolated from Maclura pomifera, which when it binds divalent
metals shifts from a red to green emission peak, and thus it allows
visualization of the distribution of Mn2+ at a cellular level underfluorescence microscopy. To demonstrate the detectability of Mn
in tissue, mice were exposed to Mn2+ for 5 weeks by way of drinking water containing 50mM MnCl2. The brains of these mice were
harvested and cryo-sectioned using the standard procedures. Tissue sections were stained with 20mM Morin hydrate in 60% ethanol solution, embedded in a mixture of glycerol, ethanol and water (5:3:2), and imaged using two-photon microscopy. The
histogram indicates that Mn accumulation (pixels with highgreen signal) appeared most prominently in amygdala. The procedure we developed successfully delineated the spatial distribution of Mn and evaluated the accumulation of Mn with drinking
water exposure.

Immune responses modelling
We showed that regulatory T cells (Treg) cells enhance cell-interactions which stabilize the tolerant state with both theoreticaland experimental studies. Modeling showed that T cell interaction with APC is crucial for maintaining stable unresponsivenessin addition to inhibition of activation-signaling when ligands forstimulation are limited and there is competition among T cells
40(Fig. 1). To robustly inhibit T cell proliferation in the presence ofspecific Treg cells even where individual T cells behave stochastically, Treg cells were required to inhibit two processes; dissociation and activation. Supporting this theory, in vitro experiments
showed antigen-specific Treg cells actually enhance the expression of adhesion molecules and inhibit the expression of costimulatory ligands on APC. Notably, the model optimized to discriminate reactivity under self and non-self conditions well reproduced
the proliferation patterns of T cells under various conditions examined in in vitro experiments. Based on the predictions of themodel, we also found a way to manipulate immune response invivo. Transient reduction of T cell number enhanced the proliferation of antigen specific T cells in draining lymph nodes. Thus, thecombined studies of theoretical modeling and immunologicalexperiments revealed a novel ability of Treg cells to inhibit celldissociation, which gives insight into immune tolerance and into
manipulation of immune responses in various clinical settings.

Observation of single T cell activation
To answer how many T cells are activated by contact with APC,
we manipulated single T cell to interact with APC and activation
was monitored. T cell activation can be monitored using fluorescent Ca2+-reporter. By counting the number of activated T cells
attached to APC, we found that only 22% became activated whenantigen was presented by APC. On the other hand, 11% of T cells
showed activation even when antigen was absent, indicatingthat activation of T cells is not deterministic. To understand how
the immune system distinguishes between self and non-self de-Figure 2.
Counting T cell activation upon engagement with APC in the presence of regulatory T cell.
Rhod2 stained T cells (red) were held with a glass micro-needle and placed on APC andfluorescence was monitored. Regulatory T cell (blue) and conventional T cell (green) are alsopresent on APC.

Recent Publications■ Chiu LD, Ichimura T, Sekiya T, Machiyama H, Watanabe T, Fujita H, Ozawa T,■ Iwaki M, Wickham S, Ikezaki K, Yanagida T, Shih W. A programmable DNA

and Fujita K. Protein expression guided chemical profiling of living cellsby the simultaneous observation of Raman scattering and anti-Stokes
fluorescence emission. Sci. Rep. 7, 43569 (2017).

origami nanospring that reveals force-induced adjacent binding of myosin VI heads. Nature Comm. 7, 13715 (2016).
■ Fujita■ Furusawa C, Yamaguchi T. Robust and Accurate Discrimination of Self/

Non-Self Antigen Presentations by Regulatory T Cell Suppression. PLoS
One. 11(9), e0163134 (2016).

K, Iwaki M, Yanagida T. Transcriptional bursting is intrinsicallycaused by interplay between RNA polymerases on DNA. Nature Comm. 7,
13788 (2016).

■ Ichimura T, Chiu LD, Fujita K, Machiyama H, Yamaguchi T, Watanabe TM

and Fujita H. Non-label immune cell state prediction using Raman spectroscopy. Sci. Rep. 6, 37562 (2016).
41

Masaru Ishii, MD/PhD

Professor

Masaru Ishii

Associate Professor
Assistant Professor

Keizo Nishikawa
Junichi Kikuta
Hiroki Mizuno
Takao Sudo
Takahiro Matsui (SA)
Szandor Simmons (SA)Research Assistant
Visiting Scientist
Support Staff

The immune system is highly dynamic and a variety of cell
types are migrating throughout the body and mutually communicating at defined sites. The aim of our laboratory is to understand the fundamental principle controlling cellular dynamics invarious kinds of tissues and organs in vivo. By means of our advanced imaging techniques, we have investigated the dynamic
nature of different cell types in a time-dependent manner, in addition to the spatial and structural information. We are now elucidating the dynamic systems in bone biology and immunology as
well as biological science such as hematopoiesis and cell lineage
commitment and other ‘niches’, cell dynamics during inflammation in various kinds of tissues and organs (see the Figure).

Intravital bone imaging revealing osteoclast and osteoblast dynamics in vivo
Our lab has originally elaborated the novel imaging system forvisualizing inside the bones. We have first elucidated the in vivo
behaviors of bone-resorbing macrophage, osteoclast, i.e., the migration and positioning of their precursor macrophages, theirmode of bone-resorbing function in vivo and the functional andphysical coupling with bone-reforming osteoblasts. By utilizing
this methodology, we showed sphingosine-1-phosphate (S1P) asa key factor in controlling the migratory behavior of osteoclast
precursors in concert with various chemokines (Nature 2009; J
Exp Med 2010). We also showed the substantial contribution of
S1P-mediated migratory control of bone cells by S1P by generating knockout mice deficient for endogenous S1P transporter (J.
Clin. Invest. 2012). Moreover, we demonstrated that vitamin D
42

Yutaka Uchida (SA)2
13

significantly blocks bone destruction by modulating S1P-mediated migration control of osteoclast precursor monocytes (Proc.
Natl. Acad. Sci. USA 2013).
Through the improvement of bone imaging system, we succeeded in visualizing the function of mature osteoclasts in vivo (J.
Clin. Invest. 2013). This novel visualization identified two distinct
mature osteoclast functional states; i.e., bone-resorbing (R) osteoclasts firmly adhering to bones and devouring the bone matrix by secreting acids, and non-resorbing (N) osteoclasts relatively loosely attached and wriggling along the bone surface.
Th17 cells, a bone destruction-prone T cell subset, express RANKL
on their surface, although its functional role remains elusive. This
novel imaging system showed that RANKL-bearing Th17 could
stimulate osteoclastic bone destruction by contacting N state osteoclasts directly to convert them to the R state, a critical mechanism underlying bone erosion in arthritic joints. Furthermore, incollaboration with Dr. Kikuchi in the frame of IFReC, we have developed a new chemical probe for detecting proton secretion inbone resorption by osteoclasts (Nat. Chem. Biol. 2016). By using
this probe, we could grasp the real time course of osteoclastic
bone resorption which was finely regulated by cell-cell contact
with bone-forming osteoblasts and other bone cell types.

Immunometabolism of bone homeostasis
Osteoclasts dramatically alter their metabolic activity during
cell differentiation. This change in the metabolic status is termed‘metabolic reprogramming’, but its role in osteoclast is not fully
understood. Using metabolomics approach, we found that meta-

bolic reprogramming during osteoclast differentiation increased
intracellular S-adenosyle methionine (SAM), a metabolite of themethionine cycle. SAM is the universal methyl donor for methylation reactions, including histone and DNA methylation. Furthermore, we identified the de novo DNA methyltransferase 3a (Dnmt3a) as a transcription factor that couples these metabolicchanges to osteoclast differentiation. We found that SAM-mediated DNA methylation by Dnmt3a regulates osteoclastogenesis
via epigenetic repression of anti-osteoclastogenic genes. Dnmt3a-deficient osteoclast precursor cells do not differentiate efficiently into osteoclasts and that mice with an osteoclast-specificdeficiency in Dnmt3a have elevated bone mass due to a smaller
number of osteoclasts. Furthermore, inhibition of DNA methylation by theaflavin-3,3΄-digallate abrogated bone loss in models of
osteoporosis. Thus, we revealed the role of epigenetic processes
in the regulation of cellular metabolism and differentiation,

which may provide the molecular basis for a new therapeuticstrategy for a variety of bone disorders (Nat Med 2015).

Application of intravital imaging techniques for dissecting human immunology

Imaging

Immunology and Cell Biology

Intravital imaging with multi-photon microscopy is an undoubtedly powerful tool for dissecting living cellular dynamics inintact tissues and organs and thus useful for studying immune
system dynamics in vivo. However, the application is currently
limited in animal models and may not be applicable for analyzing
human samples. By collaborating with companies (supported by
AMED), we are developing a new microscopy system for application to human tissues and organs in vivo. To date, we have succeeded in visualizing non-labelled human normal and cancer tissues, and we will be able to dissect human immunology in the
future.

Figure. Intravital imaging for various immune systems.
Immune cells are high dynamic and interconnecting various tissues andorgans, by forming a ‘soft-wired’ network. We are elucidating the basic
principle controlling the dynamic nature of immune cells by visualizing invivo behaviors with advanced imaging techniques.

Recent Publications■ Kon S, et al. Cell competition with normal epithelial cells promotes apical■ Nishikawa K, et al. Dnmt3a regulates osteoclast differentiation by cou-

extrusion of transformed cells through metabolic changes. Nat. Cell Biol.
19(5), 530-541. doi: 10.1038/ncb3509 (2017).

pling to an S-adenosyl methionine-producing metabolic pathway. Nat.
Med. 21, 281-7 (2015).

■ Maeda H, et al. Real-time intravital imaging of pH variation associated■ Kikuta J, et al. Dynamic visualization of RANKL and Th17-mediated osteo-

with cell osteoclast activity and motility using designed small molecular
probe. Nature Chem. Biol. 12, 579-85 (2016).

clast function. J. Clin. Invest. 123, 866-873 (2013).
■ Kikuta J, et al. S1P-mediated osteoclast precursor monocyte migration is■ Sekimoto R, et al. Visualized macrophage dynamics and significance of

S100A8 in obese fat. Proc. Natl. Acad. Sci. USA 112, 2058-66 (2015).

a critical point of control in antibone-resorptive action of active vitamin
D. Proc. Natl. Acad. Sci. USA 110, 7009-13 (2013).
43

Imaging

Nuclear Medicine
Jun Hatazawa, MD/PhD

Astrocyte activation is representative pathology in central nervous system of multiple sclerosis (MS) patients. Acetate has been
revealed to be selectively uptaken and metabolized by astrocytes
in the CNS. We aimed to visualize and investigate activation ofastrocyte in the CNS and to clarify the activity MS in vivo bymeans of PET with C-11 acetate.
Patients with relapsing-remitting MS (6 females, 46 +/- 5.9 year
old (yo)) and healthy volunteers (HV) (6 females, 58 +/- 5.8 yo)
were enrolled in the present study. In all the subjects, MRI andPET scanning was performed. A static 20-min PET acquisition wasstarted 20 min after intra-venous injection of C-11 acetate. The
images were reconstructed using a filtered-back projection
method. Parametric images of standardized uptake value (SUV)
and the ratio of regional SUV to mean SUV in thalamus (SUVt)
were produced for analysis. Volume-of-interest (VOI) analysis forgray matter (GM) and/or white matter (WM) based on segmentation technique for co-registered MRI. Voxel-based statistical parametric analysis were performed by using Statistical Parametric
Mapping 8 (SPM8) to evaluate pathological change of regional
C-11 acetate uptake. Correlation between C-11 acetate uptake
and the lesion number in T1- and T2- weighted MR images werealso assessed.
As a result, standardized uptake value (SUV) of 11C acetate
relative to SUV in bilateral thalami (SUVt) was significantly in-

Professor

Jun Hatazawa

Associate Professor

Hiroki Kato

Assistant Professor

Tadashi Watabe

Research Assistant2

Support Staff

1

creased in both white and gray matter in MS compared with HV.
The ratio of SUV in white matter to SUV in gray matter was significantly higher in MS than HV. Voxel-based statistical analysis
elucidated significant increase in uptake in broad area of whitematter especially in subcortical white matter in MS patients. The
numbers of T2 lesions and T1 black holes recognized as a markerof tissue destruction were significantly correlated with C-11 acetate uptake in white and gray matter. It seems that the C-11 acetate uptake reflecting reactive astrocytes correlates with bothaxonal damage and cortical astrogliosis. These results imply thewidespread involvement of cortical astrocytes in MS pathology.
Inflammatory demyelination in WM is a cardinal feature in MS
pathology. In addition to the demyelinated lesions detected byordinary MRI, pathological changes exist even in normal appearing WM and GM. Astrocyte pathology precedes demyelination inan animal model; astrocyte hypertrophy occurs at the leadingedge of acute MS lesions, followed later by astrocytic scarring.
Thus, the altered astrocyte activation is presumably involved in
MS pathophysiology.
Our present study suggests that pathologic white matterchanges in patients with MS can be detected with non-invasive
static C-11 acetate PET, which may be an effective MS diagnostic
tool.

Figure 1.
A representative case of MS. (SUV: standardized uptake value,)

Figure 2.
Standardize uptake value of C-11 acetate in the MS patients and the healthy volunteers. (SUVt:
ratio of regional SUV to mean thalamic SUV.)

Figure 3.
Voxel-based statistical comparison C-11 acetate uptake between MS patients and healthy
volunteers. Colored voxels indicate T-scores representing significantly increased C-11 acetate
uptake (SUVt) in patients with MS compared to HV patients.

Recent Publications■ Watabe T, et al. Quantitative evaluation of oxygen metabolism in the in-■ Watanabe S, Kato H, Shimosegawa E & Hatazawa J. Genetic and Environ-

tratumoral hypoxia: 18F-fluoromisonidazole and 15O-labelled gases inhalation PET. EJNMMI Res 7, 16. doi:10.1186/s13550-017-0263-6 (2017).

mental Influences on Regional Brain Uptake of 18F-FDG: A PET Study on
Monozygotic and Dizygotic Twins. J. Nucl. Med. 57, 392-397. doi:10.2967/
jnumed.115.164004 (2016).

■ Kato H, Shimosegawa E, Fujino K & Hatazawa J. CT-Based Attenuation Cor-

rection in Brain SPECT/CT Can Improve the Lesion Detectability of VoxelBased Statistical Analyses. PloS one 11, e0159505, doi:10.1371/journal.
pone.0159505 (2016).
44

45

Imaging

Chemical Imaging Techniques
Kazuya Kikuchi, PhD

Multicolor imaging techniques are useful for investigating the
localization and movement of proteins in living cells. By combining these techniques with pulse-chase imaging of proteins, precise spatio-temporal information on proteins in living cells is obtained. In this type of imaging analyses, protein labeling using aprotein tag and its specific probes is an attractive approach, because target proteins are conditionally labeled by the temporal
addition of probes with different fluorophores that allow themulticolor pulse-chase imaging. Previously, we developed a protein labeling/imaging system based on photoactive yellow protein (PYP) tag and its specific probe, FCANB. PYP-tag is a novel
protein tag, which is a small protein (14 kDa) derived from purple
bacteria. The noticeable feature of this labeling method is thatthe fluorescence of FCANB is quenched and is increased upon thelabeling reaction with PYP-tag. FCANB consists of a 4-hydroxycinnamic acid ligand, a fluorophore, a linker, and a nitrobenzene(NB) quencher. This modular scaffold is attractive, since various
fluorophores emitting fluorescence at different wavelengths canbe introduced into the probe. In addition, the NB quencher decreases fluorescence intensity of the fluorophore based on a contact quenching mechanism. This fluorogenic property allows theimaging of cell-surface proteins with low background signals,
even when free probes are not washed out from cells. However,the current probes available for the no-wash imaging were created using fluorophores emitting short-wavelength fluorescenceor have limitation in the availability of fluorophore, and thus reduce the benefit in multicolor imaging analyses.
In this research, to achieve multicolor protein imaging with46

Professor

Kazuya Kikuchi

Associate Professor

Yuichiro Hori

Assistant Professor

Masafumi Minoshima

Postdoctoral Fellow

1

Research Assistant

2

Visiting Scientist

2

Support Staff

1high contrast, we synthesized new PYP-tag probes with two different long-wavelength fluorophores, which increase fluorescence intensity upon the labeling reactions. As the long-wavelength fluorophores, we chose sulfonated cyanine dyes, Cy3 andCy5, which emit fluorescence with the maximum wavelengths of
570 nm and 670 nm, respectively. These fluorophores are brightand have been applied to a number of biological studies. As afluorescence quencher, 3,5-(dinitrobenzyl)acetamide (DNB) was
employed as a quencher that reduces fluorescence intensity
based on contact quenching. We introduced DNB into the scaffold of the probe to develop Cy3DNB2 and Cy5DNB2 (Fig 1). SDSA-PAGE analyses revealed that both the probes bind to PYP-tag
covalently. As we expected, fluorescence intensity of the probes
enhanced upon labeling reactions of PYP-tag (Fig 2). The probes
labeled PYP-tag more rapidly than the previously reported probe,
FCANB. It turned out that DNB increased the labeling reactions aswell as fluorescence intensity. Both the probes specifically imaged EGFR in living cells (Fig 3). Particularly, Cy5DNB2 showed
the good fluorogenic and kinetic properties and allowed nowash imaging of the cell-surface proteins with high contrast. The
strategy based on DNB quenching would offer a promising approach for design of fluorogenic probes with long-wavelength
fluorophores. These probes thus will be attractive tools for in vivo
or in situ imaging of various proteins.

Figure 1.
Principle of fluorogenic PYP-tag labeling system using Cy5DNB2.

Figure 2.
Fluorescence enhancement of Cy5DNB2 upon labelingreactions with PYP-tag. Red and blue lines represent
fluorescence spectra of Cy5DNB2 in the presence andabsence of PYP-tag, respectively.

Figure 3.
Live-cell imaging of PYP-EGFR using Cy5DNB2.

Recent Publications■ Hirayama S, Hori Y, Benedek Z, Suzuki T, Kikuchi K. Fluorogenic Probes Re-■ Kamikawa Y, Hori Y, Yamashita K, Jin L, Hirayama S, Standley D.M, Kikuchi K.

veal a Role of GLUT4 N-glycosylation in Intracellular Trafficking, Nat.
Chem. Biol. 12, 853-859 (2016).

Design of a Protein tag and Fluorogenic Probe with Modular Structure forLive-Cell Imaging of Intracellular Proteins, Chem. Sci., 7, 308-314 (2016).

■ Maeda H, Kowada T, Kikuta J, Furuya M, Shirazaki M, Mizukami S, Ishii M,

Kikuchi K. Real-time Intravital Imaging of pH Variation Associated with
Osteoclast Activity. Nat. Chem. Biol. 12, 579-585 (2016).

47

Nicholas Isaac Smith, PhD

Associate Professor

Nicholas Isaac Smith

Assistant Professor

Alison Hobro
Nicolas Pavillon

Support Staff

The Biophotonics lab research theme is to develop opticaltechniques and apply them to study biological and immunological processes. Most of our research involves using optical measurements, without fluorescent labels, to provide images, spectral data, or both. Several different physical interactions of light
can be exploited to measure samples and extract cellular information. We rely heavily on Raman scattering based measurements, using a high intensity laser beam, and measuring the shift
in the wavelength of the scattered light. With different molecules
expressed at different locations in the cell, we can produce an image where the image contrast is defined by the spatial and chemical distributions of endogenous molecules in a living cell or tissue sample. We also investigate how other light-based methods
can be used in a complementary manner to the Raman-based
measurements. With several years of development already underway, we are now moving more towards applying the toolsthat we have developed to study processes in immune cells, suchas analysis of cell phenotype, and to discriminate cell activationlevels, usually in lymphocytes and macrophages.
For T and B cells, the usual features for discrimination of immunological function are the presence or absence of surface
markers. We recently completed a study using Raman scattering
to discriminate between different lymphocyte cell lines (Hobro etal., 2016). Of note is the finding that the Raman signature of eachcell type was distinct enough that it could be used to predict thecell type in a mixed group of cells. The Raman analysis is based ona measurement of the endogenous molecules, but depending on
the nature of the measurements, most crucially the laser focus
48

1

and optical sectioning, it should not detect contributions fromsurface markers. Therefore the Raman signature should instead
be more related to the genome or transcriptome-based components of the cell. This gives a complementary approach to themore usual wet-lab methods of cell phenotyping. While the implications of this approach go beyond a simple discrimination
method, using it as a method of discriminating T vs B cells and/or
individual cell types from one another, resulted in high sensitivity
and specificity (Hobro et al., 2016). Figure 1 shows a PLS-DA separation model of individual cell lines.
We also used Raman analysis in a non-imaging mode, where
we can measure purified samples of interest (e.g. mRNA andsmall molecule interactions). The interactions between small
molecules such as mRNA, and proteins can be measured by Raman with some advantages over other methods. We built a portable Raman detection system which can be used for analysis of
cuvette-based samples, or other samples where imaging is not
required. This includes detection of pathogens in blood or serum
samples (e.g. Hobro et al., 2013), evaluation of tissue, or analysis
of drugs in solution.
For the ultimate sensitivity (approaching single-molecule level) in the cell, however, Raman scattering alone can not generatesufficient signal, but by using plasmonic Raman enhancement,
the scattering signal is amplified at a metallic surface and/or bychemical bonds that form between target molecules and the surface. The enhancement factor can then be in the order of 1 million times. These particular experiments require the introductionof a metal surface in the cell, and are not strictly label-free but thetradeoff is often worthwhile, depending on the target. Conceptually similar to the more well-known SPR sensors, our approach
uses only the interface enhancement to detect endogenous molecules without any conjugated targeting. One difficulty for theseenhanced Raman experiments is the need to introduce the metal
surface inside the cell, usually an inert metal nanoparticle. Thiscan be difficult since some cells do not readily uptake nanoparticles, and even for those that do, the particles do not typically pass
the nuclear membrane, restricting the measurements to cytosolic
regions. A novel method to solve this is to introduce gold salt solution, which can pass through the entire cell, and to form thenanoparticles in-situ by photoreduction (Smith et al., 2014),
thereby creating the Raman enhancement directly inside the cell.
Due to the challenges of Raman imaging, we considered how
best to optimize the measurements, and completed our first project on compressed sensing, using sub-sampling and optimized
reconstruction models to reduce the amount of data points
needed to form an image (Pavillon et al., 2016). One of the main

problems in Raman microscopy is the signal-to-noise ratio. Higher laser power is usually not possible due to the desire to avoid
sample damage. Therefore, relatively long imaging times are required. However, due to the inherent blur in the microscope imaging system, there is some amount of redundancy between
measurements in typical imaging conditions. By using a reduced
set of measurements, in combination with a model of the imaging properties, we can significantly speed up the Raman imaging
process. As shown in figure 2, using up to 5 times less sampling,
the image can still be reconstructed with minimal information
loss. Although our lab is primarily focused on label-free imaging,
we showed that the compressed sensing approach can be applied to laser scanning microscopy in general, such as confocal or
multiphoton fluorescence imaging. This can then lead to a significant improvement in imaging speed and/or reduction in photobleaching which is very useful for collaborations with othergroups that use fluorescent probes.

Figure 1.
B- and T-cell line discrimination by label-free Raman measurement. Eachsample represents one Raman spectra from an individual cell, where eachcell line is denoted by different symbols described in the figure key. The
separation model is built from part of the data, with a portion of the data
set aside and used to validate the model. The red dashed line represents
a dividing line between the B- and T-cell spectra. Figure from Hobro et al.
2016.

Figure 2.
Raman measurements on MEF cells, showing how compressed sensing
can significantly improve Raman imaging speed by reducing the required
measurements. Image (a) shows the point spread function of the Raman
microscope, measured with 100 nm fluorescent beads. The Raman images
are then measured at full resolution (b), and then reconstructed using
only 1/3 (c1) or 1/5 (c2) of the original data, showing that up to five times
reduction in sampling still produces usable images when combined withthe compressed sensing method. The color contrast is generated from a
C-H stretching band (2925 cm−1, green) and lipids (2853 cm−1, red). Figure
from Pavillon et al 2016.

Imaging

Biophotonics

Recent Publications■ Pavillon N and Smith NI. Compressed sensing laser scanning microscopy.

Opt. Express 24(26), 30038-300052 (2016).

■ Smith NI, Mochizuki K, Niioka H, Ichikawa S, Pavillon N, Hobro AJ, Ando J,

Fujita K and Kumagai Y. Laser-targeted photofabrication of gold nanoparticles inside cells. Nat. Commun. 5(5144), 1-9 (2014).

■ Hobro AJ, Kumagai Y, Akira S and Smith NI. Raman spectroscopy as a tool

for label-free lymphocyte cell line discrimination. Analyst 141, 3756-3764(2016).

■ Pavillon N, Hobro AJ and Smith NI. Cell Optical Density and MolecularComposition Revealed by Simultaneous Multimodal Label-Free Imaging.
Biophys. J. 105(5), 1123-1132 (2013).

■ Hobro AJ, Pavillon N, Fujita K, Ozkan M, Coban C and Smith NI. Label-free

Raman imaging of the macrophage response to the malaria pigment hemozoin. Analyst 140, 2350-2359 (2015).
49

Kazuhiro Suzuki, MD/PhD

Professor

Kazuhiro Suzuki

Assistant Professor

Akiko Nakai

Research Assistant2

Support Staff

2

Future perspectives
Our findings have added a novel layer of immune regulationby the nervous system. However, a few questions remain unsolved. First, the molecular mechanism of the crosstalk between
β2ARs and chemokine receptors is unclear. Second, it remains to
be visualized how adrenergic nerves interact with lymphocytes
and influence their behaviors in LNs. In the short term, we are going to address these questions to reveal the whole picture of ad-

renergic control of lymphocyte trafficking. The activity of thesympathetic nervous system and thereby adrenergic nerves isinfluenced by psychological conditions. In the longer term, weplan to clarify cellular and molecular mechanisms by which brain
functions are reflected in immune responses through adrenergic
nerves. Currently, the neuro-immune interactions attract particular attention in biomedical science. We are going to carve a nichefor ourselves in this rapidly growing field.

Imaging

Immune Response Dynamics

Figure 1. Control of lymphocyte egress from LNs through β2ARs.
Inputs from adrenergic nerves stimulate β2ARs expressed on lymphocytes. Activation oflymphocyte β2ARs enhances signals through CCR7 and CXCR4, chemokine receptors that
promote lymphocyte retention in LNs, and inhibits lymphocyte egress from LNs.

Control of lymphocyte trafficking through β2-adrenergic receptors
It has long been proposed that various aspects of immune responses are influenced by nervous system activity. However, thecellular and molecular basis for neural regulation of immunity islargely unclear. Adrenergic nerves constitute the efferent arc of
the sympathetic nervous system and produce noradrenaline thatinduces cellular responses through α1, α2, β1, β2 and β3 adrenergic
receptors (ARs). Like other vital organs, lymphoid organs receivea rich supply of adrenergic nerves. However, it was unclear how
the inputs from adrenergic nerves affect lymphocyte migration
among lymphoid organs. Therefore, we have been studying therole of adrenergic nerves in the control of lymphocyte trafficking
and adaptive immune responses.
After spending several hours in a lymph node (LN), lymphocytes exit from the LN into lymph, return to blood flow, and travel
to other lymphoid organs to continue antigen surveillance. We
recently reported that inputs from adrenergic nerves control lymphocyte egress from LNs through β2ARs (Nakai et al., 2014). Activation of lymphocyte β2ARs enhances the responsiveness of
CCR7 and CXCR4, chemokine receptors that promote LN retention of lymphocytes, and consequently inhibits their LN egress(Fig. 1). However, the physiological role of this mechanism in
adaptive immune responses was unclear.
Physiological significance of adrenergic control of lymphocyte trafficking
The activity of adrenergic nerves displays a circadian rhythm
50

that is synchronized with the rest-activity cycle of the species. The
noradrenaline release from adrenergic nerves increases during
the daytime in humans, whereas it reaches a peak at night in rodents. Indeed, the noradrenaline content in LNs was elevated toward the night time in mice. The night time surge of adrenergic
nerve activity in LNs was accompanied by an increase of lymphocyte numbers in LNs and their concomitant decrease in lymph
and blood. Consistent with these observations, we found thatlymphocyte egress from LNs was restricted during the night timein mice. The diurnal variation of LN egress was dependent onneural inputs to lymphocyte β2ARs (Suzuki et al., 2016).
We hypothesized that the accumulation of lymphocytes in LNs
during the period of high adrenergic nerve activity may increase
the chance of antigen encounter and potentiate adaptive immune responses. We found in mice that immunization in thenight time, when lymphocyte numbers in LNs were high, induced
more robust humoral immune responses than immunization in
the daytime. The diurnal variation of humoral immune responses
was dependent on β2AR-mediated neural signals and was diminished when lymphocyte recirculation through LNs was stopped.
These findings suggest that the β2AR-mediated control of lymphocyte trafficking contributes to the daily fluctuation of adaptive immune responses (Suzuki et al., 2016). The time-dependent
differences in the magnitude of adaptive immune responses may
have evolved to maximize the efficiency of host defense when
encounters with pathogens are more likely to occur (Fig. 2).

Figure 2. Diurnal control of adaptive immunity by adrenergic nerves.
During the period of high adrenergic nerve activity, lymphocyte egress from LNs is restricted,
which leads to an increase of lymphocyte numbers in LNs. Immunization during the period oflymphocyte accumulation in LNs promotes adaptive immune responses. This diurnal variationof lymphocyte trafficking may have evolved to maximize the efficiency of host defense against
pathogens during the active phase.

Recent Publications■ Suzuki K and Nakai A. Control of lymphocyte trafficking and adaptive im-munity by adrenergic nerves. Clin. Exp. Neuroimmunol. 8, 15-22 (2017).

■ Suzuki K and Nakai A. Autonomic control of inflammation. Clin. Exp. Neu-roimmunol. 7, 10-17 (2016).

■ Suzuki K, Hayano Y, Nakai A, Furuta F and Noda M. Adrenergic control of■ Nakai A, Hayano Y, Furuta F, Noda M and Suzuki K. Control of lymphocyte

the adaptive immune response by diurnal lymphocyte recirculation
through lymph nodes. J. Exp. Med. 213, 2567-2574 (2016).

egress from lymph nodes through β2-adrenergic receptors. J. Exp. Med.
211, 2583-2598 (2014).
51

Ben Seymour, MD/PhD

tigue (indexed by the animals burrowing behavior) induced byinflammatory signals.
In other work, Aya Nakae has been developing a novel rodent
model of post-operative pain, and has begun to characterize the

Ben Seymour

Associate Professor

Aya Nakae

Assistant Professor

Masaki Maruyama

Postdoctoral Fellow

1

My lab studies interactions between the immune system andthe brain, and combines immunology, neuroscience and information science. The research focuses on two main questions

gion that seems critical for sensing inflammation and using thisinformation to guide protective responses orientated to minimise further injury. This is the first demontsration that the brain
can specifically plan behaviour based on inflammation or injury.

1. How does the brain respond to peripheral neurogenic inflammation?
2. Can we isolate brain-based biomarkers for fatigue and pain
in inflammatory arthritis (humans and rodent models)?

Biomarkers for fatigue and pain in inflammatory arthritis.
Patients with inflammatory arthritis report that fatigue andpain are the most severe symptoms they experience as part oftheir disease. Critically, these symptoms often respond poorly to
immunologic therapies such as anti TNF, despite the fact the inflammatory markers often respond well. This highlights how little
we understand about the nature of fatigue in inflammatory disease, and how it relates to immune activation. Part of the problem is that we have no objective biomarkers for these symptoms,
which is a major problem not just for human and clinical studies,but also for translation with animal studies.
Using bioinformatic approaches, we have developed tools thatcan predict both pain and depression with reasonable accuracyby ‘decoding’ patterns of brain activity for patients with osteoarthritis, and we are now applying these to patients with rheumatoid arthritis. Furthermore, our cohort involves patients about tostart anti-TNF therapy, and longitudinally following up these patients, we aim to prospectively predict their treatment response
in terms of symtpoms of fatigue and pain. In parallel, we havebeen studying brain activity following joint inflammation (CFA
model) in rodents (in collaboration with Shionogi). This has identified a brain region (the anterior cingulate cortex) that is active
during joint inflammation, and is associated with the level of fa-

Brain responses during neurogenic inflammation.
The nervous system plays a critical role in the local tissue inflammatory response, by sensing bacterial antigens (immunoception), modulating the immune response (locally and systemically), and regulating autoimmunity (e.g. as in psoriasis). One way
to probe the role of the nervous system in tissue inflammation is
to study neurogenic inflammation, which can be done in humans
by topical application the compound capsaicin. This sensitizes
peripheral and spinal cord circuits that transmit information
about injury to the brain, and modulate the local tissue inflammatory response through descending brain-to-spinal cord pathways.
We have been studying capsaicin inflammation using (Magnetoencephalography (MEG) and functional Magnetic ResonanceImaging (fMRI)) in humans. By combining the two imaging methodologies, we have been able to chart the dynamic signature of
the brain response that uses information about peripheral inflammation to control behaviour. We have identified a brain re52

Professor

Figure 1.
Overview of our study program identifying the brain response toneurogenic inflammation.

immunological, endocrinological and neurophysiological basisof the persistent pain state that results. A vision for this researchis to understand how immunological factors may contribute thechronic pain state that results from numerous common surgicalprocedures.

Imaging

Brain-Immune Interaction

Figure 2.
Sequential images through the rat brain showing functional connectivityof the anterior cingulate cortex associated with fatigue in the CFA
inflammatory arthritis model.

Recent Publications■ Seymour B, Barbe M, Dayan P, Shiner T, Dolan R, Fink GR. Deep brain stim-■ Lancaster J, Mano H, Callan D, Kawato M, Seymour B. Decoding Acute Pain

ulation of the subthalamic nucleus modulates sensitivity to decision outcome value in Parkinson's disease. Sci. Rep. 6, 32509. doi: 10.1038/
srep32509 (2016).

with Combined EEG and Physiological Data. IEEE EMBS Neural Engineering 2017.
■ Yasuda Y, Hashimoto R, Nakae A, Kang H, Ohi K, Yamamori H, Fujimoto M,■ Lawson R, Nord C, Seymour B, Thomas D, Dayan P, Pilling S, Roiser J. Dis-rupted habenula function in major depression. Mol. Psychiatry 22(2), 202208. doi: 10.1038/mp.2016.81 (2016).

Hagihira S, Takeda M. Sensory cognitive abnormalities of pain in autism
spectrum disorder: a case-control study. Ann. Gen. Psychiatry 15, 8. doi:
10.1186/s12991-016-0095-1 (2016).

■ Yanagisawa T, Fukuma R, Seymour B, Hosomi K, Kishima H, Shimizu T,■ Granovsky Y, Anand P, Nakae A, Nascimento O, Smith B, Sprecher E,

Yokoi H, Hirata M, Yoshimine T, Kamitani Y, Saitoh Y. Induced sensorimotor
brain plasticity controls pain in phantom limb patients. Nat. Commun.7,
13209. doi:10.1038/ncomms13209 (2016).

Valls-Solé, J. Normative data for Aδ contact heat evoked potentials in
adult population: a multicenter study. Pain. 157(5), 1156-63 (2016).
■ Nakanishi M, Nakae A, Kishida Y, Baba K, Sakashita N, Shibata M, Yoshikawa H,■ Koizumi

A, Amano K, Cortese A, Shibata K, Yoshida W, Seymour B,
Kawato M, Lau H. Fear reduction without fear through reinforcement ofneural activity that bypasses conscious exposure. Nat. Hum. Behav. 1,0006 (2016).

Hagihara K.Go-sha-jinki-Gan (GJG) ameliorates allodynia in chronic constriction injury-model mice via suppression of TNF-α expression in the
spinal cord. Mol. Pain 12, 1-16 (2016).

53

Informatics

Information Systems
Yutaka Hata, PhD

Professor

Yutaka Hata

Associate Professor

Syoji Kobashi
Shugo Yasuda
Shinichiro Shima

Assistant Professor

Manabu Nii

Support Staff

1
Figure 1.
3-D tracking result in the time-lapse MR images. (Dots are brain surfaceof the mouse. Lines are trajectories of macrophages. Different colors aredifferent macrophages).

Three dimensional analysis of macrophage learning-tracking
algorithm
It is effective to analyze 3-D dynamics of macrophages. Last
year, we proposed an automated method based on Hungarian
method in 11.7 time-lapse 2-D MR images. In this year, we extend
the method to 3-D processing and evaluated the performance
numerically. The method is based on background subtraction
and the Hungarian algorithm. It was validated using mouse brain
MR images and artificially synthesized images. The results withartificially synthesized images showed that the method tracked
the macrophages well. The method successfully tracks the macrophages in the real mouse brain and visualizes the 3-D trajectory. A limitation of the proposed method is that it might suspend
due to the noise influence.
A Monte Carlo simulation method of chemotactic motile cells
A Monte Carlo simulation method for chemotactic motile cells
is developed based on a kinetic chemotaxis equation for motile
cells coupled with reaction-diffusion equations for chemical cues.
The Monte Carlo method is applied to the traveling pulse andFigure 2.
The snapshot of Monte Carlo simulation for the traveling pulse of
chemotactic bacteria in micro channel. The particles represent eachchemotactic bacterium and their colors represent the moving direction.
The inset shows the snapshot of the population density of bacteria andconcentration of chemical cue along the channel.

pattern formation problems. It is demonstrated that the Monte
Carlo method can reproduce the experimental results of the traveling pulse of chemotactic bacteria. Comparison of the Monte
Carlo simulations and theoretical analysis on the pattern formation problem of chemotactic bacteria proves a solid mathematical foundation to the Monte Carlo method.
Region Extraction Algorithm
Chronic thromboembolic pulmonary hypertension (CTEPH) is
one of the lung diseases caused by thrombi occurring in pulmonary arteries. By measuring the size of a region dominated by anarterial subtree which has thrombi, physicians find a higher treatment effect point. This research proposes an automated method
to extract the lung region dominated by an arterial subtree from
MDCT images as the basis of a region extraction algorithm. The
method extracts an arterial subtree associated with a seed point
and a region dominated by the extracted arterial subtree and visualizes them. The results show a clinical ability of visualizationand extraction of dominant region from MDCT Images.

Figure 3.
Extraction result of dominant region (red region denotes pointed pulmonary artery and purple region is theextracted region corresponding to the pulmonary artery).

Recent Publications■ Perthame B. and Yasuda S. Self-organized pattern formation of run-and-54

tumble chemotactic bacteria: Instability analysis of a kinetic chemotaxis
model. arXiv:1703.08386 (preprint).

■ Yukawa A,

Kono A, Nishii T, Kamiura N, Kobash S, & Hata Y. Pulmonary artery domain region extraction from MDCT image, Int. J. Appl. Electromagnetics and Mechanics, 52, 479-486 (2016).

■ Yasuda S. Monte Carlo simulation for kinetic chemotaxis model: an appli-■ Tashita A, Kobash, S, Nii M, Mori Y, Yoshioka Y, & Hata Y. Macrophage learn-cation to the traveling population wave. J. Comput. Phys. 330, 1022-1042(2017).

ing-tracking algorithm in time-lapse MR images, Int. Conf. on Machine
Learning and Cybernetics (2016).

55

Daron M Standley, PhD

In 2016, the systems immunology lab began to shift from general bioinformatics to several specific topics in computational immunology. Although we still develop general bioinformatics
methods, and collaborate with a wide range of experimentalgroups in other areas, our priority is to deliver state-of-the art
software targeting three main areas: (1) structural modeling ofprotein-RNA complexes involved in post-translational regulationof immune responses; (2) Functional analysis of large-scale immune receptor sequence data; and (3) multiple sequence alignment methods, both as publicly available software (MAFFT) andas curated databases for sequence and structures of interest (e.g.,
RBPs, BCRs, TCRs, pathogens, etc.).
RNA-binding proteins (RBPs) are essential for proper immune
responses, as they effect the strength and duration of activation(e.g., inflammatory responses). As an example, Arid5a, Regnase-1
and Roquin each target interleukin-6 (IL6) through a conserved
structural motif; however, each of these proteins shapes the IL6mediated response in different ways. There are likely to be otherimportant RBPs and RNA targets affecting a wide range of biochemical pathways. However, the lack of structural information
makes it difficult to generalize the modes of action of known
RBPs to other RBPs with similar domains. For these reasons, we
are working on the prediction of protein-nucleotide complexes
starting from sequence information. This approach is challenging
due to the flexibility of single-stranded nucleotides as well as thenoisiness of some RNA binding sites. Our initial goal to addressthese issues was to integrate information from multiple sequence
56

Professor

Daron M Standley

Associate Professor

Kazutaka Katoh

Assistant Professor

Songling Li

Postdoctoral Fellow

1

Research Assistant2

Support Staff

1

challenge, however, as there are few well annotated BCR or TCR
sequences with which to compare the data. Given the fact thatthe human immune system can theoretically produce hundredsof billions of unique BCRs and hundreds of millions of unique
TCRs, comparing receptors conventially at the sequence level between donors or between a donor and a reference set of annotated sequences, is unlikely to yield many functional hits. Moreover, even if a significantly similar sequence is detected, little is
known about how small sequence variations affect the sensitivity, specificity or affinity to known antigens. To address these issues, we have developed a structural modeling approach that is
efficient enough to handle large-scale sequence data sets and yet
robust enough to identify hits across multiple donors (Shirai, Yamashita). In 2016, we succeeded in speeding up the structuralmodeling step to within five seconds per structure by utilizing
several new features in MAFFT and deriving a new knowledgebased scoring function. Preliminary tests indicate that pairs ofalignments of known protein-RNA complexes in order to predict
nucleotide binding propensities for new RBPs. This strategy has
been largely successful and our resulting tool, aaRNA (Li et al),and its companion, aaDNA, have been validated in third-party
benchmarks as well as in small-scale collaborative studies withexperimental groups (Masuda et al., Mino et al, Yokogawa et al).
The next goal was to utilize predicted binding propensities todrive docking simulations of fully flexible single-stranded nucleotides. This strategy has also paid off, as evidenced by recent collaborative projects (Nyati, et al., Fukuda et al., Sakakibara, et al.)Our flexible docking simulations utilize a coarse-grained representation of the nucleotide chain and thus lack the resolution required to predict base specificity of a given RBP. To address thislimitation, we are now developing an extension that can generate all-atom representations of the coarse-grained docked models (manuscript in preparation). With these tools in place we aim
to identify new RBP-RNA interactions that are likely to play a role
in regulation of immune reponses.
B and T cell repertoires are shaped by various factors, both
intrinsic and extrinsic to the host, including genetics, age and antigen exposure. B and T cell repertoires thus contain an imprint of
past and present antigens as well immune responses to theseantigens. With the emergence of novel high-throughput B cell
receptor (BCR) and T cell receptor (TCR) sequencing along withproteomics-based technologies, it is now possible to collect BCR
and TCR sequences from individual donors on a large scale. Functional interpretation of the resulting Big Sequence Data is a major

BCR or TCR models within the same structural cluster are far more
likely than random pairs to target the same antigen (manuscript
in preparation).

Informatics

Systems Immunology

Multiple sequence alignment (MSA) is an important step incomparative analyses of biological sequences. MAFFT is one of
the most popular programs for building MSAs. Since the first release of MAFFT in 2002, we have continuously improved its accuracy, speed and utility in practical situations, providing different options for newly emerging types of data and analyses.
Recent features include: Inclusion of secondary structural information of non-coding RNAs and proteins, interactive selection ofsequences for phylogenetic tree inference, etc. We are working
on an extension to make more accurate options applicable to alarger number of sequences, in responding to the demands oflarge-scale analyses.

Figure.
A. Putative binding mode of mouse AUF-1 protein with the homology model of AUF-1 (Nyati et al).
B, Flexible docking of ssDNA to SLE antibody (Sakakibara et al).

Recent Publications■ Nyati K, et al. TLR4-induced NF-κB and MAPK signaling regulate the IL-6

■ Yamada K.D, Tomii K and Katoh K. Application of the MAFFT sequencemRNA stabilizing protein Arid5a. Nucleic Acids Res. 45, 2687-2703 (2017).

alignment program to large data—reexamination of the usefulness of
chained guide trees. Bioinformatics 32, 3246-3251 (2016).

■ Fukuda H, et al. Structural insight of APOBEC3G N-terminal domain re-veals critical residues for its RNA association. Submitted (2016).
■ Sakakibara S, et al. Clonal evolution and antigen-recognition of anti-nu-■ Katoh K and Standley D.M. A simple method to control over-alignment in

the MAFFT multiple sequence alignment program. Bioinformatics 32,
1933-1942 (2016).

clear antibodies in acute systemic lupus erythematosus. Submitted(2016).
57

Associate Professor

Diego Diez

Assistant Professor

Yutaro Kumagai

Postdoctoral Fellow

1

Research Assistant1

Support Staff

2tion and disease progression are irreversible even after removing
exposure to the harmful components (tobacco for COPD and silica for silicosis). Combining ATAC-seq and RNA-seq measurements of transcriptional activity in a mouse model of silicosis, westudy the regulatory pathways associated with irreversible inflammation.
We are integrating a novel de novo RNA motif search algorithm with RNA degradation time course in mouse dendritic cells
after LPS stimulation. Transcriptomic data is used to determine
degradation kinetics by clustering the time course data into patterns with similar degradation kinetics. Then, de novo sequenceand structure motif identification is applied to the 3’ UTR sequences of each cluster. We identified known structural motifs,
including the stem-loop structure containing motifs bound by
the RNA-binding proteins Roquin and Regnase-1 (Kumagai et al.
2016). We also identified novel motifs for which their role in controlling RNA degradation during the immune response is being
validated.
Mathematical modeling
The accurate quantification of immunological responses andthe integration of massive data open the door to approach the

The Quantitative Immunology Research Unit is a team of researchers with expertise in different scientific fields including immunology, bioinformatics and theoretical physics. Our aim is tounderstand how the immune system works by combining threedifferent but closely interconnected approaches; (1) quantitative
measurement of molecular dynamics, (2) integration of “bigdata” from multiple sources into network models, and (3) development of mathematical frameworks to understand the immune
system’s dynamics through the analysis of these massive datasets. These approaches are combined in several projects that aim
to get insight into specific questions related to the immune system. Some of these projects are described below to highlightspecific topics.
Quantitative approaches
Accurate quantification of biological responses is critical forunderstanding the dynamics of complex systems. Previously, we
have developed a fluorescent protein reporter system for thequantitative monitoring of IFN-a6 (Kumagai et al. 2007). Now weare trying to increase the “dimension” of the observation in two
ways: time and perturbation. Time lapse imaging of type I interferon expression under microscope is combined with multiple
fluorescent protein knock-in cells to monitor genes induced
upon antiviral responses. We are also developing automatedcomputational algorithms to extract important quantities to understand interferon regulation from such time lapse imaging
data.
Receptor molecule dynamics such as dimerization and cluster58

ing with downstream molecules is important for immune systemsignaling. However, because of limitations in biochemical assay
resolution, the details of this process are still poorly understoodfor most immune signaling pathways. To address this problem,
we are applying, in collaboration with RIKEN QBiC and other laboratories in IFReC, Total Internal Reflection Fluorescent Microscopy(TIRFM) to monitor the dynamics of single immune molecules.
We are using this technique to study TLR signaling, and successfully monitored TLRs and their adaptors at the single molecularlevel. We developed a novel algorithm to quantify the diffusion
dynamics of single molecules without bias, even under high molecular density conditions. This highly quantitative technique canbe used to describe the dynamics of the immune system’s signaling pathways.
Data integration
The accumulation of high-throughput (“omics”) datasets hasbrought biology into the big data era and the need for approaches that integrate, summarize and extract relevant informationthat reveals the relation between biological components.
We are developing methods that integrate measurements oftranscription factor binding with transcriptome data from different experimental conditions, and with protein-protein interaction data from public databases, to obtain insight into signaling
and gene regulatory immune networks. We apply these methods
to study the mechanisms behind respiratory diseases, such as
chronic obstructive pulmonary disease (COPD) and silicosis (Diezet al. 2015). A common feature of these diseases is that inflamma-

immunology research from the theoretical perspective. We aredeveloping novel mathematical frameworks for the quantitativedescription of the immune system.
A common obstacle for constructing dynamical models of cellular signaling is the biochemical determination of many parameters. To circumvent this problem, we have developed a mathematical framework called Stochastic Binary Modeling (SBM),
which also allows us to represent the stochastic and heterogeneous nature of cell populations (Teraguchi et al. 2011). We havedeveloped a system to automatically identify the structure andparameters of the network of regulatory pathways from multidimensional data. Now we are applying this system to infer the
network dynamics of immune signaling.

Units for Combined Research Fields

Quantitative Immunology

In another attempt to utilize the power of mathematics in immunological research, we are developing a hierarchical model ofthe immune system. In this framework, we derive the macroscopic dynamics of the immune system from the stochastic and heterogeneous behavior of the individual cells involved in antigen
presentation. This framework allows us to address one of themain questions in immunology, “how the balance between protective immunity and immune tolerance is achieved?”.

Figure.
The Quantitative Immunology Research Unit combines
quantitative, integrative and theoretical approaches.

Recent Publications■ Kumagai Y, Vandenbon A, Teraguchi S, Akira S and Suzuki Y. Genome-wide■ Patil A, Kumagai Y, Liang KC, Suzuki Y and Nakai K. Linking transcriptional

map of RNA degradation kinetics patterns in dendritic cells after LPS
stimulation facilitates identification of primary sequence and secondary
structure motifs in mRNAs. BMC Genomics 17, 1032, doi:10.1186/s12864016-3325-7 (2016).

changes over time in stimulated dendritic cells to identify gene networks
activated during the innate immune response. PLoS Comp. Biol. 9,e1003323, doi:10.1371/journal.pcbi.1003323 (2013).
■ Teraguchi S, Kumagai Y, Vandenbon A, Akira S and Standley DM. Stochas-■ Bahrini I, Song JH, Diez D and Hanayama R. Neuronal exosomes facilitate

synaptic pruning by up-regulating complement factors in microglia. Scientific reports 5, 7989, doi:10.1038/srep07989 (2015).

tic binary modeling of cells in continuous time as an alternative to biochemical reaction equations. Phys. Rev. E, Stat. Nonlin. Soft Matter Phys.
84, 062903 (2011).

■ Diez D, Agusti A and Wheelock CE. Network analysis in the investigationof chronic respiratory diseases. From basics to application. Am. J. Respir.
Crit. Care Med. 190, 981-988, doi:10.1164/rccm.201403-0421PP (2014).
59

At the same time, we are developing a new network analysis
methodology in which biological networks are inferred simultaneously over extensive parts of the differentiation lineage tree aswell as over stimulus-induced signaling pathways. The method
leverages known relationships between progenitor cells and differentiated cell types as well as similarities in signaling pathways
between different cell types. A conceptual scheme is shown in
Figure 1.
Assistant Professor

Alexis Vandenbon
Hiromasa Morikawa

The ultimate goal of immunology is health care, elucidation of
causes of diseases, and their treatment in human patients. To
achieve this goal, a complete understanding of the immune network, the interactions and regulatory principles between cells
and between gene products is required. In the Immuno-Genomics Research Unit, our aim is to establish and apply methodologies for extracting the maximum amount of information possible
from limited experimental data, using integrative bioinformatics
approaches. Here, we briefly introduce some of the research projects we are involved in.
The control of stimulus-induced dynamics in histone modifications in dendritic cells
The importance of transcription factors (TFs) and epigenetic
modifications in the control of gene expression is widely accepted. However, the causal relationships between the dynamics inhistone modifications, TF binding and gene induction in thecourse of a short-term response to pathogens are not well understood.
In collaboration with Dr. Kumagai of the Quantitative Immunology Research Unit, we have been studying the ordering ofthese events in dendritic cells (DCs) in response to lipopolysaccharide (LPS) stimulation. Genome-wide patterns in histone
modification changes and gene induction were studied throughbioinformatics analysis of newly obtained ChIP-seq, RNA-seq and
TSS-seq time series data.

60

Research Assistant

1

Visiting Scientist

1

Interdisciplinary collaborative research projects
In addition to projects that are mostly bioinformatics-driven,
we are involved in several interdisciplinary projects covering several aspects of regulation of the immune system.

In collaboration with the Quantitative Immunology Research
Unit we have analyzed the genome-wide patterns of RNA degradation in response to LPS stimulation of DCs, and employedthese for the prediction of RNA secondary structure prediction(Kumagai et al., BMC Genomics, 2016). We also contributed to thefurther analysis of post-transcriptional regulation by Regnase-1(Mino et al., Cell, 2015) and circulating serum micro-RNAs (Nosirov et al., Advances and Applications in Bioinformatics andChemistry, 2016), to the elucidation of the role of Satb1 in establishing super-enhancers during the development of regulatory T
cells (Kitagawa et al., Nature Immunology, 2017), and to the analysis of single-cell gene expression data of subsets of migratory T
cells (Ikebuchi et al., Scientific Reports, 2016).

Units for Combined Research Fields

Immuno-Genomics

Although researchers often assume that activating histone
modifications are a prerequisite for inducing transcription, ourdata only partly supported this view. Rather, LPS-induced accumulation of several histone modifications at promoters occurred
within well-defined time windows, independent of the transcriptional induction of the promoters. For example, LPS-induced
acetylation of H3K9K14 occurs rapidly, followed by methylationof H3K4, and finally methylation of H3K36.
Through integrative analysis with binding data for >20 TFs in
LPS-treated DCs, we revealed potential links between the timingof TF activation and the timing of accumulation of histone modifications. Especially, binding by STAT1/2 coincided with induction of H3K9K14ac, and was followed by increases in H3K4me3.
Analysis of TRIF, IRF3, and IFNR knock-out data further confirmed
the link between STAT1/2 binding and the establishment of these
histone modifications.
Establishment of a comprehensive cell type-specific network
inference framework
Last year, we published Immuno-Navigator, a gene co-expression database for cells of the human and mouse immune system(Vandenbon et al., PNAS, 2016). The current database includes
4,639 human microarray samples, obtained from 19 cell types
from 191 studies, and 3,434 mouse microarray samples, obtained
from 24 cell types from 261 studies in total. We are currently preparing a larger dataset including >44,900 mouse and >38,700 human RNA-seq samples, covering more than 60 cell types.

Figure.
Conceptual flow of our comprehensive cell type-specific network inference framework. A large amount of gene
expression data (1) is used to infer biological networks using default, naïve methodologies (2). Using the priorknowledge of cell type differentiation, our methodology improves the inferred networks (3). These high quality
networks can finally be used for downstream interdisciplinary collaborations (4).

Recent Publications■ Kitagawa, Y,. et al. Guidance of regulatory T cell development by Satb1-■ Ikebuchi, R., et al. A rare subset of skin-tropic regulatory T cells expressing

dependent super-enhancer establishment. Nat. Immunol. 18, 173–183(2017).

Il10 / Gzmb inhibits the cutaneous immune response. Sci. Rep. 6, 35002(2016).

■ Kumagai, Y., Vandenbon, A., Teraguchi, S., Akira, S. & Suzuki, Y. Genome-■ Mino, T., et al. Regnase-1 and Roquin Regulate a Common Element in In-

wide map of RNA degradation kinetics patterns in dendritic cells after LPS
stimulation facilitates identification of primary sequence and secondary
structure motifs in mRNAs. BMC Genomics 17, 1032 (2016).

flammatory mRNAs by Spatiotemporally Distinct Mechanisms. Cell 161,
1058–1073 (2015).

■ Vandenbon, A., et al. Immuno-Navigator, a batch-corrected coexpression

database, reveals cell type-specific gene networks in the immune system.
Proc. Natl. Acad. Sci. USA. 113, E2393–E2402 (2016).
61

IFReC's New PIs

New Principal Investigators from FY2017
IFReC welcomed four new principal investigators with a new framework for governing structure in 2017.
We expect them to add new impetus to IFReC.

Laboratory of Stem Cell Biology and Developmental Immunology

Laboratory of Molecular Immunology
Mincle, MCL, Dectin-2 and DCAR are C-type lectin receptors thatrecognize M. tuberculosis. Our objective is to identify and study
novel immunoreceptors and their ligands, in order to elucidate themechanisms underlying ligand recognition as well as their potential roles in immune disorders. Based on these results, we also aim
to design new methods to orient the immune responses.
To this end, our research centers on the following axes:
1) Significance and mechanisms of recognition of “aberrant self” or“non-self pathogens” by C-type lectin family receptors.
2) Identification of “self” through the TCR as well as its role in T cell
development.
3) Study of newly discovered T cell subsets involved in autoimmune diseases.

Additionally, we identified a population of reticular cells expressing CXCL12 at high levels, termed CXCL12-abundant reticular(CAR) cells within bone marrow (Immunity 2006) and indicatedthat CAR cells are adipo-osteogenic progenitors and the major producer of CXCL12 and SCF, creating the special microenvironment(niche) for HSCs and B cells (Immunity 2010). Recently, we foundthat the transcription factor Foxc1 was preferentially expressed in
CAR cells in the marrow, enhancing CXCL12 and SCF expression
and was essential for inhibiting adipogenic processes in CAR cellprogenitors, and development and maintenance of niches for
HSCs and immune cells (Nature 2014). We are studying the roles of
CXCL12-CXCR4 signaling and CAR cells in the spatiotemporal regulation of lymphohematopoiesis during homeostasis and diseases.

Takashi Nagasawa, MD/PhD

Sho Yamasaki, PhD

We isolated a chemokine, CXCL12 (SDF-1/PBSF) as a moleculethat stimulates the growth of B cell precursors (PNAS 1994) andfound that CXCL12 and its receptor CXCR4 are essential for colonization of bone marrow by hematopoietic stem cells (HSCs)(Nature
1996; Immunity 2003), maintenance of a pool of HSCs in bone marrow (Immunity 2006), development of immune cells, including B
cells, pDCs and NK cells (Nat. Rev. Immunol. 2006), vascular formation and cardiogenesis (Nature 1998).

Conceptual Figure

Laboratory of Statistical Immunology

Yukinori Okada, MD/PhD
Statistical genetics is a research field that evaluates causality ofhuman genetic variations on diseases, using statistical and bioinformatics approaches. Recent developments of sequencing technologies have provided human genome data of hundreds of thousands of the subjects, and successfully identified comprehensive
catalogues of genetic susceptible loci of immune-related diseases.
62

However, little is known regarding how to develop methodology to integrate large-scale human genome data with diverse biological and immunological resources. The theme of our laboratory
is to develop such methods and apply to the latest large-scale disease genome and omics data. We have demonstrated that the disease risk genes were significantly enriched in overlap with the target genes of the drugs currently used for treatment of the diseases,
and could be promising resources of drug repositioning (e.g.
CDK4/6 inhibitors for rheumatoid arthritis, Okada Y et al. Nature2015). Construction of the novel analytical methodologies of statistical genetics, such as the HLA
imputation method and themiRNA network-based GWAS
analysis (Okada Y et al. Nat
Genet 2015, Sci Rep 2016), is
also the focus of our laboratory. Through such translational approaches, we would
like to empirically show thevalue of statistical genetics todissect disease immunology
and novel drug discovery.

The immune system uses a wide range of receptors for the protection against pathogens and their clearance. Innate immune receptors elicit rapid responses, while a more complex one is triggered by adaptive immune receptors.
The analysis of the C-type lectin family of innate immune receptors has made significant progress in the past decade.
We have found that these receptors can sense both “damaged
self” and “non-self pathogens”. Notably, our team showed that

Laboratory of Molecular Neurosciencecess from the perspective of the functional network of biologicalsystems. Further, we analyzed the mechanism by which the spatiotemporal dynamics in those biological systems control a series ofprocesses.
Particularly, the ultimate goal of this study was to elucidate thecontrol mechanism exerted by the associations among the nervous system, immune system, and vascular system. Additionally,
we aimed to elucidate the principles involved in the operation ofliving organisms with neural network disorders within the central
nervous system by observing such disorders and their functional
recovery process with respect to the dynamics of the entire biological system and by conducting a comprehensive analysis of theassociation between each system.

Toshihide Yamashita, MD/PhD
Disorders of the central nervous system, such as cerebrovasculardiseases, cerebrospinal trauma, and encephalomyelitis, oftencause spatiotemporal changes in the nervous system and in various biological systems, such as the immune system and vascularsystem. In this study, we analyzed disorders of the neural networks
in the central nervous system and the subsequent restoration pro63

Chugai Lab

Joint Research Chair of Innovative Drug
Discovery in Immunology

Kunihiro Hattori (Visiting Professor)
Ryusuke Omiya (Visiting Associate Professor)
Junichi Hata (Visiting Researcher)
As well as other researchers and technicians.

Symposia & Seminars
Members of Chugai Lab
Drs. Kunihiro Hattori, Junichi Hata, and Ryusuke Omiya (left to right)

The laboratory was set up on the basis of the comprehensive
collaboration agreement for cutting-edge research formed between Osaka University and Chugai Pharmaceutical Co., Ltd.
The Chugai researchers in this laboratory and IFReC researchers will promote close exchanges of information and comprehen-sive collaboration including performing joint feasibility studies
prior to advancing joint research. The synergy of IFReC’s recent
research results with Chugai’s proprietary antibody engineering
technologies and molecular library etc. has high potential for the
discovery of seeds for new drugs.

Interview with Dr. Hattori
I don’t know if it is the tradition of Osaka University largely influenced by Professor Kishimoto, but the researchers at IFReC are
all thoroughly devoted to pure basic research. It may be that they
have not had the opportunity to view their research from the perspective of drug discovery.
I hope that this new laboratory, set up under the comprehensive collaboration agreement between Chugai and Osaka University, will act as a pathway to drug discovery based on IFReC’s
cutting-edge research.
Our job is to not simply receive experiment data from the university and make drugs but is to utilize the drug discovery tech-64

nologies developed over many years at Chugai to collaborate
with and accelerate research as well as create a win-win relationship in which we strategically acquire intellectual property andshare knowhow on efficient application methods and research
processes.
Following the global success of the monoclonal antibody
Actemra® commercialized under the direction of Professor Kishimoto, we seek second and third drugs from seeds originatingfrom Osaka University and in the near future expect to expand
the laboratory located here to 20 researchers.

Nov. 1
Opening remarks
Shojiro Nishio (President of Osaka University)
Hiroo Imura (Former President of WPI Program)
Shizuo Akira (Director of IFReC)

Speaker
Shimon Sakaguchi

Title

Symposia & Seminars

International Symposium on Advanced Immunology

Control of immune responses by regulatory T cells(Osaka University)

Vijay K. Kuchroo

Single cell genomics identifies novel regulators of autoimmunity andanti-tumor immunity( Harvard University)

Richard Flavell

Non-coding RNAs in the regulation of inflammation( Yale University)

Tasuku Honjo

Cancer immunotherapy by PD-1 blockade( Kyoto University)

Tak W. Mak( Princess Margaret Cancer Centre, Canada)

Atsushi Kumanogoh(Osaka University)

Josef S. Smolen( Medical University of Vienna)

Tadamitsu Kishimoto(Osaka University)

Beyond Checkpoint Blockade: Emerging Strategies in Immunotherapy
Involvement of semaphorins in pathogenesis of autoimmune, allergy andinflammatory diseases
Insights into pathways to inflammatory rheumatic diseases - novel therapies andresponse prediction
IL-6, Tocilizumab and Arid5a; Immunopathology and Therapy of Autoimmune Diseases

Nov. 2
Speaker
Frederic Alt(Harvard University)

Tomohiro Kurosaki(Osaka University)

Max Cooper(Emory University)

Kiyoshi Takeda■ Date :November 1-2, 2016

(Osaka University)■ Venue :Conference Room 1003, 10 th floor of Osaka International

Gabriel Nuñez

Convention Center(University of Michigan)This symposium was held for commemorating the 10th anniversary of IFReC andthe 77th anniversary of Prof. Tadamitsu Kishimoto’s birth. In the symposium, theworld’s leading scientists discussed current progress in elucidating immune reactions and mechanisms that mediate and regulate immune responses. The participants had an excellent opportunity to exchange information and ideas to accelerate
and further the progress of immunology to the future discovery of new therapies for
immune diseases.

66

Tadatsugu Taniguchi(University of Tokyo)

Ruslan Medzhitov(Yale University)

Shizuo Akira(Osaka University)

Title
Recurrent DNA Break Cluster Genes in Neural Development, Diversification and
Disease: Potential analogies to Lymphocyte Rearrangement Processes
Fate decisions of germinal center B cells into the memory B cell or plasma cellcompartment
Evolution of Lymphocyte Lineages

Regulation of intestinal homeostasis by epithelial barriersRole of the Microbiota and Immunity in the Control of Pathogens at the Intestinal
Barrier
Recent advances in the regulation of inflammation and its associated diseases

Inflammation, Homeostasis and Disease

Identification of a novel monocyte subset involved in lung fibrosis

Closing talk & remarks by Fritz Melchers

67

IFReC Seminars
Speaker

Affiliation

Title

2016
Apr. 28

Klas Kärre

Karolinska Institutet, Sweden

MHC class I dependent education of NK cells: implications for immunotherapy to tumors

Jun. 17

Masaru Kanekiyo

NIAID, NIH, USA

Self-assembling nanoparticle-based immunogendesign: mimicry of virus and beyond

Jul. 8

Stephan Gasser

National University of Singapore

DNA damage and immune recognition of cancer
cells

Sep. 27

Suresh Kumar VermaCenter for Translational Medicine,
Temple University, USA

Interleukin 10 in regulation of cardiac hypertrophyand heart failure

Oct. 20

James Di Santo

Institut Pasteur, France

Transcriptional regulation of innate lymphoid cell
development and plasticity

Oct. 31

Gabriel Nunez

University of Michigan School of Medicine, USA

Role of the microbiota in the regulation of immunity
against systemic infections

Nov. 4

Richard A. Koup

NIAID, NIH, USA

Impact of HIV and SIV on lymph node structure andfunction

Nov. 15

Jian Han

HudsonAlpha Institute for Biotechnology iCubateInc., and iRepertoire Inc., USA

Towards the ultimate diagnostics: learning how todiagnose diseases from our immune system by NGS
of immune repertoire

Gaetan BurgioJohn Curtin School of Medical Research,
Australian National University

A voyage into the discovery of the genetic
mechanisms for host resistance to malaria

Richard Culleton

NEKKEN, Nagasaki University, Japan

A voyage into the discovery of the genetic
mechanisms for malaria parasite resistance to hosts

Jan. 26

Dennis Klinman

The National Cancer Institute (NCI), NIH, USA

Intra-tumoral delivery of TLR agonists for cancer
therapyJan. 27

Daniela Verthelyi

The Food and Drug Administration (FDA), USA

Modeling Zika virus in mice: what have we learned
so far?

Feb. 1

Mikael Martino

European Molecular Biology Laboratory /Australian Regenerative Medicine Institute,
Monash University, Australia

Promoting tissue regeneration by modulating theimmune system an example with bone regeneration

Feb. 2

Anne O'Garra

The Francis Crick Institute, UK

Systems approaches to studying the immune
response in tuberculosis: strategies to improvemouse models of human disease

Feb. 3

Lewis L. Lanier

University of California, San Francisco, USA

Tracking the fate of memory Natural killer cells

Feb. 22

Ye Htun Oo

University of Birmingham Liver and Hepatobiliary
Unit, UK

Exploring human intrahepatic Treg and IL-17
secreting cells to develop translational Treg celltherapy

John C. Reed

Global Head, Roche Pharm Research &Early Development

Bolstering immune responses

David M. Lee

Global head, Immunology,
Inflammation & Infectious Diseases,
Roche Pharma Research & Early Development

Restoring immune homeostasis

Haner Direskeneli

School of Medicine Hospital, Marmara University,
Istanbul, Turkey

Immunopathogenesis of Behcet's Disease

Junya Masumoto

Ehime University Proteo-Science Center andGraduate School of Medicine, Japan

Inflammatory signaling in familial Behcet's Disease

Gunnur Deniz

Aziz Sancar Institute of Experimental Medicine,
Istanbul University, Turkey

Natural Killer cells in Behcet's Disease

Symposia & Seminars

Date

2017
Jan. 13Mar. 23

IFReC holds seminars throughout the year with speakers from a variety of disciplines
including immunology, imaging and informatics with the aim of promoting collaborative research, as well as to inspire and educate the next generation of scientists.
Since its establishment, IFReC has held more than a hundred seminars, which have
served as a forum for effective interaction between researchers beyond national borders
and academic disciplines. This program has certainly contributed to IFReC’s mission ofpromoting internationalization and interdisciplinary research.

68

Mar. 2469

Speaker

25st

26st

27st

28rd

Kosuke Fujimoto (Mucosal Immunology)

Regulation of gut homeostasis by the ulcerative colitis-associatedgene RNF186

Alexis Vandenbon (Immuno-Genomics Unit)

Immuno-Navigator: a co-expression database for cell type-specificnetwork inference in the imune system

Yoshiko Murakami (Immunoglycobiology)

Paroxysmal nocturnal hemoglobinuria caused by PIGT
mutations : Atypical PNH

Miwa Sasai (Immunoparasitology)

Some ATG proteins play a critical role in non-autophagic cell-autonomous
immunity to vacuolar pathogens

Alison J. Hobro (Biophotonics)

Multimodal label-free imaging of cellular responses to stressTakashi Satoh (Host Defense)

NF-IL6 licenses pro-fibrotic monocyte differentiation from its progenitor

Akiko Nakai (Immune Response Dynamics)

A novel molecular mechanism of chemoattractant receptor signaling

Syoji Kobashi (Information Systems)

In vivo 3-D macrophage labeling and tracking method using time-lapse
MR images

Michelle Sue Jann Lee (Malaria Immunology)

Plasmodium infection modulates bone homeostasis

David Millrine (Immune Regulation)

The anti-inflammatory properties of thalidomide

Mohammad Mahabub-Uz-Zaman (Immune Regulation)

Pathogenic role of Arid5a in septic shock

Hironori Kawahara (Immune Network)

Regulation of glial inflammatory responses by neuronal exosomes

James Wing (Experimental Immunology)

A distinct subset of CD25 negative T-follicular regulatory cells localizes ingerminal centers

Kazuya Takeda (Immune Regulation)

Tracking allergen-reactive IgE+ B cells in patients with eosinophilicchronic rhinosinusitis

Aya Nakae (Brain-Immune Interaction)

Newly developed animal post-operative persistent pain model and its
behavior and pituitary changes after experimental pain in humans

Masaki Maruyama (Brain-Immune Interaction)

Dynamics of capsaicin-induced skin inflammation and its influence on
decision behaviors

Tristan Nakagawa (Brain-Immune Interaction)

The representation of capsaicin-induced tissue inflammation in the brain

Hiroki Kato (Nuclear Medicine)

11

29th

■ Date :25 th Colloquium : April 20, 2016
26 th Colloquium : June 8, 2016

Title

Symposia & Seminars

IFReC Colloquia

C-acetate PET in patients with multiple sclerosis: a pilot study

27 th Colloquium : August 10, 2016
28 th Colloquium : December 14, 2016
29 th Colloquium : February 8, 2017
■ Venue : Taniguchi Memorial Hall, Osaka University
IFReC colloquia are a series of discussion meetings for IFReC members held once
every other month since FY2011. At each colloquium, three speakers from IFReC
laboratories give talks about their latest research progress followed by intensive
discussion. After the colloquium, a small social gathering is held to further the
discussions and encourage the exchange among IFReC members in an informal
setting. These events serve as a platform to promote fusion researches and deepen
understanding of researches conducted in IFReC.

70

71

Events

The 6th NIF Winter School on Advanced Immunology
Shizuo Akira
IFReC, Japan

Veronique Angeli
National University of Singapore

Burkhard Becher
University of Zurich, Switzerland

Antonio Bertoletti
Duke-NUS Medical School, Singapore

Subhra Biswas
Singapore Immunology Network

Ana Cumano
Institut Pasteur, France

Wendy Havran
The Scripps Research Institute, USA

Tomohiro Kurosaki
IFReC, Japan

Paul Macary
National University of Singapore

Laura Mackay
Doherty Institute, Australia

Evan Newell
Singapore Immunology Network

Christiane Ruedl
Nanyang Technological University, Singapore

Shimon Sakaguchi
IFReC, Japan

Federica Sallusto
Institute for Research in Biomedicine, Switzerland

Kazuhiro Suzuki
IFReC, Japan

Title
Functional diversity of macrophage/monocyte subsets revealed
by gene targeting

Events

Lecturer

Homeostatic function of arterial macrophages

How T cells talk to myeloid cells in inflammation

Immunotherapy with T-cell receptor redirected T cell targeting viral
antigen in viral related tumors

Immuno-metabolic re-programming of monocytes/macrophages

Lymphocyte commitment during development

Immune crosstalk in the skin

Selection of germinal-center B cells into memory B cell or plasma
cell compartment
Analyzing the impact of antibody-mediated BloT5 neutralization on
airway hypersensitivity

Development and Function of Tissue-Resident Lymphocytes

High-dimensional cellular immune profiling in health and disease

Intestinal dendritic cells

Control of immune responses by regulatory T cells

Challenges and opportunities of human immunology

Control of lymphocyte trafficking and adaptive immune responses
by adrenergic nerves

Filip Swirski
Harvard Medical School and
Massachusetts General Hospital, USA■ Date :January 22-26, 2017

■ Venue :Grand Copthorne Waterfront Hotel, Singapore

Unleashing the inflammatory cascade in cardiovascular disease

The sixth Winter School on Advanced Immunology was jointly organized with Singapore Immunology Network (SIgN).
Forty-four young researchers, who were competitively selected from 85 applicants and 16 world leading immunologists,
got together in Singapore on 22-26 January 2017. Five young IFReC researchers participated in the school. The participants shared intriguing insights and findings in immunology, discussed new ideas and forged friendships that will fuelnetworking and future collaborations.
( Visit the following website for details; http://ifrec-sign-winterschool.org )74

75

Japanese Lessons
Japanese language classes are held for overseas researchers / students to alleviate any stress and inconvenience in research or daily
life that may be caused by the language barrier.

Outreach Activities
We offer two lecture-style classes, "Class A: Elementary to
Pre-intermediate" and "Class B: Intermediate to Advanced".
Class members are expected to learn verb and adjective
conjugations to have basic knowledge of Japanese grammar
in Class A , to learn grammar, vocabulary of intermediate/
advanced level, Kanji etc. to improve each member of skills
in Class B.

Comment by Ms. Kaori Tajima (Japanese instructor)
Many researchers attend the classes because at IFReC Japanese is necessary when carrying out
daily research activities in the laboratory. In order for the participants to start using Japanese sooner
in communication with their colleagues, we learn words such as experiment, analysis, business trip,
thesis/paper etc. at an early stage. Many of the participants eke out time for the classes from their fullschedule so words such as busy, overtime, deadline, are often used in class. The participants find theclasses a welcome break from the laboratory and emphasis is placed on listening and speaking using
pair and group work to encourage networking with researchers from other laboratories.
At the end of a two year course, most communication in the class is in Japanese. This is the time
when the participants can really start to enjoy their life in Japan by taking what they have learnt in
the classes and using it to broaden their weekend activities.

76

Science Cafe

Super Science High School Students Fair
IFReC science cafe was held in Osaka University Suita campus
in FY2016. About 70 participants enjoyed a novel topic in immunology in a relaxing atmosphere.

Outreach Activities

The series of science cafes is a long-lasting IFReC outreachactivity to promote communication among researchers andthe general public. It also enhances people’s understanding of
immunology researches and the researchers involved in them.

■ Date :August 10 -11, 2016

■ Venue :Kobe International Exhibition Hall

■ Host :MEXT, JST■ Support : Boards of Education ( Kobe Prefecture and Kobe City)
Super Science High Schools (SSH) are selected high schools in Japan, which promoteadvanced math/science education as well as collaborative researches with universities,
and activities to develop international perspectives.
The SSH Student Fair FY2016 was held in Kobe and more than 200 schools, including
several schools from overseas, held booths with posters to present their researches. WPI
institutes held a collaborative booth and introduced the research activities of each institute using posters, booklets and demonstrations.

“Science Café on the Edge” at 2016 Icho Festival<Forefront of immune therapy! Development of vaccine and adjuvant >■ Date :May 1, 2016

■ Venue :Techno Alliance Building (Osaka University, Suita Campus)■ Guest :Etsushi Kuroda (Vaccine Science, IFReC)

Students Visit
Nara Prefecture super science high school,
science tour
■ Date : August 18, 2016
IFReC welcomed thirty students from Nara Prefectural Nara Senior High Schooland Unebi Senior High School. They heard a lecture by Prof. Kurosaki (Lymphocyte Differentiation), and visited Akira Lab., Arase Lab. and Kishimoto Lab. to try
some experiments and to talk with researchers.

Osaka Prefectural Tennoji High School■ Date : October 7, 2016
IFReC welcomed seventeen students from Osaka Prefectural Tennoji HighSchool. They heard a short lecture from an IFReC Assistant Professor, Yutaro Kumagai (Quantitative Immunology), introducing immunology and innate immunity
and tried some experiments using nucleotide database. They also visited theCenter for Information and Neural Networks (CiNet), and listened to IFReC Professor Yoshichika Yoshioka (Biofunctional Imaging) and Associate Professor Aya
Nakae (Brain-Immune Interaction) explain their research.

78

79

In celebration of the 10th anniversary of WPI program, a lecture
meeting for general citizens was organized by MEXT, JSPS, and
WPI institutes. The nine leading researchers from all the WPI
institutes were invited as the speakers, and the meeting was
highly acclaimed by the audience.

■ Date :December 17, 2016

■ Venue :Lecture Hall, 3F MEXT East Building

AAAS 2017 Annual Meeting
Outreach Activities

The Forum Commemorating the 10th Anniversary of WPI“Toward the future of Science in Japan”

Greetings
Yasunao Seki ( MEXT )13:30−13:45

Yuichiro Anzai ( JSPS)
Hiroo Imura ( Former WPI President )Keynote Lecture
13:45−14:00

Lectures Part 1“Toward the free cross-border science”
Toshio Kuroki ( WPI Program Director )“Mathematics, Physics, and Earth Science”

14:00−14:20

Hitoshi Murayama ( Director, Kavli-IPMU, University of Tokyo )14:20−14:40

Shigeru Ida ( Vice Director, WPI-ELSI, Tokyo Tech )14:40−15:00

Motoko Kotani ( Director, WPI-AIMR, Tohoku University )Lectures Part 2“Life Science”

15:20−15:40

Ryuichiro Kageyama ( Vice Director, WPI-iCeMS, Kyoto University )15:40−16:00

Masashi Yanagisawa ( Director, WPI-IIIS, University of Tsukuba )16:00−16:20

Shimon Sakaguchi ( Vice Director, WPI-IFReC, Osaka University )Lectures Part 3“Chemistry and Material Science”

16:40−17:00

Masakazu Aono ( Director, WPI-MANA, National Institute for Materials Science)

17:00−17:20

Kenichiro Itami ( Director, WPI-ITbM, Nagoya University)

17:20−17:40

Seiji Ogo ( Group Leader, WPI-I2CNER, Kyushu University)■ Date :February 16-20, 2017

■ Venue :Hynes Convention Center (Boston, U.S.A)

The American Association for the Advancement of Science (AAAS) is the biggest international scientific society in the world
and its mission is to “advance science and serve society”. The AAAS 2017 Annual Meeting was held in Boston and it offered abroad range of activities including lectures, symposia, seminars and exhibits, with the theme of "Serving society through science policy ".
WPI institutes held a collaborative booth to introduce the WPI program and the institutes’ activities using posters, booklets
and a demonstration experiment. More than 300 participants visited the booth and gained interest in the WPI program andworld leading researches in Japan.

Closing
17:40−17:50

17:50−18:30

80“The future of Japan led by Science”
Ryoji Noyori ( President of WPI Program, Nobel Laureate 2001)
Freewheeling discussion by audience and speakers

81Research Projects

Papers in Fusion Research Fields
Fusion Fields

One of the missions of the WPI program is to generate novel
research fields through fusion of existing research fields. Under
this mission, IFReC has been committed to the creation of innovative immunology fields through integration with imaging
and bioinformatics technologies for comprehensive understanding of immune dynamics. To facilitate fusion research,
IFReC launched the Research Support Program for Combined
Research Fields in October 2009. This program provides financial support for research proposals combining different
research areas. The project teams must consist of researchers
from different fields or backgrounds. The IFReC board of representatives screened the projects based on proposals and the
projects selected are given ¥3 million a year for three years.
IFReC also started the Dual Mentor Program in October FY2012
with the aim of helping young researchers engage in fusion
research at IFReC. In a selected project, a graduate student ora young postdoc, who obtained a Ph. D degree less than two
years previous, is supervised by two mentors, whose specialization lies in different research fields, and is provided financial
support of ¥3 million a year for three years by IFReC.
These programs have effectively encouraged IFReC researcher’s interest in fusion research. By FY2016, a total of 27 projects
had been selected for the program. Many interdisciplinary approaches were made and 36 papers successfully produced, contributing to the advancement of the fusion research at IFReC.
The papers of fusion research published by IFReC members, including those produced from this program, accounted for morethan 30% of the total papers produced at IFReC in FY2015. This
demonstrates that our continued efforts for the developmentof fusion research have come to fruition over the years of WPI
program.

Other Fields% of Fusion Fields

250

26%
200

23%

26%

28%

30%

18%
150

16%

Research Projects

Support Programs for Fusion Researches

14%
143

100

131
106

111

126

39

38

49

52

22

46

2010

2011

2012

2013

2014

2015

99
50

67
42
8

0

2008

11
2009

2013 − 2016

Project Leader

84

Collaborators

Yutaro Kumagai

Visualizing information processing of immune cells via combination offluorescent reporter and single molecule imaging

Jun Kozuka, RIKEN QBiC
Shunsuke Teraguchi, IFReC

Kazuhiro Suzuki

Visualizing activation of germinal center B cells using genetically encoded
calcium indicators

Yoshihiro Baba, IFReC

Naganari Okura

Development of an epigenome-based computational classification system
for the treatment of autoimmune diseases

Alexis Vandenbon, IFReC
Shota Nakamura, RIMD

Alexis Vandenbon

Identification of key factors for inducing functionally stable regulatory
T cells

Shimon Sakaguchi, IFReC
Hiromasa Morikawa, IFReC
Naganari Ohkura, IFReC

Diego Diez

The dynamics of novel signaling networks of macrophages exposed topathogens

Rikinari Hanayama, IFReC
Yutaro Kumagai, IFReC
Shunsuke Teraguchi, IFReC

Evaluation Workshop
All the Combined Research Program and Dual Mentor Program projects are required to present their results once a year to IFReC members. At an annual Evaluation Workshop, IFReC PIs evaluate each project on the basis of specific criteria,such as “Achievement of the interdisciplinary research project mission “, Innovationof methodology” and “Prospects”. The evaluators have sometimes added severe
comments to encourage improvement. Each project leader is given the evaluation
result after the workshop. In the workshop, a comment sheet was provided to theaudience and the comments from the audience were also given to the project leaders.
In the 2016 workshop, five project leaders of the Research Support Program presented their research outcomes in front of IFReC members. The projects that gained
a high score from IFReC PIs were awarded “Best Project Award.” Also, two of the past
project leaders, selected by the director according to their outcome, reported on
their outstanding interdisciplinary research achievements in the workshop.

Title

85

Young Scientist Support Program for Research Abroad
To strengthen our international research network and our basis for international collaborative research, IFReC has provided
financial support to young researchers who wish to participate
in research activities at overseas institutions. The program aims

to develop the practical skills and abilities of young researchers in international collaborative research and to develop their
network with researchers overseas. Seven researchers used this
support program in FY2016.

Young Scientist Support Program for Research Abroad
Name
Kazuya Masuda

CountryUSA

Conferences attended
Gene expression and signaling in the immune system

Michelle Lee Sue Jann

Australia

International Congress of Immunology 2016

Szandor Simmmons

Germany

1. Institutes Seminar Series of the DRFZ
2. 46th Annual Meeting of the DGFI
3. Collaboration meetings with scientist of the Max Planck InstituteAlexis Vandenbon

Singapore

15th International Conference on Bioinformatics (InCoB2016)

Kouyuki Hirayasu

Italy

16 Annual Meeting of the Society for Natural Immunity

Alison Jane Hobro

USA

SCIX conference

Yutaro KumagaiSingaporeth

15th International Conference on Bioinformatics (InCoB2016)

Since the start of this program, IFReC has provided support for 49 visits overseas by young researchers.

Support provided for 43 overseas visits by young researchers in the past five years

Europe 15

America 20

Asia 4Africa 1
Australia 3

IFReC has supported the active participation of young scientists in research activities overseas

86

Data

Kishimoto Foundation Fellowship

IFReC and its parent institution, the Research Institute for Microbial Diseases (RIMD) are located on the same site, constituting a large research complex. It contains the Core Instrumentation Facility, the Animal Resource Center and the Network
Administration Office, all of which are jointly operated by IFReC
and RIMD. The Core Instrumentation Facility is equipped withvarious highly advanced instruments and skilled technicians
provide in-house services to IFReC and RIMD researchers. The

Animal Resource Center consists of three buildings for specificpathogen-free (SPF) animals and the live immuno-imaging
facility. With a large capacity animal-breeding facility in IFReC,
researchers are able to choose animal rooms suitable for theirexperiment purpose.
Using these common facilities, IFReC researchers are able toeffectively and smoothly carry out their experiments to promote their world-leading research at IFReC.

IFReC–RIMD Research Complex at Suita Campus of Osaka University

The Kishimoto Foundation was established in 2008 in honorof Tadamitsu Kishimoto, who, during the 1980s and 90s, elucidated the function of interleukin-6 (IL-6), a key molecule forstimulating immune responses. He later developed the anti-IL6
receptor-based therapy, tocilizumab, to treat immune disorders
such as Castleman's disease or rheumatoid arthritis.

FY2016 Kishimoto Fellowship Recipients

1
6

IFReC launched the Kishimoto Foundation Fellowship programfor researchers in various fields of immunology in 2010. The
program is supported by the Kishimoto Foundation and designed to support overseas researchers in order to promote anddevelop immunological research and international exchanges
at IFReC. The fellowships are open to international postdoctoral
researchers who seek to collaborate with IFReC researchers. The
recipients are provided with a salary and an airfare to Japan.

Data

Common Facilities

Position of recipient

Name (initials)

Nationality

Host researcher

PeriodSpecially Appointed Researcher

T. N.

Germany

Seymour

Apr. 1, 2015 - Mar. 31, 2017

Specially Appointed Researcher

P. L.

Australia

Coban

Oct. 1, 2015 - Sep. 30, 2016

Specially Appointed Researcher

D. H.

Australia

Sakaguchi

Oct. 16, 2015 - Oct 15, 2016

Specially Appointed Researcher

J. P.

Vietnam

KishimotoApr. 1, 2016 - Mar. 31, 2018

Specially Appointed Researcher

N.T.

Slovenia

Quantitative
Immunology
Research Unit

Oct. 1, 2016 - Mar. 31, 2018

2

3
5

4

Photo : S. Higashiyama

1. IFReC Research Building
2. Integrated Life Science Building
3. Main Building, Research Institute for Microbial Diseases
4. South Building, Research Institute for Microbial Diseases
5. Cutting-edge Research Building for Infectious Diseases
6. Animal Resource Center for Infectious Diseases

Animal Resource Center for Infectious Diseases

Network Administration Office■ Specific pathogen-free (SPF) animal facility■ Provision and maintenance of network infrastructure:■ Sperm/ embryo freezing and preservation

LAN system and servers (web, mail, mailing lists, etc.)■ In vitro fertilization and embryo transplantation■ Intracytoplasmic sperm injection

Core Instrumentation Facility■ Transgenic and knock-out animals■ Genome editing in experimental animals■ Basic and advanced instruments■ In-house service

Live Immuno-Imaging Facility■ SPF animal experiment facility with 11.7T MRI, in-vivo imager& two-photon microscope.

88■ DNA sequencing, cell sorting, electron microscopy, mass

spectrometry and next-generation DNA sequencing analysis■ Radio isotope facility■ DNA chip center

89

Shimon Sakaguchi

Five IFReC researchers

The Crafoord Prize

Highly Cited Researchers 2016

Highly Cited Researchers are researchers with papers thathave a large number of citations from all over the world asselected by Thomson Reuters. In 2016, some 3,000 researchers
from 21 fields were selected as being some of the most influential researchers in the world.
Six selections were from IFReC for a total of five researchers(Director Akira were selected for two categories).

Shimon Sakaguchi was awarded the Crafoord Prizein Polyarthritis 2017 for his discoveries relating toregulatory T cells, which counteract harmful immune
reactions in arthritis and other autoimmune diseases.
The award ceremony was held at the Royal Swedish
Academy of Sciences.
Sakaguchi is the fourth Japanese prize winner, of
which three are currently or used to be IFReC researchers.

Kiyoshi Takeda

Biology & Biochemistry• Shizuo Akira, Director of IFReC

Data

Major Awards

Immunology• Shizuo Akira, Director of IFReC• Shimon Sakaguchi, Deputy Director of IFReC• Ken Ishii, Vaccine Science, IFReC• Kiyoshi Takeda, Mucosal Immunology, IFReC• Masahiro Yamamoto, Immunoparasitology, IFReC

Erwin von Bälz Prize/Osaka Science Prize

Shimon Sakaguchi, other laureates, and crown princess
Victoria at the ceremony on May 18, 2017.

Tadamitsu Kishimoto

The King Faisal International Prize
Kiyoshi Takeda (second left) at the award
ceremony of the Bälz Prize(Photo : Katsumi Yanagiya)

Tadamitsu Kishimoto was awarded the King Faisal
International Prize for Medicine 2017. The awarded
topic is “Biologic Therapeutics in Autoimmune Diseases”. The award ceremony was held at Riyadh, theKingdom of Saudi Arabia.
The King Faisal International Prize is launched by
the King Faisal Foundation, and is an award including Science and Medicine. Many prize winners havegone on to receive other prestigious prizes, such as
the Nobel Prize.

Kazuhiro Suzuki
Medical Research Encouragement Prize of theJapan Medical Association
Astellas Awards for the Best Biomedical Research

King Salman bin Abdulaziz Al-Saud of Saudi Arabia and Tadamitsu
Kishimoto at the award ceremony on April 4, 2017.

Kazuhiro Suzuki with the certificates ofcommendation for the prizes

9091Finance

Number of IFReC Staff

Breakdown of total expenditure at IFReC in FY2016

300

Data

Composition

28
Administrative Staff
Research Support Staff
Researchers

250

24

28
29
74

25

82

63

200

Individual Research
Project

89
28

45

25.7%

28

Personnel

32.3%

21
77

31

150
15
23

100

171

173

183

3,423,108,667

50( JPY )

186

135
13
3

Total Expenditure

74

179

127

115

89

Equipment

Project
Activities

21.9%

52

19.9%

0
Mar.2008

Mar.2009

Mar.2010

Mar.2011

Mar.2012

Mar.2013

Mar.2014

Mar.2015

Mar.2016

Mar.2017

Travel0.3%

Number of Researchers
Travel200
30%
From Overseas
Japanese

32%

31%
55

150

31%

54

26%
48

0.6%

23%

Equipment

42

54

20.3%

40
27%
24

50

117

15%
8

119

From the
University

20.8%

31%

42
100

Grant-in-Aid for
Scientific Research

5.6%

128

138

29%

Project
Activities

33

36.9%

137

Other Sources

1,241,101,000( JPY )

Personnel

57.0%
87

93

WPI Grant

82

Contract
Research

2,182,007,667( JPY )

19.9%

Management
Expenses Grant

24.5%
Donation

14.4%

65
44
0
Mar.2008

92

Mar.2009

Mar.2010

Mar.2011

Mar.2012

Mar.2013

Mar.2014

Mar.2015

Mar.2016 Mar.201793Two FOXP3+CD4+ T-cell subpopulations distinctly control the prognosis of colorectal cancers.

Regulated selection of germinal center cells into the memory B cell compartment.

Nat Med. 22:679-84 (2016). doi: 10.1038/nm.4086.

Nat Immunol. 17:861-869 (2016). doi: 10.1038/ni.3460.

Takuro Saito T, Nishikawa H, Wada H, Nagano Y, et al.

Shinnakasu R, Inoue T, Kometani K, Moriyama S, et al.

CD4+ T cells that express the forkhead box P3 (FOXP3) transcription factor function as regulatory T (Treg) cells and hinder
effective immune responses against cancer cells. Abundant
Treg cell infiltration into tumors is associated with poor clinical outcomes in various types of cancers. However, the role ofTreg cells is controversial in colorectal cancers (CRCs), in which
FOXP3+ T cell infiltration indicated better prognosis in somestudies.
Shimon Sakaguchi and his group showed that CRCs, which
are commonly infiltrated by suppression-competent FOXP3hi
Treg cells, can be classified into two types by the degree ofadditional infiltration of FOXP3lo nonsuppressive T cells. Functionally distinct subpopulations of tumor-infiltrating FOXP3+ T
cells contribute in opposing ways to determining CRC prognosis. Depletion of FOXP3hi Treg cells from tumor tissues, which
would augment antitumor immunity, could thus be used as
an effective treatment strategy for CRCs and other cancers,

How memory cells are selected and generated during germinal-center (GC) reactions remains unclear.
Tomohiro Kurosaki and his group found that light-zone (LZ)
GC B cells with B cell antigen receptors (BCRs) of lower affinity
were prone to enter the memory B cell pool. Mechanistically,cells in this memory-prone fraction had higher expression of thetranscriptional repressor Bach2 than that of their counterparts
with BCRs of higher affinity.Haploinsufficiency of Bach2 resulted
in reduced generation of memory B cells, independently of suppression of the gene encoding the transcription factor Blimp-1.
Bach2 expression in GC cells was inversely correlated with thestrength of help provided by T cells. Thus, they propose an instructive model in which weak help from T cells maintains relatively high expression of Bach2, which predisposes GC cells toenter the memory pool.

Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2.

Real-time intravital imaging of pH variation associated with osteoclast activity.

Nat Microbiol. 1:16054 (2016). doi: 10.1038/nmicrobiol.2016.54.

Nat Chem Biol. 12:579-585 (2016). doi: 10.1038/nchembio.2096.

Hirayasu K, Saito F, Suenaga T, et al.

Hisashi Arase group found that immunoglobulins disrupted
by microbial pathogens are specifically detected by leukocyte
immunoglobulin-like receptor A2 (LILRA2), an orphan activating receptor expressed on human myeloid cells. The microbiallycleaved immunoglobulins but not normal immunoglobulins
stimulated human neutrophils via LILRA2. In addition, stimulation of primary monocytes via LILRA2 inhibited the growth of L.
pneumophila. When mice were infected with L. pneumophila,
immunoglobulins were cleaved and recognized by LILRA2.
More importantly, cleaved immunoglobulins were detectedin patients with bacterial infections and stimulated LILRA2expressing cells.
Their findings demonstrate that LILRA2 is a type of innate immune receptor in the host immune system that detects immunoglobulin abnormalities caused by microbial pathogens.

94

whereas strategies that locally increase the population of FOXP3lo non-Treg cells could be used to suppress or prevent tumor
formation.

Data

Selected Articles

Maeda H, Kowada T, Kikuta J, Furuya M, Shirazaki M, Mizukami S, et al

Intravital imaging by two-photon excitation microscopy(TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell
imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, aswell as their undesirable physicochemical properties for TPEM
imaging.
Kazuya Kikuchi and Masaru Ishii groups designed and developed a functional SMP with an active-targeting moiety, higher
photostability, and a fluorescence switch and then imaged
target cell activity by injecting the SMP into living mice. The
combination of the rationally designed SMP with a fluorescentprotein as a reporter of cell localization enabled quantitation of
osteoclast activity and time-lapse imaging of its in vivo function
associated with changes in cell deformation and membrane
fluctuations. Real-time imaging revealed heterogenic behaviors
of osteoclasts in vivo and provided insights into the mechanismof bone resorption.

95

Identification of an atypical monocyte and committed progenitor involved in fibrosis.

Proc Natl Acad Sci USA. 113:11543-11548 (2016).

Nature 541: 96-101(2017). doi : 10.1038/nature20611.

Zaman MM, Masuda K, Nyati KK, Dubey PK, et al.

Adenine-thymine (AT)-rich interactive domain containing protein 5a (Arid5a) is an RNA-binding protein that has been shown
to play an important immune regulatory function via the stabilization of IL-6 and STAT3 mRNA. However, the role of Arid5ain the overwhelming and uncontrolled immune response that
leads to septic shock is unknown.
Tadamitsu Kishimoto group reported that Arid5a-deficient
mice are highly resistant to lipopolysaccharide (LPS)-induced
endotoxic shock and secrete lower levels of major proinflammatory cytokines, including IFN-γ, IL-6, and TNF-α, than WT micein response to LPS. Their previous study suggests that Arid5a
control the IL-6 level in vivo in response to LPS by stabilizationof IL-6 mRNA. They also observed that neutralization of IFN-γ
and IL-6 significantly recovered the mice from endotoxic shock.

They conclude that Arid5a regulates the augmentation of IL-6
and IFN-γ in response to LPS, which possibly works synergistically for amplification of various other cytokines that ultimately
cause the development of septic shock in mice.

“Pulmonary fibrosis is one of a family of related diseases calledinterstitial lung diseases. As the lung tissue becomes scarred, itinterferes with a person's ability to breathe. Most cases of pulmonary fibrosis have no known cause. These cases are called
idiopathic pulmonary fibrosis.” (American Lung Association)
Monocytes and macrophages comprise a variety of subsetswith diverse functions. It is thought that these cells play a crucialrole in homeostasis of peripheral organs, key immunologicalprocesses and development of various diseases. Among these
diseases, fibrosis is a life-threatening disease of unknown aetiology.
Shizuo Akira group identified a new type of macrophage, Segregated nucleus Atypical Monocytes (SatM). They found SatM
have a bi-lobed segmented nuclear shape, and are ‘disorderspecific monocyte/macrophage subtypes’ corresponding tofibrosis. It may also now be possible to develop novel, more specific therapeutic targets for this intractable disease in the future.

Inhaled fine particles induce alveolar macrophage death and interleukin-1α release to promoteinducible bronchus-associated lymphoid tissue formation.

J Exp Med. 213:2567-2574 (2016).

Immunity 45:1299-1310 (2016). doi: 10.1016/j.immuni.2016.11.010.

It has long been proposed that various aspects of immune
responses are influenced by nervous system activity. However,the cellular and molecular basis for neural regulation of immunity are largely unclear.
Kazuhiro Suzuki group revealed that neural inputs to β2adrenergic receptors (β2ARs) expressed on lymphocytes generate the diurnal variation in the frequency of lymphocyte egressfrom lymph nodes, which is reflected in the magnitude of theadaptive immune response. During the period of high adrenergic nerve activity, lymphocyte egress from LNs is restricted,
which leads to an increase of lymphocyte numbers in LNs. Immunization during the period of lymphocyte accumulation in
LNs promote adaptive immune responses. This diurnal variationof lymphocyte trafficking may have evolved to maximize theefficiency of host defense against pathogens.

96

Satoh T, Nakagawa K, Sugihara F, et al.

Adrenergic control of the adaptive immune response by diurnal lymphocyte recirculation
through lymph nodes.

Suzuki K, Hayano Y, Nakai A, Furuta F, and Noda M, et al.

Data

Arid5a exacerbates IFN-γ-mediated septic shock by stabilizing T-bet mRNA.

Kuroda E, Ozasa K, Temizoz B, Ohata K, Koo CX, et al.

Particulate pollution is thought to function as an adjuvantthat can induce allergic responses. However, the exact cell
types and immunological factors that initiate the lung-specificimmune responses are unclear.
Ken Ishii group found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) andcaused inducible bronchus-associated lymphoid tissue (iBALT)
formation in the lung. IL-1α release continued for up to 2 weeks
after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1αrelease and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, whichcoincided with subsequent immunoglobulin E responses, andIL-1-receptor-deficient mice failed to induce iBALT formation.
Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

97

1

Barman, S; Kayama, H; Okuzaki, D; Ogino, T; Osawa, H; Matsuno,
H; Mizushima, T; Mori, M; Nishimura, J; Takeda, K. Identificationof a human intestinal myeloid cell subset that regulates gut
homeostasis. International Immunology 28, 533-545 (2016).

2

Brewitz, A; Eickhoff, S; Dahling, S; Quast, T; Bedoui, S; Kroczek,
RA; Kurts, C; Garbi, N; Barchet, W; Iannacone, M; Klauschen, F;
Kolanus, W; Kaisho, T; Colonna, M; Germain, RN; Kastenmuller, W.
CD8(+) T Cells Orchestrate pDC-XCR1(+) Dendritic Cell Spatial
and Functional Cooperativity to Optimize Priming. Immunity
46, 205-219 (2017).

3

4

5

6

7

8

Chiu, LD; Ichimura, T; Sekiya, T; Machiyama, H; Watanabe, T;
Fujita, H; Ozawa, T; Fujita, K. Protein expression guided chemical profiling of living cells by the simultaneous observation ofRaman scattering and anti-Stokes fluorescence emission. Scientific Reports 7, 43569 (2017).
Ebata, T; Mitsui, Y; Sugimoto, W; Maeda, M; Araki, K; Machiyama,
H; Harada, I; Sawada, Y; Fujita, H; Hirata, H; Kawauchi, K. Substrate Stiffness Influences Doxorubicin-Induced p53 Activation
via ROCK2 Expression. Biomed Research International , 5158961(2017).
Edvardson, S; Murakami, Y; Nguyen, TTM; Shahrour, M; St-Denis,
A; Shaag, A; Damseh, N; Le Deist, F; Bryceson, Y; Abu-Libdeh,
B; Campeau, PM; Kinoshita, T; Elpeleg, O. Mutations in thephosphatidylinositol glycan C (PIGC) gene are associated withepilepsy and intellectual disability. Journal of Medical Genetics
54, 196-201 (2017).
Fujimoto, K; Kinoshita, M; Tanaka, H; Okuzaki, D; Shimada,
Y; Kayama, H; Okumura, R; Furuta, Y; Narazaki, M; Tamura, A;
Hatakeyama, S; Ikawa, M; Tsuchiya, K; Watanabe, M; Kumanogoh,
A; Tsukita, S; Takeda, K. Regulation of intestinal homeostasis by
the ulcerative colitis-associated gene RNF186. Mucosal Immunology 10, 446-459 (2017).
Fujiwara, S; Yoshioka, Y; Matsuda, T; Nishimoto, H; Ogawa, A;
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Yoshida, Y; Mikami, N; Matsushima, Y; Miyawaki, M; Endo, H;
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Yoshioka, Y; Kuroda, E; Hirai, T; Tsutsumi, Y; Ishii, KJ. Allergic
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Zaman, MMU; Masuda, K; Nyati, KK; Dubey, PK; Ripley, B; Wang,
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Zaretsky, AG; Konradt, C; Depis, F; Wing, JB; Goenka, R; Atria,
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145

131

The articles were published between April, 2016 and March, 2017. The data were acquired using Web of Science Core CollectionTM on May 12,

132

Sugawara, R; Lee, EJ; Jang, MS; Jeun, EJ; Hong, CP; Kim, JH; Park, A;
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2017, and sorted by alphabetical order of the first authors.

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Accounting : +81-6-6879-4917
Research Planning & Management Office :+81-6-6879-4777+81-6-6879-4272
ifrec-office@ifrec.osaka-u.ac.jp

Access Map

ADDRESS

Graduate School of Medicine
Handaiguchi

Ibaraki Mihogaoka

Senri Gate

Osaka Monorail
Handai-byoin-mae Station
Handai Honbumae

Graduate School of Engineering

HankyuKita-Senri Station

Main Gate

Access from the nearest station
By Hankyu Railway
15 min. walk east from Kita-Senri Station on the
Hankyu Senri Line.

Graduate School of Frontier Biosciences

By Osaka Monorail
25 min. walk from Handai Byoin Mae Station on
the Osaka Monorail.
By Hankyu Bus
Route1:
Get on the bus bound for "Handai Honbu Mae" or"Ibaraki Mihogaoka" from Senri-Chuo Station. Get
off at "Handai Honbu Mae", walk 15 min.
Route 2 :Get on the bus bound for "Onohara Higashi, Fuji
Kasai " from Senri-Chuo Station. Get off at "Handaiguchi", walk 5 min.

Kita Fire
Station

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By Kintetsu Bus
Get on the bus bound for "Handai Honbu Mae"or "Ibaraki Mihogaoka" from Hankyu Ibarakishi
Station (via JR Ibaraki Station). Get off at "Handai
Honbu Mae", walk 15 min.

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