3.6

World Premier International
Research Center

Osaka University
WPI Immunology Frontier Research Center FY 2017

Immunology
Frontier
ResearchCenterAnnual Reportof IFReC
FY 2017

Edit : Research Planning & Management Office, IFReC
Published in June, 2018

Osaka University

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

Message from the Director

CON T E N T S
Message from the Director ・
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・ 1

Organization
Organization Chart ・
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・ 4

Super Science High School Students Fair / Students Visit ・
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・ 73

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

ers at IFReC have been making an increased ef-

am very pleased to present the IFReC annual re-fort to make this innovative attempt successful inport for fiscal 2017.

Japan.

Since its inception in 2007, IFReC has estab-

Although the governing structure has changed,

lished itself as a Visible International Research

our most important mission “Constructing a

Center of Immunology with the support of many

world-top immunology research center” remains

people including the WPI Program Director andthe same. We will make unceasing efforts to de-

the Program Officer.

velop immunology research to ensure translation

In the April of 2017, IFReC became one of the

AAAS 2018 Annual Meeting ・
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・ 75

first members of the “WPI Academy”. Further-

We are committed to continuing contributions

Naturejobs Career Expo ・
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・ 76

more, IFReC created a new mark in its history

to scientific advances through research and edu-

with a novel academic-industry partnership

cation and evolvement as a world top immunol-

agreement. This governance system is an ambi-

ogy research center.

Research Projects
Advanced Postdoc System ・
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・ 78

Laboratories

tious program without precedent. The research-

WPI Science Symposium ・
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・ 74

Committee and Advisory Board for IFReC ・
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Administrative Office of IFReC ・
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As the Director of the Immunology Frontier Re-

to medical science.

Young Scientist Support Program for Research Abroad ・
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Immunology Group ・
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Imaging Group ・
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Data

Informatics Group ・
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Common Facilities ・
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Unit for Combined Research Fields ・
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・ 60

Kishimoto Foundation Fellowship ・
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Major Awards ・
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Events & Outreach Activities

Composition ・
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・ 86

The 7 th NIF Winter School on Advanced Immunology ・
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Finance ・
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・ 87

The 9 th International Symposium of IFReC

Selected Articles ・
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・ 88

- Immunology at the Forefront ・
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Publications ・
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IFReC Seminars ・
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IFReC Colloquia ・
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Science Café ・
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・ 72

Access Map ・
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・ 98

Shizuo Akira, MD, PhD
Director
WPI Immunology Frontier Research Center

1Organization

As of March, 2018

Immunology Group

Organization Chart

MEXT

Osaka university

WPI Initiative
Program Committee

International Advanced
Research Institute (IARI)

JSPS

Director
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
Molecular Neuroscience ....................................................................... Toshihide Yamashita
Molecular Immunology ......................................................................... Sho Yamasaki
Statistical Immunology ......................................................................... Yukinori Okada
Stem Cell Biology and Developmental Immunology .................. Takashi Nagasawa

Imaging Group
Research Groups
Management
Committee

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

Board ofRepresentatives

Administrative
Director
General Affairs Section

Informatics GroupInternational
Scientific Advisory
Board

Systems Immunology ............................................................................ Daron M Standley

Unit for Combined Research Fields

Accounting Section

Quantitative Immunology ................................................................... Yutaro Kumagai
Diego Diez

Administrative Department

Joint Research Chair of Innovative Drug Discovery in Immunology
Research PlanningandManagement Office

Chugai-pharm .......................................................................................... Kunihiro Hattori
Ryusuke Omiya
Junichi Hata

Common Facilities

Cooperative
Institutions

4Core Instrumentation Facility
Animal Resource Center for Infectious Diseases
Network Administration Office

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

Overseas

Indian Institute of Science Education and Research, India

5Committee and Advisory Board for IFReC
World Premier International Research Center Initiative (WPI)
Program Director
Akira Ukawa

As of Mar. 2018

Deputy Director, RIKEN Advanced Institute for Computational Science, Japan

Deputy Program Director
Minoru Yoshida

Chief Scientist, Chemical Genetics Laboratory, RIKEN, Japan

Program Committee Members

6

WPI Academy
In FY 2017, MEXT established the WPI Academy to be the vanguard in internationalizing and further renovating Japan’s research
environment. The WPI Academy is a much-anticipated upgrade of WPI institutes, and is expected to position Japan as a hub at the
pinnacle of international researcher circulation.
The five WPI centers including IFReC are regarded to have achieved “world-premier status”, and thus became the initial members of
the WPI Academy.
In the decade ahead, the research institutes of WPI and WPI Academy will work together to hold public relations and outreach activities.

Toshiaki Ikoma

Special Adviser, Canon Inc., Japan

Academy Director

Shinichiro Ohgaki

President, Japan Water Research Center, Japan

Toshio Kuroki

Maki Kawai

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

Academy Officer for IFReC

Kiyoshi Kurokawa

Professor Emeritus, National Graduate Institute for Policy Studies, Japan

Takehiko Sasazuki

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, Japan

Ryozo Nagai

President, Jichi Medical University, Japan

Special Advisor, Research Center for Science Systems, JSPS, Japan

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

International Scientific Advisory Board

As of Mar. 2018

Jeffrey Ravetch

The Rockefeller University, USA

Immunology

Christopher Goodnow

Australian National University, Australia

Immunology

Richard Locksley

University of California, San Francisco, USA

Immunology

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

Lewis L. Lanier

University of California, San Francisco, USA

Immunology

Hiroshi Matsumoto

President, RIKEN, Japan

Anne O'Garra

The Francis Crick Institute, UK

Immunology

Rita Colwell

Distinguished Professor, University of Maryland, USA

Kiyoshi Takatsu

Toyama Prefectural Institute for Pharmaceutical Research, Japan

Immunology

Richard Dasher

Consulting Professor, Stanford University, USA

Kazuo Sugamura

Miyagi Cancer Center Research Institute, Japan

Immunology

Victor Joseph Dzau

President, National Academy of Medicine, USA

Yale Goldman

University of Pennsylvania, USA

Imaging

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

Yasuyoshi Watanabe

Center for Life Science Technologies, RIKEN, Japan

ImagingKlaus von Klitzing

Masamitsu Iino

Nihon University School of Medicine, Japan

Imaging

Chuan Poh Lim

Chairman, Agency for Science, Technology and Research, Singapore

Akinori Kidera

Yokohama City University, Japan

Informatics

Harriet Wallberg

Former president, Karolinska Institutet, Sweden

Jean Zinn-Justin

Scientific adviser, IRFU/CEA, France7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 and Management Office・ Research Promotion and Support(Consultation for grants and patents, Fusion research program, etc.)
・ Establishing Research Environments(Facility and Safety management, Research agreement, etc.)
・ Fostering Young Scientists(Winter School, Postdoc program, Orientation, etc.)
・ Organizing Scientific Events(Symposia, Colloquia, Seminars, etc.)
・ Public Relations(Publishing, Website, Outreach to citizens, etc.)

As of April, 20188

Shizuo Akira, MD/PhD

Professor

Shizuo Akira

Associate Professor

Kazuhiko Maeda

Assistant Professor

Takashi Satohveloped spontaneous autoimmune diseases accompanied bysplenomegaly and lymphadenopathy. Regnase-1 has the endonuclease activity and destabilizes a set of mRNAs through cleavage of their 3' UTRs such as IL-6 and IL-12 p40 in macrophages
and c-Rel, Ox40, and IL-2 in CD4+ T cells. Regnase-1 itself is cleavedby Malt1 protease after T cell receptor stimulation and enhances
T cell activation. Consistently, the protease activity 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 the understanding of the precious role of Regnase-1 in immune and non-immune cells by using both mutant
and tissue-specific Regnase-1 deficient mice. We are studying toachieve the goal of a comprehensive understanding of the innate
immune system and to develop an effective treatment for immune-related inflammatory diseases.

Immunology

Host Defense

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, SatM
Macrophages consist of at least two subgroups. M1 macrophages
are pro-inflammatory and have a central role in host defense. On
the contrary, M2 macrophages are associated with responses toanti-inflammatory reactions, and tissue remodeling. Monocytes
and macrophages comprise a variety of subsets with diverse
functions. It is thought that these cells play a crucial role in homeostasis of peripheral organs, key immunological processes anddevelopment of various diseases. Among these diseases, 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 associated with activation of
monocytes and macrophages. Recently, we have identified a new
macrophage subset in that Ceacam1+Msr1+Ly6C-F4/80-Mac1+
monocytes, which we termed segregated-nucleus-containing
atypical monocytes (SatM), share granulocyte characteristics, areregulated by CCAAT/enhancer binding protein beta (C/EBPβ),10

Postdoctoral Fellow

2

Research Assistant

8

Visiting Scientist

5

Support Staff

8

and are critical for fibrosis. We are further investigating physiological role of SatM and related subsets.

Role of basic leucine zipper transcription factor ATFlike 2 (Batf2) in tumor-associated macrophages
The development of effective treatments against cancers is urgently needed, and the accumulation of CD8+ T cells within tumors is especially important for cancer prognosis. Innate immune
cells play an important role in the cancer progression through the
production of various cytokines. We found that basic leucine zipper transcription factor ATF-like 2 (Batf2) has an antitumor effect.
Subcutaneous inoculated tumor model produced fewer IL-12
p40+ macrophages and activated CD8+ T cells within the tumorsof Batf2-deficient mice compared with WT mice. In vitro studies
showed that the IL-12 p40 expression was significantly lower in
Batf2-deficient macrophages following their stimulation by TLR
ligands. Additionally, we found that BATF2 interacts with NF-kB
p50/p65 and promotes IL-12 p40 expression. BATF2 has an antitumor effect through the up-regulation of IL-12 p40 in tumor-associated macrophages, which eventually induces CD8+ T-cell activation and accumulation within the tumor. Therefore, BATF2
may be an important target in anticancer treatment with immune
checkpoint blockers and TLR agonists.

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 CCCH-typezinc finger proteins. Conventional Regnase-1 knockout mice de-Figure 1.
The morphology of SatM.

Figure 2.
Concept of disorder specific macrophages.

Recent Publications● Kanemaru H. et al. BATF2 activates DUSP2 gene expression and upregu-● Eid MMA. et al. Integrity of immunoglobulin variable region is supported

lates NF-κB activity via phospho-STAT3 dephosphorylation. Int. Immunol.
(2018) in press.

by GANP during AID-induced somatic hypermutation in germinal center
B cells. Int. Immunol. 29, 211-220 (2017).

● Nakagawa K. et al. Schlafen-8 is essential for lymphatic endothelial cell

● Maeda K & Akira S. Regulation of mRNA stability by CCCH-type zinc finger

activation in experimental autoimmune encephalomyelitis. Int. Immunol.
30, 69-78 (2018).

proteins in immune cells. Int. Immnunol. 29, 149-155 (2017).

● Kanemaru H. et al. Antitumor effect of Batf2 through IL-12 p40 up-regula-tion in tumor-associated macrophages. Proc. Natl. Acad. Sci. USA. 114,
E7331 (2017).
11

Immunology

Immunoglycobiology
PGAP3

Taroh Kinoshita, PhD
Yoshiko Murakami, MD/PhD

PGAP4

PGAP4

PGAP2

EDD
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Professor

Taroh Kinoshita
Yoshiko Murakami

Research Assistant

1

Visiting Scientist

3

Support Staff

4

(15 )

EtN
P

P EtN

(16 )

EtN
P

P EtN

P

P

(17 )

EtN
P

H

P EtN

P

P EtN

P

stem
I

P

TM3

Golgi

TM1

TM2

Fatty acidremodeling

Glucosamine
MannoseSide-chain
modification

P EtN

Ethanolamine phosphateP

1-alkyl-2-acyl or diacyl PI

Unsaturated chainP

N-acetylgalactosamine

We have been studying biosynthesis, functions and deficiencies of glycosylphosphatidylinositol (GPI) and GPI-anchored proteins (GPI-APs). In 2017, we made the following progress in thestudies on GPI biosynthesis and GPI deficiency.

Studies on GPI-AP biosynthesis
GPI-anchors in some mammalian GPI-APs are modified by aside-chain glycan. Biosynthesis and biological functions of theside-chain modification have been unclear. We identified a Golgiresident N-acetylgalactosamine transferase and termed it PGAP4.
PGAP4 transfers N-acetylgalactosamine side-chain to GPI-anchor.
This is the 17th step in the GPI-AP biosynthesis pathway occurring after fatty acid remodeling mediated by PGAP3 and PGAP2(Figure). PGAP4 has a novel structure among many Golgi-resident
glycosyltransferases: PGAP4 has three transmembrane domains(TMs) whereas many other glycosyltransferases are type II single
membrane pass proteins. PGAP4 has type II membrane proteincharacteristics in the N-terminal part (short cytoplasmic part, TM1
and stem region) and in the C-terminal portion has tandem transmembrane domains (TM2 and TM3) insertion into a typical glycosyltransferase domain as shown in the 3D structural model (Figure). We propose TM2 and TM3 act to localize a glycosyltransferase
domain close to the Golgi membrane where the substrate GPI isinserted (Hirata, T. et al., Nat. Commun., 2018).
We collaborated with Dr. Fujita in Jiangnan University, China,
and studied regulation of the first GPI remodeling reaction (Inositol deacylation) that occurs soon after attachment of GPI to proteins. We found that N-glycan attached to nascent GPI-AP regu12

lates GPI-inositol deacylation. Mechanistically, a molecularchaperone calnexin associates with GPI-APs, dependent upon Nglycan and GPI, and facilitates GPI-inositol deacylation. These results indicate that N-glycan participates in quality control of GPIAP folding before exiting the endoplasmic reticulum (Liu, Y. et al,
J. Cell Biol., 2017).

Saturated chain

Figure.
PGAP4 transfers N-acetylgalactosamine side-chain to GPI-anchor at the 17th step in GPI-anchored protein
biosynthesis pathway (left). PGAP4 has type II membrane protein characteristics in the N-terminal part(short cytoplasmic part, TM1 and stem region) and in the C-terminal portion has tandem transmembrane
domains (TM2 and TM3) insertion into a glycosyltransferase domain as shown in the 3D structural model(right).

Studies on inherited GPI deficiencies (IGDs)
In collaborations with clinicians and medical geneticists working with IGD caused by PIGO mutations, we clarified genotype phenotype correlations of PIGO deficiency with variable phenotypes from infantile lethality to mild learning difficulties (Tanigawa,J. et al., Hum. Mutat., 2017). In collaborations with Dr. Campeau
group in Canada, who identified the first cases of IGD having mutations in GPAA1 gene, we characterized functional abnormalities caused by those mutations. GPAA1 encodes a subunit of GPItransamidase that transfers GPI to precursor proteins to generate
GPI-APs. We found the patients’ mutations cause partial losses offunction of GPAA1 (Nguyen, T.T.M. et al., Am. J. Hum. Genet., 2017).
In collaboration with Dr. Pagnamenta and colleagues in UK, who
identified the first case of IGD caused by mutations in PIGH gene,
we determined functions of PIGH proteins generated from themutant gene (Pagnamenta, A.T. et al., Hum. Mutat., 2018). Pathogenic mutations that cause IGDs were found in 18 of 27 genes forbiosynthesis and maturation of GPI. Knowledge gained fromthese studies contributes to our deeper understanding of IGDs
and progress in development of better diagnostics of IGDs andeffective measures to ameliorate symptoms of IGDs.

Recent Publications● Hirata T, Mishra SK, Nakamura S, Saito K, Motooka D, Takada Y, Kanzawa N,● Makrythanasis P, Kato M, Zaki M, Saitsu H, Nakamura K, Santoni F,

Murakami Y, Maeda Y, Fujita M, Yamaguchi Y & Kinoshita T. Identificationof a Golgi GPI-N-acetylgalactosamine transferase with tandem transmembrane regions in the catalytic domain. Nat. Commun. 9, 405 (2018).

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 & Murakami Y. Pathogenic variants in PIGG
cause intellectual disability with seizures and hypotonia. Am. J. Hum.
Genet. 98, 615-626 (2016).

● Tanigawa J, Mimatsu H, Mizuno S, Okamoto N, Fukushi D, Tominaga K,

Kidokoro H, Muramatsu Y, Nishi E, Nakamura S, Motooka D, Nomura N,
Hayasaka K, Niihori T, Aoki Y, Nabatame S, Hayakawa M, Natsume J, Ozono K,
Kinoshita T, Wakamatsu N & Murakami Y. Phenotype-genotype correlations of PIGO deficiency with variable phenotypes from infantile lethality
to mild learning difficulties. Hum. Mutat. 38, 805-815 (2017).

● Hirata T, Fujita M, Nakamura S, Gotoh K, Motooka D, Murakami Y, Maeda Y& Kinoshita T. Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport. Mol. Biol. Cell. 26, 30713084 (2015).

● Lee G-H, Fujita M, Takaoka K, Murakami Y, Fujihara Y, Kanzawa N, Murakami K,

Kajikawa E, Takada Y, Saito K, Ikawa M, Hamada H, Maeda Y & Kinoshita T.
A GPI processing phospholipase A2, PGAP6, modulates Nodal signaling inembryos by shedding CRIPTO. J. Cell Biol. 215, 705-718 (2016).
13

Immunology

Immunopathology
Atsushi Kumanogoh, MD/PhD

Professor

Atsushi Kumanogoh

Assistant Professor

Shohei Koyama

Support Staff

5

Figure.
During IL-4 induced M2 activation, mTORC1 is activated and upregulates Sema6D,that in turn activates c-Abl kinase to PPARγ expression. Activated PPARγ mediates
fatty acid uptake and metabolic reprogramming, which are essential events forfully M2 activation. This Sema6D- PPARγ axis signaling confers anti-inflammatory
properties on CX3CR1hi intestinal macrophage and prevent development of colitis.

Our research team is involved in two approaches, that is, basic
and clinical immunology. As basic aspects of our projects, ourproposed 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
in immune 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 the diagnosis/therapy for human immunological disorders, such as autoimmunity, allergy, immune deficiency, cancer/metastasis, and neurodegenerative diseases. We focus hereon the functions of Semaphorin 6D (Sema6D) in neuronal, immune and metabolic systems.
Polarization of macrophages into the pro-inflammatory M1 oralternative M2 states has distinct metabolic requirements. Emerging evidence points to a critical role of mTORC (mechanistic target of rapamycin complex) pathway in macrophage polarization.
However, it remains unclear how metabolic status regulates macrophage activation state. In this study, we found that following
exposure to exogenous stimuli, mTORC activity–dependent Sema6D expression plays an essential role in M2 polarization via
regulation of fatty acid uptake and metabolism. Loss of Sema6D
in macrophages was refractory to Th2 cytokine–induced M2 macrophage activation, but promoted pro-inflammatory cytokine
14

production. Moreover, Sema6D-mediated PPARγ expression isindispensable not only for optimal M2 macrophage polarization,
but also for uptake of fatty acid and reprogramming of related
metabolism. Specifically, we found that upon IL-4 stimulation, association with the tyrosine kinase c-Abl via the SH3 domain of
Sema6D contributes to PPARγ expression through reverse signaling. Furthermore, we found that the Sema6D–PPARγ signaling
axis governs the anti-inflammatory function of intestinal resident
CX3CR1hi macrophages and prevents development of colitis invivo and in vitro. Collectively, these findings highlight a crucialrole for Sema6D reverse signaling in modulating macrophage
polarization, and provide mechanistic insights into metabolic reprogramming with implications for macrophage homeostasis.

Recent Publications● Kang S, Nakanishi Y, Kioi Y, Okuzaki D, Kimura T, Takamatsu H, Koyama S,● Kimura T,Nada S, Takegahara N, Okuno T, Nojima S, Kang S, Ito D, Morimoto K,

Nojima S, Nishide M, Hayama Y, Kinehara Y, Kato Y, Nakatani T, Shimogori T,
Takagi J, Toyofuku T and Kumanogoh A. Semaphorin 6D reverse signaling
controls macrophage lipid metabolism and anti-inflammatory polarization. Nat. Immunol. (2018) in press.

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. Nat. Commun. 7, 13130 (2016).

● Nishide M and Kumanogoh A. The role of semaphorins in immune re-sponses and autoimmune rheumatic diseases. Nat. Rev. Rheumatol. 14,
19-31 (2018).
● Hosen N, Matsunaga Y, Hasegawa K, Matsuno H, Nakamura Y, Makita M,

Watanabe K, Yoshida M, Satoh K, Morimoto S, Fujiki F, Nakajima H, Nakata J,
Nishida S, Tsuboi A, Oka Y, Manabe M, Ichihara H, Aoyama Y, Mugitani A,
Nakao T, Hino M, Uchibori R, Ozawa K, Baba Y, Terakura S, Wada N, Morii E,
Nishimura J, Takeda K, Oji Y, Sugiyama H, Takagi J and Kumanogoh A. The
activated conformation of integrin β7 is a novel multiple myeloma-specific target for CAR T cell therapy. Nat. Med. 23, 1436-1443 (2017).
15

Hisashi Arase, MD/PhD

Professor

Hisashi Arase

Associate Professor

Tadahiro Suenaga

Assistant Professor

Masako Kohyama

Immunology

Immunochemistrycorrelated with susceptibility to autoimmune disease. This suggested that misfolded proteins, which normally would not be
exposed to the immune system, can be targets for autoantibodies as ‘neo self’ antigens, which are involved in the pathogenicity
of autoimmune diseases (Figure 2).

Kouyuki Hirayasu

We have been working on the interactions between pathogens and various paired receptors. In addition, we have foundthat MHC class II molecules function as molecular chaperons totransport cellular misfolded proteins to the cell surface. Analysesof misfolded proteins transported to the cell surface revealed
that these proteins are involved in autoimmune diseases as a target for autoantibodies.

Host pathogen interaction mediated by paired receptor
Paired receptors are composed with activating and inhibitory
receptors. PILRα is one of paired inhibitory receptors that are expressed on various immune cells. We have shown that PILRα
plays an important role in the regulation of immune response(Wang et al. Nat. Immunol. 2012; Kishida et al. Int. Immunol. 2015;
Kohyama et al. Eur. J. Immunol. 2016). We also 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). Similarly, Siglec-4(MAG, myelin associated glycoprotein), one of paired receptors,
associates with varicella zoster virus (VZV) gB and mediates VZV
infection (Suenaga et al. Proc. Natl. Acad. Sci. USA. 2010; Suenaga
et al. J. Biol. Chem. 2015). These findings suggested that paired
receptors are involved in viral infection.
LILR is another type of paired receptor family. We found that
activating LILRA2 recognizes abnormal immunoglobulins
cleaved by microbial proteases but not normal immunoglobu16

Postdoctoral Fellow

4

Research Assistant3

lins. LILRA2 seems to be a sensor to detect immunoglobulin abnormalities in microbial infection (Hirayasu et al. Nature Microbiology 2016). On the other hand, we found that RIFINs, products ofthe multigene family of Plasmodium falciparum, bind to inhibitory LILRB1 and downregulate immune response. Furthermore, expression of RIFINs was associated with severe malaria. These findings suggest that binding of RIFIN to LILRB1 plays an importantrole in immune evasion by Plasmodium falciparum (Figure 1. Saito
et al. Nature 2017).

Figure 1. Immune evasion of Plasmodium falciparum by RIFIN via
inhibitory receptors.
Plasmodium falciparum induces the expression of RIFINs on the surface of
infected erythrocytes. Individual RIFINs may have evolved to target host
inhibitory receptors, thus facilitating escape from host immune systems,
which may lead to inefficient development of immunity against malaria
parasites (Satio et al. Nature 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 the 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).

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). Similarly, we also found thatmyeloperoxidase/HLA class II complex is a target for autoantibodies in ANCA-associated vasculitis (Hiwa et al. Arthritis. Rheumatol. 2017). Autoantibody binding to misfolded proteins transported to the cell surface by MHC class II molecules was strongly

Recent Publications● Saito F, Hirayasu K, Satoh T, Wang CW, Lusingu J, Arimori T, Shida K,● Jin H, Arase N, Hirayasu K, Kohyama M, Suenaga T, Saito F, Tanimura K,

Palacpac NMQ, Itagaki S, Iwanaga S, Takashima E, Tsuboi T, Kohyama M,
Suenaga T, Colonna M, Takagi J, Lavstsen T, Horii T, Arase H. Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors. Nature.
552, 101-105 (2017).

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).

● Hiwa R, Ohmura K, Arase N, Jin H, Hirayasu K, Kohyama M, Suenaga T,

Saito F, Terao C, Atsumi T, Iwatani H, Mimori T, Arase H. Myeloperoxidase/
HLA Class II Complexes Recognized by Autoantibodies in Microscopic
Polyangiitis. Arthritis. Rheumatol. 69, 2069-2080 (2017).

● Wang J, Shiratori I, Uehori J, Ikawa M, Arase H. Neutrophil infiltration dur-ing inflammation is regulated by PILRα via modulation of integrin activation. Nat. Immunol. 14, 34-40 (2012).

● Hirayasu K, Saito F, Suenaga T, Shida K, Arase N, Oikawa K, Yamaoka T, Murota H,

Chibana H, Nagai H, Nakamura Y, Katayama I, Colonna M, Arase H. LILRA2
is an innate immune sensor for microbially cleaved immunoglobulins.
Nat. Microbiol. 1, 1-7 (2016).
17

the CNS. Thus, our findings suggest that Arid5a-OX40 axis in Th17
cells plays important roles in the pathogenesis of EAE.

Tadamitsu Kishimoto, MD/PhD

Professor

Tadamitsu Kishimoto

Associate Professor

Shigeru Hashimoto

Assistant Professor

Sujin Kang

Postdoctoral Fellow

1

Research Assistant

3

Visiting Scientist

3

Support Staff

3

Regulation of inflammatory responses by dynamic
subcellular localization of RNA-binding protein Arid5a(published)
Although Arid5a plays important roles in immune regulation,
how its subcellular localization impacts immune regulation has
remained unclear. We found that IL-6 was substantially produced
by LPS stimulation in Arid5a overexpressing-transgenic mice, but
not in an unstimulated condition, indicating that the dynamics
and subcellular localization of Arid5a might be important for its
function in the immune response. In addition, Arid5a was imported into the nucleus via a classical importin-α/β1-mediated pathway. On stimulation, nuclear Arid5a levels decreased whereas
cytoplasmic Arid5a levels increased. Moreover, Arid5a was associated with up-frameshift protein 1, and its nuclear export was
regulated by a nuclear export receptor, chromosomal region
maintenance 1. Taken together, our results suggest the importance of the nucleocytoplasmic localization of Arid5a in inflammation through a change in the Arid5a:Regnase-1 ratio (Figure 1).

Analysis of anti-inflammatory effects of lenalidomide(ongoing)

therapeutic action in several autoimmune/inflammatory diseases. However, it is difficult to study the mechanism of IMiD action
in murine disease models because murine cereblon—the substrate receptor for IMiDs action—is resistant to some of IMiDs’
therapeutic effects. To overcome this difficulty, we generated humanized cereblon mice, thereby providing a novel animal modelto unravel complex IMiD mechanisms of action in mouse physiological setup. In our current study, we investigated potential IMiD
degradative effects towards Ikaros family zinc finger protein 1/3(IKZF1/3), a potential target substrate of lenalidomide. Unlike
wild type mice, which were resistant to Lenalidomide, T lymphocytes from humanized cereblon mice responded with a higher
degree of IKZF1/3 protein degradation. Furthermore, IMiD degradation of IKZF1/3 resulted in an increase in intereukin-2 among
humanized cereblon mice but not in the wild type group. Additionally, we assessed the potential therapeutic effects of IMiDs in
dextran sulfate sodium (DSS) induced colitis. In both WT and humanized mice lenalidomide showed a significant therapeutic effect in DSS model of colitis, while the effect of pomalidomide wasless pronounced. We conclude that lenalidomide offers therapeutic opportunities against inflammatory diseases independentof cereblon whereas its effect on IKZF1/3 and interleukin-2 is totally dependent of cereblon.

Immunology

Immune Regulation

Lenalidomide is an immunomodulatory drug (IMiD) having a

Introduction
Interleukin-6 (IL-6) is an important cytokine in the early phaseof acute immune responses, as well as activation of lymphocytes,
hematopoietic cells and vascular endothelial cells to protect the
body against invasion of pathogens. On the other hand, persistent production of IL-6 leads to development of various chronic
diseases such as rheumatoid arthritis (RA) and juvenile idiopathic
arthritis (JIA). The significance of IL-6 in the pathogenesis of chronic diseases, has been successfully proven by tocilizumab, a humanized anti-IL-6 receptor antibody, when used for those chronic immune disorders. In addition, a high level of IL-6 expression in
the sera of patients has also been responsible for the induction ofsystemic inflammatory responses syndrome (SIRS) and cytokine
release syndrome (CRS) To pursue the molecular mechanisms ofoverproduction of IL-6, we focused on IL-6 gene expression in activated macrophages under TLR signaling. IL-6 mRNA is tightly
regulated by RNA binding proteins, the dysfunction of whichleads to sustained IL-6 production. Previously, we identified an
RNA-binding protein, AT-rich interactive domain 5a (Arid5a), whichbinds to the 3'UTR of IL-6 mRNA. Arid5a is highly expressed inmacrophages in response to lipopolysaccharide (LPS) and stabilizes Il6 mRNA by binding to its 3'-UTR. Additionally, Arid5a alsostabilizes either Stat3 or T-bet mRNAs in T cells and is involved in
T cell-fate regulation. Moreover, Arid5a-deficient (Arid5a−/−) mice
are highly resistant to endotoxic shock, experimental autoimmune
encephalomyelitis (EAE), and bleomycin-induced lung injury.
These findings suggest that Arid5a plays important roles in posttranscriptional regulation and inflammatory disease control.
18

TLR4-induced NF-κB and MAPK signaling regulate IL-6
mRNA-stabilizing protein Arid5a (published)
Arid5a mRNA expression is induced in response to LPS stimulation, however, the mechanisms of the Arid5a protein during stabilization of IL-6 mRNA has remained unclear. We identified that
NF-κB and an NF-κB-triggered IL-6-positive feedback loop activate Arid5a gene expression, increasing IL-6 expression via stabilization of the IL-6 mRNA during the early phase of TLR4 stimulation. Subsequently, AU-rich element RNA-binding protein 1 binds
to the Arid5a mRNA 3'-UTR to destabilize it, which involves MAPK
phosphatase-1. During the late phase of TLR4-stimulation, p38
phosphorylates Arid5a, leading to the degradation of Arid5a
through the E3 ubiquitin ligase, WWP1. Inhibition of Arid5a phosphorylation increased IL-6 expression. Thus, the regulation of
Arid5a by NF-κB and MAPK signaling is required to maintain thebalance of IL-6 mRNA expression.

Arid5a stabilizes OX40 mRNA in murine CD4+ T cells byrecognizing a stem-loop structure in its 3'-UTR (published)
The roles of Arid5a in Th17 cells and its association with autoimmunity remain unknown.
Deficiency of Arid5a in T cells decreased OX40 expression levels and repressed IL-17 production upon OX40 ligation. Notably,
Arid5a stabilized OX40 mRNA by recognizing the alternative decay element (ADE)-like stem-loop (SL) in the 3'-UTR. Arid5a−/−mice showed resistance to EAE by either decreased OX40 expression in CD4+ T cells or reduction of CD4+CD45+ T cell numbers in

Figure.
Model of inflammatory response by Arid5a/Regnase-1/UPF1.

Recent Publications● Higa M. et al. Regulation of inflammatory responses by dynamic subcel-● Millrine D. et al. Rabex-5 is a lenalidomide target molecule that negatively

lular localization of RNA-binding protein Arid5a. Proc. Natl. Acad. Sci. U S A.
115(6), E1214-E1220 (2018).

regulates TLR-induced type 1 IFN production. Proc. Natl. Acad. Sci. USA.
20,113(38),10625-30 (2016).

● Hanieh H. et al. Arid5a stabilizes OX40 mRNA in murine CD4<sup>+</● Masuda K. et al. Arid5a regulates naive CD4+ T cell fate through selectivesup> T cells by recognizing a stem-loop structure in its 3'UTR. Eur. J. Immunol. 48(4), 593-604 (2018).

stabilization of Stat3 mRNA. J. Exp Med. 213(4), 605-19 (2016).

● Nyati KK. et al. TLR4-induced NF-κB and MAPK signaling regulate the IL-6

mRNA stabilizing protein Arid5a. Nucleic Acids Res. 45(5), 2687-2703(2017).
19

Kiyoshi Takeda, MD/PhD

Professor

Kiyoshi Takeda

Associate Professor

Eiji Umemoto

Assistant Professor

Hisako Kayama

Immunology

Mucosal Immunology
porter Asbt, which is expressed in epithelial cells, restored Mdr1deficient Teff cell homeostasis and attenuated ileitis. Furthermore, a subset of ileal Crohn’s disease patients exhibited
MDR1loss of function. Together, these results suggest that coordinated interaction between Teff cells and CBAs in the ileum
regulates intestinal immune homeostasis (Fig. 2).

Ryu Okumura
Mari Murakami

Regulation of protozoa-induced immune response by
BATF2
Chagas’ disease, characterized by chronic cardiomyopathy, iscaused by infection with the intracellular protozoan parasite Trypanosoma cruzi. Although IL-17 confers protection against lethal
T. cruzi infection, inappropriate IL-17 responses are implicated inchronic tissue inflammation. IL-23 contributes to T. cruzi-specificIL-17 production, but the molecular mechanisms underlying theregulation of the IL-23/IL-17 axis during T. cruzi infection arepoorly understood. The Toll-like receptor (TLR)–MyD88/TRIF adaptor molecule pathways initiate both innate and adaptive immune
responses and thus contribute to the resistance of the host to T.
cruzi. In addition, the mechanisms underlying the TLR-independent host defense against T. cruzi infection have recently been
demonstrated. We found that transcription factor BATF2 is induced in Mф and DCs in a TLR-independent and IFN-γ-dependent
manner. Therefore, we analyzed the roles of IFN-γ-inducible
BATF2 in T. cruzi-infected innate immune cells. In the spleens andlivers of T. cruzi-infected Batf2–/– mice, IL-17, but not IFN-γ, wasmore highly produced by CD4+ T cells compared to those of wildtype mice. In this context, Batf2–/– mice showed severe multi-organ pathology, despite reduced levels of parasitemia. In Batf2–/–
innate immune cells, T. cruzi-induced production of IL-23, but not
IL-6, was increased. The T. cruzi-induced enhanced Th17 response
was abrogated in Batf2–/–Il23a–/– mice. The interaction of BATF2
with c-JUN prevented c-JUN/ATF-2 complex formation, thereby
inhibiting Il23a expression. Taken together, IFN-γ-inducible BATF2
expressed in the innate immune cells suppresses IL-23 produc20

Visiting Scientist

1

Support Staff

3

tion by preventing the formation of the c-JUN/ATF-2 heterodimer, resulting in the inhibition of excessive Th17 responses during T. cruzi infection (Fig. 1).

Intestinal metabolites that regulate mucosal inflammation
Pro-inflammatory CD4+ T effector (Teff) cells continually circulate in intestinal tissue to provide immune surveillance, as intestinal mucosal is constantly exposed to potentially pathogenic
stimuli. However, an excess of pro-inflammatory cytokines is implicated in the pathogenesis of inflammatory bowel diseases including Crohn’s disease and ulcerative colitis. Therefore, Teff cells
are tightly regulated by innate myeloid cells, such as CD103+ DCs
and CX3CR1+ phagocytes, and microbiota in the intestine. But
whether intestinal T cells interface with host-derived metabolites
is less clear. Mucosa-associated taurine-/glycine-conjugated bile
acids (CBAs) in the ileum are present at high micro-molar concentrations. We found that the xenobiotic transporter, Mdr1, is expressed in mouse Teff cells at steady-state, where it is highest in
the ileum. In addition, CD103+ dendritic cells in the small intestine
induced Mdr1 expression in Teff cells. Wild-type Teff cells upregulated Mdr1 in the ileum, but those lacking Mdr1 displayed mucosal dysfunction and induced Crohn’s disease-like ileitis in naïve T
cells-transferred Rag1–/– mice. Both in vitro and in the small intestine in vivo, Mdr1 mitigated oxidative stress and pro-inflammatory cytokines expression and then enforced homeostasis in Teff
cells during CBAs exposure. Blocking ileal CBA reabsorption in
Rag1–/– mice lacking an apical sodium-dependent bile acid trans-

Figure 1.
Transcription factor BATF2 induced by IFN-γ in Mф and DCs prevents Th17mediated multi-organ pathology by suppressing IL-23 production throughprevention of the c-JUN/ATF-2 heterodimer formation during T. cruzi
infection.

Figure 2.
CD4+ T effector (Teff) cells in the ileum upregulates Mdr1 expression in a
CD103+ DCs-dependent mechanism. Mdr1 protects Teff cells from bile aciddriven oxidative stress and pro-inflammatory cytokines expression in theileum.

Recent Publications● Cao W, Kayama H, Chen ML, Delmas A, Sun A, Kim SY, Rangarajan ES, McK-

● Okumura R, Kurakawa T, Nakano T, Kayama H, Kinoshita M, Motoka D,evitt K, Beck AP, Oikonomopoulos A, Lacey PN, Martinez GJ, Izard T, Lorenz
RG, Rodriguez-Palacios A, Cominelli F, Abreu MT, Hommes DW, Koralov SB,
Takeda K, Sundrud, MS: The xenobiotic transporter Mdr1 permits T cell
adaptation to bile acid reabsorption in the ileum. Immunity 47, 11821196 (2017).

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, 117-121 (2016).
● Tsai SH, Kinoshita M, Kusu T, Kayama H, Okumura R, Ikeda K, Shimada Y,● Kitada S, Kayama H, Okuzaki D, Koga R, Kobayashi M, Arima Y, Kumanogoh A,

Murakami M, Ikawa M & Takeda K. BATF2 inhibits immunopathologicalTh17 responses by suppressing Il23a expression during Trypanosoma
cruzi infection. J. Exp. Med. 214, 1313-1331 (2017).

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).

● Maeda Y, Kurakawa T, Umemoto E, Motooka D, Ito Y, Gotoh K, Hirota K,

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).
21

Hitoshi Kikutani, MD/PhD

Structural basis for antigen recognition of anti-DNA
antibody in systemic lupus erythematosus (SLE)
Anti-DNA antibody is the established criterion for SLE diagnosis and its contribution to clinical symptoms in SLE has been
widely accepted. However, 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-bound Fab of one mAb clone (designated as 71F12) was solved (Protein Data Bank [PDB] ID: 5GKR).
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. The structure revealed that this antibody recognizes thymine-containing oligonucleotides in a manner that is only compatible with ssDNA unlike previously
proposed models. We speculate that high affinity anti-dsDNA
antibodies target “loosened” single-stranded segments, which
may be present in a released dsDNA upon cell death.

22

Professor

Hitoshi Kikutani

Assistant Professor

Shuhei Sakakibara

Postdoctoral Fellow

1

Research Assistant1

Support Staff

1

ated through rigorous clonal expansion and affinity maturation,in which low-affinity anti-ssDNA B cells can acquire high-affinity
to both ss- and dsDNA by only one or two mutations (Figure 2).
Now, the findings raise the questions of how low-affinity anti-ssDNA precursor B cells escape from tolerance checkpoints and inwhat conditions they undergo clonal expansion and affinity mutation to differentiate into high affinity anti-dsDNA antibodyproducing cells. To address these questions, we generated a sitedirected knock-in (KI) mouse line, G9gl, which carries unmutated
IgH and L chains derived from one of the SLE anti-DNA antibody
clones. The number of G9gl-expressing B cells was reduced inblood and lymphoid organs of the KI mice. In the G9gl heterozygotes, about 60% of B cells expressed endogenous BCR and their
KI heavy chain alleles were inactivated via aberrant receptor editing. Although G9gl+ B cells displayed reduced levels of surface
BCR, they were not functionally anergic and could respond efficiently to LPS, anti-CD40 or BCR crosslinking. Moreover, ssDNA
could induce vigorous activation and proliferation in G9gl B cells.
A fraction of G9gl mice exhibited high titer of serum autoantibodies such as anti-DNA IgG with spontaneous germinal center formation in spleen. Taken together, low affinity precursors of SLEderived anti-DNA B cells can escape from tolerance check point
during early B cell development and retain the capability to trigger self-reactive germinal center reaction.

sal 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 IgEproduced 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 the sinonasal cavity, such as Streptococcus pyogenes. Next, to compre-

hend the development pathway of the antigen-specific IgE, deep
sequencing of NP-associated BCR repertoires was performed(Figure 3). It showed that major clonal lineages of IgE were often
found in IgG or IgA1 clonal lineages, indicative of sequential classswitch 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 1. X-ray crystallography of anti-DNA antibody 71F12.
The Fab binds to thymines through a stacking interaction (W101 of theheavy chain) and several hydrogen bonds. It should be noted that mutatedF33 of the heavy chain locates at the antigen recognition site.

Figure 2.
Working model of generation process of anti-dsDNA antibodies in SLE.
Unmutated low-affinity anti-ssDNA precursors cannot bind to dsDNA. After
SHM, high-affinity anti-DNA clones acquire binding capability to both ss
and dsDNA at high affinity.

Immunology

Immune Regulation

Figure 3. Clonal connectivity between isotypes in NP-associatedimmunoglobulins.
Deep sequencing analysis of expressed BCR repertoires unraveled theclone distribution across immunoglobulin isotypes. Large IgE clones werestrongly connected with IgG and IgA lineages.

Recent Publications

Characterization of low-affinity progenitor B cells forpathogenic anti-dsDNA antibody-producing cells derived from SLE

Tracking allergy-related IgE in chronic rhinosinusitis
with nasal polyposis (CRSwNP)Our previous study has demonstrated that SLE ANA are gener-

CRSwNP is characterized by eosinophilic inflammation and na-● Sakakibara S. et al. Clonal evolution and antigen recognition of anti-nu-

clear antibodies in acute systemic lupus erythematosus. Sci Rep 7(1),16428 (2017).
23

Shimon Sakaguchi, MD/PhD

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. In the previous years, westudied how Treg cells controlled humoral immune responses
and showed that CTLA-4-expressing T follicular regulatory (Tfr)
cells suppressed B cell expression of CD80 and CD86, which wasessential for T follicular helper (Tfh) cell formation and germinal
center (GC) development (Wing and Sakaguchi, Int. Immunol.
2013; Wing et al., Immunity 2014). This year, we have further explored which type of Tfr cells controls Tfh cells and germinal center formation (Wing et al., PNAS, 2017). Tfh cells differentiate
through a multistep process, culminating in GC-localized GC-Tfh
cells that provide support to GC B-cells. Tfh are known to be inhibited by IL-2 which suppresses expression of the canonical Tfh
transcription factor BCL6. On the other hand, we initially identified Tregs on the basis of their expression of the IL-2 receptor alpha chain (CD25) and broadly speaking Treg survival and proliferation is dependent on IL-2 (Sakaguchi et al., J. Immnol, 1995).
Since Tfr are a type of Treg but must also express BCL6 this puts
them in a unique position of needing to balance the IL-2 depen24

Professor

Shimon Sakaguchi

Associate Professor

Wing James Badger

Assistant Professor

Kenji Ichiyama

Postdoctoral Fellow

3

Research Assistant4

Support Staff

5dent Treg identify with IL-2 inhibited BCL6 expression. While investigating this issue we have found a novel CD25-negative subpopulation within both murine and human PD1+CXCR5+Foxp3+
Tfr cells. Downregulation of CD25 appears to be a marker of Tfr
development with CD25+Tfr forming initially before later formation of more highly differentiated CD25-Tfr. During the response
to protein vaccination CD25+Tfr formation occurs earlier ataround days 3-5 whereas CD25-Tfr form later at around day 6-7
and coincide with peak germinal center formation. Microscopic
analysis of Tfr in the spleens and draining lymph nodes of vaccinated mice reveals that while the majority of Tfr resident in thefollicle and near the T-B border express CD25, almost all germinal
center resident Tfr lack CD25 expression. Accordingly, detailed
analysis of chemokine receptor and cell adhesion molecules
demonstrates that, in keeping with their germinal enter localization, CD25-Tfr express significantly increased levels of CXCR5,
while reducing expression of molecules responsibly for maintainlocalization in the T-cell zones such as CCR7 and PSGL-1. Further
characterization by flow cytometry and RNA-sequencing showsthat while CD25+Tfr are more similar to effector Tregs, CD25-Tfr
have shifted their gene expression signature to a point equidistant between Tfh and effector Tregs displaying an extreme levelof flexibility in their phenotype. Despite this they retain stable
expression of Foxp3, maintain a characteristic Treg epigenetic
signature and express key Treg suppressive molecules such as
CTLA-4, allowing them to suppress both T-cells and B-cells duringin vitro co-culture. We found that circulating CD45RO+ Tfr in human blood also downregulate CD25, while in tonsils a fraction of

BCL6hiCXCR5hiFoxp3+ Tfr could be found that lacked CD25 expression. Surprisingly, a significant fraction of CD45RA+ Tregs inhuman blood also express CXCR5 a point that we are now investigating in more detail.
We suggest that, similar to Tfh cells, Tfr cells follow a differentiation pathway generating a mature GC localized subpopulation, CD25-Tfr cells. This represents a significant split from previously described BLIMP-1 dependent effector Tregs (eTregs). Tfr
and eTregs are both highly activated but have distinct effector

markers (PD1 and ICOS for Tfr), CD103 and KLRG1 for eTregs (Figure 1). From these findings we also infer that CD25+Tfr located at
the T-B border and CD25-Tfr located in the germinal center may
have different roles in the control of Tfh responses at each phaseof the Tfh formation process. These findings can be exploited for
effective control of vaccine-induced humoral immune responses
and also treatment of autoantibody-dependent autoimmune diseases.

Immunology

Experimental Immunology

Figure.
Upon activation Naïve CD25+ Tregs bifurcate into either IL-2 dependent
BLIMP1 positive effector Tregs in the T-cell zone or non-lymphoid tissues
or early follicular resident CD25+Tfr. These can them downregulate CD25
expression causing the loss of BLIMP1 expression and higher level BCL6
and CXCR5 expression, allowing these CD25-Tfr to travel to the germinal
center. Adapted from Wing et al. Clin. Immunol. and Allergology. 2018.

Recent Publications● Wing JB, Kitagawa Y, Locci M, Hume H, Tay C, Morita T, Kidani Y, Matsuda K,● Maeda Y, Nishikawa H, Sugiyama D, Ha D, Hamaguchi M, Saito T, Nishioka M,

Inoue T, Kurosaki T, Crotty S, Coban C, Ohkura N, Sakaguchi S. A distinct
subpopulation of CD25- T-follicular regulatory cells localizes in the germinal centers. Proc. Natl. Acad. Sci. U S A. 114, E6400-E6409 (2017).

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).

● Kitagawa Y, Ohkura N, Kidani Y, Vandenbon A, Hirota K, Kawakami R,● Wing JB, Ise W, Kurosaki T, & Sakaguchi S. Regulatory T-cells control anti-

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. Nat. Immunol. 18, 173-183 (2017).

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

● Saito T, Nishikawa H, Wada H, Nagano Y, Sugiyama D, Atarashi K, Maeda Y,

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,& Sakaguchi S. Two FOXP3+CD4+ T-cell subpopulations distinctly control
the prognosis of colorectal cancers. Nat. Med. 22, 679-684 (2016).
25

Immunology

Cell Signaling
Takashi Saito, PhD

Professor

Takashi Saito

Figure. STING stimulation induces growth arrest of and type I-IFN production in T
cells.
Stimulation of T cells through TCR (antigen) and STING (STING ligand such as
cGAMP) induces growth arrest of T cells by inhibiting mTORC1 and simultaneously
produces type-I IFN from T cells. Effector T cells such as Th1 cells and activated CD8 T
cells produce high level of type-I IFN, which may contribute to anti-tumor immunityand protection from viral infection.

T cells play central roles in immune regulation. They initiate immune responses, induce activation and generate various effector
T cells, which protect against infection and oncogenesis. Aberrancy of T cell function results in infectious and autoimmune diseases and cancer. Our laboratory aims to determine the molecular mechanism of T cell activation, differentiation and homeostasis,
particularly from a signaling perspective.
T cell activation is induced through TCR-microclusters (MC), thesignaling clusters generated by recruiting TCR and proximal signaling molecules. We have analyzed function and dynamics of
TCR-MC and downstream signal molecules such as a downstream
adaptor molecule CIN85. Analysis of T cell-specific CIN85 deficient mice revealed that CIN85 functions in negatively regulating
TCR signals by recruiting several negative regulators includingSts-2 phosphatase as a feedback regulation of activation.
We have also investigated the regulation of T cell activation by
innate signals. We have already analyzed the function of TLRs andnucleic acid recognition in T cells. Since we found that STING, amajor intracellular DNA sensor in innate immune cells, is highly
expressed in T cells, the function of STING in T cell activation was
analyzed. STING activation in T cells induced growth arrest andproduction of type I-interferon (IFN), in mTORC1-dependent
manner. The level of type I-IFN produced in effector T cells wasmuch higher than innate immune cells. Together with our observation that STING in T cells contributes to anti-tumor immune
response, STING activation in T cells can be utilized for modulating proliferation of T cells as well as anti-tumor immunity (Figure).
One of the final aims of our diverse approaches is to elucidate26

the mechanisms of inducing autoimmune diseases through aberrant T cell function in order to be able to modulate them and inhibit/prevent autoimmunity and allergic inflammation. We haveanalyzed the regulation of T cell function by phosphatases(PTPN22, PTPN2), whose deficiency is related to the induction of
autoimmune diseases. We generated and analyzed T cell-specificdeficient mice of these phosphatases, and the function of these
phosphatases in T cell activation by analyzing associated proteins
and imaging analysis. The onset of autoimmunity will be eventually analyzed in these mouse models.

Recent Publications● Hayashi M. et al. Advax, a Delta Inulin Microparticle, Potentiates In-built● Hashimoto-Tane A. et al. Micro adhesion-ring surrounding TCR microclus-

Adjuvant Property of Co-administered Vaccines. EBioMedicine 15, 127136 (2017).

ters are essential for T cell activation. J. Exp. Med. 213, 1609-1625 (2016).
● Takeuchi A. et al. CRTAM determines the CD4+ cytotoxic T lymphocyte● Hashimoto-Tane A, Yokosuka T & Saito T. Analyzing the Dynamics of Sig-

lineage. J. Exp. Med. 213, 123-138 (2016).

naling Microclusters. Methods Mol. Biol. 1584, 51-64 (2017).
● Takeuchi A & Saito T. CD4 CTL, a Cytotoxic Subset of CD4+ T Cells, Their

Differentiation and Function. Front. in Immunol. 8,194 (2017).
27

Tomohiro Kurosaki, MD/PhD

Professor

Tomohiro Kurosaki

Associate Professor

Wataru Ise
Takeshi Inoue

Assistant Professor

Ryo Shinnakasu

Research Assistant2

Support Staff

3

Immunology

Lymphocyte Differentiation
However, the physiological significance of SFK regulation by Csk
in vivo remains unclear. To define this, we generated and analyzed Csk kinase-dead knock-in mutant mice. Histological examination of multiple tissues revealed the smaller splenic white pulp
in mutant mice, and flow cytometric analysis showed significant
decrease of mature B cells, most notably marginal zone (MZ) B
cells, in mutant spleen. In addition, the mutant mice showed impaired GC and memory B cell generation upon immunization
with TD antigen. These results suggested that the regulation of
SFK activity by Csk plays important roles in B cell development
and function.

acute AgA)

Tet2/Tet3+/+

CD86

B

B)

CD28

CD4 T

Tet2/3

Tet2/Tet3о/о

Tet2/3HDAC

chronic Ag (auto)

Action mechanisms of DNA demethylases, Tet2 andTet3, on B cell tolerance
As B cell intrinsic tolerance mechanisms, it is known that evenmodest alterations in B cell signaling thresholds can break tolerance, promoting autoimmunity in the appropriate environmental settings. In addition to the B cell signaling components, thecontribution of epigenetic factors has been proposed. One of thepreviously observed epigenetic abnormalities associated with
autoimmune diseases is altered DNA metylation, prompting us tolook for the roles of Tet proteins in B cell tolerance. Ten-eleventranslocation proteins (Tet1, Tet2, and Tet3) were recently discovered to act as DNA demetylase by catalyzing the conversion of
5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine(5hmc) and other intermediate products in the DNA metylation
pathway.
We found that specific deletion of both Tet2 and Tet3 in B cells(using CD19-Cre) resulted in spontaneous hyper T cell activity,
autoantibody production, and lupus nephritis. Treatment of antiCD20 or anti-CD4 depletion antibodies ameliorated aberrant activation of CD4+ T cells or B cells, respectively, suggesting that apositive feed-forward loop between activated B and T cells is operating. Mechanistically, by employing the hen egg lysozyme Ig
transgene model system, we demonstrated that self-tolerant B
cells express low levels of CD86 (B7.2), whereas ablation of Tet2/
28

Tet3 rendered upregulation of CD86, thereby switching the B cellfate from peripheral tolerance to proliferation through T-B interaction. We also found that Tet2 and Tet3 recruited HDAC2, thereby repressing transcription of CD86 in self-tolerant B cells. Together, our data demonstrate that Tet2/Tet3 contributes toperipheral B cell tolerance by suppressing CD86 expression in achromatin-modified manner.

Roles of DNA demetylases, Tet2 and Tet3, in foreignantigen induced immune responses
As mentioned above, chronic depletion of Tet2/Tet3 in B cells
resulted in breaking B cell tolerance and the resultant T cell hyper-activation. Therefore, to address the function of Tet2 andTet3 in immune responses, we acutely deleted Tet2/Tet3 in B
cells. We found that initial proliferation took place normally evenin the absence of Tet2 and Tet3, whereas the deficient B cells
could not differentiate into germinal center (GC) B cells or plasma
cells. Because a transcription factor Bcl6 or IRF4 is known to be
critical for GC B cell or plasma cell differentiation, respectively,
our data demonstrate the importance of Tet2/Tet3 in inductionof Bcl6 and IRF4. We are now analyzing how Tet2/Tet3 regulates
transcription of Bcl6 and IRF4.

Roles of a Src-family regulator Csk in B cell development and function
The Src family kinases (SFKs) play key roles in regulating signaltransduction by a diverse set of surface receptors, and their activation is negatively regulated by C-terminal Src Kinase (Csk).

CD86

ʹ

Ac

Ac

Ac

ON

CD86

(Self-tolerant)

Tet2/Tet3+/+

The epigenetic modifications, such as chromatin modification
and DNA metylation, influence the transmission of genetic information and regulate cellular differentiation and proliferation.

OFF

HDAC

Tet2/Tet3+/+

chronic Ag (auto)

Tet2/Tet3о/о(breaking tolerant)

Figure. Tet2 and Tet3 contribute to peripheral tolerance of B cells.
(A)In the presence of Tet2 and Tet3, acute antigen stimulation induces a co-stimulatory molecule, CD86,
which in turn, evokes cytokine production by T cells, thereby B cell proliferation (upper panel).
When this antigen stimulation continues like auto-antigen stimulation, such CD86 upregulation ceases.
Therefore, T cell activation and subsequent B cell proliferation stop (self-tolerant state).
But in the absence of Tet2 and Tet3, such chronic antigen stimulation cannot downregulate CD86
expression, thereby changing the B cell fate from peripheral tolerance to proliferation.
(B)Tet2 and Tet3 recruit HDAC to the CD86 locus in the chronic state, thereby dampening transcription of
CD86.

Recent Publications● Inoue T, Moran I, Shinnakasu R, Phan TG, Kurosaki T. Generation of memo-ry B cells and their reactivation. Immunol. Rev. 283(1):138-149. (2018).

● Inoue T, Shinnakasu R, Ise W, Kawai C, Egawa T, Kurosaki T. The transcrip-tion factor Foxo1 controls germinal center B cell proliferation in response
to T cell help. J. Exp. Med. 214(4), 1181-1198 (2017).

● Ise W, Fujii K, Shiroguchi K, Ito A, Kometani K, Takeda K, Kawakami E,

Yamashita K, Suzuki K, Okada T, Kurosaki T. T Follicular Helper Cell-Germinal Center B Cell Interaction Strength Regulates Entry into Plasma Cell or
Recycling Germinal Center Cell Fate. Immunity. 48(4), 702-715.e4. (2018).

● Shinnakasu R, Inoue T, Kometani K, Moriyama S, Adachi Y, Nakayama M,

Takahashi Y, Fukuyama H, Okada T, Kurosaki T. Regulated selection of germinal center cells into the memory B cell compartment. Nat. Immunol.
17(7), 861-869 (2016).

● Herndler-Brandstetter D, Ishigame H, Shinnakasu R, Plajer V, Stecher C,

Zhao J, Lietzenmayer M, Kroehling L, Takumi A, Kometani K, Inoue T,
Kluger Y, Kaech SM, Kurosaki T, Okada T, Flavell RA. KLRG1+ Effector CD8+
T Cells Lose KLRG1, Differentiate into All Memory T Cell Lineages, andConvey Enhanced Protective Immunity. Immunity. 48(4), 716-729.e8.
(2018).
29

Cevayir Coban, MD

Research in our laboratory focuses on the elucidation of themechanisms involved in the host and Plasmodium parasites. Malaria is still an important disease and continues to kill half-a million people every year. Why and how malaria infection causes
sudden and life-threatening complications such as cerebral malaria, has not been fully clarified and is extensively investigated in
our lab. However, after an initial systemic infection, most malaria
patients develop only partial immunity over the years and sufferfrom mild symptoms with low parasitemia. Hence, the ‘chronic’
illness caused by incomplete immunity to malaria may cause
long-term ‘hidden’ pathologies such as Burkitt lymphoma, physical growth retardation in children and a detrimental effect on thebone tissue environment (Nature Reviews Immunology, 2018;
International Immunology, 2018).
In the above mentioned context, we have investigated the pathology of cerebral malaria by using an experimental cerebral
malaria model. Deep investigation of the brain by ultra-high field
MRI showed that the olfactory bulb is physically and functionallydamaged 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 provided evidence that olfactory functional impairment (lossof smell) could be a valuable early diagnosis marker for cerebral
malaria (Cell Host Microbe, 2014).
There is increasing compelling evidence that malaria survivors
experience long-term ‘hidden’ pathologies. Using well-estab30

Professor

Cevayir Coban

Assistant Professor

Michelle Sue Jann LEE

Postdoctoral Fellow

2

Research Assistant

1

Visiting Scientist

2

Support Staff

1

lished mouse models mimicking various aspects of human Plasmodium infection, we recently showed that infection causes significant and long term 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 the bone marrow niche induces MyD88-dependent inflammatory responses in osteoclast and osteoblast
precursors, leading to increased RANKL expression and overstimulation 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 bone loss. Importantly, supplementation of alfacalcidol, a vitamin D3 analog, could prevent the bone loss. These
results highlight the risk of bone loss in malaria-infected patientsand the potential benefits of coupling bone therapy with antimalarial treatment (Figure 1, Science Immunology, 2017).
These recent studies from our lab have led us to hypothesizethat systemic infection by Plasmodium parasites causes local, but
tissue specific immunopathology during blood stage of infection,
mainly via the manipulation of inter-tissue interactions between
the blood and other host tissues that often result in dysfunctionof certain organs (Nature Reviews Immunology, 2018). Understanding these pathologies might be critical for the developmentof novel adjunct therapies to be used in combination with current anti-malarials.
The final goal of our research is to translate our understandingof host-Plasmodium interactions into vaccines or drugs to treat
malaria. Our strategy is to focus on the adjuvants, because if ratio-

Immunology

Malaria Immunology
nally 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 (Vaccine, 2016).

Naive
Convalescent/chronic phase

Malaria-infected

Figure. Malaria induces chronic bone loss due to sustained accumulation of Plasmodium products evenafter the cure of malaria disease (Science Immunology, 2017).
Although blood stage Plasmodium infection causes deadly complications, most people develop partial
immunity and suffer from mild symptoms. Recent evidence suggests that the incomplete recovery from
infection causes chronic illnesses such as bone loss or growth retardation.
During acute malaria infection, heme is one of the factors released abundantly due to hemolysis ofinfected and non-infected red blood cells and strongly inhibits both osteoclasts and osteoblasts. Cytokines
such as IL-4, IL-10, IL-13 and IFNγ released from activated T cells in response to Plasmodium infection,
disrupt the signaling in osteoclastogenesis. During convalescence and chronic phase of Plasmodium
infection, although parasites are cleared systemically, parasite products remain and gradually accumulate
abundantly in the bone marrow for long periods leading to MyD88-dependent chronic inflammation in
immature and mature osteoclasts and osteoblasts. Plasmodium product-induced inflammatory responses
upregulate RANKL expression on osteoblasts. The inflammatory cytokines (IL-1α, IL-1β, IL-6 and TNFα) andRANKL synergistically induce osteoclastogenesis. The robust activation of OCs after clearance of parasites
skews bone remodeling toward bone resorption and eventually leads to bone loss.

Recent Publications● Coban C, Lee MSJ, Ishii KJ. Tissue-specific immunopathology during ma-

laria infection. Nat. Rev. Immunol., doi:10.1038/nri.2017.138 (2018).

● Zhao H et al. Lipocalin 2 bolsters innate and adaptive immune responses

to blood-stage malaria infection by reinforcing host iron metabolism. Cell
Host Microbe 12(5), 705-16 (2012).

● Lee MSJ, Maruyama K, Fujita Y, Konishi A, Lelliott PM, Itagaki S, Horii T, Lin JW,

Khan SM, Kuroda E, Akira S, Ishii KJ, Coban C. Plasmodium products persist
in the bone marrow and promote chronic bone loss. Sci. Immunol. 2(12),pii: eaam8093 (2017).

● Coban C et al. Immunogenicity of Whole Parasite Vaccines Against Plas-

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).
31Ken J. ISHII, MD/PhD

Professor

Ken J. ISHII

Associate Professor

Etsushi Kuroda
Norifumi Iijima

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.

An Antigen-Free, Plasmacytoid Dendritic Cell-Targeting Immunotherapy To Bolster Memory CD8(+) T Cells
in Nonhuman Primates
The priming, boosting, and restoration of memory cytotoxic
CD8+ T lymphocytes by vaccination or immunotherapy in vivo is
an area of active research. Particularly, nucleic acid-based compounds have attracted attention due to their ability to elicit
strong Ag-specific CTL responses as a vaccine adjuvant. Nucleic
acid-based compounds have been shown to act as anticancer
monotherapeutic agents even without coadministration of cancer Ag(s); however, so far they have lacked efficacy in clinical trials. We recently developed a second-generation TLR9 agonist, ahumanized CpG DNA (K3) complexed with schizophyllan (SPG),
K3-SPG, a nonagonistic Dectin-1 ligand. K3-SPG was previously
shown to act as a potent monoimmunotherapeutic agent against
established tumors in mice in vivo. In this study we extend themonoimmunotherapeutic potential of K3-SPG to a nonhumanprimate model. K3-SPG activated monkey plasmacytoid dendritic
cells to produce both IFN-α and IL-12/23 p40 in vitro and in vivo.
32

Assistant Professor

Natsuko Kishishita

Postdoctoral Fellow

4

Research Assistant

4

Visiting Scientist

6

Support Staff

2

of CARD14, suggesting that CARD14 is acting downstream of IL23, not TLR7 or TLR9. Instead, a bone marrow chimera study suggested that CARD14 in radio-sensitive hematopoietic cells was
required for IMQ-induced psoriasiform skin inflammation, controlling the number of Vγ4+ T cells producing IL-17 or IL-22 infiltrating through the dermis to the inflamed epidermis. These data
indicate that CARD14 is essential and a potential therapeutic target for psoriasis.

CD63-Mediated Antigen Delivery into Extracellular
Vesicles via DNA Vaccination Results in Robust CD8+ T
Cell Responses
DNA vaccines are attractive immunogens for priming humoral
and cellular immune responses to the encoded Ag. However,their ability to induce Ag-specific CD8+ T cell responses requires
improvement. Among the strategies for improving DNA vaccine
immunogenicity are booster vaccinations, alternate vaccine formulations, electroporation, and genetic adjuvants, but a few,
such as extracellular vesicles (EVs), target natural Ag delivery systems. By focusing on CD63, a tetraspanin protein expressed on
various cellular membranes, including EVs, we examined whether a DNA vaccine encoding an Ag fused to CD63 delivered into
EVs would improve vaccine immunogenicity. In vitro transfection
with plasmid DNA encoding an OVA Ag fused to CD63 (pCD63OVA) produced OVA-carrying EVs. Immunizations with the puri-

A single injection s.c. or i.v. with K3-SPG significantly increased
the frequencies of activated memory CD8+ T cells in circulation,
including Ag-specific memory CTLs, in cynomolgus macaques.
This increase did not occur in macaques injected with free CpG K3or polyinosinic-polycytidylic acid. Injection of 2 mg K3-SPG induced mild systemic inflammation, however, levels of proinflammatory serum cytokines and circulating neutrophil influx werelower than those induced by the same dose of polyinosinic-polycytidylic acid. Therefore, even in the absence of specific Ags, weshow that K3-SPG has potent Ag-specific memory CTL responseboosting capabilities, highlighting its potential as a monoimmunotherapeutic agent for chronic infectious diseases and cancer.

Future prospect
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 will continue innovative research anddevelopment vaccines against these diseases together closelywith National Institute of Biomedical Innovation, Health and Nutrition (NIBIOHN) accompanying active exchanges of researchers.

Exosome-targeting DNA vaccine
Extracellular Vesicles (EV) / exosome contains
target antigen

Dendritic cells or muscle cells

CD63

DNA vaccination(in vivo transfectionof plasmid DNA
encoding antigen)antigen
CD63

Essential Role of CARD14 in Murine Experimental Psoriasis
Caspase recruitment domain family member 14 (CARD14) was
recently identified as a psoriasis-susceptibility gene, but its immunological role in the pathogenesis of psoriasis in vivo remains
unclear. In this study, we examined the role of CARD14 in murine
experimental models of psoriasis induced by either imiquimod(IMQ) cream or recombinant IL-23 injection. In all models tested,
the psoriasiform skin inflammation was abrogated in Card14-/mice. Comparison of the early gene signature of the skin between
IMQ-cream-treated Card14-/- mice and Tlr7-/-Tlr9-/- mice revealed not only their similarity, but also distinct gene sets targeted by IL-23. Cell type-specific analysis of these mice identified
skin Langerinhigh Langerhans cells as a potent producer of IL-23,
which was dependent on both TLR7 and TLR9 but independent

fied OVA-carrying EVs primed naive mice to induce OVA-specificCD4+ and CD8+ T cells, whereas immunization with EVs purified
from cells transfected with control plasmids encoding OVA protein alone or a calnexin-OVA fusion protein delivered into theendoplasmic reticulum failed to do so. Vaccinating mice with
pCD63-OVA induced potent Ag-specific T cell responses, particularly those from CD8+ T cells. CD63 delivery into EVs led to betterCD8+ T cell responses than calnexin delivery into the endoplasmic reticulum. When we used a mouse tumor implantation model to evaluate pCD63-OVA as a therapeutic vaccine, the EV-delivered DNA vaccination significantly inhibited tumor growthcompared with the control DNA vaccinations. These results indicate that EV Ag delivery via DNA vaccination offers a new strategy
for eliciting strong CD8+ T cell responses to the encoded Ag,
making it a potentially useful cancer vaccine.

Immunology

Vaccine Science

Plasmid DNANaïve T cells

APCs

CD8 T cells

Th1

Patent pending“EXOSOME-TARGETED DNA VACCINE”(WO2017175253) PCT/JP2016/001900
Kanuma T et al J. Immunol. 2017

Recent Publications● Temizoz B, Kuroda E, Ishii KJ. Combination and inducible adjuvants targeting

nucleic acid sensors. Curr Opin Pharmacol. 2018 Jun 2;41:104-113.
● Masuta Y, Yamamoto T, Natsume-Kitatani Y, Kanuma T, Moriishi E, Kobiyama K,

Mizuguchi K, Yasutomi Y, Ishii KJ. An Antigen-Free, Plasmacytoid Dendritic
Cell-Targeting Immunotherapy To Bolster Memory CD8(+) T Cells in Nonhuman Primates. J. Immunol. 200(6), 2067-2075 (2018).
● Tanaka M, Kobiyama K, Honda T, Uchio-Yamada K, Natsume-Kitatani Y,

Mizuguchi K, Kabashima K, Ishii KJ. “Essential Role of CARD14 in Murine
Experimental Psoriasis.”J. Immunol. 200(1), 71-81 (2018).

● Kanuma T, Yamamoto T, Kobiyama K, Moriishi E, Masuta Y, Kusakabe T,

Ozasa K, Kuroda E, Jounai N, Ishii KJ. CD63-Mediated Antigen Delivery
into Extracellular Vesicles via DNA Vaccination Results in Robust CD8(+) T
Cell Responses. J. Immunol. 198(12), 4707-4715 (2017).
● Hayashi M, Aoshi T, Haseda Y, Kobiyama K, Wijaya E, Nakatsu N, Igarashi Y,

Standley DM, Yamada H, Honda-Okubo Y, Hara H, Saito T, Takai T, Coban C,
Petrovsky N, Ishii KJ. Advax, a Delta Inulin Microparticle, Potentiates Inbuilt Adjuvant Property of Co-administered Vaccines. EBioMedicine. 15,
127-136 (2017).

33

Masahiro Yamamoto, PhD

Professor

Masahiro Yamamoto

Associate Professor

Miwa Sasai

Assistant Professor

Jisu Ma

unexpectedly dispensable, Gate-16 uniquely and critically participates in IFN-inducible GTPase-dependent host defense
against vacuolar pathogens. The mechanism of action of Gate-16
involves Arf1 activation and distribution of IFN-γ-induced GTPases throughout the cytosol and subsequently to the vacuole containing the intracellular pathogen.

Immunology

Immunoparasitology

Youngae Lee
Postdoctoral Fellow

Macroautophagy (herein, autophagy) is a fundamental eukaryotic cellular system to sustain homeostasis to degrade old/damaged cytoplasmic components or unwanted pathogens in double membranous autophagosome and autolysosome involving
autophagy-related (Atg) proteins. Although yeast has only one
Atg8, which is essential for autophagy, mammals harbor at least
five Atg8 orthologues consisting of LC3 and Gabarap subfamilies:
LC3a, LC3b, LC3C, Gabarap, Gabarapl1 and Gate-16 (also known
as Gabarap2). Autophagic stimuli, such as nutrient starvation, increase LC3 lipidation in an Atg3/Atg5/Atg7/Atg16L1-dependent
manner and induce accumulation of the lipidated LC3 on autophagosomal membrane, which is a widely accepted marker for
autophagy activity assessment 7. LC3 subfamily members play animportant role in the elongation of phagophore membrane. In
contrast, Gabarap subfamily members are required for autophagosomal maturation, suggesting that both LC3 and Gabarap
Atg8 subfamilies differentially participate in autophagy. Furthermore, LC3C plays an important role in anti-bacterial autophagy(xenophagy) in human cells. Thus, conserved canonical roles ofAtg8 in eukaryotic autophagy have been established so far.
Noncanonical functions of Atg8 have been recently reported.
LC3 is detected on single membranous phagosome, whose process is known as LC3-associated phagocytosis (LAP) and is involved in autoimmunity and host defense against fungus andbacteria. Although LAP requires the LC3 conjugation machinery
such as Atg5, Atg7 and Atg16L1, it does not require Atg14, another essential canonical autophagy regulator. In addition, maturation and functions of osteoclasts are associated with LC3. Al34

1

though formation of the ruffled border by osteoclasts and theirsecretory functions in bone resorption requires Atg5, Atg7 andlapidated LC3, the maturation of osteoclasts is not associatedwith autophagic activity.
An unconventional Atg8 conjugation system is also reported incell-autonomous host defense stimulated by interferons (IFNs).
Many intracellular pathogens create pathogen-containing vacuoles (PCVs), which protect the microbe from sensing by cytoplasmic innate receptors and allows efficient proliferation. Against
infection by vacuolar pathogens, the host immune system generates IFNs, which further stimulate production of immunity-related GTPases called IFN-inducible GTPases. These GTPases are accumulated on PCV membranes, leading to the PCV disruption,
death of vacuolar pathogens and subsequent various inflammatory responses. The mechanism by which type II IFN (IFN-γ) stimulation induces recruitment of IFN-inducible GTPases to PCV ofToxoplasma gondii, an obligatory protozoan pathogen that causes
life-threatening toxoplasmosis in human and animals, has been
extensively studied and shown to require Atg3, Atg5, Atg7 andAtg16L1 but not Atg9a, Atg14 and ULK1/2, indicating the unconventional usage of the Atg8 conjugation system other than canonical autophagy. In particular, T. gondii PCV is highly decorated
with LC3 as well as IFN-inducible GTPases26, suggesting the major
function. However, physiological relevance of the Atg8 conjugation system and the biological significance of LC3 in the IFN-γinduced response are not fully understood.
Here we have analyzed mice lacking individual Atg8 family
members and found that, although LC3 subfamily members are

Figure.
GABARAP subfamily of mammalian ATG8 plays a critical role in IFN-γ-induced cell-intrinsic immunity.

Recent Publications● Sasai M, Sakaguchi N, Ma JS, Nakamura S, Kawabata T, Bando H, Lee Y,

Saitoh T, Akira S, Iwasaki A, Standley DM, Yoshimori T, Yamamoto M. Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense. Nat. Immunol. 18, 899-910 (2017).
35

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 flippases, that translocates PtdSer
and phosphatidylethanolamine from outer to inner leaflets. Whencells undergo apoptosis, or platelets are activated, the asymmetrical distribution of phospholipids is disrupted by a scramblase(s)that non-specifically scramble phospholipids between the innerand outer leaflets of plasma membranes, leading to PtdSer-exposure. The PtdSer, thus exposed to the cell surface, works as an “eatme” signal for apoptotic cells, and as a scaffold on the activatedplatelets for blood clotting factor. The PtdSer-exposure is also observed in activated lymphocytes, capacitated sperm, aged erythrocytes, exosomes, and enveloped virus.
We 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. The CDC50A works as a chaperone
for ATP11A and ATP11C to escort them from ER to plasma membranes, and is required for their flippase activity at plasma membranes.
36

Professor

Shigekazu Nagata

Associate Professor

Katsumori Segawa

Postdoctoral Fellow

4

Research Assistant

3

Visiting Scientist

2

Support Staff

1

There are two families of membrane proteins that support
non-specific scrambling of phospholipids at plasma membranes.
Five members (TMEM16C, 16D, 16F, 16G and 16J) of the TMEM16
family, that contain 10 transmembrane segments, function as
Ca2+-dependent scramblases (Figure 1). In a microarray system ofmembrane bilayers in which phospholipids are asymmetrically
localized, we recently showed that a single dimeric molecule of
TMEM16F can scramble phospholipids between two membrane
bilayers.
TMEM16F is ubiquitously expressed in various cells, while other members are specifically expressed in the brains or intestines.
Human and mouse platelets express only TMEM16F as a 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 a loss offunction 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 on the dead cell’s surface is recognized by macrophages for engulfment. Xkr4 and Xkr9 are rather tissue-specifical-ly expressed in the brains and intestines, respectively. Whereas,
Xkr8 is ubiquitously expressed in various tissues. Mouse thymocytes, lymphocytes, and neutrophils express only Xkr8, and when
they undergo apoptosis, they expose PtdSer in an Xkr8-dependent manner. Thus, Xkr8-null thymocytes and neutrophils are notefficiently engulfed by macrophages. The Xkr8-null female micedevelop systemic lupus erythematosus-type autoimmune disease. It is possible that unengulfed dead cells undergo secondary

necrosis, and intracellular materials released from dead cells activate the immune system.
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.
A flippase comprised of P4-ATPase (ATP11A or ATP11C) and CDC50A, and a
Ca2+-dependent scramblase (TMEM16F) is schematically shown. In activatedplatelets, the intracellular Ca 2+ concentration increases and activates
TMEM16F to scramble phospholipids, while it inactivates P4-ATPases andreduces their flipping activity. When the Ca 2+ concentration returns tonormal level, TMEM16F stops scrambling phospholipids, while P4-ATPases
resume flipping PtdSer and PtdEtn. Thus, PtdSer is only transiently exposedto the cell surface in this process, and likely depends on the intracellularconcentration of ATP and Ca2+. The constant flipping of PtdSer prevents the
PtdSer-exposing cells from being engulfed by macrophages.

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. Apoptosis and clearance of apoptotic cells. Annu. Rev. Immu-

nol. 36, 489-517 (2018).

● Segawa K, Kurata S and Nagata S. The CDC50A extracellular domain is re-● Watanabe R, Sakuragi T, Noji H and Nagata S. Single-molecule analysis ofquired for forming a functional complex with and chaperoning phospholipid flippases to the plasma membrane. J. Biol. Chem. 293, 2172-2182(2018).

phospholipid scrambling by TMEM16F. Proc. Natl. Acad. Sci. USA 115,
3066-3071 (2018).

● Nagata S and Tanaka M. Programmed cell death and the immune system.

Nat. Rev. Immunol. 17, 333-340 (2017).
● Kawano M and Nagata S. Lupus-like autoimmune disease caused by a lackof Xkr8, a caspase-dependent phospholipid scramblase. Proc. Natl. Acad.
Sci. USA 115, 2132-2137 (2018).
37

Immunology

Molecular Neuroscience
Toshihide Yamashita, MD/PhD

Professor

Toshihide Yamashita

Associate Professor

Rieko Muramatsu

Assistant Professor

Yuki Fujita
Shogo Tanabe
Hiroshi Tsujioka
Ken Matoba

Postdoctoral Fellow

3

Research Assistant3

Support Staff

4

Figure 1.
The mechanism of spontaneous functional recovery.

Disorders of the central nervous system, such as cerebrovascular diseases, cerebrospinal trauma, and encephalomyelitis, oftencause spatiotemporal changes in the nervous system and in various biological systems, such as the immune system and vascularsystem. We have analyzed disorders of the neural networks in thecentral nervous system and the subsequent restoration processfrom the perspective of the functional network of biological systems (Fig. 1). Further, we have analyzed the mechanism by which
the spatiotemporal dynamics in those biological systems control
a series of processes (Fig. 2). Particularly, the ultimate goal of thisstudy is to elucidate the control mechanism exerted by the associations among the nervous system, immune system, and vascular system. Additionally, we aim to elucidate the principles involved in the operation of living organisms with neural network
disorders within the central nervous system by observing suchdisorders and their functional recovery process with respect tothe dynamics of the entire biological system and by conducting acomprehensive analysis of the association between each system.
We perceive the central nervous system as a single organ within a biological system; further, studies from the perspective of theinvolvement of the entire biological system in disorders and recovery of neural networks are scarce. By perceiving disorders in
the neural networks and the biological reactions during the subsequent recovery process as a “scrap-and-build” strategy, we aim
to elucidate the mechanisms behind a series of reactions as wellas their significance that may potentially create a new and neverseen-before trend in Life Sciences.

38

Figure 2.
Biological systems that regulate rewiring of neural network after CNS injury.

Recent Publications● Tanabe S & Yamashita T. B-1a lymphocytes promote oligodendrogenesis

during brain development. Nat. Neurosci. (2018) in press.
● Kuroda M, Muramatsu R, Maedera N, Koyama Y, Hamaguchi M, Fujishima

H, Yoshida M, Konishi M, Itoh N, Mochizuki H & Yamashita T. Promotion ofcentral nervous system remyelination by peripheral FGF21. J. Clin. Invest.
127, 3496-3509 (2017).
● Fujita Y, Masuda K, Nakato R, Katou Y, Tanaka T, Nakayama M, Takao K,● Fujitani M, Zhang S, Fujiki R, Fujihara Y & Yamashita T. A chromosome

16p13.11 microduplication causes hyperactivity through dysregulationof miR-484/protocadherin-19 signaling. Mol. Psychiatry 22, 364-374(2017).
● Hayano Y, Takasu K, Koyama Y, Ogawa K, Minami K, Asaki T, Kitada K,

Kuwabara S. & Yamashita T. Dorsal horn interneuron-derived Netrin-4
contributes to spinal sensitization in chronic pain via Unc5B. J. Exp. Med.
213, 2949-2966 (2016).

Miyakawa T, Shirahige K & Yamashita T. Cohesin regulates formation ofneuronal networks in the brain. J. Exp. Med. 214, 1431-1452 (2017).
39

Sho Yamasaki, PhD

Professor

Sho Yamasaki

Assistant Professor

Eri Ishikawa

Immunology

Molecular Immunology
Mincle gene attenuated inflammatory responses. These results
suggest that β-GlcCer is an endogenous Mincle ligand and acts as
an immunostimulatory factor upon cell damage (Figure 2).

Chihiro Motozono

Our bodies are continuously exposed to external and internal
insults caused by infections and stresses, most of which are primarily sensed by immune receptors to maintain tissue homeostasis. However, the molecular mechanism by which these receptors
discriminate diverse insults to elicit suitable immune responses
remains elusive. To illustrate the molecular basis behind this regulation, our laboratory is focusing on the following areas; 1) immune sensing of insults via C-type lectin receptors, 2) T cell responses induced by self-peptides, and 3) innate T cell subsets
related to autoimmune diseases.

Immune sensing of pathogens and damaged-self via
C-type lectin receptors
C-type lectin receptors (CLRs) comprise a large family of proteins that share a common structural motif and are involved invarious immune responses. Among them, ITAM-coupled CLRs arerecently identified as pattern recognition receptors (PRRs) for
pathogens. We found that macrophage-inducible C-type lectin(Mincle/Clec4e) is an FcRγ-coupled activating receptor for Mycobacterium tuberculosis. A widely-known mycobacterial adjuvant,cord factor (also called trehalose-6,6’-dimycolate, TDM), wasidentified as a Mincle ligand. Another CLR, MCL (Clec4d), also recognizes mycobacterial cord factor and promotes Mincle expression. Indeed, innate and acquired immune responses induced by
TDM were abrogated in both Mincle- and MCL-deficient mice. In
addition, we found that another CLR Dectin-2 (Clec4n) also recognizes mycobacteria through a hydrophilic lipoglycan, lipoalabinomannan (LAM). This interaction triggered unique adjuvant
40

Research Assistant1

Support Staff

2

activities that are distinct from those induced by TDM-Mincle/
MCL axis. Furthermore, DCAR (Clec4b1), a CLR located in thesame gene cluster, was identified as a receptor for phosphatidylinositol mannosides of mycobacteria. DCAR was selectively expressed in monocyte-derived inflammatory cells and triggered
Th1 protective immunity against mycobacteria. Thus, these four
CLRs clustered on the same chromosome are found to be activating receptors for mycobacteria (Figure 1). In addition to mycobacteria, Mincle also recognized pathogenic fungi and gramnegative bacteria that lack TDM. Instead of TDM, several novel
glycolipids, glycosylglycerolipids and glycosyldiacylglycerols
were identified as Mincle ligands from these pathogens. These
ligands possessed adjuvant activity in mice, as the injection of thepurified ligands augmented T cell responses. These findings shedlight on CLRs as an emerging immune receptor family for wide
spectrum of pathogens, and thus CLRs could be potential targets
for the development of a novel adjuvant.
Sensing tissue damage is a crucial function of pattern recognition receptors (PRRs). However, endogenous ligand recognition
by PRRs is not well documented. We have previously reported
that Mincle recognizes damaged cells. To elucidate endogenous
ligands derived from damaged cells, we fractionated supernatants from damaged cells and identified β-glucosylceramide(GlcCer). β-GlcCer induced inflammatory and acquired immune
responses via Mincle on myeloid cells. Accumulation of β-GlcCer
leads to Gaucher disease, a disorder characterized mainly by systemic inflammation. In a Gaucher model in which mice are deficient in the β-GlcCer–degrading enzyme, further deletion of the

Figure 1.
Protective immunity against mycobacteria initiated through C-type lectin
receptors. Mycobacterial components initiate host immune responses
through CLRs. CLRs trigger signal cascades via immune-receptor tyrosinebased activation motifs (ITAMs) by directly recognizing multivalentglycolipids.

Figure 2.
Sensing pathogens and damaged tissue by CLRs. Mincle and related CLRs
expressed on myeloid cells can sense ligands come from pathogens anddamaged self. Myeloid cells are activated upon ligand binding, leading to
the secretion of cytokines as well as antigen presentation by MHC class II,
and consequently activate adaptive immunity. On the other hand, activatedmyeloid cells could also secrete some effectors. Through these responses,
Mincle functions in host defense and dead tissue clearance. These
procedures are precisely controlled in a healthy immune system; however,
the overreaction of any step may induce harmful responses to our body,
generating disorders such as inflammatory diseases, autoimmune or tissue
injury.

Recent Publications● Nagata M, Izumi Y, Ishikawa E, Kiyotake R, Doi R, Iwai S, Omahdi Z,● Yonekawa A, Saijo S, Hoshino Y, Miyake Y, Ishikawa E, Suzukawa M, Inoue H,

Yamaji T, Miyamoto T, Bamba T, Yamasaki S. Intracellular metabolite
β-glucosylceramide is an endogenous Mincle ligand possessing immunostimulatory activity. Proc. Natl. Acad. Sci. USA. 114, E3285-94 (2017).

Tanaka M, Yoneyama M, Oh-hora M, Akashi K, Yamasaki S. Dectin-2 is adirect receptor for mannose-capped lipoarabinomannan of mycobacteria. Immunity 41, 402-13 (2014).

● Toyonaga K, Torigoe S, Motomura Y, Kamichi T, Hayashi JM, Morita YS,● Miyake Y, Toyonaga K, Mori D, Kakuta S, Hoshino Y, Oyamada A, Yamada H,

Noguchi N, Chuma Y, Kiyohara H, Matsuo K, Tanaka H, Nakagawa Y, Sakuma T,
Ohmuraya M, Yamamoto T, Umemura M, Mastuzaki G, Yoshikai Y, Yano I,
Miyamoto T, Yamasaki S. C-type lectin receptor DCAR recognizes mycobacterial phosphatidyl-inositol mannosides to promote a Th1 response
during infection. Immunity 45, 1245-57 (2016).

Ono K, Suyama M, Iwakura Y, Yoshikai Y, Yamasaki S. C-Type lectin MCL isan FcRγ-coupled receptor that mediates the adjuvanticity of mycobacterial cord factor. Immunity 38, 1050-62 (2013).

● Ishikawa E, Kosako H, Yasuda T, Ohmuraya M, Araki K, Kurosaki T, Saito T,

Yamasaki S. Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1. Nat. Commun. 7, 12756 (2016).
41

Yukinori Okada, MD/PhD

Goal of our laboratory
The genetic backgrounds of individuals have a substantial impact on the risk for a wide range of immune-related diseases. Statistical immunology is a research field that evaluates causality ofhuman genetic variations on immune-related diseases, using statistical and bioinformatics approaches. Recent developments ingenome sequencing technologies have provided the human genome data of millions of subjects, and successfully identifiedcomprehensive catalogues of genetic risk loci of immune-relateddiseases. However, little is known regarding how to develop
methodology to integrate large-scale human genome data withdiverse biological and immunological resources. The goal of ourlaboratory is to develop such methods and apply them to the latest large-scale disease genome and multi-layer omics data.

Identification of causal immune cells on human complex traits using trans-layer multi-omics analysis
We conducted large-scale genome-wide association studies(GWAS) of >160,000 Japanese subjects on 58 clinical measurements, including hematological measurements (e.g., counts of
red blood cells, white blood cell subtypes, and platelets), biochemical measurements (blood sugar, serum lipids, liver functions, kidney functions, and electrolytes), and clinical tests (e.g.,
blood pressure and echocardiography). Our study identified
1,400 genetic variants associated with these phenotypes, ofwhich half were novel findings. We further obtained additional
GWAS results of 30 human diseases including immune-relateddiseases such as rheumatoid arthritis and Graves’ disease. We
42

Professor

Yukinori Okada

Research Assistant

1

Visiting Scientist

1

Support Staff

1

Chemical (ATC) Classification System. Application of genetic risk
variants on stroke to the newly developed in silico drug repositioning pipeline successfully identified a strong network with antithrombotic therapy as the most strongly associated clinical indication (Figure 2). This result is considered as evidence thatutilization of genetic risk variants should contribute to novel drug
discovery (Rainer M et al. Nat Genet 2018).

Figure 1.
Network among human complex traits and cell types identified by trans-layer omics
analysis. The most relevant human disease with regulatory T cells was Graves’ disease (GD;
human autoimmune thyroiditis).

conducted trans-layer omics analysis to integrate the GWAS results of human complex traits with the epigenetic signature ofpromoter and enhancer information from >200 cell types. We
adopted the newly developed statistical method named “linkage
disequilibrium score regression (LDSC)” which quantifies genetic
correlation among the traits according to each of the cell types.
Our study successfully elucidated a hidden network among complex human traits and cell types (Figure 1). For example, the most
relevant human diseases to the regulatory T cells was Graves’ disease and autoimmune thyroiditis. This finding should support
the original feature of the regulatory T cells in mice that depletioncauses autoimmune thyroiditis. Our in silico study successfully extracted the biological pathophysiology of human immune-related diseases by integrating genetic and epigenetic data (Kanai M
et al. Nat Genet, 2018).

ᵱᶒᶐᶍᶉᶃ ᶐᶇᶑᶉᴾᵱᵬᵮ

ᵱᶒᶐᶍᶉᶃᴾᶐᶇᶑᶉᴾᶅᶃᶌᶃᵄᵢᶐᶓᶅᴾᶒᵿᶐᶅᶃᶒᴾᶅᶃᶌᶃᵢᶐᶓᶅᶐᶑᵔᵖᵐᵓᵒᵓᵒᵤᵥᵟᵟᶊᶒᶃᶎᶊᵿᶑᶃ
ᵲᶃᶌᶃᶁᶒᶃᶎᶊᵿᶑᶃ
ᵟᶌᶇᶑᶒᶐᶃᶎᶊᵿᶑᶃ
ᵰᶃᶒᶃᶎᶊᵿᶑᶃ

ᶐᶑᵕᵑᵎᵒᵖᵒᵏᵮᵢᵣᵑᵟᵡᶇᶊᶍᶑᶒᵿᶘᶍᶊ

ᶐᶑᵓᵔᵑᵗᵑᵓᵎᵔ

ᵮᵪᵥ

ᵫᶍᶑᶒᴾᶑᶒᶐᶍᶌᶅᶊᶗᴾᵿᶑᶑᶍᶁᶇᵿᶒᶃᶂ
ᶁᶊᶇᶌᶇᶁᵿᶊᴾᶇᶌᶂᶇᶁᵿᶒᶇᶍᶌ

ᵟᶌᶒᶇᶒᶆᶐᶍᶋᶀᶍᶒᶇᶁ
ᶒᶆᶃᶐᵿᶎᶗ

ᵫᶃᶊᵿᶅᵿᶑᶒᶐᵿᶌ
ᵱᶒᶐᶃᶎᶒᶍᶉᶇᶌᵿᶑᶃ

Figure 2.
Application of in silico drug repositioning method identified network between human
genetic variants with stroke risk and drugs currently used for antithrombotic therapy.

In silico drug repositioning based on the large-scale
human disease genome study
Utilization of large-scale human disease genetic studies fornovel drug discovery is one of the most featured topics in thepharmaceutical field. We previously developed a statistical method to screen novel candidate drugs for drug repositioning byconnecting genetic information with drug databases (e.g., repositioning of CDK4/6 inhibitors for treatment of rheumatoid arthritis). Through international collaboration with trans-ethnic genetic studies on stroke comprising 520,000 individuals, we newly
developed a method to additionally integrate the disease anddrug classification curated by WHO Anatomical Therapeutic

Immunology

Statistical Immunology

Recent Publications● Malik R. et al. Tyrosine kinases Multiancestry genome-wide association● Akiyama M. et al. Genome-wide association study identifies 112 new locistudy of 520,000 subjects identifies 32 loci associated with stroke andstroke subtypes. Nat. Genet. doi:10.1038/s41588-018-0058-3 (2018).

for body mass index in the Japanese population. Nat. Genet. 49, 14581467 (2017).

● Kanai M. et al. Genetic analysis of quantitative traits in the Japanese pop-

● Ishigaki K. et al. Polygenic burdens on cell-specific pathways underlie theulation links cell types to complex human diseases. Nat. Genet. 50, 390400 (2018).

risk of rheumatoid arthritis. Nat. Genet. 49, 1120-1125 (2017).

● Hirata J. et al. Variants at HLA-A, HLA-C, and HLA-DQB1 confer risk of pso-

riasis vulgaris in Japanese. J. Invest. Dermatol. 138, 542-548 (2018).
43Immunology

Stem Cell Biology and Developmental Immunology
Takashi Nagasawa, MD/PhD

Professor

Takashi Nagasawa

Associate Professor

Tatsuki Sugiyama

Assistant Professor

Yoshiki Omatsu

Support Staff

1

1mm

1mm

Seike, M. et al., Genes Dev 2018
Figure 1. A progression of occlusion of the marrow cavities in the absence of Ebf3
Femurs in aged Lepr-Cre; EBF3f/f mice revealed that the bone marrow spaces werefilled with bone (Seike et al. Genes Dev. 2018).

Hematopoietic stem cells (HSCs), which give rise to all blood
cells, including immune cells, are maintained by special microenvironments known as niches within bone marrow cavities. We
isolated a chemokine, CXCL12 (SDF-1/PBSF) as a molecule that
stimulates the growth of B cell precursors (Nagasawa et al. PNAS
1994) and found that CXCL12 and its receptor CXCR4 are essential for colonization of bone marrow by hematopoietic stem cells(HSCs)(Nagasawa et al. Nature 1996; Ara et al. Immunity 2003),
maintenance of a pool of HSCs in bone marrow (Sugiyama et al.
Immunity 2006), and development of immune cells, including B
cells, plasmacytoid dendritic cells (pDCs) and NK cells (Nagasawa.
Nat. Rev. Immunol. 2006) as well as vascular formation and cardiogenesis (Tachibana et al. Nature 1998). Subsequently, we
identified a population of reticular cells expressing CXCL12 athigh levels, termed CXCL12-abundant reticular (CAR) cells withinbone marrow (Sugiyama et al. Immunity 2006) and indicated that
CAR cells are adipo-osteogenic progenitors and the major producer of CXCL12 and SCF, creating the special microenvironment(niche) for HSCs and B cells (Omatsu at al. Immunity 2010). Furthermore, we found that 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 cell progenitors, and development
and maintenance of niches for HSCs and immune cells (Omatsu
et al. Nature 2014). We are studying the roles of CXCL12-CXCR4
signaling and CAR cells in the spatiotemporal regulation of lymphohematopoiesis during homeostasis and diseases.

44

Hematopoietic stem cell niche-specific Ebf3 maintains
the bone marrow cavity
Bone marrow is the tissue filling the space between bone surfaces. CAR cells, which are a major cellular component of HSC
niches, give rise to osteoblasts in bone marrow. However, it hadremained unclear how osteogenesis is prevented in most CAR
cells to maintain HSC niches and marrow cavities. Recently, we
find that the transcription factor Ebf3 is preferentially expressedin CAR cells and that Ebf3-expressing cells are self-renewing mesenchymal stem cells, which give rise to all osteoblasts and adipocytes in adult bone marrow, using lineage-tracing. When Ebf3
was deleted in CAR cells, HSC-niche function was severely impaired and bone marrow was osteosclerotic with increased bonein aged mice (Figure 1). In mice lacking Ebf1 and Ebf3, CAR cells
exhibiting a normal morphology were abundantly present but
their niche function was markedly impaired with depleted HSCs
in infant marrow. Subsequently, the mutants become progressively more osteosclerotic, leading to the complete occlusion of
marrow cavities in early adulthood. CAR cells differentiate intobone-producing cells with reduced HSC-niche factor expression
in the absence of Ebf1/Ebf3. Thus, HSC cellular niches express
Ebf3 that is required to create HSC niches, to inhibit their osteoblast differentiation, and to maintain spaces for HSCs (Seike et al.
Genes Dev. 2018).

Figure 2. The development and functions of CAR cells.
The transcription factors Foxc1 and Ebf3, and HSC niche factors CXCL12 and SCF
were preferentially and abundantly expressed in CAR cells within the bone marrow.

Recent Publications● Seike M, Omatsu Y, Watanabe H, Kondoh G and Nagasawa T. Stem cell

● Pitulescu ME, Schmidt I, Giaimo BD, Antoine T, Berkenfeld F, Ferrante F,

nich-specific Ebf3 maintains the bone marrow cavity. Genes Dev. 32(5-6),
359-372 (2018).

Park H, Ehling M, Biljes D, Rocha SF, Langen UH, Stehling M, Nagasawa T,
Ferrara N, Borggrefe T and Adams RH.
Dll4 and Notch signalling couples sprouting angiogenesis and artery formation. Nat. Cell Biol. 19(8), 915-927, (2017).

● Shimoto M, Sugiyama T and Nagasawa T. Numerous niches for hemato-

poietic stem cells remain empty during homeostasis. Blood 129(15),2124-2131 (2017).
● Shimba A, Cui G, Tani-Ichi S, Ogawa M, Abe S, Okazaki F, Kitano S, Miyachi

H, Yamada H, Hara T, Yoshikai, Y, Nagasawa T, Schütz G, Ikuta K.
Glucocorticoids Drive Diurnal Oscillations in T Cell Distribution and Responses by Inducing Interleukin-7 Receptor and CXCR4. Immunity 48(2),286-298 (2018).
45

Imaging

Single Molecule Imaging (Yanagida – Seymour Group)
Toshio Yanagida, PhD
Ben Seymour, MD/PhD
Professor

Toshio Yanagida
Ben Seymour

Visiting Scientist

1

Support Staff

1

Figure 1.
Sensorimotor brain regions showing modular reorganization in chronic
musculoskeletal back pain.

Recent advances in imaging methodologies provide a unique
opportunity to interrogate biological process at multiple levels.
Our goal is to integrate imaging methods across levels and species, with new intelligent approaches to data analysis, to provide
translational insight into the multi-system interactions in the
body that support physiological and pathophysiological functions.
A particular interest of our lab is pain and fatigue, which are thetwo dominant symptoms of immune diseases such as inflammatory arthritis. Their relative resistance to treatment by disease
modifying drugs such as immunologics represents a primary
limitation to the clinical efficacy of these drugs. Our research aims
to identify the adaptive changes that occur in the brain that allow
these symptoms to develop and persist after peripheral inflammation, using brain imaging in rodent, human pre-clinical andclinical models of pain and inflammation. In particular, we combine data-driven and hypothesis-driven analyses to understandthe complex brain changes in inflammation. Data-driven methods utilize machine learning and deep learning tools to interrogate classifiable changes in complex brain networks, and the useof graph theory and other network topological analyses to characterize differences between brain states. Hypothesis-driven
analyses use computational models of how behavior changes after peripheral injury, to probe the normative basis for illness behavior ('cognitive neuroimmunology').
We know from our previous research that inducing changes in46

the function of the sensorimotor cortex can modulate pain following nerve injury in human patients [5], but we haven't known
if significant sensorimotor reorganization actually occurs in thebrain after injury. Now, we have been able to identify and characterize substantial reorganization of the sensorimotor cortices inchronic musculoskeletal back pain, using a new method that
identifies changes in the brain modular architecture [1]. In particular, these changes correlate with activity in a specific brain
region - the pregenual anterior cingulate cortex (pgACC). This issignificant as in another study, using a model of heat injury inhealthy humans, we have shown that pgACC modulates pain
when people try to engage in behavior to protect potentially inflamed skin: specifically, it appears to modulate incoming sensory
signals, and control outgoing autonomic signals, to optimize behavioral responses [2]. The reason this is significant is because it
provides a mechanistic link between the modulation of pain andthe injured state, reflecting a process that could in principle cause
pain to develop in the transition from acute to chronic inflammation. We have now started a large translational project to address
this in inflammatory arthritis - in both humans (rheumatoid arthritis) and rodent models (CFA joint injection) [5], using combined molecular and brain imaging methods. In summary, thisresearch suggests that pgACC may represent a new target to understand how pain and fatigue develop in chronic inflammatory
disease, potentially providing a way to predict symptomatic response to treatment, and innovate new treatments that could
modulate pain and fatigue as an adjunct to immune modulators.

Figure 2.
Localization of pregenual anterior cingulate cortex (pgACC), which supports
behavioral 'resilience' after injury.

Figure 3.
Structural images showing brain changes in anterior cingulate cortex after
joint inflammation in rodents.

Recent Publications● Mano H, Kotecha G, Leibnitz K, Matsubara T, Nakae A, Shenker N, Shibata M,

Voon, Yoshida W, Lee M, Yanagida T, Kawato M, Rosa M, Seymour B. Classification and characterization of brain network changes in chronic backpain: A multicenter study. Wellcome Open Research 3:19 (2018).

● Fujita

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

● Yanagisawa T, Fukuma R, Seymour B, Hosomi K, Kishima H, Shimizu T, Yokoi H,● Zhang S, Mano H, Lee M, Yoshida W, Robbins T, Kawato M, Seymour B. The

Control of Tonic Pain by Active Relief Learning. eLife 2018;7:e31949(2018).

Hirata M, Yoshimine T, Kamitani Y, &amp; Saitoh Y. Induced sensorimotor
brain plasticity controls pain in phantom limb patients. Nat. Commun. 7,13209 (2016).

● Morris L, Sprenger C, Koda K, de la Mora M, Yamada T, Mano H, Kashiwagi Y,

Yoshioka Y, Morioka Y, Seymour B. Anterior cingulate cortex connectivity
is associated with suppression of behaviour in a rat model of chronic pain.
Brain and Neuroscience Advances (2018) in press.
47

Masaru Ishii, MD/PhD

Professor

Masaru Ishii

Associate Professor

Keizo Nishikawa

Assistant Professor

Junichi Kikuta

doubtedly powerful tool for dissecting live 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 for analyzing humansamples. By collaborating with companies (supported by AMED)

we are developing a new microscopy system for applying humantissues and organs in vivo. Currently, we have succeeded in visualizing non-labelled human normal and cancer tissues, which canbe used for differential diagnosis (Sci Rep, 2017), and we will beable to dissect human immunology in future.

Imaging

Immunology and Cell Biology

Hiroki Mizuno
Takao Sudo
Takahiro Matsui
Szandor Simmons
Yutaka Uchida

The major aim of our laboratory is to understand the fundamental principle controlling cellular dynamics in various kinds oftissues and organs in vivo, by means of advanced imaging techniques. Recent advances in optical imaging technology have enabled us to visualize the dynamic nature of different cell types ina spatiotemporal manner (see the Figure).

Intravital bone imaging revealing bone cell dynamics
in vivo
By using intravital multiphoton microscopy, we have originallyelaborated the novel imaging system for visualizing inside thebones. We previously succeeded in visualizing the function of
fully differentiated osteoclasts adhering to bone surfaces 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. In
order to further analyze the actual event of bone resorption invivo, in collaboration with Dr. Kikuchi in the frame of IFReC, we
have developed a new chemical probe for detecting proton secretion in bone resorption by osteoclasts (Nat Chem Biol, 2016).
Furthermore, we could detect an in vivo mode of dynamic
communication between mature osteoblasts (mOBs) and mature
osteoclasts (mOCs), and found that the mOBs and mOCs were
distributed mainly in a segregated fashion, although some directcell-to-cell contact was detected between mOBs and mOCs inspatiotemporally limited areas. A pH-sensing fluorescence probe48

Research Assistant

2

Visiting Scientist

1

Support Staff

2

we developed revealed that mOCs secreted protons for bone resorption when they were not in contact with mOBs, whereas
mOCs contacting mOBs were non-resorptive, suggesting thatmOBs could inhibit the bone resorption activity of mOCs by direct cell–cell contact. This study is the first to use intravital imaging techniques to reveal spatiotemporal intercellular interactions
between mOBs and mOCs, thus contributing to our understanding of bone homeostasis in vivo (Nat Commun, 2018).
Moreover, we have shown that our intravital imaging technology turned out to be a powerful tool for dissecting in vivo pharmacological actions of various drugs. For example, of the biologic
agents used in clinic for treating rheumatoid arthritis, anti-IL-6R
and anti-TNFα mAbs affected mature osteoclasts and switchedbone-resorbing osteoclasts to non-resorbing cells. CTLA4-Ig had
no action on mature osteoclasts but mobilized osteoclast precursors, eliminating their firm attachment to bone surfaces. In agreement with these results, CD80/86 (the target molecules of CTLA4Ig) were prominently expressed only in osteoclast precursor cells,
being suppressed during osteoclast maturation. Intravital imaging revealed that various biologic DMARDs acted at specific therapeutic time-points during osteoclastic bone destruction, withdifferent efficacies. These results enable us to grasp the realmodes of action of drugs, optimizing the usage of drug regimens(Ann Rheum Dis, 2018).

Application of intravital imaging techniques for dissecting human immunology
Intravital imaging with multi-photon microscopy is an un-

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● Matsuura Y. et al. In vivo visualization of different modes of action of bio-

● Iwamoto Y. et al. Intercellular communication between keratinocytes andlogic DMARDs inhibiting osteoclastic bone resorption. Ann. Rheum. Dis.
(2018) in press.

fibroblasts induces local osteoclast differentiation: a mechanism underlying cholesteatoma-induced bone destruction. Mol. Cell Biol. 6, 1610-20(2016).

● Furuya M. et al. Direct cell-cell contact between mature osteoblasts andosteoclasts dynamically controls their functions in vivo. Nat. Commun. 9,300 (2018).

● Nishikawa K. et al. Dnmt3a regulates osteoclast differentiation by coupling

to an S-adenosyl methionine-producing metabolic pathway. Nat. Med. 21,
281-287 (2015).

● Maeda H. et al. Real-time intravital imaging of pH variation associated withcell osteoclast activity and motility using designed small molecular probe.
Nat. Chem. Biol. 12, 579-85 (2016).
49

Jun Hatazawa, MD/PhD

The aim of our lab is to visualize and quantify in-vivo dynamics
using molecular imaging techniques and eventually lead to theproper management and treatment of the patients. Theranostics
is now focusing attention on the oncology field and tumor selective targeting is essential using the same molecule for both diagnostic imaging and targeted alpha or beta therapy. It is also important to evaluate the resistant factors for the chemo-radiation
therapy against cancer such as low perfusion and hypoxia to improve personalized precision medicine.

F-FBPA as a tumor-specific probe of L-type aminoacid transporter 1 (LAT1)

18

L-type amino acid transporter 1 (LAT1) is the major amino acidtransporter which is strongly expressed in many cancer cells
while being expressed at lower levels in normal tissues. Inhibitionof LAT1 was associated with an antitumor effect in non-small celllung cancer, suggesting possible application of LAT1 inhibitors tocancer therapy. 18F-fluorodeoxyglucose (18F-FDG) is a major PET
tracer used worldwide for the evaluation of cancer patients. However, the glucose transporter is up-regulated not only in cancer
cells, but also in inflammatory cells, which may lead to false-positive findings, such as non-specific accumulation in the mediastinal lymph nodes. 18F-FBPA was shown to have low physiologicaluptake in most normal organs, except the kidney, which is a great
advantage to use it as a reliable tracer for cancer diagnosis. We
evaluated the in-vitro cellular uptake of 18F-FBPA focusing on its
selectivity for LAT1 and the usefulness of 18F-FBPA as a tumorspecific tracer in rat xenograft and inflammation models. As a re50

Professor

Jun Hatazawa

Associate Professor

Hiroki Kato

Assistant Professor

Tadashi Watabe

Research Assistant2

Support Staff

2

Imaging

Nuclear Medicine
creased oxygen extraction, suggesting the need for a treatment
strategy to normalize the blood flow for oxygen-avid active tumor cells in hypoxic regions.

Figure 1.
Static PET images of 18F-FDG, 11C-Methionine (Met), and 18F-FBPA. All tracers showed
high uptakes in the tumors on the PET images. In contrast, low uptakes were observedin the inflammatory lesions for 18F-FBPA and 11C-Met PET, whereas the uptake values of18
F-FDG PET remained high in the inflammatory lesions as well.

sult, the cellular analyses revealed the higher selectivity of 18FFBPA for LAT1 and in-vivo 18F-FBPA PET showed the high uptake
in the glioma tumor and low uptake in the inflammatory lesion(Figure 1). We could demonstrate the utility of 18F-FBPA as a tumor-specific probe of LAT1 with low accumulation in the inflammatory lesions.

The relationship among intratumoral hypoxia, bloodflow and oxygen metabolism
Intratumoral hypoxia is one of the resistant factors in radiotherapy and chemotherapy for cancer. Although it is detected by
18
F-fluoromisonidazole (FMISO) PET, the relationship between
intratumoral hypoxia and oxygen metabolism has not been studied. We evaluated the intratumoral perfusion and oxygen metabolism in hypoxic regions using the rat xenograft model. Coregistered images of 18F-FMISO and tumoral blood flow showedthat intratumoral hypoxic regions with FMISO uptakes were located as lining the inside of the high blood flow area and there
was no accumulation in the central area inside the FMISO uptake,suggesting a necrotic region (Figure 2). Quantitative analysis revealed that there were significant negative correlations between
18
F-FMISO SUV and tumoral blood flow. In comparison between
moderate and severe hypoxia, significant differences were observed in oxygen extraction fraction and tumoral blood volume.
Oxygen extraction and vascular density were considered as key
factors to define the severity of intratumoral hypoxia. Using thein-vivo molecular PET imaging technique, we showed that intratumoral hypoxic regions showed decreased blood flow with in-

Figure 2.
Comparison among pimonidazole staining, 18F-FMISO PET, tumoral blood flow (TBF),
and fusion image. Pimonidazole staining corresponded to 18F-FMISO uptake regions,surrounded by blood flow preserved regions.

Recent Publications● Horitsugi G. et al. Oxygen-15 labeled CO2, O2, and CO PET in small animals:● Watabe T. et al. (18)F-FBPA as a tumor-specific probe of L-type amino acidevaluation using a 3D-mode microPET scanner and impact of reconstruction algorithms. EJNMMI Res. 7, 91 (2017).

transporter 1 (LAT1): a comparison study with (18)F-FDG and (11)C-Methionine PET. Eur. J. Nucl. Med. Mol. Imaging 44, 321-331 (2017).

● Watabe T. et al. Quantitative evaluation of oxygen metabolism in the intra-

tumoral hypoxia: (18)F-fluoromisonidazole and (15)O-labelled gases inhalation PET. EJNMMI Res. 7, 16 (2017).
51

Kazuya Kikuchi, PhD

Intracellular protein-labeling probes for multicolor
single-molecule imaging of immune receptor-adaptor
molecular dynamics
Multicolor single-molecule imaging (SMI) enables tracking of
hundreds or thousands of individual molecules over a period ofseveral seconds, and provides direct information about molecular dynamics and interactions in the cellular context, and consequently, about dynamic and kinetic parameters of biological reactions in living cells. Genetically encoded fluorescent proteins(FPs) are widely used as fluorescence reporters. However, theability to use FPs for SMI and other photon-intensive imaging applications is limited because of their poor photostability. Moreover, FPs have common limitations in strictly controlling theirexpression levels. Dual-color SMI using two FPs is particularly
challenging, as it requires optimization of the expression levels ofboth FPs to determine conditions under which FPs display similar
fluorescence intensities within a cell. To address this issue, we developed near-infrared (NIR) fluorescent probes for protein-labeling, which specifically binds to a mutant β-lactamase tag (BL-tag).
Although NIR fluorophores have many advantages in biologicalapplications such as their low spectral overlap with cellular autofluorescence, the intrinsic high hydrophobicity of the dyes caninterfere SMI due to the significant background signals.
Here, we developed NIR fluorescent probes conjugated with acell-permeable BL-tag ligand for intracellular protein-labelingand application to multicolor SMI. We selected SiR-Me and SiRcarboxyl as the NIR fluorophores, which possess excellent bright52

Professor

Kazuya Kikuchi

Associate Professor

Yuichiro Hori

Assistant Professor

Masafumi Minoshima

Postdoctoral Fellow

1

Research Assistant2

Support Staff

1

ness, photo-stability, and cell permeability, and bacampicillin as
the BL-tag ligand for intracellular protein-labeling. In addition,novel bacampicillin-based labeling probes by introducing a hydrophilic oligoethylenoxy linker, increasing hydrophilicity andthe distance between the fluorescent dye and the BL-tag ligand
in a stepwise manner: SiRcB(n) (n = 2, 4, 6) (Figure 1a). To evaluate
its capabilities for intracellular SMI, we used SiRcB(n) (n = 2, 4, 6)
for SMI of the BL-tag anchored to the inner leaflet of the plasma
membrane. Tracking analysis revealed that a significantly larger
number of fluorescent spots with higher diffusion coefficientswas detected upon labeling with SiRcB(n) (n = 2, 4, 6) than with
SiRcB not possessing a hydrophilic linker. Considering the cellpermeability and the minimized non-specific binding, we selected SiRcB4 as an NIR-fluorescent probe for multicolor SMI experiments in combination with a HaloTag-based red-fluorescentprobe.
In the multicolor SMI study, we focused on the receptor-adaptor interaction between Toll-like receptor 4 (TLR4) and Toll-interleukin-1 receptor (TIR) domain-containing adaptor protein (TIRAP) upon lipopolysaccharide (LPS) stimulation to analyze theirmolecular dynamics upon innate immune response in living cells.
TIRAP was selected as the target adaptor protein because it localizes in the plasma membrane, where it interacts with TLR4. Thus,
the dynamic interactions between TLR4 and TIRAP occur at theappropriate depth for SMI using total internal reflection fluorescence microscopy (TIRFM). To analyze the TLR4-TIRAP dynamics,
the BL-tag protein-labeling system was used in combination witha commercial HaloTag system. BL-tag and Halo-Tag were attached to the N-terminus of TIRAP (BL-TIRAP) and the C-terminus
of TLR4 (TLR4-Halo), respectively (Figure 1d).
We used SiRcB4 and the HaloTag-TMR ligand to perform multicolor SMI in living cells expressing BL-TIRAP and TLR4-Halo, after
stimulation with 10 μg/mL LPS, and quantitatively analyzed theprotein dynamics by statistical analysis. Analysis of protein dynamics showed an increase in the population of slow-moving
TLR4 and TIRAP upon ligand stimulation by LPS (Figure 1e-f). This
result indicates that the mobility of both proteins was decreasedby their interaction. Another SMI experiment using TIR-domainlacking TLR4 mutant (∆TIR) revealed that the relative ratio ofslow-moving TIRAP molecules did not change even after ligand
stimulation (Figure 1g-i), indicating that since TIRAP did not interact with TLR4(∆TIR), binding of TIRAP to TLR4 through the TIR domain is essential for changing its mobility. This result is consistent
with the current model of TLR4 dynamics in response to ligand
stimulation, which demonstrates the reliability of our imaging
system for detecting protein-protein interactions. Our multicolor
SMI system enabled quantitative analysis of LPS-induced TLR4TIRAP interactions in living cells at the single-molecule level forthe first time. This system may also be useful for quantitative
analysis of other receptors in response to various PAMPs and investigation of the immune response of downstream adaptors localized at the cytoplasmic membrane.

Imaging

Chemical Imaging Techniques

Figure.
(a) Structures of bacampicillin-based probes. SiRcB2, SiRcB4, and SiRcB6 contain di-, tetra-, and hexa-ethylene glycol, respectively. (b) Imaging of HEK293T
cells expressing TLR4-Halo and BL-TIRAP by confocal microscopy. The cells were incubated with 100 nM HaloTag-TMR Ligand and 500 nM SiRcB4 at 37 ºC
for 30 min. Images were acquired before and after 10 min stimulation with 10 μg/mL of LPS. Scale bar: 20 μm. (c) Multicolor SMI of HEK293T cells expressing
TLR4-Halo and BL-TIRAP. The cells were incubated with 50 pM HaloTag-TMR Ligand and 100 pM SiRcB4 for 30 min at 37 °C. Scale bar: 5 μm. (d, e) Fluorescentlabeling of TLR4 and TIRAP (d), and TLR4(∆TIR)-Halo and BL-TIRAP (e) with the HaloTag-TMR ligand and SiRcB4, respectively. (f–i) Transient response of the
diffusion of TLR4-Halo (f), and BL-TIRAP (g) in TLR4-Halo and BL-TIRAP expressing cells, and TLR4(∆TIR)-Halo (h), and BL-TIRAP (i) in TLR4(∆TIR)-Halo and BLTIRAP expressing cells, upon LPS stimulation (10 μg/mL). N > 140,000 (5 cells).

Recent Publications● Hori Y, Otomura N, Nishida A, Nishiura M, Umeno M, Suetake I & Kikuchi K.

● Matsui Y, Funato Y, Imamura H, Miki H, Mizukami S & Kikuchi K. Visualiza-

Synthetic-Molecule/Protein Hybrid Probe with Fluorogenic Switch forLive-Cell Imaging of DNA Methylation. J. Am. Chem. Soc. 140, 1686-1690(2018).

tion of Long-term Mg2+ Dynamics in Apoptotic Cells with a Novel Targetable Fluorescent Probe. Chem. Sci. 8, 8255-8264 (2017).
● Matsui Y, Sadhu K K, Mizukami S & Kikuchi K. Highly Selective Tridentate● Matsui Y, Mizukami S & Kikuchi K. Ratiometric Imaging of Intracellular

Mg2+ Dynamics Using a Red Fluorescent Turn-off Probe and a Green Fluorescent Turn-on Probe. Chem. Lett. 47, 23-26 (2018).

Fluorescent Probes for Visualizing Intracellular Mg2+ Dynamics without
Interference from Ca2+ Fluctuation. Chem. Commun. 53, 10644-10647(2017).

● Sato R, Kozuka J, Ueda M, Mishima R, Kumagai Y, Yoshimura A, Minoshima M,

Mizukami S & Kikuchi K. Intracellular protein-labeling probes for multicolor single-molecule imaging of immune receptor-adaptor molecular
dynamics. J. Am. Chem. Soc. 139, 17397-17404 (2017).
53

Nicholas Isaac Smith, PhD

Associate Professor

Nicholas Isaac Smith

Assistant Professor

Alison Hobro

to be correlated with activation. The results showed that the label-free methods could provide an accurate estimation of activation. In fact, the separation of the two classes (activated vs nonactivated) was found to be superior in some cases to thefluorescent measurements. See Figure 2, where the discrimination of LPS-exposed macrophages against control cells is shown,using a fluorescence iNOS signal, compared to the Raman andphase measurements. See Pavillon et al 2018 for details of the
project. While this study initially used cell lines, we are now extending the work using primary-cultured murine macrophages.

Nicolas Pavillon

An interesting finding was that although both the Raman andphase modalities could detect activation, they are based on very
different physical features of the cell. The independent measurements based on composition and morphology, respectively,
gave highly correlated indicators of activation in paired measurements on the same single cells. When activation was inhibitedusing progesterone, the samples comprised four different classes

Our research is aimed at developing label-free imaging methods which can be used to help understand the immune response.
In particular, single-cell level analysis is a main aim, coinciding
with recent increased awareness of the importance of cell-to-celldiversity. The development of label-free single cell analytic tools
means that immune cell type and function can be characterized
without any modification of the cells of interest. For T and B cells,
the discrimination of the cell type is usually performed by labelling the proteins expressed on the surface. If we can instead develop tools to optically measure the features that best characterize lymphocyte type without labelling, it provides several
advantages. One is that the variability in the labelling process itself is removed, and another is that it becomes possible to characterize more of the cell features that emerge during differentiationor activation. In essence, the process may then measure the upstream events that occur before the expression of the surface
markers. Along this direction, we successfully used Raman scattering to discriminate between numerous lymphocyte cell lines(Hobro et al 2016), where the identification of a cell line could be
performed in a fully label-free optical measurement.
In many types of immunofluorescence imaging, fixation andpermeabilization is required. Our lab interests are in non-invasive, non-fixed methods. Nevertheless, since Raman imaging
measures the molecular contents of the cell without a-priori
knowledge or labelling, it is possible to use Raman imaging toascertain the changes that occur during fixation to provide insight for other studies that do require fixation. Figure 1 shows
54

data from this project. The Raman mapping of composition ofindividual cells was performed using line-scanning Raman imaging and principal component analysis (PCA) so that each cell isdirectly comparable in terms of components to the others. The
results show that even common cross-linking fixatives such asparaformaldehyde, although assumed to maintain overall cellstructure and contents, actually show a reduction in intensity in
the Raman image generated from the first two principal components, showing that the process reduces the components in thecell (Figure 1). See Hobro et al 2017 for details.
As well as characterizing phenotype or for studying large-scalebiochemical changes in the cells, we also explored the use of two
different label-free techniques for characterizing macrophage activation. Using Raman spectroscopy as well as quantitative phaseimaging, we studied the potential for both of these label-freemodes to evaluate the changes occurring in macrophages whenstimulated by lipopolysaccharides. The quantitative phase imaging mode provides the spatial distribution of refractive index in
the cell. Following stimulation, macrophage morphology was observed to change sufficiently so that it is possible to evaluate theactivation state of a single cell using only the phase information.
In addition to this, the Raman modality gives us quantitative information on changes in cellular composition. Measured Raman
data, as well as the quantitative phase, could both be used togenerate feature vectors that relate to the activation. We validated these modalities by comparing the Raman and phase information with the signals of inducible NOS in the cell, which is known

Figure 1.
Use of Raman imaging and principal component analysis to evaluatechanges occurring during fixation. Cultured MEF cells were imaged live,
then fixed with paraformaldehyde at 4 degrees C. The principal component
analysis was performed on the whole dataset so that comparison ofchanges can be made between the groups of cells. The color channels are
generated from the first two spectral principal components. See Hobro etal. 2017 for original figure and details.

of cells: control, LPS-exposed, progesterone-exposed, and LPS +progesterone-exposed. The Raman measurements could successfully distinguish all four classes, implying that the effects ofprogesterone on resting macrophages creates a sufficient difference in molecular composition of the cell to be discriminated. In
contrast, the phase imaging modality could not reliably distinguish between control cells and progesterone exposed cells, indicating that the cellular changes due to progesterone are mostly
in the overall composition of the cell, and that there are insufficient downstream effects to exhibit in the cell morphology. Thisillustrates how a multimodal approach to single-cell analysis canbe highly useful, providing insights that are difficult to achievewith other techniques.

Imaging

Biophotonics

As well as the main targets of label-free imaging and analysis,
we also aimed to expand the applications of these technologies
to the spectroscopic analysis of structure and composition ofsmaller targets such as RNA, virii, and blood/serum samples.

Figure 2.
Evaluating the changes in Raw264 cells under LPS stimulation. Cells werefirst measured live, without modification in Raman and phase (“morph”
denotes morphology measured by phase imaging), and then immunolabelled for iNOS, allowing us to compare our methods to more typical
protocols in immunology. Panel A shows a comparison between different
cells in iNOS. Panels B, C, D show the combined predictions of Raman andphase measurements for the macrophage state, and comparisons between
the Raman, phase, and iNOS signal measurements. Control cells are in black,and LPS-exposed cells are in red. The separation of the two groups showsthat the label-free methods are highly effective at estimating macrophageactivation.

Recent Publications● N. Pavillon, A. J. Hobro, S. Akira and N. I. Smith, "Noninvasive detection of● A. J. Hobro and N. I. Smith, "An evaluation of fixation methods: spatial andmacrophage activation with single-cell resolution through machine learning". Proc. Natl. Acad. Sci. USA 115(12), pp. E2676-E2685 (2018).

compositional cellular changes observed by Raman imaging". Vib. Spectrosc. 91, pp. 31-45 (2017).

● A. J. Hobro and N. I. Smith, "Vibrational spectroscopic imaging of patho-● A. J. Hobro, Y. Kumagai, S. Akira and N. I. Smith, "Raman spectroscopy as agens, microorganisms, and their interactions with host systems". Opt. Commun. (2018) in press.

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

● N. Pavillon and N. I. Smith, "Compressed sensing laser scanning microsco-py". Opt. Express 24(26), pp. 30038-300052 (2016).
55

Kazuhiro Suzuki, MD/PhD

sponses may have evolved to maximize the efficiency of hostdefense when encounters with pathogens are more likely to occur (Fig. 2).
Our findings have added a novel layer of immune regulationby the nervous system. However, some questions remain unsolved. First, the molecular mechanism of the crosstalk between

Professor

Kazuhiro Suzuki

Assistant Professor

Akiko Nakai

Research Assistant2

Support Staff

2

β2ARs and chemokine receptors is unclear. Second, it remains to
be visualized how adrenergic nerves interact with lymphocytes
and influence their behaviors in LNs. Third, the effect of β2AR activation on other lymphocyte functions, including their metabolic
activity, is poorly understood. We are going to address thesequestions in future studies to reveal the whole picture of adrenergic control of adaptive immune responses.

Imaging

Immune Response Dynamics

Figure 1. Control of lymphocyte egress from LNs through β2ARs.
Activation of lymphocyte β2ARs enhances signals through CCR7 and CXCR4, chemokine receptors that promote lymphocyte
retention in LNs, and inhibits lymphocyte egress from LNs.

During the last century, extensive studies were performed on
individual organ systems and yielded huge progress to biomedical science. However, because the organ systems in our body mutually communicate to cross-regulate their functions, it is nearly
impossible to precisely predict responses of one organ system
without knowing the states of other organ systems. Thus, the importance of studies that clarify interconnections among multiple
organ systems has been increasingly recognized. In this regard,
recent studies that have revealed the cellular and molecular basis
for connections between the nervous and immune systems represent the new trend. Studies during the last decade have demonstrated that the autonomic and somatosensory nervous systems regulate both the development and functions of immune
cells through multiple modes of action.
Adrenergic nerves constitute the efferent arc of the sympathetic nervous system and produce noradrenaline that induces
cellular responses through α1, α2, β1, β2 and β3 adrenergic receptors (ARs). Like other vital organs, lymphoid organs receive a rich
supply of adrenergic nerves. However, it had been 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
56

demonstrated that inputs from adrenergic nerves control lymphocyte egress from LNs through β2ARs. 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). Additionally, inmurine models of inflammatory diseases, including multiple sclerosis and allergic dermatitis, we showed that activation of β2ARs
ameliorates the diseases, which is accompanied by reduced LN
egress of pathogenic T cells and their recruitment to inflammatory sites (Nakai et al., 2014).
The activity of adrenergic nerves displays a circadian rhythm
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 found to cause restricted LN egress oflymphocytes and an increase of their numbers in LNs. The accumulation of lymphocytes in LNs during the night time was accompanied by more robust humoral immune responses thanthose induced in the daytime. The diurnal variation of humoral
immune responses was dependent on β2AR-mediated neural signals and was diminished when lymphocyte recirculation throughLNs was stopped. These findings suggest that the β2AR-mediatedcontrol of lymphocyte trafficking contributes to the daily rhythmof adaptive immune responses (Suzuki et al., 2016). The time-dependent differences in the magnitude of adaptive immune re-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 of lymphocyte accumulation in LNs enhances adaptive immune
responses.

Recent Publications● Suzuki K and Nakai A. Immune modulation by neuronal electric shockwaves. J. Allergy Clin. Immunol. (2018) in press.
● 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. Neuro-

immunol. 7, 10-17 (2016).
● Nakai A, Hayano Y, Furuta F, Noda M. and Suzuki K. Control of lymphocyte

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

● Suzuki K, Hayano Y, Nakai A, Furuta F and Noda M. Adrenergic control of theadaptive immune response by diurnal lymphocyte recirculation throughlymph nodes. J. Exp. Med. 213, 2567-2574 (2016).
57

Daron M Standley, PhD

Professor

Daron M Standley

Associate Professor

Kazutaka Katoh

Using both 3D modeling and molecular docking we modeled SPP
inhibitors of HCV (Hirano, J. et al 2017). We also helped to model
the complex and novel structural arrangement of the RagulatorRag GTPase complex (R Yonehara, et al. 2017).

Error (Root-mean square deviation) of TCR and BCR models

Shunsuke Teraguchi
Assistant Professor

Informatics

Systems Immunology

Songling Li
Floris Van Eerden

Repertoire Builder

Abody Builder

Pigs Pro

Lyra

BCR

1.89

1.99

2.06

2.06

BCR H3

4.20

4.67

4.76

4.94

Postdoctoral Fellow

2

TCR

2.04

NA

NA

2.32

Research Assistant2
2

TCR B3

3.17

NA

NA

3.75

Support Staff

Significantly high error (compared to Repertoire Builder):-log(p) > 5-log(p) > 10

Figure 1. Repertoire Builder BCR and TCR models significantly more accurate than other methods.
The table shows root-mean square deviations of BCR models, BCR CDRH3 regions, TCR models, andTCR CDR alpha-3 regions for Repertoire Builder and three popular methods. The background color
indicates statistical significance.

Flexible DNA Docking
In 2017, our lab continued to pursue two major research
themes: functional analysis of B cell receptor (BCR) and T cell receptor (TCR) repertoires and protein-nucleotide interactions.
Both of these themes have the potential to make a strong contribution to basic and applied immunology research at IFReC. To
this end, we develop bioinformatics methods in our lab, including methods for multiple sequence alignment, machine learningand 3D structural modeling. We also collaborate intensively with
wet immunology groups in order to fine-tune our bioinformatics
methods.

based tool for predicting RNA binding sites on proteins (aaRNA;
Li, S. et al. 2014) and have extended this tool to DNA and for docking the nucleotides onto the proteins. Such flexible nucleotide
docking has been validated in the case of Regnase-1 (Yokogawa
et al. 2016), Arid5a-OX40 (H Hanieh et al. 2017), AUF1-AU-rich repeat interactions (Nyati, K.K. et al. 2017), APOBEC3G-RNA (Izumi
et al) and for the anti-nuclear antibody project mentioned above(Figure 2). We are currently applying aaRNA to a larger range ofRNA-binding proteins, with emphasis on those proteins that interact with Regnase-1 in order to understand the basis for target
specificity.

A. Docking revealed two possible binding modes

Mode 2

Alignment Accuracy

B. Mode 1 confirmed by X-ray crystallography

Mode 1

Functional analysis of BCR and TCR repertoires
Functional analysis of BCR and TCR repertoires can be defined
as identification of the target antigen. In 2017, we focused on prediction of BCR or TCR structure from sequence. We developed a
web-based tool (Repertoire Builder) for modeling BCR and TCR
structure from sequence accurately in a high-throughput manner(Schritt, D. and Li, S. et al.). To our knowledge, Repertoire Builder
is the fastest and most accurate tool available (Figure 1). In a typical run, 10,000 sequences can be modeled in under 30 minuteswith an average error of under 2Å. We are currently pursuing thechallenge of antigen binding. For BCRs, we have applied this approach to the successful modeling of anti-nuclear antibodies associated with acute systemic lupus erythematosus (Sakakibara, S.
et al. 2017).

Multiple sequence alignment

Protein-nucleotide interactions

Structural modelingIn the second research direction, we have improved a web58

We are continuously improving the performance and usabilityof the MAFFT multiple sequence alignment software. As one example, a MAFFT option for high-quality alignment has been extended to large-scale sequence data (Figure 3). The accuracy ofthis method was well demonstrated in previous benchmarks but
was impractical for large-scale data sets. By using parallel programming techniques, the calculation speed was improved significantly (Nakamura et al, 2018). Other new features have been
added to our online service in response to user demands (Katoh,
K. et al. 2017). We also carried out sequence and structural analysis of the autophagy-related 8 (Atg8) protein family (Sasai, M. et
al. 2017).

We supported structural modeling for several wet labs in 2017.

Figure 2. ssDNA docked to Anti-nuclear antibody model.
A, Docking before x-ray structure determined revealed two possible DNA
bining models. B, X-ray crystallography confirmed model 1.

Figure 3. Improvements in MAFFT.
Over a wide-range of data sizes, new options of MAFFT consistently perform
more accurately than alternative methods.

Recent Publications● Yonehara R. et al. Structural basis for the assembly of the Ragulator-Rag

GTPase complex. Nat. commun. 8, 1625 (2017).

● Nyati KK. et al. TLR4-induced NF-kappaB and MAPK signaling regulate theIL-6 mRNA stabilizing protein Arid5a. Nucl. Acids Res. 45, 2687-2703(2017).

● Sasai M. et al. Essential role for GABARAP autophagy proteins in interfer-

on-inducible GTPase-mediated host defense. Nat. Immunol. 18, 899-910(2017).

● Hirano J. et al. Characterization of SPP inhibitors suppressing propagationof HCV and protozoa. Proc. Natl. Acad. Sci. USA. 114, E10782-E10791(2017).

● Sakakibara S. et al. Clonal evolution and antigen recognition of anti-nu-

clear antibodies in acute systemic lupus erythematosus. Sci. Rep. 7, 16428(2017).
59

Associate Professor

Diego Diez

Assistant Professor

Yutaro Kumagai

Research Assistant1

Support Staff

2

et al. 2015). A common feature of these diseases is that inflammation 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.

Transcriptional regulatory networks are important determinants of cell identity and function. In this process thousands ofregulators, including transcription factors, determine the expression level of their target genes. We model these processes using
linear regression methods. We assume that the expression levelof each gene depends on the activity of a small number of regulators. Furthermore, regulators contribute in an additive way to theexpression level of their target genes. We validate the feasibilityof these assumptions using simulated networks resembling different types of real networks. Using the expression level of theregulators as a proxy for their activities we apply these methods
to several immune datasets.

Units for Combined Research Fields

Quantitative Immunology

Mathematical modeling

The Quantitative Immunology Research Unit is a team of researchers with expertise in different scientific fields including immunology and bioinformatics. Our aim is to understand how theimmune system works by combining three different but closelyinterconnected approaches; (1) quantitative measurement ofmolecular dynamics at single cell and single molecule level, (2)integration of “big data” from multiple sources into networkmodels, and (3) development of mathematical frameworks to understand the immune system’s dynamics through the analysis ofthese 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 highlight specific 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 cluster60

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 (Sato et al, 2017). We developed a novel algorithm to quantify the diffusion dynamics of single molecules without bias, evenunder high molecular density conditions (Teraguchi and Kumagai, in press). This highly quantitative technique can be used todescribe the dynamics of the immune system’s signaling pathways.

Data integration
High-throughput (“omics”) technologies have brought biology
into the big data era and the need for approaches that integrate,summarize and extract relevant information that 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 (DiezThe accurate quantification of immunological responses andthe integration of massive data open the door to approach the
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.

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

Recent Publications● Teraguchi S & Kumagai Y. Estimation of diffusion constants from single● Bahrini I, Song JH, Diez D & Hanayama R. Neuronal exosomes facilitate

molecular measurement without explicit tracking. BMC Syst. Biol. 12(Suppl. 1), 15 (2018).

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

● Sato R, Kozuka J, Ueda M, Mishima R, Kumagai Y, Yoshimura A, Minoshima M,● Diez D, Agusti A & Wheelock CE. Network analysis in the investigation of

Mizukami S & Kikuchi K. Intracellular Protein-Labeling Probes for Multicolor Single-Molecule Imaging of Immune Receptor-Adaptor Molecular
Dynamics. J. Am. Chem. Soc. 139(48), 17397-17404 (2017).

chronic respiratory diseases. From basics to application. Am. J. Respir. Crit.
Care Med. 190, 981-988, doi:10.1164/rccm.201403-0421PP (2014).

● Kumagai Y, Vandenbon A, Teraguchi S, Akira S & Suzuki Y. Genome-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, doi:10.1186/s12864016-3325-7 (2016).
61

Events & Outreach Activities

Lecturer
Jinmiao Chen
(SIgN, Singapore)

James Di Santo(Institut Pasteur, France)

Title
Constructing Cell Lineages From Single-Cell Transcriptomes

Developmental Options and Functional Plasticity of Human Innate Lymphoid Cells

Events & Outreach Activities

The 7th NIF Winter School on Advanced Immunology

Markus Feuerer(The University of Regensburg / German Cancer ResearchCenter, Germany)

Florent Ginhoux(SIgN, Singapore)

Kenya HONDA(Keio University, Japan)

Takashi Nagasawa(IFReC, Osaka University, Japan)

Lai Guan Ng(SIgN, Singapore)

Yukinori Okada(IFReC, Osaka University, Japan)

Hai Qi
The 7th NIF Winter School on Advanced Immunology was held with Singapore Immunology
Network (SIgN) at The Awaji Yumebutai International Conference Center in Japan on 21-26 January 2018. The program comprised a four-day lecture course at Awaji Island and a one-day international symposium at Suita Campus of Osaka University. Fifty-four young researchers fromaround the world, selected from 213 applicants, learned cutting-edge immunology from 15
world-renowned senior researchers at the course and three up-and-coming researchers at the
symposium. Three young researchers from companies collaborating with IFReC also attended
and joined in the discussions. The Winter School was highly evaluated by the participants andthe lecturers as an excellent opportunity for networking with young peers through active discussions and communications as well as its high scientific level.

(Tsinghua University, China)

Laurent Rénia(SIgN, Singapore)

Brigitta Stockinger(The Francis Crick Institute, UK)

Georg Stoecklin(Heidelberg University, Germany)

Hiroshi Takayanagi(The University of Tokyo, Japan)■ Date : January 21- 26, 2018
■ Venue : Awaji Yumebutai International Conference Center, Hyogo
Icho-Kaikan, Suita Campus, Osaka University, (The 9th International Symposium of IFReC)

Henrique Veiga-Femandes(Champalimaud Center for the Unknown, Portugal)

Toshihide Yamashita(IFReC, Osaka University, Japan)

64

Specialization of regulatory T cells in tissues

Human Dendritic Cells: From Development to Functions

Modulation of the immune system by the gut microbiota

Microenvironmental niches for hematopoietic stem cells and immune cells

Watching The Great Leukocyte Migration

Statistical genetics, disease biology, and drug discovery

T-B cell interactions and the germinal center reaction

Immune-mediated Pathologies During Malaria Infection

Environmental influences on intestinal homeostasis and inflammation

Translation regulation during macrophage activation

Osteoimmunology and autoimmunity

Innate Lymphoid Cells and the Second Brain
The spatiotemporal dynamics in the biological systems regulate the neuronal
network in the central nervous system

65

Speaker

Title

Opening remarks by Director Shizuo Akira
Takashi Shichita(Tokyo Metropolitan Institute of Medical Science, Japan)

Daniel Mucida(Rockefeller University, USA)

Events & Outreach Activities

The 9th International Symposium of IFReC- Immunology at the Forefront

The resolution of sterile inflammation after ischemic stroke

Tissue adaptation: implications for gut tolerance and immunity

Etsushi Kuroda(Osaka University/ National Institutes of Biomedical Innovation, Health and Nutrition, Japan)

Michelle Linterman(University of Cambridge, UK)

Thomas Korn
(Technische Universität München, Germany)

Naganari Ohkura(Osaka University, Japan)

Takao Hashiguchi(Kyushu University, Japan)

Cevayir Coban(Osaka University, Japan)

Mechanisms of allergic inflammation induced by inhaled fine particles

CXCL13 recruits B cells to the lung and initiates tertiary germinal centre formation

Modes and outcome of IL-6 signaling into T cells
Significance of the regulatory T cell-specific epigenetics in autoimmune diseasesusceptibility
Molecular dissection of virus entry and its inhibition by antibodies/inhibitors

Host-Plasmodium interactions at tissue level

Closing Remarks by Deputy Director Kiyoshi Takeda
IFReC invited many talented scientists who are future leaders in various research fields
as speakers. It was an excellent opportunity to share ideas and expertise in order to contribute to further development in Immunology.

■ Date :January 26, 2018

■ Venue : Icho kaikan, Suita campus, Osaka University

66

67

Events & Outreach Activities

IFReC Seminars
2017
Special Lecture
Jul. 28

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 asa forum for effective interaction between researchers beyond national borders and academic
disciplines. This program has certainly contributed to IFReC’s mission of promoting internationalization and interdisciplinary research.
On July 20, 2017, a seminar by Mr. Osamu Nagayama (CEO and Chairman of Chugai Pharmaceutical Co.) was held in commemoration of the Comprehensive Collaboration Agreement
between Osaka University and Chugai Pharmaceutical Co., Ltd.

68

Osamu Nagayama

CEO, Chairman of Chugai Pharmaceutical Co., Ltd.

Date

Speaker

Affiliation

Title

Sep. 4

Christophe J. Desmet

University of Liège / FRS-FNRS, Belgium

Lost in translation: of the importance of proteintranslation for normal function of hematopoietic
stem and progenitor cells

Sep. 4

Andrea Reboldi

University of Massachusetts, USA

Mechanisms controlling intestinal IgA production

Sep. 4

Meirav Pevsner- Fischer

Weizmann Institute of Science, Israel

Host-microbiome interactions in health and disease

Oct. 3Claudia Mauri

University College London, UK

Regulatory B cells in health: what goes wrong inpatients with autoimmunity?

Oct. 20

Axel Kallies

University of Melbourne/Walter and Eliza Hall
Institute of Medical Research, Australia

Molecular regulation of tissue-resident regulatory T
cell development

Oct. 26

Warren J. Leonard

NHLBI, NIH, USA

Fine tuning IL-2: superenhancers, STAT5 tetramerization, and partial agonists

Nov. 7

Hidehiro Yamane

NIAID, NIH, USA

Mechanisms underlying the fine-tuning of CD4+
T cell positioning during T follicular helper cell differentiation69

Speaker
Etsushi Kuroda(Vaccine Science)

Toshihide Yamashita(Molecular Neuroscience)

Shogo Tanabe
30th(Molecular Neuroscience)

Hiroshi Tsujioka(Molecular Neuroscience)

Yukinori Okada(Statistical Immunology)

Sayuri Gyobu(Biochemistry and Immunology)

Keiichi Namba(Graduate School of Frontier Biosciences / Riken QBiC)

Sho Yamasaki
31st(Molecular Immunology)

Songling Li(Systems Immunology)

Takashi Nagasawa(Stem Cell Biology and Developmental Immunology)

Takashi Saito(Cell Signaling)

Yuichiro Hori
32nd

(Chemical Imaging Techniques)

Naoki Hosen(Immunopathology)

Wataru Ise
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 anddeepen understanding of researches conducted in IFReC.

(Lymphocyte Differentiation)

Szandor Simmons(Immunology and Cell Biology)
33 rd(Immunoglycobiology)

Eiji Umemoto■ Date :30th Colloquium : April 19, 2017
31st Colloquium : June 21, 2017
32nd Colloquium : August 30, 2017
33rd Colloquium : October 11, 201734th Colloquium : December 27, 201735th Colloquium : February 21, 2018
■ Venue : Taniguchi Memorial Hall, Osaka University

Yoshiko Murakami(Mucosal Immunology)

Hisashi Kanemaru(Host Defense)
34 th

Patrick Michael Lelliott(Malaria Immunology)

Kazuki Kishida(Immunochemistry)

Nicolas Pavillon(Biophotonics)

Jisu Ma
35 th(Immunoparasitology)

Diego Diez
(Quantitative Immunology Research Unit)

Akiko Nakai(Immune Response Dynamics)

70

Title
Cyclic GMP-AMP exacerbates asthma via IL-33
The spatiotemporal dynamics in the biological systems regulate theneuronal network in the central nervous system
B-1 cells promote oligodendrogenesis during brain development
Interleukin-11 induces and maintains progenitor cells of various tissues
during tail regeneration of Xenopus laevis tadpoles

Events & Outreach Activities

IFReC Colloquia

Statistical genetics, disease biology, and drug discovery
Mechanism and physiological role of phospholipid scrambling by
TMEM16 family
Recent progress of electron cryomicroscopy for structural analysis ofprotein assemblies at atomic level
Immune recognition of pathogens and damaged-self via ITAM receptors
Quantifying structural and functional convergence in B cell receptor
repertoires
Microenvironmental niches for hematopoietic stem cells and immune
cells
Regulation of T cell activation and function by innate signals-STING
activation induces growth arrest and IFN production in T cellsDevelopment of Chemical Tool for Imaging DNA Methylation
The activated conformation of integrin β7 as a target for multiple
myeloma–specific chimeric antigen receptor T-cell therapy
Selection of germinal center B cells into the plasma cell compartment
High-endothelial cell-derived sphingosine-1-phosphate regulates
dendritic cell localization and vascular integrity in the lymph node
Autoinflammation-Paroxysmal nocturnal hemoglobinuria (PNH)
syndrome
Identification of bacteria-induced intestinal metabolites that influenceimmune responses
The antitumor effect of BATF2 through IL-12 p40 up-regulation intumor-associated macrophages
A novel technique to quantify extracellular trap release

Antigen-specific immune regulation mediated by TCR-like antibodies
Label-free monitoring of activation and inhibition of macrophage
responses
A family member of phospholipase C is specifically involved in CD8
T cell-mediated immune responses
Inferring gene regulatory networks from omics data
The COMMD3/8 complex dictates the specificity of GRK recruitment tochemoattractant receptors

71

The series of science Cafés is a long-term IFReC outreach activity to promote communication between researchers and thegeneral public. It also enhances people’s understanding of immunology researches and the researchers involved in them. In

Super Science High School Student Fairthe two science cafés organized by IFReC in FY2017, a total of
about 150 participants enjoyed novel topics in immunology ina relaxing atmosphere.

“Science Café on the Edge” at 2017 Icho Festival■ Speaker : Takashi Satoh (Assistant Professor, Host Defense, IFReC/RIMD)
April 30, 2017
■ Date :■ Venue : Techno Alliance Hall, Suita Campus, Osaka University

Super Science High Schools (SSH) are selected high schools by MEXT in Japan, which
promote advanced math/ science education as well as collaborative researches with universities, and activities to develop international perspectives.
In the SSH Student Fair 2017 held in Kobe, 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 instituteusing posters, booklets and demonstrations.

■ Date :■ Venue :■ Host :■ Support :Events & Outreach Activities

Science Café

August 9-10, 2017
Kobe International Exhibition Hall
MEXT, JST
Boards of Education (Kobe prefecture and Kobe city)Students Visit and Facility tour
“Science Café on the Edge” as part of “Lab Café”
by Osaka University■ Speaker : Jun Sakanoue (Associate Professor, RPMO, IFReC)
March 9, 2018
■ Date :■ Venue : Art Area B1 at Naniwabashi station (Keihan Electric Railway Co.)72

During the summer vacation, IFReC welcomed 30 SSH students from Nara Prefecture. They heard a lecture by Dr. Yutaro
Kumagai (Quantitative Immunology Research Unit), and visited Akira Lab and the Core Instrumental Facility to try someexperiments and to talk with researchers.
We also welcomed 55 junior high-school students and their parents. They also enjoyed the experiments and a tour of
the RIMD History Museum.

■ Date : August 3, 2017 (Junior High School)
August 11, 2017 (High School)73

AAAS 2018 Annual Meeting

Events & Outreach Activities

WPI Science Symposium

IFReC co-organized the 6th WPI Science Symposium titled; Starting the Future!
At IFReC’s booth, we introduced the images and movies acquired through IFReC researches, and provided leaflets of IFReC and WPI. Interaction with the visitors was quite
stimulating for the WPI staff.

■ Date : February 11, 2018
■ Venue : National Museum of Emerging Science and Innovation (Miraikan), Tokyo

The American Association for the Advancement of Science (AAAS) is the biggest international scientific society in theworld and its mission is to “advance science and serve society”. The AAAS 2018 Annual Meeting was held in Austin, and it
offered a broad 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 400 participants visited the booth and gained interest in the WPI
program and world leading researches in Japan.

■ Date : February 15-19, 2018
■ Venue : Austin Convention Center (Austin, Texas, USA)74

75

Naturejobs Career Expo

Research Projects

Naturejobs is a worldwide career resource for scientists, providing a wide range of career advice andinformation across Nature Publishing Group journals as well as centrally on the website.
In 2017, Nature Publishing Group held their “Career Expo” in UK, USA and Germany. IFReC participatedin “Naturejobs Career Expo in Dusseldorf”. IFReC staff carried out recruiting activities for the Advanced
Postdoc position starting in FY2017 by holding an exhibition booth and an oral session.

■ Date : November 17, 2017
■ Venue : Classic Remise, Dusseldorf, Germany

76

For IFReC, fostering the education of young researchers is a
responsibility as a global research institution. It is also vital forthe institution to continually incorporate the original ideas ofyoung researchers and to promote international brain circulation in order to further develop IFReC’s research. Around theworld, leading research institutions are in fierce competition to
discover excellent young researchers. In order to employ young
researchers who are expected to be internationally active at IFReC and to strengthen IFReC’s function as a hub of international brain circulation, it is necessary to improve the conditions forresearchers in Japan, to an international standard.

Young Scientist Support Program for Research Abroad
IFReC has, therefore, established the Advanced Postdoc
system this fiscal year. It offers outstanding young researchers
in the fields of immunology and cell biology opportunities towork with field-leading researchers in IFReC as well as to conduct their own research and publish under their own merit.
IFReC has selected three excellent postdocs for three years
under this system out of 78 applicants. They were assigned tolaboratories in IFReC with an international standard level salaryand research funds (3 million yen per year) to conduct original
research.

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. 4 researchers used this support
program in FY2017. Since the start of this program, IFReC has
provided support for 53 visits overseas by young researchers.

Research Projects

Advanced Postdoc System

Young Scientist Support Program for Research Abroad
Name
Rouaa Beshr

CountryUSA

Conference Attended
Society of Nuclear Medicine and Molecular Imaging (SNMMI)

Romanov Victor

Australia

Annual congress of the european association of nuclear medicine (EANM'2017)

LIM Ee Lyn

Switzerland

World Immune Regulation Meeting XIITaba Fargol

France17th Annual Congress of the European Society for Photodynamic Therapy

Support provided for 36 overseas visits by young researchers in the past five years

EU 15

America 15

Asia 2Africa 1
Australia 3

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

78

79

Data

Kishimoto Foundation Fellowship

IFReC and its parent institution, the Research Institute forMicrobial Diseases (RIMD) are located on the same site, constituting a large research complex. The complex contains theCore Instrumentation Facility, the Animal Resource Centerand the Network Administration Office, all of which are jointly
operated by IFReC and RIMD. The Core Instrumentation Facility is equipped with various highly advanced instruments andskilled technicians provide in-house services to IFReC andRIMD researchers. The Animal Resource Center consists ofthree buildings for specific pathogen-free (SPF) animals andthe live immuno-imaging facility. With a large-capacity animalbreeding facility in IFReC, researchers are able to choose animalrooms suitable for their experiment 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

1
6

3

2

IFReC launched the Kishimoto Foundation Fellowship program for researchers in various fields of immunology in 2010.
The program is supported by the Kishimoto Foundation anddesigned to support overseas researchers in order to promoteand develop 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.
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.

FY2017 Kishimoto Fellowship Recipients
Position of Recipient

Name (initials)

Nationality

Host researcher

PeriodSpecially Appointed Researcher

D. H.

Australia

Sakaguchi

Oct. 16, 2015 - Oct 15, 2018

Specially Appointed Researcher

J. P.

Vietnam

KishimotoApr. 1, 2016 - Mar. 31, 2019

Specially Appointed Researcher

N. T.

Slovenia

Quantitative
Immunology
Research Unit

Oct. 1, 2016 - Jul. 15, 2017

Specially Appointed Researcher

H. J.

China

Arase

May 1, 2017 - Apr. 30, 2018

Visiting Researcher

H. H.

JordanKishimotoJun. 26, 2017 - Aug. 18, 2017

Visiting Researcher

P. K.

TurkeyCoban

Mar. 1, 2018 - May 28, 2018

Visiting Researcher

E. E.

TurkeyCoban

Mar. 1, 2018 - May 28, 2018

Visiting Researcher

N. N.

Vietnam

KishimotoMar. 10, 2018 - Mar. 22, 2018

Visiting Researcher

D. L.

Vietnam

KishimotoMar. 10, 2018 - May 8, 201854Photo : S. Higashiyama

1. IFReC Research Building

Data

Common Facilities

2. Integrated Life Science Building
3. Main Building, Research Institute for Microbial Diseases, RIMD
4. South Building, Research Institute for Microbial Diseases, RIMD
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
・ Transgenic and knock-out animals
・ Genome editing in experimental animals

Live Immuno-Imaging Facility
・ SPF animal experiment facility with 11.7T MRI, in-vivo imager

Core Instrumentation Facility
・ Basic and advanced instruments
・ In-house service
・ DNA sequencing, cell sorting, electron microscopy, mass

spectrometry and next-generation DNA sequencing analysis
・ Radio isotope facility& two-photon microscope.

82

83

Data

Major Awards
Shimon Sakaguchi

Cevayir Coban

Person of Cultural Merit of Japan

Cevayir Coban (PI, Malaria Immunology) was awarded the 4th Japanese Society for Immunology (JSI) Prize for Women Immunologists. The awarded title is "Revealing the mechanism of host-Plasmodium parasite interactions".

The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan announced that Prof. Shimon Sakaguchi, Deputy Director of IFReC was nameda Person of Cultural Merit. MEXT commented that Prof. Sakaguchi was awarded for
his outstanding achievements in studies of Regulatory T cell (Treg); discovering andclarifying the roles of Treg in autoimmune diseases and allergies. This is the third time
for the scientist of IFReC to be honored as a Person of Cultural Merit, others include
Shizuo Akira (Director of IFReC) in 2009 and Toshio Yanagida in 2013.

Four IFReC researchers

JSI Women Immunologist Award

Taroh Kinoshita

Takeda Prize for Medical Science, JSI Human Immunology Research Award

This prize is given to scientists who have made remarkable contributions in the field of medical science. Kinoshita was honored forresearch on GPI-anchored proteins and mechanisms of its deficiencies.

Highly Cited Researchers 2017

Highly Cited Researchers are researchers with papers that have a large number of citations from all over the world as selected by©Clarivate Analytics. They are thought to be leading the way in solving the world’s biggest challenges. In 2017, some 3,300 researchers from 21 fields were selected as Highly Cited Researchers. Eight researchers in three fields were selected from Osaka University,and four of them are from IFReC.

Cevayir Coban (L) and Taroh Kinoshita (R)at the venue of the commemorative lectures

Michelle Sue Jann Lee• Shizuo Akira
Director of IFReC

Satoh and Segawa• Shimon Sakaguchi
Deputy Director of IFReC• Kiyoshi Takeda
Deputy Director of IFReC• Masahiro Yamamoto
Immunoparasitology, IFReC

JSPS Ikushi Prize

IFReC Assistant Professor Michelle Sue Jann Lee was awarded the 8th JSPS
Ikushi Prize. The 18 prize winners including Dr. Lee were unequivocally recognized for their excellent results in each research area. The award ceremony washeld at the headquarters of Japan Academy (Gakushi-in) in the presence of
their Imperial Highnesses Prince and Princess Akishino.

The Young Scientists' Prize by MEXT

Takashi Satoh (Host Defense) and Katsumori Segawa (Biochemistry & Immunology) were given the Young Scientists' Prize ofCommendation for Science and Technology by the Minister of MEXT. The prize is given to young scientists who have been recognized to have a high level of research ability and development in the science and technology field in Japan.

Takashi Satoh (L) and
Katsumori Segawa (R).

84

85

Finance
Data

Composition

Breakdown of total expenditure at IFReC in FY2017

Number of IFReC Staff
350
Administrative Staff
Research Support Staff
Researchers

300

28

250

Individual Research
Project

24

28
29
74

25
200

45

89

63

82

28

28

30.3%

62

Total Expenditure

21
150
15
23

100

50

77

31

13
3

135

171

173

183

3,072,273,597

74( JPY )

186
179

89

168
127

Personnel

30.1%

28

Equipment

115

23.3%

52

Project
Activities

16.0%

0
Mar.2008 Mar.2009 Mar.2010 Mar.2011 Mar.2012 Mar.2013 Mar.2014 Mar.2015 Mar.2016 Mar.2017 Mar.2018

Travel0.2%

Number of Researchers
200
From Overseas
Japanese

32%

31%

30%

31%

54

48

54

40

117
93

119

128

138

969,467,150

33

137

Project
Activities
124

87

82

31.7%

From the
University

20.4%

Comprehensive
collaborationagreement Grant

29%

24
50

18.8%

44

31%

27%

15%
8

0.4%

Grant-in-Aid for
Scientific Research

Equipment

42

42
100

Travel

23%
26%

55

150

26%

( JPY )

Other Sources

Personnel

49.0%

13.7%

Contract
Research

2,102,806,447( JPY )

22.3%

Management
Expenses Grant

26.7%

Donation

16.8%

65
44
0
Mar.2008 Mar.2009 Mar.2010 Mar.2011 Mar.2012 Mar.2013 Mar.2014 Mar.2015 Mar.2016 Mar.2017 Mar.2018

86

87

Data

Selected Articles

BATF2 inhibits immunopathological Th17 responses by suppressing Il23a expression during
Trypanosoma cruzi infection.

Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated hostdefense.

J. Exp Med. 214(5):1313-1331, 2017. doi: 10.1084/jem.20161076.

Nature Immunology. 18(8):899-910, 2017. doi: 10.1038/ni.3767.

Kitada S, Kayama H, Okuzaki D. et al.

Sasai M, Sakaguchi N, Ma JS. et al.

Kiyoshi Takeda's group and others have revealed the regulation of immune response after Chagas disease causing Trypanosoma cruzi (T. cruzi) infection.
T. cruzi infection induces IL-23 production by host immune
cells, and antigen-specific Th17 responses are then promoted.
However, whether the IL-23-Th17 axis is controlled by IFNγ-dependent mechanisms during T. cruzi infection remains
unclear. In this study, they have demonstrated that IFN-γinducible BATF2 exerts its immunoregulatory function throughthe suppression of Il23a by interacting directly with c-JUN in
innate immune cells, thereby preventing immunopathologicalTh17 responses during T. cruzi infection.

Masahiro Yamamoto's group and others revealed evealed aunique role of GABARAPs, in particular Gate-16, in interferon-γ(IFN-γ)-mediated antimicrobial response. The group showedthat Gate-16 specifically associates with Arf1 to mediate uniform
distribution of IFN-inducible GTPases, and that lack of Gate-16
reduces Arf1 activation, leading to formation of IFN-inducible
GTPase-containing aggregates, hampering their function. Furthermore, mice lacking Gate-16 alone are susceptible to Toxoplasma. Thus, GABARAPs are uniquely required for antimicrobial
host defense through cytosolic distribution of IFN-inducible
GTPases.
These results will help Gate-16 targeted strategy for the treatment of toxoplasmosis and alimentary intoxication caused by
Toxoplasma and Salmonella.

Plasmodium products persist in the bone marrow and promote chronic bone loss

The activated conformation of integrin β7 is a novel multiple myeloma-specific target for CAR T
cell therapy.

Science Immunology. 2(12) eaam8093, 2017. doi: 10.1126/sciimmunol. aam8093.

Nature Medicine. 23(12):1436-1443, 2017. doi: 10.1038/nm.4431.
Lee MSJ, Maruyama K, Fujita Y. et al.
Hosen N, Matsunaga Y, Hasegawa K. et al.
Cevayir Coban's group revealed that malaria infection induces robust immune activation and invasion of parasite byproducts into the bone marrow leading to harmful outcomeson bone homeostasis.
The Coban Team has used mouse malaria models to showthat even after one time malaria infection, Plasmodium products gradually accumulate in the bone marrow niche andare "eaten-up" by bone resorbing cells osteoclasts. These accumulated malarial products in bone marrow niche induce
MyD88-dependent inflammatory responses in osteoclast andosteoblast 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 bone loss. Importantly,

88

malaria-mediated bone loss was found to be more severe in
those of younger age, leading to growth retardation.

Naoki Hosen, Athushi Kumanogoh and their research group
showed that the active conformer of an integrin can serve asa specific therapeutic target for multiple myeloma (MM). They
screened >10,000 anti-MM mAb clones and identified MMG49
as an MM-specific mAb specifically recognizing a subset of
integrin β7 molecules. The MMG49 epitope, in the N-terminal
region of the β7 chain, is predicted to be inaccessible in theresting integrin conformer but exposed in the active conformation. Elevated expression and constitutive activation of integrin
β7 conferred high MMG49 reactivity on MM cells, whereas
MMG49 binding was scarcely detectable in other cell types
including normal integrin β7+ lymphocytes. T cells transducedwith MMG49-derived chimeric antigen receptor (CAR) exerted
anti-MM effects without damaging normal hematopoietic cells.

Thus, MMG49 CAR T cell therapy is promising for MM, and a
receptor protein with a rare but physiologically relevant conformation can serve as a cancer immunotherapy target.

89

Data

Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors.
Nature. 552(7683):101-105, 2017. doi: 10.1038/nature24994.

Regulation of inflammatory responses by dynamic subcellular localization of RNA-binding
protein Arid5a.
Proc Natl Acad Sci USA. 115(6):E1214-E1220, 2018. doi: 10.1073/pnas.1719921115.

Saito F, Hirayasu K, Satoh T. et al.
Higa M, Oka M, Fujihara Y. et al.
Malaria, one of the world's three major infectious diseases
alongside tuberculosis and AIDS, has been reported to affectapproximately 300 million people every year, accounting forabout 500,000 deaths, and there is still no effective vaccine.
Hisashi Arase and others showed that P. falciparum uses immune inhibitory receptors to achieve immune evasion. RIFIN
proteins are products of a polymorphic multigene family comprising approximately 150–200 genes per parasite genome3
that are expressed on the surface of infected erythrocytes. We
found that a subset of RIFINs binds to either leucocyte immunoglobulin-like receptor B1 (LILRB1) or leucocyte-associatedimmunoglobulin-like receptor 1 (LAIR1). LILRB1-binding RIFINs
inhibit activation of LILRB1-expressing B cells and natural killer(NK) cells. Furthermore, P. falciparum-infected erythrocytes
isolated from patients with severe malaria were more likely to

Tadamitsu Kishimoto and his research group revealed theregulatory mechanism of subcellular localization of Arid5a inresponse to inflammation. It has been known that an inflammatory accelerator, Arid5a, is localized in the nucleus, and an
inflammatory brake, Regnase-1, is localized in the cytoplasm. In
this study, they showed that 1) Arid5a translocates to the cytoplasm from the nucleus in response to inflammation, 2) bimax,
which inhibit cNLS-dependent nuclear import via high-affinity
interactions with NLS-binding sites of importin-α, inhibits thenuclear import of Arid5a, 3) CRM1 inhibitor, Leptomycin B, inhibits the nuclear export of Arid5a after LPS stimulation.

Direct cell–cell contact between mature osteoblasts and osteoclasts dynamically controls theirfunctions in vivo.

Combination of multiple optical measurements can assess macrophage activation at single-celllevel without any contrast agent.

Nat Commun. 9(1):300, 2018. doi: 10.1038/s41467-017-02541-w.

Proc Natl Acad Sci USA. 115(12):E2676-E2685, 2018. doi: 10.1073/pnas.1711872115.

Furuya M, Kikuta J, Fujimori S. et al.
Masaru Ishii and his group showed, by using an intravital
imaging technique, that mOB and mOC functions are regulated
via direct cell–cell contact between these cell types. The mOBs
and mOCs mainly occupy discrete territories in the steady state,although direct cell–cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probereveals that mOCs secrete protons for bone resorption when
they are not in contact with mOBs, whereas mOCs contacting
mOBs are non-resorptive, suggesting that mOBs can inhibit
bone resorption by direct contact. Intermittent administrationof parathyroid hormone causes bone anabolic effects, whichlead to a mixed distribution of mOBs and mOCs, and increasecell–cell contact. This study reveals spatiotemporal intercellularinteractions between mOBs and mOCs affecting bone homeostasis in vivo.

90

interact with LILRB1 than erythrocytes from patients with nonsevere malaria, although an extended study with larger sample
sizes is required to confirm this finding. Our results suggest thatP. falciparum has acquired multiple RIFINs to evade the host immune system by targeting immune inhibitory receptors.

Pavillon N, Hobro AJ, Akira S, and Smith N.
Nicholas I. Smith and the collaborators developed a labelfree multimodal microscopy platform that allows the non-invasive study of cellular preparations without the need of any additional chemicals or contrast agent. The parameters extracted
from these measurements, coupled with machine algorithms,
enable the study of fine cellular processes such as macrophagecells activation upon exposure to lipopolysaccharide (LPS). The
authors demonstrate that activation, as well as partial activation inhibition, can be observed at single-cell level throughphenotypic and molecular characterization purely throughnon-invasive optical means.

91

1

2

Ando, T; Kashiwakura, J; Itoh-Nagato, N; Yamashita, H; Baba, M;
Kawakami, Y; Tsai, SH; Inagaki, N; Takeda, K; Iwata, T; Shimojo, N;
Fujisawa, T; Nagao, M; Matsumoto, K; Kawakami, Y; Kawakami,
T. Histamine-releasing factor enhances food allergy. Journal of
Clinical Investigation 127, 4541-4553 (2017).

3

Andrabi, M; Hutchins, AP; Miranda-Saavedra, D; Kono, H; Nussinov, R; Mizuguchi, K; Ahmad, S. Predicting conformational
ensembles and genome-wide transcription factor binding sites
from DNA sequences. Scientific Reports 7, 4071 (2017).

4

Arase, N; Tanimura, K; Jin, H; Yamaoka, T; Kishibe, M; Nishioka,
M; Kiyohara, E; Tani, M; Matsuoka, S; Ohmura, K; Takasugi,
K; Yamamoto, T; Murota, H; Arase, H; Katayama, I. Novel autoantibody against the beta 2-glycoprotein I/human leucocyte
antigen-DR complex in patients with refractory cutaneous ulcers. British Journal of Dermatology 178, 272-275 (2018).

5

6

7

Arima, Y; Ohki, T; Nishikawa, N; Higuchi, K; Ota, M; Tanaka,
Y; Nio-Kobayashi, J; Elfeky, M; Sakai, R; Mori, Y; Kawamoto, T;
Stofkova, A; Sakashita, Y; Morimoto, Y; Kuwatani, M; Iwanaga, T;
Yoshioka, Y; Sakamoto, N; Yoshimura, A; Takiguchi, M; Sakoda, S;
Prinz, M; Kamimura, D; Murakami, M. Brain micro-inflammation
at specific vessels dysregulates organ-homeostasis via the activation of a new neural circuit. Elife 6, e25517 (2017).
Atsumi, T; Suzuki, H; Jiang, JJ; Okuyama, Y; Nakagawa, I; Ota,
M; Tanaka, Y; Ohki, T; Katsunuma, K; Nakajima, K; Hasegawa,
Y; Ohara, O; Ogura, H; Arima, Y; Kamimura, D; Murakami, M.
Rbm10 regulates inflammation development via alternative
splicing of Dnmt3b. International Immunology 29, 581-591(2017).
Biering, SB; Choi, J; Halstrom, RA; Brown, HM; Beatty, WL; Lee,
S; McCune, BT; Dominici, E; Williams, LE; Orchard, RC; Wilen, CB;
Yamamoto, M; Coers, J; Taylor, GA; Hwang, S. Viral Replication
Complexes Are Targeted by LC3-Guided Interferon-Inducible
GTPases. Cell Host & Microbe 22, 74-85 (2017).

8

Carrascosa, LC; Klein, M; Kitagawa, Y; Luckel, C; Marini, F;
Konig, A; Guralnik, A; Raifer, H; Hagner-Benes, S; Radler, D; Bock,
A; Kang, C; Lohoff, M; Garn, H; Schaub, B; Berberich-Siebelt, F;
Sakaguchi, S; Bopp, T; Huber, M. Reciprocal regulation of the Il9locus by counteracting activities of transcription factors IRF1
and IRF4. Nature Communications 8, 15366 (2017).

9

Coban, C; Yamamoto, M. Introduction: Interactions Between
the Immune System and Parasites Special Issue. International
Immunology 30, 91-91 (2018).

10

Cossarizza, A; Chang, HD; Radbruch, A; Akdis, M; Andra, I;
Annunziato, F; Bacher, P; Barnaba, V; Battistini, L; Bauer, WM, etal. Guidelines for the use of flow cytometry and cell sorting in
immunological studies. European Journal of Immunology 47,
1584-1797 (2017).

11

Dai, HS; Griffin, N; Bolyard, C; Mao, HC; Zhang, JY; Cripe, TP;
Suenaga, T; Arase, H; Nakano, I; Chiocca, EA; Kaur, B; Yu, JH;
Caligiuri, MA. The Fc Domain of Immunoglobulin Is Sufficient to
Bridge NK Cells with Virally Infected Cells. Immunity 47, 159-170(2017).

12

92

Akiyama, M; Okada, Y; Kanai, M; Takahashi, A; Momozawa, Y;
Ikeda, M; Iwata, N; Ikegawa, S; Hirata, M; Matsuda, K; Iwasaki, M;
Yamaji, T; Sawada, N; Hachiya, T; Tanno, K; Shimizu, A; Hozawa,
A; Minegishi, N; Tsugane, S; Yamamoto, M; Kubo, M; Kamatani,
Y. Genome-wide association study identifies 112 new loci forbody mass index in the Japanese population. Nature Genetics
49, 1458-1467 (2017).

Ebina-Shibuya, R; Matsumoto, M; Kuwahara, M; Jang, KJ; Sugai,
M; Ito, Y; Funayama, R; Nakayama, K; Sato, Y; Ishii, N; Okamura,
Y; Kinoshita, K; Kometani, K; Kurosaki, T; Muto, A; Ichinose, M;
Yamashita, M; Igarashi, K. Inflammatory responses induce anidentity crisis of alveolar macrophages, leading to pulmonary
alveolar proteinosis. Journal of Biological Chemistry 292, 1809818112 (2017).

13

Eid, MMA; Shimoda, M; Singh, SK; Almofty, SA; Pham, P; Goodman,
MF; Maeda, K; Sakaguchi, N. Integrity of immunoglobulin variable regions is supported by GANP during AID-induced somatic
hypermutation in germinal center B cells. International Immunology 29, 211-220 (2017).

26

Hemmi, M; Tachibana, M; Fujimoto, N; Shoji, M; Sakurai, F;
Kobiyama, K; Ishii, KJ; Akira, S; Mizuguchi, H. T helper 17 Promotes induction of antigen-specific gut-Mucosal cytotoxic
T lymphocytes following adenovirus Vector Vaccination. Frontiers in Immunology 8, 1456 (2017).

14

Endo, T; Freinkman, E; de Rooij, DG; Page, DC. Periodic production of retinoic acid by meiotic and somatic cells coordinates
four transitions in mouse spermatogenesis. Proceedings of the
National Academy of Sciences USA 114, E10132-E10141 (2017).

27

Higa, M; Oka, M; Fujihara, Y; Masuda, K; Yoneda, Y; Kishimoto, T.
Regulation of inflammatory responses by dynamic subcellular
localization of RNA-binding protein Arid5a. Proceedings of the
National Academy of Sciences USA 115, E1214-E1220 (2018).

15

Foster, AJ; Nagata, M; Lu, XY; Lynch, AT; Omandi, Z; Ishikawa, E;
Yamasaki, S; Timmer, MSM; Stocker, BL. Lipidated Brartemicin
Analogues Are Potent Th1-Stimulating Vaccine Adjuvants. Journal of Medicinal Chemistry 61, 1045-1060 (2018).

28

Hill, A; DeZern, AE; Kinoshita, T; Brodsky, RA. Paroxysmal nocturnal haemoglobinuria. Nature Reviews Disease Primers 3, 17028(2017).

29

Hirata, J; Hirota, T; Ozeki, T; Kanai, M; Sudo, T; Tanaka, T; Hizawa,
N; Nakagawa, H; Sato, S; Mushiroda, T; Saeki, H; Tamari, M;
Okada, Y. Variants at HLA-A, HLA-C, and HLA-DQB1 Confer Riskof Psoriasis Vulgaris in Japanese. Journal of Investigative Dermatology 138, 542-548 (2018).

30

Hirata, T; Mishra, SK; Nakamura, S; Saito, K; Motooka, D; Takada,
Y; Kanzawa, N; Murakami, Y; Maeda, Y; Fujita, M; Yamaguchi, Y;
Kinoshita, T. Identification of a Golgi GPI-N-acetylgalactosaminetransferase with tandem transmembrane regions in the catalytic domain. Nature Communications 9, 405 (2018).

16

17

18

19

Fuchs, A; Gliwinski, M; Grageda, N; Spiering, R; Abbas, AK; Appel,
S; Bacchetta, R; Battaglia, M; Berglund, D; Blazar, B; Bluestone,
JA; Bornhauser, M, et al. Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization.
Frontiers in Immunology 8, 1844 (2018).
Fujitani, M; Sato, R; Yamashita, T. Loss of p73 in ependymal cells
during the perinatal period leads to aqueductal stenosis. Scientific Reports 7, 12007 (2017).
Furukawa, A; Kakita, K; Yamada, T; Ishizuka, M; Sakamoto, J;
Hatori, N; Maeda, N; Ohsaka, F; Saitoh, T; Nomura, T; Kuroki, K;
Nambu, H; Arase, H; Matsunaga, S; Anada, M; Ose, T; Hashimoto,
S; Maenaka, K. Structural and thermodynamic analyses reveal
critical features of glycopeptide recognition by the human PILR
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Sakakibara, S; Arimori, T; Yamashita, K; Jinzai, H; Motooka,
D; Nakamura, S; Li, SL; Takeda, K; Katayama, J; El Hussien,
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Sarashina-Kida, H; Negishi, H; Nishio, J; Suda, W; Nakajima, Y;
Yasui-Kato, M; Iwaisako, K; Kang, SJ; Endo, N; Yanai, H; Asagiri,
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Sasai, M; Pradipta, A; Yamamoto, M. Host immune responses
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Sasai, M; Sakaguchi, N; Ma, JS; Nakamura, S; Kawabata, T; Bando,
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Tanaka, M; Kobiyama, K; Honda, T; Uchio-Yamada, K; NatsumeKitatani, Y; Mizuguchi, K; Kabashima, K; Ishii, KJ. Essential Role ofCARD14 in Murine Experimental Psoriasis. Journal of Immunology 200, 71-81 (2018).

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Sasaki, E; Momose, H; Hiradate, Y; Furuhata, K; Takai, M; Kamachi,
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Uchida, T; Ueta, H; Xu, XD; Hirakawa, J; Tahara, K; Zhou, S; Sawanobori,
Y; Simmons, S; Kitazawa, Y; Kawashima, H; Matsuno, K. Rapid
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Sasaki, E; Momose, H; Hiradate, Y; Ishii, KJ; Mizukami, T; Hamaguchi,
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Uchiyama, R; Yonehara, S; Taniguchi, S; Ishido, S; Ishii, KJ; Tsutsui,
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Sato, K; Omahdi, Z; Shibata, K; Sonoda, KH; Yamasaki, S; Tanaka,
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123

Takahama, M; Akira, S; Saitoh, T. Autophagy limits activation of
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124

Takano, T; Motozono, C; Imai, T; Sonoda, KH; Nakanishi, Y; Yamasaki,
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125

Takemura, N; Kurashima, Y; Mori, Y; Okada, K; Ogino, T; Osawa, H;
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135

Yamazaki, S; Odanaka, M; Nishioka, A; Kasuya, S; Shime, H; Hemmi,
H; Imai, M; Riethmacher, D; Kaisho, T; Ohkura, N; Sakaguchi,
S; Morita, A. Ultraviolet B-Induced Maturation of CD11b-Type
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136

Yanagihashi, Y; Segawa, K; Maeda, R; Nabeshima, Y; Nagata, S.
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137

Ye, BQ; Liu, BY; Yang, LL; Huang, GL; Hao, L; Xia, PY; Wang, S;
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138

Yokokawa, H; Higashino, A; Suzuki, S; Moriyama, M; Nakamura,
N; Suzuki, T; Suzuki, R; Ishii, K; Kobiyama, K; Ishii, KJ; Wakita, T;
Akari, H; Kato, T. Induction of humoural and cellular immunity
by immunisation with HCV particle vaccine in a non-humanprimate model. Gut 67, 372-379 (2018).

139

Yokota, M; Tamachi, T; Yokoyama, Y; Maezawa, Y; Takatori, H;
Suto, A; Suzuki, K; Hirose, K; Takeda, K; Nakajima, H. I kappa BNS
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140

Yoshinaga, M; Nakatsuka, Y; Vandenbon, A; Ori, D; Uehata, T;
Tsujimura, T; Suzuki, Y; Mino, T; Takeuchi, O. Regnase-1 Maintains Iron Homeostasis via the Degradation of Transferrin Receptor 1 and Prolyl-Hydroxylase- Domain-Containing Protein 3
mRNAs. Cell Reports 19, 1614-1630 (2017).

141

Zwack, EE; Feeley, EM; Burton, AR; Hu, BF; Yamamoto, M; Kanneganti,
TD; Bliska, JB; Coers, J; Brodsky, IE. Guanylate Binding Proteins
Regulate Inflammasome Activation in Response to Hyperinjected Yersinia Translocon Components. Infection and Immunity
85, e00778-16 (2017).

Data

108

Saito, S; Tanoue, M; Masuda, K; Mori, Y; Nakatani, S; Yoshioka,
Y; Murase, K. Longitudinal observations of progressive cardiac
dysfunction in a cardiomyopathic animal model by self-gatedcine imaging based on 11.7-T magnetic resonance imaging.
Scientific Reports 7, 9106 (2017).

The articles were published between April, 2017 and March, 2018. The data were acquired using Web of Science Core CollectionTM on May 25,
2018, and sorted by alphabetical order of the first authors.

97

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