WPI Immunology Frontier Research Center 2019-2020

World PremierInternational
Research CenterO s a k a Un iv e r s it y

Immunology
Frontier
Research
Cent er

Annual Reportof IFReC
2019-2020

Edit: Research Planning & Management Office, IFReC
Published in July, 2020

O s a k a U n i v e r s it y

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

Message from the Director ............................................................... 1

Message from the Director

Organization
Organization Chart ........................................................................... 4
Committees & Advisory Board for IFReC ................................... 6Contents

Laboratories
Laboratories of IFReC ................................................................... 10▪ Immunology Group ................................................................... 12▪ Imaging Group ........................................................................... 60▪ Bioinformatics Group ................................................................ 72▪ Combined Research Fields .................................................... 76

Kiyoshi TAKEDA, MD/PhD
Director

Events & Outreach Activities

WPI Immunology Frontier Research Center

The 9th NIF Winter School on Advanced Immunology ......... 80
Immunology at the Forefront- The 11th International Symposium of IFReC ....................... 82
The 1st UCL-OU Joint Symposium on Immunology .............. 84
IFReC Seminars .............................................................................. 86
IFReC Colloquia .............................................................................. 88
Science Café on the Edge ........................................................... 90
Visits ................................................................................................... 91
Collaboration with KNOWLEDGE CAPITAL ............................. 92
Osaka University Co-Creation Day /“Kagaku Zanmai” in Aichi ............................................................. 94
Super Science High School Student Fair /WPI Science Symposium...................................................... 95
Nature Jobs Career Expo in London & NY ............................... 96
AAAS 2020 Annual Meeting ......................................................... 97

As the Director of the Immunology Frontier(Germany) and the University College of London (UK),

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

and we also promoted cooperation with institutions

am very pleased to present the IFReC annual reportin Asia. The NIF Winter School, which began as anfor the year 2019-2020.

international educational program, will celebrate

Since its inception in 2007, IFReC has establishedits 10th anniversary next year. We are proud of ouritself as a high-profile international research center ofgraduates who have taken up prominent positions at

immunology with the broad support of many people.

world-famous institutions. We hope that our WinterFurthermore, since the establishment of the WPI

School contributes to the worldwide circulation of

Academy in 2017, IFReC has strived to create a newtalented young researchers, which is one of the aims

mark in its history through a novel academic-industryof the WPI.

partnership agreement.

S t a r t i n g f ro m t h e l a t t e r p a r t o f l a s t y e a r,

In 2019, I assumed the directorship of IFReC fromcoronavirus disease 2019 (COVID-19) caused by aDr. Shizuo Akira. Fortunately, in 2019-2020, we were

coronavirus called severe acute respiratory syndrome

able to make great strides in our research resulting

coronavirus 2 (SARS-CoV-2) has spread throughout

in many academic achievements. In addition, we

the world and turned into a pandemic. As various

Major Awards ............................................................................... 100

embarked on a couple of new initiatives. The first

countries deal with this ongoing crisis, we continue

Common Facilities ....................................................................... 102

is the emphasis on human immunology research.

our efforts in basic immunology research and look for

Composition & Finance .............................................................. 103

The IFReC Human Immunology Laboratory wasways in which we can contribute to the society in the

Advanced Postdoc Program /Grant for Next Generation Principal Investigators ............... 104

launched in 2019 and two new PIs have joined. Infight against COVID-19.

this laboratory, the human immune system will be

We are totally committed to continuing ourUnique Support Programs for Young Researchers ............. 105

analyzed through the collaboration of the many

contributions to scientific advances through research

Selected Articles .......................................................................... 106

research colleagues in IFReC. The second is theand education and our evolvement into a world-classpromotion of international cooperation. We started

immunology research center.

Japanese Language Class .......................................................... 98
Information

Publications ................................................................................... 110
Access Map ...................................................................................... 116

mutual visits and joint symposiums with overseas
partner institutions, such as the University of Bonn

1

Organization

Organization Chart
Osaka University

MEXT

International Advanced
Research Institute (IARI)WPI Initiative Program Committee
JSPS

As of June, 2020

Joint Research Chairs with the Pharmaceutical Companies
International
Scientific
Advisory
Board

Research
GroupsImmunology Group• Innovative Drug Discovery in Immunology (Chugai Pharmaceutical)• Immune-therapeutic Drug Discovery (Otsuka Pharmaceutical)• Innate Immunity• Innovative Drug Discovery in Host Defense

Imaging Group

DeputyDirectors

Bioinformatics Group
Combined Research Fields

Core Instrumentation Facility

Common Facilities

Network Administration OfficeDirector
Management
Committee

Board of Representatives

Administrative
Director

Administrative
Department• General Affairs Section• Accounting Section

Research Planning and Management Office

Animal Resource Center for Infectious Diseases

Functions of Administrative Office
General Affairs Section• Employment, acceptance, and management of work hours for researchers and staff• Social insurance (for part-time staff), employment insurance• Various research supports (MTA, patents, animal experiments, safety etc.)• Support for international researchers and students• IFReC Kishimoto Foundation Fellowship Program

Accounting Section• Budget drafting / implementation / management• Acceptance and implementation of third-party funding• Purchasing and payment of payroll, travel expense and honorarium• Health insurance procedures• Management of facilities

Cooperative Institutions• Institute for Frontier Life and Medical Sciences, Kyoto University, Japan• RIKEN Center for Integrative Medical Sciences, Japan• National Institute of Biomedical Innovation, Health and Nutrition, Japan• ImmunoSensation, Clusters of Excellence, the Rheinische Friedrich-Wilhelms-University of Bonn,
Germany

4

Research Planning and Management Office• Research promotion and support (Consultation for grants and patents, etc.)• Establishing research environments (Facility and Safety management, research
agreement, etc.)• Fostering young scientists (Winter School, advanced postdoc program, orientation, etc.)• Organizing scientific events (Symposia, colloquia, seminars, etc.)• Public relations (Publishing, website, outreach to citizens, etc.)5Committees & Advisory Board for IFReC
World Premier International Research Center Initiative (WPI)● Program Director
Akira Ukawa

As of Mar. 2020
Director, Center for World Premier International Research Center Initiative, JSPS, Japan● Deputy Program Director
Minoru Yoshida

6

Group Director, Chemical Genomics Research Group, RIKEN, Center for Sustainable
Resource Science, JapanWPI 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 Japanas a hub at the pinnacle of international researcher circulation.
In the decade ahead, the research institutes of WPI and WPI Academy will work together to hold public relations andoutreach activities.

● Academy Director

As of Mar. 2020

● Program Committee Members

Toshio Kuroki

Rita Colwell

Distinguished University Professor, University of Maryland, USA● Academy Officer for IFReC

Richard Dasher

Director, US-Asia Technology Management Center, Stanford University, USA

Takehiko Sasazuki

Victor Joseph Dzau

President, National Academy of Medicine, USA

Michinari Hamaguchi

President, Japan Science and Technology Agency (JST), Japan

Maki Kawai

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

Klaus von Klitzing

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

Makoto Kobayashi

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

Kiyoshi Kurokawa

Professor Emeritus, National Graduate Institute for Policy Studies, Japan

Chuan Poh Lim

Chairman, Singapore Food Agency, Singapore

Hiroshi Matsumoto

President, RIKEN, Japan

Ryozo Nagai

President, Jichi Medical University, Japan

Michiharu Nakamura

Counselor to the President, JST, Japan〈Chairperson〉Ryoji Noyori

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

Norihiko Suzuki

Chair of the Board/President, Akita International University, Japan

Harriet Wallberg

Professor, Karolinska Institutet, Sweden

Jean Zinn-Justin

Scientific Adviser, IRFU/CEA, France

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

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

International Scientific Advisory Board from abroad

As of Mar. 2020

Jeffrey Ravetch

The Rockefeller University, USA

Immunology

Christopher Goodnow

The Garvan Institute of Medical Research, Australia

Immunology

Richard Locksley

University of California, San Francisco, USA

Immunology

Lewis L. Lanier

University of California, San Francisco, USA

Immunology

Anne O'Garra

The Francis Crick Institute, UK

Immunology

Yale E. Goldman

University of Pennsylvania, USA

Imaging7

Laboratories

Host Defense

Laboratories
of
IFReC

Immunochemistry

Immunoglycobiology

Immunopathology

Stem Cell Biology andDevelopmental Immunology

Molecular Immunology

Aging Biology

Oncogene Research

Shizuo Akira

Taroh Kinoshita
Yoshiko Murakami

Atsushi Kumanogoh

Takashi Nagasawa

Sho Yamasaki

Eiji Hara

Masato Okada#InnateImmunity#PathogenRecognition#macrophage#GPI-anchor#ParoxysmalNocturnalHemoglobinuria#ImmuneSemaphorin#AutoimmuneDiseases#T-cellActivation#CARCell #StemCell#niche#lectin#NovelImmuneReceptor#aging #SASP#cancer#mTOR #SRC
#cancer

Cutaneous Immunology

Innate Immune Systems

Immune Regulation

Immune Regulation

Mucosal Immunology

Signal Transduction

Human Immunology

James B. Wing
Single Cell Immunology

Hisashi Arase

Tadamitsu Kishimoto

Hitoshi Kikutani

Kiyoshi Takeda

Nobuyuki Takakura

Manabu Fujimoto

Kazuyo Moro

#MHC #neo-self#MisfoldedProtein#malaria#rheumatism #IL-6#Th17Differentiation#SLE#Anti-nuclearAntibody(ANA)#GutImmunity#InflammatoryBowelDisease(IBD)#microbiota#BloodVessels #StemCell#cancer#IntractableSkinDiseases#allergy#ILC2#AutoimmuneDiseases

Experimental Immunology

Cell Signaling

Lymphocyte Differentiation

Malaria Immunology

Single Molecule Imaging

Immunology andCell Biology

Nuclear Medicine

Daisuke Okuzaki
Single Cell Genomics#HumanDiseases#SingleCell #genomics

Biophotonics

Shimon Sakaguchi

Takashi Saito

Tomohiro Kurosaki

Cevayir Coban

Toshio Yanagida
Ben Seymour

Masaru Ishii

Jun Hatazawa

Nicholas Isaac Smith#Treg #ImmuneTolerance#CancerImmunology#T-cellActivation#TCRSignal#MemoryB-cell#AntibodyProduction#MalariaParasite#vaccine#SingleMoleculeImaging#MembraneProtein#osteoclast #LiveImaging#CancerMetastasis#PET/ MRI #FBPA#CancerTherapy#LabelFree #RamanScattering#IntraCellImaging

Immune Response Dynamics

Systems Immunology

Statistical Immunology

Vaccine Science

Immunoparasitology

Biochemistry and
Immunology

Molecular Neuroscience

Chemical Imaging
Techniques

Ken J. Ishii

Masahiro Yamamoto

Shigekazu Nagata

Toshihide Yamashita

Kazuya Kikuchi

Kazuhiro Suzuki

Daron M. Standley

Yukinori Okada#vaccine#adjuvant#parasite #toxoplasma#ImmuneEvasion#macrophage#CellDeathSignal#apoptosis#CentralNervousSystem#encephalomyelitis#ChemicalBiology#FluorescentProbe#AdrenergicRceptor#LymphocyteTrafficking#ImmuneRepertoire#ReceptorModeling#StatisticalGenetics #BigData#DiseaseRiskGenes

understanding of the precious roles of Regnase-1 in immune
and non-immune cells by using tissue-specific Regnase-1deficient mice and mutant mice.

Shizuo Akira, MD/PhD
Professor

Shizuo Akira

Associate Professor

Kazuhiko Maeda
Takashi SatohHiroki Tanaka
Kanako Kuniyoshi
2575Assistant Professor
Postdoctoral Fellow
Research Assistant
Visiting Scientist
Support Staff

We are focused on the innate immune system, which
is an evolutionarily conserved host defense mechanism
against various pathogens. Innate immune responses are
initiated by pattern recognition receptors, which recognize
specific structures of microorganisms. Toll-like receptors(TLRs) are capable of sensing organisms ranging frombacteria to fungi, protozoa and viruses, and play a majorrole in innate immunity. Individual TLRs recognize different
microbial components, and give rise to different patterns ingene expression.

Role of SatM, an atypical monocyte and committedprogenitor involved in fibrosis
Macrophages consist of at least two subgroups. M1
macrophages are pro-inflammatory and have a central rolein host defense. On the contrary, M2 macrophages areassociated with responses to anti-inflammatory reactions
and tissue remodeling. Monocytes and macrophages
comprise a variety of subsets with diverse functions. It isthought that these cells play a crucial role in homeostasisof peripheral organs, key immunological processes,
and development of various diseases. Among these
diseases, fibrosis is a life-threatening disease of unknown
aetiology. Its pathogenesis is poorly understood, andthere are few effective therapies. The development offibrosis is associated with activation of monocytes andmacrophages. We identified a new macrophage subset that
Ceacam1+Msr1+Ly6C-F4/80-Mac1+ monocytes, which
12

Regnase-1 is inactivated in response to external stimuli
through posttranslational modifications, yet the precise roleof phosphorylation largely remains unknown. We showedthat IL-17 induces the phosphorylation of Regnase-1 inan Act1-TBK1-IKKi-dependent manner, especially in nonhematopoietic cells. Phosphorylated Regnase-1 is released
from the endoplasmic reticulum into the cytosol, thereby
losing its mRNA degradation function, which leads toexpression of IL-17 target genes. IL-17-induced Regnase-1
phosphorylation is completely blocked in two Regnase-1mutant (Regnase-1AA/AA and Regnase-1ΔCTD/ΔCTD) mice. Thus,
Regnase-1 plays a critical role in the development of IL-17mediated inflammatory diseases via the Act1-TBK1-IKKi
axis. Blockade of Regnase-1 phosphorylation sites may bepromising for treatment of T helper 17-associated diseases.
Damage to intestinal epithelial cell (IEC) layers duringintestinal inflammation is associated with inflammatory
bowel disease. We showed that Regnase-1 controls colon

epithelial regeneration by regulating protein kinase mTOR
(mechanistic target of rapamycin kinase) and purine
metabolism. During dextran sulfate sodium-induced
intestinal epithelial injury and acute colitis, Regnase-1deficiency in IECs (Regnase-1ΔIEC) mice were resistant tobody weight loss, maintained an intact intestinal barrier,and showed increased cell proliferation and decreased
epithelial apoptosis. Chronic colitis and tumor progression
were also attenuated in Regnase-1ΔIEC mice. Regnase-1

Immunolo g y

Host Defenseof T cell activation. Based on these findings, we promotepredominantly regulates mTORC1 signaling. Metabolic
analysis revealed that Regnase-1 participates in purine
metabolism and energy metabolism during inflammation.
Increased expression of ectonucleotidases contributed to
the resolution of acute inflammation in Regnase-1ΔIEC mice.
Regnase-1-deficiency in IECs has beneficial effects on
the prevention and/or blocking of intestinal inflammatory
disorders.
We are further studying to achieve the goal of acomprehensive understanding of the innate immune systemand to develop an effective treatment for immune-related
inflammatory diseases.

we termed SatM (segregated-nucleus-containing atypicalmonocytes), share granulocyte characteristics, are regulated
by C/EBPβ (CCAAT/enhancer binding protein beta), andare critical for fibrosis. To investigate the physiologicalrole of SatM and related subsets, we recently identified
an RNA-binding protein RBM7 that is a component of theNEXT (nuclear exosome targeting) complex. We found thatthe expression of Rbm7 is increased in the fibrotic phase.
Rbm7-deletion in nonhematopoietic cells suppresses
fibrosis. Dysregulated expression Rbm7 triggers apoptosis
via nuclear degradation of noncoding RNA Neat1. Rbm7
in epithelial cells plays a critical role in the developmentof fibrosis by regulating ncRNA decay, thereby producing
chemokines that recruit SatMs. Inhibition of RBM7 would
provide an effective treatment of fibrosis in patients.

Molecular mechanism of endoribonuclease Regnase-1
in inflammation
Regnase-1 is a member of CCCH-type zinc finger
proteins. Regnase-1-deficient mice develop spontaneous
autoimmune diseases accompanied by splenomegaly andlymphadenopathy. Regnase-1 has endonuclease activity
and destabilizes a set of mRNAs through cleavage of their
3’UTRs such as IL-6 and IL-12 p40 in macrophages andc-Rel, Ox40, and IL-2 in CD4+ T cells. Regnase-1 protein iscleaved by MALT1 protease after T cell receptor stimulation,
resulting in the enhancement of T cell activation. Dynamic
control of Regnase-1 expression is critical for modulation

Figure 1. Dysregulated expression of the nuclear exosome
targeting complex component Rbm7 in non-hematopoietic cells
licenses the development of fibrosis.

Figure 2. IL-17 induces Regnase-1 phosphorylation in an Act-1/
TBK1/IKKi –dependent manner. Phosphorylation of Regnase-1
is released from the endoplasmic reticulum into the cytosols,
resulting in loss of the mRNA degradation function.

Recent Publications
⿎⿎ Fukushima K, Satoh T., et al. Dysregulated expression of the nuclear
exosome targeting complex component Rbm7 in non-hematopoietic
cells licenses the development of fibrosis. Immunity 52, 542-556(2020).

USA. 115, 11036-11041 (2018).
⿎⿎ Satoh T., et al. Identification of an atypical monocyte and committedprogenitor involved in fibrosis. Nature 541, 96-101 (2017).

⿎⿎ Tanaka H., et al. Phosphorylation-dependent Regnase-1 release fromendoplasmic reticulum is critical in IL-17 response. J. Exp. Med. 216,
1431-1449 (2019).
⿎⿎ Maeda K., et al. Innate immunity in allergy. Allergy 74, 1660-1674(2019).
⿎⿎ Nagahama H., et al. Regnase-1 controls colon epithelial regeneration
via regulation of mTOR and purine metabolism. Proc. Natl. Acad. Sci.

13

Studies on inherited GPI deficiencies (IGD)

Studies on GPI biosynthesis
Some GPI-APs have an N-acetylgalactosamine(GalNAc)-side chain (Figure 2). The GalNAc side-chain

In collaborations with Dr. Campeau’s group in Canada

Taroh Kinoshita, PhD
Yoshiko Murakami, MD/PhD(Co-PI)
Professor

Taroh Kinoshita
Yoshiko Murakami

Research Assistant
Visiting Scientist
Support Staff

1
3
4

and many clinicians and medical geneticists in several othercountries, we reported the first group of individuals who
suffer from IGD caused by bi-allelic hypomorphic mutationsin the PIGB gene (Murakami et al. Am. J. Hum. Genet.
2019). We also contributed to a study on the first group ofindividuals with IGD caused by PIGU mutations (Knaus etal. Am. J. Hum. Genet. 2019).

can be elongated with β1-3-linked galactose (Gal). The
Gal transferase (GPI-GalT) that mediates this elongation
has been unknown. Through a CRISPR genome-wide
knockout screen we identified B3GALT4, which is known
as ganglioside GM1 synthase, as GPI-GalT (Figure 2). We
also found that lactosylceramide is required for efficient GPI
galactosylation. These results revealed close interactions of
the GPI-AP biosynthetic pathway with the glycosphingolipid
biosynthetic pathway in the Golgi apparatus (Wang Y et al.
Nat. Commun. 2020).

Immunolo g y

Immunoglycobiology

PNH is a distinct form of PNH (Figure 1).

Taroh Kinoshita

We have been studying biosynthesis, functions anddeficiencies of glycosylphosphatidylinositol (GPI) andGPI-anchored proteins (GPI-APs). In 2019, we made thefollowing advances in studies on GPI deficiencies andbiosynthesis.

Studies on paroxysmal nocturnal hemoglobinuria caused
by PIGT mutations (PIGT-PNH)
I n c o l l a b o r a t i o n s w i t h D r. K r a w i t z ’s a n d D r.
Schrezenmeier’s groups in Germany and Dr. Kohara’s and
Dr. Kanakura’s groups in Kobe and Osaka, respectively,
we reported genetic, biochemical, immunological andclinical characteristics of patients with PNH caused by
PIGT mutations, and compared them with those of classicalPNH caused by mutations in PIGA (Hochsmann, Murakami,
Osato et al. J. Clin. Invest. 2019). PNH, characterizedby complement-mediated hemolysis and thrombosis, iscaused by generation and clonal expansion of GPI-AP
deficient hematopoietic stem cells. Single somatic mutationin X-linked PIGA causes GPI-AP-deficiency in classical
PNH, whereas two mutations are required for PIGT on
chromosome 20q to cause GPI-AP-deficiency. Patients with
PIGT-PNH had a germline mutation in the maternal PIGT andin their PNH cells, the paternal PIGT was lost by a somatic,
Mb-size deletion including the entire PIGT gene. We foundthat the Mb-size deletion always included a region called“myeloid common deleted region (CDR)” implicated in 20qmyeloproliferative neoplasm (Figure 1). The deletion results
14

in loss of tumor-suppressive genes in the myeloid CDR
because those in the maternal myeloid CDR are imprinted,
suggesting causality to clonal expansion in PIGT-PNH. PIGA
protein is essential for the initial step in GPI biosynthesis,
so a PIGA defect does not cause accumulation of any GPIintermediate. In contrast, PIGT is essential for the transfer
of preformed GPI to proteins. We showed accumulationof un-protein-linked free GPI in PIGT-defective cells(Figure 1). Clinically, patients with PIGT-PNH had recurrent
autoinflammatory symptoms such as urticaria, arthralgia
and aseptic meningitis, as well as typical PNH symptoms.
Moreover, the patients had the autoinflammation for many
years before the onset of PNH. Eculizumab, an anticomplement C5 antibody drug, was effective in preventing
intravascular hemolysis, thrombosis, and inflammatory
symptoms, suggesting that the autoinflammation wascomplement-dependent. Being consistent with the
autoinflammation, elevation of serum IL-18 was documented.
To clarify the mechanistic basis of autoinflammation, weused macrophages derived from PIGT-KO and PIGA-KO
THP-1 cells. The PIGT-KO cells activated the lectin pathway
of complement and bound more C4- and C3-fragments
and C5b-9 membrane attack complexes than those on the
PIGA-KO cells. The elevated activation of complement on
the PIGT-KO cells was associated with higher levels of C5b9 dependent IL-1β secretion by the PIGT-KO cells than the
PIGA-KO cells. These results suggest that overactivation ofcomplement and inflammasome in PIGT-defective cells isthe basis of autoinflammation seen in PIGT-PNH and PIGT-

Figure 1. Mechanistic basis of PIGT-PNH. Patients’ maternal
chromosomes 20q have a germline loss-of-function PIGT
mutation and imprinted myeloid common deleted region (CDR).
Mutant hematopoietic stem cells in those patients lost paternal
normal PIGT and active myeloid CDR because of a megabasesize deletion. The mutant stem cell expands similar to thoseof 20q- myeloproliferative neoplasm and generates PNH-type
erythrocytes and monocytes. Intravascular hemolysis and recurrent
autoinflammation occur because of uncontrolled activation ofcomplement and inflammasomes. Anti-C5 antibody eculizumab iseffective for both hemolysis and inflammatory symptoms. (Adopted
from Hochsmann, B., Murakami, Y., Osato, M., et al., J. Clin.
Invest., 2019, 129:5123-5136)

Figure 2. Identification of B3GALT4, previously known asganglioside GM1 synthase, to be a galactosyltransferase thatmodifies N-acetylgalactosamine side chain of GPI anchors.

Recent Publications
⿎⿎ Wang Y, Maeda Y, Liu Y-S, Takada Y, Ninomiya A, Hirata T, Fujita M,
Murakami M and Kinoshita T. Cross-talks of glycosylphosphatidylinositol
biosynthesis with glycosphingolipid biosynthesis and ER-associateddegradation. Nat. Commun.11, 860 (2020).
⿎⿎ Hochsmann B, Murakami Y, Osato M, Knaus A, Kawamoto M, Inoue
N, Hirata T, Murata S, Anliker M, Eggerman T, Jaeger M, Floettmann
R, Hoellein A, Murase S, Ueda Y, Nishimura J, Kanakura Y, Kohara
N, Schrezenmeier H, Krawitz PM and Kinoshita T. Complement
and inflammasome overactivation mediates paroxysmal nocturnal
hemoglobinuria with autoinflammation. J. Clin. Invest.129, 5123-5136(2019).
⿎⿎ Wang Y, Hirata T, Maeda Y, Murakami Y, Fujita M and Kinoshita,
T. Free, unlinked glycosylphosphatidylinositols on mammalian cellsurfaces revisited. J. Biol. Chem. 294, 5038-5049 (2019).
⿎⿎ Hirata T, Mishra SK, Nakamura S, Saito K, Motooka D, Takada
Y, Kanzawa N, Murakami Y, Maeda Y, Fujita M, Yamaguchi Y andKinoshita T. Identification of a Golgi GPI-N-acetylgalactosaminetransferase with tandem transmembrane regions in the catalytic
domain. Nat. Commun. 9, 405 (2018).
⿎⿎ 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 and Murakami Y. Phenotypegenotype correlations of PIGO deficiency with variable phenotypes
from infantile lethality to mild learning difficulties. Hum. Mutat.38, 805815 (2017).

15

Immunolo g y

Immunopathology

Atsushi Kumanogoh, MD/PhD

16

Professor

Atsushi Kumanogoh

Assistant Professor
Research AssistantSupport Staff

Yoshimitsu Morita
５５

Our research team is involved in two approaches, thatis, basic and clinical immunology. As basic aspects of our
projects, our proposed study is the regulation of immune
cell motility and migratory behavior in vivo by soluble andmembrane-bound ‘immune guidance molecules’ suchas semaphorins and their receptors. Semaphorins wereoriginally identified as axon-guidance molecules that

other paranasal diseases and serum SEMA4D levels werepositively correlated with disease features. The expressionof membrane SEMA4D on eosinophils was downregulated
in ECRS patients, whereas downregulation of SEMA4D was
not detected in other leukocytes, suggesting that increasingof serum SEMA4D in ECRS patients is due to proteolytic
cleavage of membrane-bound SEMA4D from eosinophils.

function during neuronal development. However, cumulative
evidence indicates that semaphorins also participate inimmune responses, both physiological and pathological,
and they 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 forhuman immunological disorders, such as autoimmunity,
allergy, immune deficiency, cancer/metastasis, andneurodegenerative diseases. We here focus on the clinicalimplications of Sema4D in Eosinophilic chronic rhinosinusitis(ECRS). ECRS is characterized by intractable nasal polyps
with eosinophilic infiltration based on Th2 inflammation.
Conservative treatments for ECRS are endoscopic
sinus surgery (ESS) and systemic administration of
glucocorticoids. However, ECRS frequently recurs after ESS,
and glucocorticoid therapy occasionally causes multipleside effects. Therefore, it is necessary to identify not only thedisease pathogenesis, but also potentially novel therapeutictargets and/or predictive biomarkers for ECRS.
We f o u n d s e r u m s o l u b l e S E M A 4 D l e v e l s w e resignificantly higher in ECRS patients than in patients with

Furthermore, we showed that MMP-9 levels were elevated in
ECRS patients and MMP-9 proteolytically cleaved SEMA4D
from the eosinophil surface, yielding its soluble form. Soluble
SEMA4D promotes transendothelial migration of eosinophils,
causing them to infiltrate nasal polyps, in addition, SEMA4D
can increase nasal epithelium permeability, thereby
facilitating allergen infiltration. Investigations using animalmodels demonstrated that loss of SEMA4D made ECRS
milder. Furthermore, treatment with anti-SEMA4D antibody
significantly reduced the inflammation. Collectively, thesedata show that the SEMA4D plays pivotal roles in ECRS
pathogenesis. Moreover, our results reveal the potential
utility of SEMA4D not only as a predictive biomarker, butalso as a potentially novel therapeutic target in ECRS.

Figure. Serum soluble SEMA4D levels were elevated in patients with ECRS and positively correlated with disease severity. Cell surface
expression of SEMA4D on eosinophils from ECRS patients was reduced, which was due to MMP-9-mediated cleavage of membrane
SEMA4D. Soluble SEMA4D induced eosinophil transendothelial migration. Treatment with anti-SEMA4D antibody ameliorated eosinophilic
infiltration in sinus tissues and nasal lavage fluid in the ECRS animal model.

Recent Publications
⿎⿎ Tsuda T, Nishide M, Maeda Y, Hayama Y, Koyama S, Nojima S,
Takamatsu H, Okuzaki D, Morita T, Nakatani T, Kato Y, Nakanishi
Y, Futami Y, Suga Y, Naito Y, Konaka H, Satoh S, Naito M, Izumi M,
Obata S, Nakatani A, Shikina T, Takeda K, Hayama M, Inohara H, andKumanogoh. Pathological and therapeutic implications of eosinophilderived semaphorin 4D in eosinophilic chronic rhinosinusitis. J. Allergy
Clin. Immunol. 145(3), 843-854 (2020).
⿎⿎ Kang S, Nakanishi Y, Kioi Y, Okuzaki D, Kimura T, Takamatsu H,
Koyama S, 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. Nature Immunol. 19,
561-570 (2018).

⿎⿎ Nishide M, and Kumanogoh A. The role of semaphorins in immune
responses and autoimmune rheumatic diseases. Nat. Rev. Rheumatol.
14, 19-31 (2018).
⿎⿎ 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 β. Nat. Med.
23,1436-1443 (2017).

17

(Arase et al. Br. J. Dermatol. 2017). Similarly, we also foundthat myeloperoxidase/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 correlated with susceptibility

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

Immunolo g y

Immunochemistry

Hisashi Arase, MD/PhD
Professor

Hisashi Arase

Associate Professor Tadahiro Suenaga（-Dec 31, 2019）
Masako Kohyama（Mar 1, 2020-）
Assistant Professor Masako Kohyama（-Feb 28, 2020）
Wataru Nakai（Mar 1, 2020-）
Postdoctoral Fellow 3
Research Assistant 4
Visiting Scientist
1
Support Staff
3

We have been working on the interactions between
pathogens and various paired receptors. In addition,
we have found that MHC class II molecules function as
molecular chaperones to transport cellular misfolded
proteins to the cell surface. Analyses of misfolded proteinstransported 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 of activating andinhibitory 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 theregulation 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 withglycoprotein B (gB), an envelope protein of herpes simplex
virus-1 (HSV-1), and the interaction between PILRα andgB 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 ofpaired 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 involvedin viral infection.
LILR is another type of paired receptor family. We
18found that activating LILRA2 recognizes abnor mal
immunoglobulins cleaved by microbial proteases but notnormal immunoglobulins. LILRA2 seems to be a sensor todetect immunoglobulin abnormalities in microbial infection(Hirayasu et al. Nature Microbiology 2016). On the other
hand, we found that RIFINs, products of multigene familyof Plasmodium falciparum, bind to inhibitory LILRB1 anddownregulate 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 inefficientdevelopment 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 not transported to outside the cells. Therefore, misfolded
proteins transported to the cell surface by MHC class II molecules
may be recognized as ‘neo-self’ antigens by immune system,
which might initiate aberrant immune response to self-antigens(Jiang et al. Int. Immunol. 2013; Jin et al. Proc. Natl. Acad. Sci.
USA. 2014; Tanimura et al. Blood 2015, Arase Adv. Immunol.
2016; Arase et al. Br. J. Dermatol. 2017; Hiwa et al. Arthritis
Rheumatol. 2017, Shimizu et al. Int Immunol. 2019).

Misfolded proteins complexed with MHC class II
molecules are targets for autoimmune diseases.
MHC class II allelic polymorphisms are associated withsusceptibility to many autoimmune diseases. However,it has remained unclear how MHC class II molecules are
involved in autoimmune disease susceptibility. We foundthat cellular misfolded autoantigens are rescued fromprotein degradation by MHC class II molecules (Jiang et al.
Int. Immunol. 2013). Furthermore, we found that misfolded
proteins complexed with MHC class II molecules are targets
for autoantibodies in autoimmune disease patients (Jin etal. Proc. Natl. Acad. Sci. USA. 2014; Tanimura et al. Blood.
2015; Shimizu et al. Int, Immunol. 2019). In addition, we
could detect autoantibodies against β2GPI/HLA class II
complex in the patients with refractory cutaneous ulcers

Recent Publications
⿎⿎ Shimizu Y, Kohyama M, Yorifuji H, Jin H, Arase N, Suenaga T, Arase
H. FcγRIIIA-mediated activation of NK cells by IgG heavy chain
complexed with MHC class II molecules. Int. Immunol. 31, 303-314(2019).
⿎⿎ Saito F, Hirayasu K, Satoh T, Wang CW, Lusingu J, Arimori T, Shida
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).
⿎⿎ 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 byautoantibodies in microscopic polyangiitis. Arthritis. Rheumatol. 69,
2069-2080 (2017).
⿎⿎ 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. Nature Microbiology 1, 1-7 (2016).
⿎⿎ Jin H, Arase N, Hirayasu K, Kohyama M, Suenaga T, Saito F, Tanimura
K, Matsuoka S, Ebina K, Shi K, Toyama-Sorimachi N, Yasuda S,
Horita T, Hiwa R, Takasugi K, Ohmura K, Yoshikawa H, Saito T, Atsumi
T, Sasazuki T, Katayama I, Lanier LL, Arase H. Autoantibodies to
IgG/HLA-DR complexes are associated with rheumatoid arthritis
susceptibility. Proc. Natl. Acad. Sci. USA. 111, 3787-3792 (2014).

19and augments the production of some chemokines leading
to recruitment of Tregs and gMDSCs. Moreover, Arid5astabilized Ido1 and chemokine mRNAs.
Taken together, our results indicate that Arid5a acts as

Tadamitsu Kishimoto, MD/PhD
Professor

Tadamitsu Kishimoto

Associate Professor
Assistant Professor
Postdoctoral Fellow
Research Assistant
Visiting Scientist
Support Staff

Shigeru Hashimoto
Sujin Kang
4222

Figure. Scheme of proinflammatory cytokines production throughThr749 STAT1 phosphorylation downstream of TLR4 endocytosis.
LPS–TLR4 ligation on cell surface induces MYD88-dependent
signaling cascades resulting in early phase transcriptional
induction of proinflammatory cytokines. LPS–induced TLR4
endocytosis induces the transcription of antiviral genes throughIFNβ–Tyr701 STAT1 phosphorylation. Moreover, LPS–induced TLR4
endocytosis promotes Thr749 STAT1 phosphorylation through TBK1/
IKKβ kinase complex at the late phase of LPS stimulation. In
turn, phosphorylated STAT1 at Thr749 induces ARID5A and IL12B
transcription resulting in augmented production of proinflammatory
cytokines. Red arrows indicate the noncanonical signaling pathway.

Arid5a orchestrates the immunosuppressive
microenvironment of pancreatic cancer

IL-6 induced vascular endothelial cell activation plays a
key role in cytokine storms

STAT1 phosphorylation confers distinct DNA-binding andgene-regulatory properties

The cytokine storm is a systemic inflammatory response,
caused by CAR-T cell therapy, infections including sepsis
or COVID-19. Cytokine storms are characterized byexcessive proinflammatory cytokines production. These
proinflammatory cytokines including interleukin-6 (IL6) elicit an increase in vascular permeability and theinflux of fluid, resulting in respiratory failure. However, theprecise mechanism as to how IL-6 signaling induces thedysregulation of endothelial cells remains to be elucidated.
We found that vascular endothelial cell-driven IL-6
promptly activates IL-6 receptor (IL-6R) in a trans-signaling
pathway, and this process accelerated the formation of theproinflammatory cytokine network, including IL-6, IL-8, andMCP-1, plasminogen activator inhibitor-1, which reflects
endothelial injury. Furthermore, treatment with tocilizumab
which is an IL-6R antagonist, substantially abolished theformation of this cytokine network in LPS-stimulated vascularendothelial cells, suggesting that endothelial cells activatedby IL-6 signaling form a positive loop during a cytokine
storm. Taken together, these results indicate that blockadeof IL-6 signaling is a potential biological therapy for cytokine
storm.

The LPS–induced endocytosis of TLR4 is essential in
the production of IFN-β, which activates the transcriptionof antiviral response genes by STAT1 phosphorylated at
Tyr701. In human macrophages, TLR4 endocytosis activatedthe noncanonical phosphorylation of STAT1 at Thr749, which
subsequently promoted the production of IL-6 and IL-12p40
through distinct mechanisms. Noncanonical phosphorylationof STAT1 activated the expression of gene encoding ATrich interaction domain-containing protein 5A (ARID5A),
which stabilizes IL6 mRNA. Moreover, noncanonicalphosphorylation of STAT1 enhanced the transcriptionof gene encoding IL-12p40 (IL12B). Phosphorylation of
Thr749 facilitated the binding of STAT1 to a noncanonicalDNA motif in the promoter regions of ARID5A and IL12B.
In LPS-stimulated macrophages, endogenous STAT1
bound to the ARID5A and IL12B promoters, respectively.
Endocytosis of TLR4 induced the formation of a complex
between the kinases TBK1 and IKKβ through noncanonicalphosphorylation of STAT1. Collectively, our study indicatesthat phosphorylation of STAT1 at different sites confers
distinct DNA binding and gene regulation activities, inwhich canonical phosphorylation at Tyr 701 and Ser 727
facilitates STAT1 anti-viral gene transcription. On the other
hand, noncanonical phosphorylation at Thr749 confers theproinflammatory functions of STAT1.

20

Arid5a was identified as an RNA-binding protein thatplays crucial roles in the development of inflammatory
diseases via the augmentation of IL-6 signaling. However,
the roles of Arid5a in cancer remain elusive. We foundthat although KPC cells derived from pancreatic ductal
adenocarcinoma (PDAC) mouse model lacking Arid5a(Arid5a-KO), had similar growth rates as wild-type KPC cells
when subcutaneously inoculated into immunodeficient mice,
Arid5a-KO KPC cells had substantially reduced growth insyngeneic mice. Using mass cytometry, we observed thatin tumors derived from Arid5a-KO mice, the infiltration of
granulocytic myeloid-derived suppressor cells (gMDSCs)
and regulatory T cells (Tregs) is suppressed, whereas therecruitment and activation of CD8+T lymphocytes is induced.
Interestingly, Arid5a-KO KPC cells showed significantly
lower expression of indoleamine 2,3-dioxygenase
1 (Ido1), which augments the production of kynurenine,

a dual regulator in PDAC to generate immunosuppressive
tumor microenvironments; first, Arid5a-Ido1 axis controls
Treg differentiation/activation; and second, the increasein chemokine levels induced by Arid5a recruits Treg and
gMDSCs, in the tumor microenvironment. Thus, our findings
provide insights into the molecular basis of the immune
evasion of PDAC via Arid5a, and indicate that Arid5a is apromising druggable target for tumor immunotherapy.

Immunolo g y

Immune Regulation

an immunoregulatory metabolite of tryptophan catabolism,

Assessment of the physiological action of
immunomodulatory drugs in humanized cereblon mice
Thalidomide and its derivatives are immunomodulatory
drugs (IMiDs) that have therapeutic actions on hematologicalmalignancies and inflammatory diseases. However, themolecular basis of these pleiotropic activities of IMiDs is still
unknown.
IMiDs bind directly to cereblon (CRBN), which is a
substrate receptor of the CRL4-CRBN E3 ligase complex
6, which results in the ubiquitination and degradation of
specific protein targets. Using mice expressing humanized
CRBN, which mimics the action of IMiDs in human cells,
we found that T-lymphocytes from humanized CRBN micetreated with a novel IMiD result in the increased productionof IL-22, IL-2, and IL-9 via the degradation of Aiolos andinterferon regulatory factor 4, whereas WT mice did not showsuch an increase. Moreover, immunodeficient mice, whichwere engrafted with the myelodysplastic syndrome (MDS)
patient-derived cell line MDS-L, and treated with IMiDs
showed a longer survival rate and less infiltration of MDS
cells into their bone marrow than WT mice. Consistently,
IMiDs showed significant suppressive effects on the growthof MDS-L cells in vitro. Assessment of protein expression inhumanized CRBN mice treated with IMiDs is currently underinvestigation.

Recent Publications
⿎⿎ Kang S, Narazaki M, Metwally H, Kishimoto T. Historical overview
of the interleukin-6 family cytokine. J. Exp. Med. 217, pii: e20190347(2020).
⿎⿎ Choy E, Benedetti F, Takeuchi T, Hashizume M, Markus RJ, Kishimoto
T. Translating IL-6 biology into effective treatments. Nat. Rev.
Rheumatol 23, 1-11 (2020).
⿎⿎ Metwally H, Tanaka T, Li S, Parajuli G, Kang S, Hanieh H, Hashimoto
S, Chalise JP, Gemechu Y, Standley DM, Kishimoto T. Noncanonical
STAT1 phosphorylation expands its transcriptional activity into
promoting LPS-induced IL-6 and IL-12p40 production. Sci Signal. 13,
pii: eaay0574 (2020).
⿎⿎ Nyati KK, Zaman MM, Sharma P, Kishimoto T. Arid5a, an RNA-binding protein in immune regulation: RNA stability, inflammation, andautoimmunity. Trends Immunol. 41, 255-268 (2020).
⿎⿎ Nyati KK, Agarwal RG, Sharma P, Kishimoto T. Arid5a regulation andthe roles of Arid5a in the inflammatory response and disease. Front.
Immunol. 10, 2790 (2019).
⿎⿎ Chalise JP, Hashimoto S, Parajuli G, Kang S, Singh SK, Gemechu Y,
Metwally H, Nyati KK, Dubey PK, Zaman MM, Nagahama Y, Hamza
H, Masuda K, Kishimoto T. Feedback regulation of Arid5a and Ppar-γ2
maintains adipose tissue homeostasis. Proc. Natl. Acad. Sci. USA.
116, 15128-15133 (2019).
⿎⿎ Kang S, Tanaka T, Narazaki M, Kishimoto T. Targeting interleukin-6
signaling in clinic. Immunity, 50(4), 1007-1023 (2019).

21

LMP1+ B cells through IDO1 represents a novel mechanism
for immune evasion of EBV (Tsai C-Y et al. Int. Immunol.
2018).

Hitoshi Kikutani, MD/PhD

Self-reactive and polyreactive B cells are generated andselected in the germinal center during γ-herpesvirus
infection
Murine γ-herpesvirus 68 (MHV68) is closely related to
Epstein–Barr virus (EBV) and induces splenomegaly andhypergammaglobulinemia at 2–3 weeks after infection inmouse inbred strains. Although a humoral immune response
is initiated in the acute phase, production of neutralizing
antibodies is delayed and a high titer of IgG autoantibody
is produced instead. Nevertheless, it has remained unclear
how self-reactive B cells are selectively generated. To
determine how self-reactive B cells are generated during
MHV68 infection, we analyzed the reactivity of recombinant
antibodies obtained from single, isolated germinal center(GC) B cells, transient short-lived plasmablasts, and longlived plasma cells (PCs). We revealed that about a quarterof IgG + GC B cells emerge as polyreactive to multiple
self-antigens and/or virion antigens. The self-reactivityof most polyreactive clones was dependent on somatic
hypermutation (SHM), but not due to intrinsic alteration ofvirally infected B cells. Our findings suggest that MHV68
infection generates polyreactive B cells through the GC
reaction.
Furthermore, both virus-mono-specific and polyreactive
clones were selected to differentiate into B220lo CD138+
plasma cells (PCs). However, the representation of GCderived polyreactive clones was reduced and that of virusmono-specific clones was markedly increased in terminally22

Professor

Hitoshi Kikutani

Associate Professor
Assistant Professor
Research Assistant
Visiting Scientist
Support Staff

Shuhei Sakakibara
Chao-Yuan Tsai1
2
1

From our previous study on monoclonal anti-dsDNA
antibodies isolated from acute SLE patients (Sakakibara S
et al., 2017), we demonstrated that low-affinity anti-ssDNA
B cells can acquire high-affinity to both ss- and dsDNA
by only one or two mutations. This raises the questionsof whether low-affinity anti-ssDNA precursor B cells are
subjected to immunological tolerance and in what conditions
they undergo clonal expansion and affinity mutation todifferentiate into high affinity anti-dsDNA antibody-producing

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 SLE antiDNA antibody clones (Figure 2). The G9gl KI mice had
reduced number of splenic B cells, in which only one
third of them expressed the KI BCR and the rest of B cells
expressed endogenous BCR due to receptor editing. As
expected, the KI BCR-expressing B cells reacted to ssDNA,
but not dsDNA. These low-affinity anti-ssDNA precursor
B cells exhibited anergic phenotype such as lowered
BCR expression and shorter half-life in vitro. Furthermore,they were excluded from GC reaction induced in several
experimental settings including ssDNA-conjugated mBSA
immunization and Treg depletion. Therefore, multiple
tolerance checkpoints regulate low-affinity anti-DNA
precursor B cells not only at the early development stage
but also GC to prevent pathogenic autoantibody production.

Figure 1.

Figure 2.

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

Immunolo g y

Immune Regulationdifferentiated PCs as compared to transient plasmablasts.
Collectively, our findings demonstrate that, during acuteMHV68 infection, self-reactive B cells are generated throughSHM and selected for further differentiation into short-lived
plasmablasts but not terminally differentiated PCs (Sakaibara
S et al. Int. Immunol. 2020).

Bystander inhibition of humoral immune responses by
Epstain-Barr virus LMP1
Epstein-Barr virus (EBV) infects over 95% of adults andpersists throughout their lifespan. EBV establishes latent
infection in B cells with expression of limited viral genes.
Latent membrane protein1 (LMP1), one of the EBV genes
expressed in the latently infected B cells, is an integral
plasma membrane protein and provides constitutive
activation of signaling pathways including NF-κB, p38 andc-Jun. To elucidate the role of LMP1 in B cell function invivo, we generated transgenic mice expressing LMP1 under
the control of the CD19 promoter or the activation-induced
cytidine deaminase (AID) promoter. We found that LMP1
expression in antigen-committed B cells severely impaired
humoral immune responses not only through direct inhibitionof germinal center B cell differentiation, but also indirect
suppression of the neighboring B cells (Figure 1). Our
study further demonstrated that LMP1-expressing B cells
overexpress indoleamine 2,3-dioxygenase 1 (IDO1), andtherefore tryptophan deprivation and harmful metabolites
cause the indirect suppression. This bystander inhibition by

Recent Publications
⿎⿎ Sakakibara S., et al. Self-reactive and polyreactive B cells are
generated and selected in the germinal center during γ-herpesvirus
infection. Int. Immunol. 32, 27-38 (2020).
⿎⿎ Takeda K., et al. Allergic conversion of protective mucosal immunity
against nasal bacteria in chronic rhinosinusitis with polyposis. J.
Allergy Clin. Immunol. 143, 1163-1175 (2019).
⿎⿎ Tsai C-Y., et al. Bystander inhibition of humoral immune responses by
Epstein-Barr virus LMP1. Int. Immunol. 30, 579-590 (2018).
⿎⿎ Sakakibara S., et al. Clonal evolution and antigen recognition ofantinuclear antibodies in acute systemic lupus erythematosus.
Scientific Reports 7, 16428 (2017).

23

Kiyoshi Takeda, MD/PhD
Professor

Kiyoshi Takeda

Associate Professor

Eiji Umemoto
Hisako Kayama
Ryu Okumura
Mari Murakami
92

Assistant Professor
Research AssistantSupport Staff

Mucosal barriers including mucins and antimicrobial
peptides serve as a segregator of commensal microbes andintestinal epithelial cells (IECs) to maintain gut homeostasis.
We previously reported that Ly6/Plaur domain containing
8 (Lypd8), a highly glycosylated GPI-anchored protein
expressed on enterocytes of the colon, promotes thesegregation of commensal bacteria and colonic epithelia
by inhibiting bacterial invasion of the colonic epithelia. We
analyzed the role of Lypd8 in the host defense against
enteric bacterial pathogens such as Citrobacter rodentium,
which induces attachment and effacement (A/E) lesions oncolonic epithelia.

Lypd8-deficient mice are sensitive to C. rodentium
infection
To determine the role of Lypd8 in protection against
enteric pathogens, we first analyzed the ability of orally
infected Lypd8 −/− mice to eliminate C. rodentium. We
evaluated the numbers of C. rodentium in fecal samples
from Lypd8 −/− mice over the course of infection. C.
rodentium was more abundant in the feces of Lypd8−/− micecompared with wild-type (WT) mice at the early time points,suggesting that C. rodentium more rapidly colonized andproliferated in the colons of Lypd8−/− mice. Intimin-mediatedbacterial attachment to IECs is necessary for colonizationof A/E bacteria. Thus, we next analyzed the attachment of
C. rodentium to colonic epithelia of Lypd8−/− mice. The total
number of adherent C. rodentium was markedly increased
24

examined Lypd8 binding to these mutant strains by flow
cytometry. While no decrease in Lypd8 binding to the T3SSdeficient strain compared with the WT was observed, Lypd8
binding to the intimin-deficient strain was substantiallyreduced compared with the WT strain, suggesting that
intimin on the surface of C. rodentium is targeted by Lypd8
in the colon.

Lypd8 interferes with the interaction between intimin
and Tir of A/E bacteria by binding to intimin
To further define Lypd8 binding to intimin expressedby C. rodentium, we analyzed whether FLAG-tagged
Lypd8 recombinant proteins bind to GST-tagged intimin
recombinant proteins by an enzyme-linked immunosorbent
assay (ELISA) with anti-FLAG antibody. The Ig-like andC-type lectin (CTL) domains of intimin are pivotal for intiminbinding to Tir of A/E bacteria. Thus, we examined thebinding of Lypd8 to each intimin protein domain. Predictably,highly glycosylated Lypd8 proteins bound to the CTL domain
of intimin. In addition, Lypd8 bound to the Ig-like domain.
The interaction between intimin and Tir is indispensable forthe generation of A/E lesions. We next examined whether
Lypd8 inhibits the interaction between intimin and Tir.
High absorbance readings indicative of intimin-Tir binding
gradually decreased following the addition of increasing
concentrations of Lypd8. These data clearly indicate that
Lypd8 inhibits A/E bacteria attachment to enterocytes by
suppressing the association between intimin and Tir (Figure).
Ta k e n t o g e t h e r, o u r s t u d i e s d e m o n s t r a t e t h a tenterocytes, previously regarded as passive contributors
to the defense against invading pathogens compared withother intestinal epithelial cells such as goblet cells andPaneth cells, actively contribute to the fight against entericpathogens by expressing Lypd8.

Immunolo g y

Mucosal Immunology

in Lypd8−/− mice compared with WT mice from day 3 to day
12 post-infection. These findings indicate that Lypd8 inhibits
the colonization of C. rodentium in the colon by suppressing
bacterial attachment to colonic epithelia. In accordancewith the increased number of C. rodentium adhering tocolonic epithelia, Lypd8−/− mice showed more severe colitis,
characterized by a thicker mucosa and increased cellular
infiltration to the lamina propria. These results suggestthat Lypd8 deficiency impairs host defenses against C.
rodentium.

Lypd8 suppresses the attachment of A/E bacteria tocolonic epithelia by binding to bacterial cells
To assess the mechanism by which Lypd8 inhibits theattachment of C. rodentium to the colonic epithelia, wenext analyzed whether Lypd8 binds to C. rodentium in thecolon. We used flow cytometry with anti-Lypd8 antibody andmeasured Lypd8 binding to GFP-expressing C. rodentium
in the feces of WT mice following infection. Lypd8 boundto GFP-expressing C. rodentium in the colon. C. rodentium
attachment is strengthened by the generation of A/E
lesions involving the interaction of intimin expressed on thebacterial surface and translocated intimin receptor (Tir) on
the host cell membrane after being injected via the type III
secretion system (T3SS). To test whether Lypd8 binds tothese virulence factors, we generated eae-deficient (Δeae;
equivalent to intimin-deficient) and escN-deficient (ΔescN;
equivalent to T3SS-deficient) C. rodentium strains, and thenFigure. Lypd8, a highly glycosylated GPI-anchored protein on enterocytes, inhibits the attachment of C. rodentium to enterocytes by
suppressing the interaction of Intimin and Tir, both of which are critical virulence factors for A/E lesion.

Recent Publications
⿎⿎ Kayama H, Okumura R, Takeda K. Interaction between the microbiota,
epithelia, and immune cells in the intestine. Ann. Rev. Immunol. 38, 2348 (2020).
⿎⿎ Okumura R, Kodama T, Hsu CC, Sahlgren BH, Hamano S, Kurakawa
T, Iida T, Takeda K. Lypd8 inhibits attachment of pathogenic bacteria tocolonic epithelia. Mucosal Immunol. 13, 75-85 (2020).
⿎⿎ Morita N, Umemoto E, Fujita S, Hayashi A, Kikuta J, Kimura I, Haneda
T, Imai T, Inoue A, Mimuro H, Maeda Y, Kayama H, Okumura R,
Aoki J, Okada N, Kida T, Ishii M, Nabeshima R, Takeda K. GPR31dependent dendrite protrusion of intestinal CX3CR1+ cells by bacterial
metabolites. Nature 566,110-114 (2019).

⿎⿎ Kayama H, Kohyama M, Okuzaki D, Motooka D, Barman S, Okumura
R, Muneta M, Hoshino K, Sasaki I, Ise W, Matsuno H, Nishimura
J, Kurosaki T, Nakamura S, Arase H, Kaisho T, Takeda K. Hemeameliorates dextran sodium sulfate-induced colitis through providing
intestinal macrophages with non-inflammatory profiles. Proc. Natl.
Acad. Sci. USA 115, 8418-8423 (2018).
⿎⿎ Okumura R, Kurakawa T, Nakano T, Kayama H, Kinoshita M, Motooka
D, Gotoh K, Kimura T, Kamiyama N, Kusu T, Ueda Y, Wu H, Iijima H,
Barman S, Osawa H, Matsuno H, Nishimura J, Ohba Y, Nakamura
S, Iida T, Yamamoto M, Umemoto E, Sano K and Takeda K. Lypd8
promotes the segregation of flagellated microbiota and colonicepithelia. Nature 532, 117-121 (2016).

25

rendering them selectively susceptible to signal-deprived
apoptotis. Taken together, eTreg cell depletion by imatinib
is instrumental in evoking effective immune responses tovarious cancers (Figure 2).
Taken together, it is expected that small molecules

capable of reducing or expanding Treg cells can beclinically useful for treatment of a variety of immunologicaldiseases and control of physiological and pathologicalimmune responses.

Immunolo g yExperimental Immunology

Shimon Sakaguchi, MD/PhD
Professor

Shimon Sakaguchi

Associate Professor
Assistant Professor
Postdoctoral Fellow
Research Assistant
Visiting Scientist
Support Staff

James Badger Wing
Kenji Ichiyama
4
3
13
4

Figure 1. Induction of Foxp3 in activated conventional T cells by inhition of CDK8/19.
CDK8/19 is physiologically repressing Foxp3 expression in activated conventional T cells. Its pharmacological inhibition enables
conversion of antigen-specific effector/memory T cells into Foxp3+ Treg cells by mainly upregulating STAT5. Adapted from Akamatsu,
Mikami, et al. Sci. Immunol. 2019.

This laboratory studies: (i) the cellular and molecularbasis of immunologic self-tolerance, in particular the rolesof regulatory T cells; (ii) the strategy for eliciting effective
immune responses to autologous tumor cells, or inducing
immunologic tolerance to organ transplants, by manipulating
the mechanisms of immunologic self-tolerance; and (iii)the causes and pathogenetic mechanisms of systemic
autoimmune diseases, such as rheumatoid arthritis, byutilizing 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. Depletionof Treg cells or attenuation of their suppressive activity
can enhance immune responses such as tumor immunity,
while expansion of Treg cells or augmentation of theirsuppressive activity can induce immunological tolerance
and treat autoimmune and other immunological diseases.
This year, we have explored chemical reagents which areable to induce or deplete Treg cells and thereby suppressor enhance immune responses, respectively.
By screening libraries of tyrosine or serine/threonine
kinase inhibitors for in vitro Foxp3 inducing activity inconventional T cells, we found that chemical inhibition of
cyclin-dependent kinases (CDK) 8/19, a serine/threonine
kinase, was able to induce Foxp3 in antigen-stimulated
effector/memory as well as naïve CD4+ and CD8+ T cells.
Knockdown/ knockout of the CDK8 or CDK19 gene alsosimilarly induced Foxp3. The induction was associatedwith STAT5 activation, independent of TGF-β action, and26

not affected by inflammatory cytokines. Furthermore, invivo administration of a newly developed CDK8/19 inhibitor
along with antigen immunization generated functionallystable antigen-specific Foxp3+ Treg cells, which effectively
suppressed skin contact hypersensitivity and autoimmune
disease in animal models. The results indicate that CDK8/19
is physiologically repressing Foxp3 expression in activatedconventional T cells and that its pharmacological inhibition
enables conversion of antigen-specific effector/memory
T cells into Foxp3+ Treg cells for the treatment of various
immunological diseases (Figure 1).
In contrast to such a Treg-inducing serine/threonine
kinase inhibitor, we found that a certain tyrosine inhibitor
can deplete Treg cells. Imatinib, a tyrosine kinase inhibitorof oncogenic BCR-ABL protein expressed by chronic
myelogenous leukemia (CML) cells, possesses off-targets
including LCK expressed in T cells. We showed in humans
that imatinib-treated CML patients in complete molecularremission (CMR) exhibited selective depletion of effector
Treg (eTreg) cells and significant increase in effector/
memory CD8 + T cells while non-CMR patients did not.

Figure 2. Direct or T cell-dependent killing of CML cells by imatinib.
Imatinib is able to not only kill leukemic cells directly but also deplete Treg cells, thereby inducing immunological killing of leukemic cells.
Imatinib is therefore instrumental in immunological treatment of various other cancers as well. Adapted from Tanaka, et al. J. Exp. Med.
2020.

Imatinib at CML-therapeutic concentrations indeed induced
apoptosis specifically in eTreg cells and expanded tumorantigen-specific CD8+ T cells in vitro in healthy individuals

⿎⿎ Ohkura N, Yasumizu Y, Kitagawa Y, Tanaka A, Nakamura Y, Motooka D,
Nakamura S, Okada Y, Sakaguchi S. Treg-specific epigenomic region
variants as a key determinant of susceptibility to common autoimmune
diseases. Immunity (2020) in press.

and melanoma patients, and suppressed colon tumor
growth in vivo in mice. Mechanistically, because of FoxP3dependent much lower expression of LCK and ZAP70 in Treg cells compared with other T cells, imatinib
inhibition of LCK further reduced their TCR signal intensity,

Recent Publications
⿎⿎ Sakaguchi S, Mikami N, Wing JB, Tanaka A, Ichiyama K, and Ohkura
N. Regulatory T cells and human disease. Ann. Rev. Immunol. 38, 541566 (2020).

⿎⿎ Mikami N, Kawakami R, Chen KY, Sugimoto A, Ohkura N, Sakaguchi S.
Epigenetic conversion of conventional T cells into regulatory T cells by
CD28 signal deprivation. Proc. Natl. Acad. Sci. USA, in press.

Takeuchi Y, Ha D, Shigeta N, Kitawaki T, Maeda Y, Saito T, Shinohara
Y, Kameoka Y, Iwaisako K, Monma F, Ohishi K, Karbach J, Jäger E,
Sawada K, Katayama N, Takahashi N, Sakaguchi S. Tyrosine kinase
inhibitor imatinib augments tumor immunity by depleting effector
regulatory T cells. J. Exp. Med. pii: jem.20191009. doi: 10.1084/
jem.20191009 (2020).
⿎⿎ Akamatsu M, Mikami N, Ohkura N, Kawakami R, Kitagawa Y,
Sugimoto A, Hirota K, Nakamura N, Ujihara S, Kurosaki T, Hamaguchi
H, Harada H, Xia G, Morita Y, Aramori I, Narumiya S, Sakaguchi S.
Conversion of antigen-specific effector/memory T cells into Foxp3expressing Treg cells by inhibition of CDK8/19. Sci. Immunol. pii:
eaaw2707. doi: 10.1126/sciimmunol.aaw2707 (2019).

⿎⿎ Tanaka A, Nishikawa H, Noguchi S, Sugiyama D, Morikawa H,

27

Immunolo g y

Cell Signaling

Takashi Saito, PhD

28

Professor

Takashi Saito

Support Staff

1

The objective of our team is to determine the molecularmechanisms of T cell activation, differentiation and function,and ultimately aim to elucidate the onset of and to modulate
T cell function/activation to prevent immune diseases suchas autoimmunity and allergic inflammation. Toward thisgoal, we have analyzed regulation of T cell function from asignaling perspective.

activation and function by innate signaling. We have alreadyshown that T cell stimulation by some TLR ligands induced
co-stimulation of T cells to induce cell proliferation andcytokine production. Particularly TLR2 activates Th1 cells
but not naïve T cells without TCR stimulation. We foundthat naïve T cells failed to respond to TLR2 stimulation due
to the defective expression of TIRAP. The function of the

Our finding that TCR-microclusters (MC) initiate T
cell activation led us to analyze the dynamics of signal
molecules at the immune synapse. We have also found thatsuch microcluster formation also regulates the negative
signaling particularly through inhibitory receptors suchas CTLA4 and PD-1. Following these analyses, we thenanalyzed the dynamics of negative regulation of T cell
activation through phosphatases, particularly PTPN22 whose
SNPs are strongly related to various autoimmune diseases.
The deficiency of PTPN22 exhibited enhanced activation of
T cells and an increase in effector/memory T cells. Imaging
analysis revealed that that PTPN22 was recruited to TCRMC upon activation. MS analysis of the PTPN22-associatedproteins showed several inhibitory molecules and following
imaging analysis suggested that PTPN22 exhibited a“inhibitory complex” with other inhibitory molecules to inhibit
T cell activation. The PTPN22 mutant, susceptible to thedevelopment of autoimmune diseases, was defective of
TCR-MC recruitment, provided the inhibitory mechanisms
and a cause of the autoimmune susceptibility by themutation.
We have also analyzed the modulation of T cell

innate-sensor STING in T cells was also analyzed because
T cells express similar level of STING as innate immune
cells. STING activation in T cells by STING ligands such as
cGAMP induced growth inhibition on one hand, and type
I-IFN production on the other hand. Both functions weremediated through mTORC1 activation, and we found thereciprocal regulation between STING and mTORC1 signals
for T cell function (Figure). Furthermore, we showed that
STING activation in T cells contributes to anti-tumor immunity
probably through the great amount of type I IFN production
by tumor-specific T cells.

Figure. Molecular mechanism of reciprocal regulation of STING and TCR signaling.
Co-stimulation of T cells by STING and TCR activation can induce growth arrest by partially inhibiting mTORC1 activation, andsimultaneously induces IFN-I production though sustained activation of IRF3 and the partial activation of mTORC1. The STING-mediatedinhibition of mTORC1 signals is partly dependent on IRF3/IRF7 but not on TBK1/IKKε. cGAMP-induced STING activation also leads to theinhibition of IL-2 signaling pathways.

Recent Publications
⿎⿎ Imanishi T and Saito T. T cell co-stimulation and functional modulation
by innate signals. Trends Immunol. 41, 200-212 (2020).
⿎⿎ Sasaki T, Yajima T, Shimaoka T, Ogawa S, Saito T, Yamaoka K,
Takeuchi T and Kubo M. Synergistic effect of IgG4 antibody and CTLs
causes tissue inflammation in IgG4-related disease. Int. Immunol.
(IF:4.168) 32, 163-174 (2020).

Hoshii T, Hirao A, Miyake K, Barber GN, Arita M, Ishii KJ, Akira S, Saito
T. Reciprocal regulation of STING and TCR signaling by mTORC1 forT cell activation and function. Life Sci. Alliance 2, e201800282 (2019).
⿎⿎ Saito T. Molecular dynamics of co-signal molecules in T cell activation.
Adv. Exp. Med. Biol. 1189, 135-152 (2019).

⿎⿎ Kong MS, Hashimoto-Tane A, Kawashima Y, Sakuma M, Yokosuka
T, Kometani K, Onishi R, Carpino N, Ohara O, Kurosaki T, Phua KK
and Saito T. Inhibition of T cell activation and function by the CIN85
adaptor protein. Sci. Signal. 12, eaav4373 (2019).
⿎⿎ Imanishi T, Unno M, Kobayashi W, Yoneda N, Matsuda S, Ikeda K,

29

Tomohiro Kurosaki, MD/PhD
Professor

Tomohiro Kurosaki

Associate Professor

Wataru Ise
Takeshi Inoue
Ryo Shinnakasu
11
4Assistant Professor
Postdoctoral Fellow
Research AssistantSupport Staff(Narita) virus first and then re-infection with the H1N1

with relatively low affinities are preferentially recruited into
the memory B cell compartment. This observation led us to

A / Puerto Rico/8/1934 (PR8) virus. We demonstrate that GC-

propose that such mechanisms may prevent the memory

Narita virus are not effective in neutralizing the re-infecting

B cell population from becoming overly committed to theprimary antigen, and instead, allow them to potentiallyacquire a more diverse repertoire. Therefore, memory B
cells might be intrinsically well-suited for recognition ofand protection from secondary infection by related but
antigenically variant pathogens.
To rigorously test this proposal, we used a mouse modelof drifted viral infection with pandemic H1N1 A/Narita/2009

PR8 virus. Instead, pre-existing anti-HA stem-specificmemory B cells are activated upon PR8 virus re-infection,
thereby contributing to host protection (Figure). Furthermore,
our results suggest the requirement for maturation of antiHA stem memory B cells in the GC during primary influenza
infection, whereas GC-mediated maturation is not requiredfor protection after secondary re-infection.

experienced LLPCs generated during primary infection with

Immunolo g y

Lymphocyte Differentiation

we and others have recently demonstrated that GC B cells

Narita-infection serum transfer

Generation mechanism of memory B cells during
germinal center (GC) reactions
Memory B cells and long-lived plasma cells (PCs)are responsible for effective long-term immunity against
pathogens. The majority of these cells responding to T
cell-dependent antigens are generated from the germinal
center (GC) reaction. Indeed, memory B cells emerge
from the GC as recirculating cells and, upon a secondary
antigen challenge, they are primed to elicit rapid antibody
responses.
In contrast to these insights into the GC precursor cells
for recycling and plasma cell fates, studies of the memory
fate decision have been hampered by the lack of a known
master transcription factor for differentiation of memory B
cells. Hence, surrogate markers such as an S1PR2 reporter,
CCR6 expression, or a cell cycle reporter has been recently
employed for identification of memory precursor cells.
Although informative, these studies have not identified
key features for development of the GC precursor cells
committed to the long-lived memory B cell fate, or whatsignals regulate these key features.
After identifying a memory-prone GC population(CD38intBcl6hi/intEfnb1+), we found that this small population
exhibited lower mTORC1 activity than the recyclingprone population. Constitutive high mTORC1 activity led
to defective development of CD38 intBcl6 hi/intEfnb1 + cells,
whereas decreasing mTORC1 activity resulted in relativeenrichment in this memory-prone cell population versus the30

recycling-prone one. Moreover, the CD38intBcl6hi/intEfnb1+
cells had higher levels of Bcl2 and surface BCR, thereby
contributing to their survival. Given the positive correlation
between the strength of T cell help and mTORC1 activity,
our data support a model in which weak help from T cells
together with provision of an increased survival signal is key
for GC cells to assume the memory B cell fate.

Importance of memory B cell compartment for viral
re-infection
Two humoral memor y compar tments, long-lived
plasma cells (LLPCs) and memory B cells, co-exist in
our body. LLPCs constitutively produce an antibody (Ab)and neutralize invading pathogens immediately upon reinfection, whereas memory B cells require re-stimulation bycognate antigen for their differentiation into Ab-secreting
plasma cells. Since serum Ab titers are known to becorrelated with vaccine efficacy, the importance of LLPCs
is well-appreciated. In contrast, the importance of memory
B cells in conferring protection from re-infection has been
controversial.
The majority of, though not all, memory cells and LLPCs
are thought to arise from germinal center (GC) reactions.
Immunization with NP hapten leads to the introduction of
the high-affinity-conferring, W33L VH mutation in a large
proportion of LLPC Abs. Thus, the LLPC pool is thought
to be primarily composed of specificities possessing thehighest affinity for the primary antigen. On the other hand,

Narita-infection serum Narita transfer

Narita-infection serum PR8 infection

Leach S, Shinnakasu R, et al. Int Immunol 31, 771-779 (2019)
Figure. De novo generation of HA stem-specific antibodies from memory B cells is required for protection from heterologous infection.
Pre-existing antibodies generated in mice previously infected with Narita virus (3-5 months post infection) provided protection from weightloss in naïve recipient mice infected with Narita virus (Lower left; blue line). By contrast, transfer of serum from Narita-infected mice failed
to protect naïve recipient mice infected with the PR8 virus (Lower right; blue line). On the other hand, antibodies generated from memory
B cells, which were initially induced after the primary Narita infection, after PR8 heterologous re-infection at day 7 were able to provide
protection to naïve recipient mice infected with the PR8 virus (Lower right; green line).

Recent Publications
⿎⿎ Fukuyama H, Shinnakasu R and Kurosaki T. Influenza vaccination
strategies targeting the hemagglutinin stem region. Immunol. Rev.
(2020) in press.
⿎⿎ Tanaka S, Ise W, Inoue T, Ito A, Ono C, Shima Y, Sakakibara S,
Nakayama M, Fujii K, Miura I, Sharif J, Koseki H, Pandelakis K,
Raman I, Li Q, Kubo M, Fujiki K, Nakato R, Shirahige K, Araki H, Miura
F, Ito T, Kawakami E, Baba Y and Kurosaki T. Tet2 and Tet3 in B cells
are required to repress CD86 and prevent autoimmunity. Nat. Immunol.
(2020) in press.

reactivation by boosting. Cell 180, 92-106 (2020).
⿎⿎ Leach S, Shinnakasu R, Adachi Y, Momota M, Makino-Okamura
C, Yamamoto T, Ishii KJ, Fukuyama H, Takahashi Y and Kurosaki
T. Requirement for memory B cell activation in protection fromheterologous influenza virus reinfection. Int. Immunol. 31, 771-779(2019).
⿎⿎ Ise W and Kurosaki T. Plasma cell differentiation during the germinal
center reaction. Immunol. Rev. 288, 64-74 (2019).

⿎⿎ Mesin L, Schiepers A, Ersching J, Barbulescu A, Cavazzoni CB,
Angelini A, Okada T, Kurosaki T and Victora GD. Restricted clonality
and limited germinal center reentry characterize memory B cell

31

Immunolo g yMalaria Immunology

Cevayir Coban, MD
Professor

Cevayir Coban

Assistant Professor
Visiting Scientist
Support Staff

Michelle Sue Jann Lee
1
2
Figure 1. Heparin directly induces both lytic and vital NETs. This NET formation requires reactive oxygen species and neutrophil elastase but
not NADPH oxidase or peptidyl-arginine deiminase 4.

32

Malaria is a severe disease affecting millions of lives
every year. To solve the malaria problem, our approach is
to focus on the elucidation of the mechanisms involved in
the host and Plasmodium parasites interactions. We haverecently gained advanced understanding of the disease
pathology by using experimental mouse malaria models
which led us to reach the conclusion that Plasmodium

findings suggest the ability of heparin to directly induce NET
formation and that should be considered in the context ofheparin treatment and when heparin-related side effects
occur.
In an effort to create vaccines against malaria and otherinfectious diseases, we developed a new adjuvant called
synthetic hemozoin, a synthetic analog of Plasmodium-

parasite presence in different organs should be considered
in a tissue-specific context (Nature Reviews Immunology2018). From this understanding, our final aim is to create
successful vaccines for the elimination of malaria.
Recently we developed an automated analysis method
to rapidly acquire and characterize neutrophil extracellular
traps (NETs) by using in human PBMC as well as in whole
blood during infection of mice with the malaria parasite(Cytometry A 2019). In subsequent studies using imaging
flow cytometry, we have demonstrated an unexpected
finding that heparin, a common anticoagulant, has the ability
to induce NETs. NETs by heparin occurred with cell lysis
and death, but live neutrophils releasing extracellular DNA
strands, known as vital NETs, also occurred abundantly.
Formation of NETs was time and dose dependent, andrequired reactive oxygen species and neutrophil elastase.
Other compounds related to heparin such as low molecularweight heparin, fondaparinux and heparan sulfate
either failed to induce NETs, or did so to a much lesser
extent (International Immunology 2020). Heparin is used
extensively, however, its biological effects are not limited
to coagulation and remain incompletely understood. Our

produced hemozoin, and completed its preliminary GLP
non-clinical safety and toxicology studies in several animals
and infection models (Vaccine 2016). Adjuvants improve
the potency of vaccines, but the modes of action of mostadjuvants are not unknown. In 2019, we demonstrated byusing the T-dependent antigen NIPOVA and a synthetic
hemozoin that MyD88 is required for early GC formationand enhanced antibody class-switch recombination in
mice. Using cell-type-specific MyD88 KO mice, we foundthat IgG2c class switching, but not IgG1 class switching,
was controlled by B cell-intrinsic MyD88 signaling. Notably,
IFN-γ produced by various cells including T cells, NK cells,
and dendritic cells was the primary cytokine for IgG2c CSR
and B-cell intrinsic MyD88 is required for IFN-γ production.
Moreover, IFN-γ receptor (IFNγR) deficiency abolishedsHZ-induced IgG2c production, while recombinant IFN-γ
administration successfully rescued IgG2c CSR impairment
in mice lacking B-cell intrinsic MyD88. These findings
suggest that B cell-intrinsic MyD88 signaling is involved in
the mechanism of synthetic hemozoin adjuvants and thismay enhance our specific understanding of how adjuvantsand vaccines work. (European J. Immunology 2019).

Figure 2. Cell-intrinsic MyD88 plays differential roles in the modes of action of vaccine adjuvants. Using the synthetic hemozoin particulate
adjuvant, we found that B cell intrinsic MyD88 is required for IFNγ induction in T cells, NK cells and dendritic cells to induce IgG2c classswitching in a TLR/IL1-independent manner.

Recent Publications
⿎⿎ Lelliott PM, Momota M, Shibahara T, Lee MSJ, Smith NI, Ishii KJ,
Coban C. Heparin induces neutrophil elastase dependent vital andlytic NET formation. International Immunology, pii: dxz084. doi:
10.1093/intimm/dxz084 (2020).
⿎⿎ Lee MSJ, Natsume-Kitatani Y, Temizoz B, Igari Y, Tsukui T, Kobiyama
K, Kuroda E, Ise W, Inoue T, Kurosaki T, Mizuguchi K, Akira S, Ishii
KJ, Coban C. B cell-intrinsic MyD88 signaling controls IFNγ-mediatedearly IgG2c class switching in response to a particulate adjuvant.
European J. Immunology, doi: 10.1002/eji.201848084 (2019).

⿎⿎ Coban C, Lee MSJ, Ishii KJ. Tissue-specific immunopathology
during malaria infection. Nature Reviews Immunology, doi: 10.1038/
nri.2017.138 (2018).
⿎⿎ 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.
Science Immunology, June 2, 2 (12), pii: eaam8093 (2017).

⿎⿎ Lelliott PM, Momota M, Lee MSJ, Kuroda E, Iijima N, Ishii KJ, Coban
C. Rapid quantification of NETs in vitro and in whole blood samples byimaging flow cytometry, Cytometry A, doi: 10.1002/cyto.a.23767 (2019).

33

Ken J. Ishii, MD/PhD
Professor

Ken J. ISHII

Associate Professor
Assistant Professor

Kouji Kobiyama
Hideo Negishi
Burcu Temizoz
1
1
2

Postdoctoral Fellow
Research AssistantSupport Staff

The primary goal of our laboratory is to understand theimmunological mechanisms of the intra- and inter-cellularsignaling pathways that mediate the immunogenicity ofsuccessful vaccines, as well as the biological responses
to adjuvants. Such knowledge will enable us to develop
novel concepts, modalities and next generation immunopreventive and/or therapeutic agents against infectious
diseases, cancer and allergy as well as other noncommunicable diseases. Three recent works published2019-2020 are listed below;

1) ZBP1 governs the inflammasome-independent IL-1α
and neutrophil inflammation that play a dual role inanti-influenza virus immunity.
Influenza A virus (IAV) triggers the infected lung to
produce IL-1 and recruit neutrophil. Unlike IL-1β, however,
little is known about IL-1α in terms of its mechanism of
induction, action and physiological relevance to the hostimmunity against IAV infection. In particular, whether Z-DNA
binding protein 1 (ZBP1), a key molecule for IAV-induced
cell death, is involved in the IL-1α induction, neutrophil
infiltration, and the physiological outcome have not been
elucidated. Here we show in a murine model that the IAVinduced IL-1α is mediated solely by ZBP1, in an NLRP3inflammasome-independent manner, and is required forthe optimal IL-1β production followed by the formation ofneutrophil extracellular traps. During IAV infection, ZBP1
displays a dual role in anti-IAV immune responses mediated34

innate immune activation failed to increase eosinophils in the
BALF than that from wild type mice. Consistently, intranasal
and oral administration of amlexanox, a TBK1 inhibitor,
decreased cGAMP-induced lung allergic inflammation.
Thus, cGAMP functions as a type 2 adjuvant in the lung andcan promote allergic asthma in manners that are dependent
on the intracellular STING/TBK1/IRF3/7 signaling pathway
and the resultant intercellular signaling pathway via IL-33
and ST2 might be a novel therapeutic target for allergic
asthma.

3) IL-33 Is Essential for Adjuvant Effect ofHydroxypropyl-β-Cyclodexrin on the Protective
Intranasal Influenza Vaccination.
Vaccine adjuvants are traditionally used to augment andmodulate the immunogenicity of vaccines, although in manycases it is unclear which specific molecules contributeto their stimulatory activity. We previously reported thatboth subcutaneous and intranasal administration ofhydroxypropyl-β-cyclodextrin (HP-β-CD), a pharmaceutical
excipient widely used to improve solubility, can act asan effective adjuvant for an influenza vaccine. However,

the mechanisms by which mucosal immune pathway is
critical for the intranasal adjuvant activity of HP-β-CD havenot been fully delineated. Here, we show that intranasallyadministered HP-β-CD elicits a temporary release of IL33 from alveolar epithelial type 2 cells in the lung; notably,
IL-33 expression in these cells is not stimulated following
the use of other vaccine adjuvants. The experiments using
gene deficient mice suggested that IL-33/ST2 signaling issolely responsible for the adjuvant effect of HP-β-CD when
it is administered intranasally. In contrast, the subcutaneous
injection of HP-β-CD and the intranasal administration ofalum, as a damage-associated molecular patterns (DAMPs)inducing adjuvant, or cholera toxin, as a mucosal adjuvant,enhanced humoral immunity in an IL-33-independent
manner, suggesting that the IL-33/ST2 pathway is uniqueto the adjuvanticity of intranasally administered HP-β-CD.
Furthermore, the release of IL-33 was involved in theprotective immunity against influenza virus infection whichis induced by the intranasal administration of HP-β-CDadjuvanted influenza split vaccine. In conclusion, our results
suggest that an understanding of administration route- andtissue-specific immune responses is crucial for the design ofunique vaccine adjuvants.

Immunolo g y

Vaccine Scienceby neutrophil, resulting in either a protective or pathologicaloutcome in vivo. Thus, ZBP1-mediated IL-1α production is
the key initial step of IAV-infected NETs, owing the duality of
the consequent lung inflammation.

2) Cyclic GMP-AMP Triggers Asthma in an
IL-33-Dependent Manner That Is Blocked byAmlexanox, a TBK1 Inhibitor.
Extracellular host-derived DNA, as one type of damage
associated molecular patterns (DAMPs), is associated withallergic type 2 immune responses. Immune recognition ofsuch DNA generates a second messenger cyclic GMP-AMP(cGAMP) and induces type-2 immune responses; however,its role in allergic diseases, such as asthma, has not been
fully elucidated. This study aimed to determine whether
cGAMP could induce asthma when used as an adjuvant. We
intranasally sensitized mice with cGAMP together with house
dust mite antigen (HDM), followed by airway challengewith HDM. We then assessed the levels of eosinophils inthe broncho-alveolar lavage fluid (BALF) and serum HDMspecific antibodies. cGAMP promoted HDM specific allergic
asthma, characterized by significantly increased HDM
specific IgG1 and total IgE in the serum and infiltration ofeosinophils in the BALF. cGAMP stimulated lung fibroblast
cells to produce IL-33 in vitro, and mice deficient in IL-33 orIL-33 receptor (ST2) failed to develop asthma enhancement
by cGAMP. Not only Il-33 -/- mice, but also Sting -/-, Tbk1 -/-,and Irf3 -/- Irf7 -/- mice which lack the cGAMP-mediatedRecent Publications
⿎⿎ Toriyama M, Rizaldy D, Nakamura M, Fujita F, Okada F, Morita A, Ishii
KJ. Immunological role of primary cilia of dendritic cells in humanskin disease. bioRxiv, doi: https://doi.org/10.1101/2020.02.04.933333(2020).
⿎⿎ Kobari S, Kusakabe T, Momota M, Shibahara T, Hayashi T, Ozasa
K, Morita H, Matsumoto K, Saito H, Ito S, Kuroda E, Ishii KJ. IL-33
Is essential for adjuvant effect of hydroxypropyl-β-cyclodexrin on theprotective intranasal influenza vaccination. Front Immunol. 11, 360(2020).

anti-influenza virus immunity. Int Immunol. 32(3), 203-212 (2020).
⿎⿎ Ozasa K, Temizoz B, Kusakabe T, Kobari S, Momota M, Coban C, Ito
S, Kobiyama K, Kuroda E, Ishii KJ. Cyclic GMP-AMP triggers asthma
in an IL-33-dependent manner that is blocked by amlexanox, a TBK1
inhibitor. Front Immunol. 10, 2212 (2019).

⿎⿎ Momota M, Lelliott P, Kubo A, Kusakabe T, Kobiyama K, Kuroda E,
Imai Y, Akira S, Coban C, Ishii KJ. ZBP1 governs the inflammasomeindependent IL-1α and neutrophil inflammation that play a dual role in35

Masahiro Yamamoto, PhD
Professor

Masahiro Yamamoto

Associate Professor
Assistant Professor

Miwa Sasai
Ji Su Ma
Youngae Lee11
1
1

Postdoctoral Fellow
Research Assistant
Visiting Scientist
Support Staff

36

The 2017 World Health Organization report estimatesthat nearly 440,000 deaths were caused by infection
with the obligatory protozoan human malaria parasite,
Plasmodium. Malaria, one of the most important vectorborne diseases, occurs when Plasmodium parasites aretransmitted via Anopheles mosquitoes to mammalian hosts
such as humans. The life cycle of Plasmodium parasites isdispensable for liver stage parasite development in vitro,since traverse-deficient parasites form normal liver stage
development in hepatocytes, suggesting that sporozoite
infection into hepatocytes rather than the cell traverse isimportant for the liver stage development. Sporozoites fromthe rodent malaria parasite Plasmodium berghei invade
rodent hepatocytes causing hepatocyte growth factor

divided into three stages, the vector stage in the mosquito,
and the liver and blood stages in the host. During the vector
stage, Plasmodium forms oocysts in the mosquito midgut
from where it moves in the next life stage (as sporozoites) tosalivary glands and acquires infectivity to mammalian hosts.
Infection starts with the delivery of sporozoites to the hostby the bite of a Plasmodium infected mosquito. Sporozoites
first enter the blood circulation and then the liver, where
they invade hepatocytes. After this, they develop further
as exoerythrocytic forms (EEFs). Each EEF generates therelease of thousands of merozoites into the blood stream
where they invade erythrocytes, initiating the blood stagesof infection and causing clinical malarial disease. Thus,
the development of Plasmodium parasites in the liver isan obligatory stage for the successful infection of theirmammalian hosts.
After their arrival at the liver, sporozoites traverse thehost cells such as Kupffer cells and hepatocytes and form
parasitophorous vacuoles (PVs) in the finally-invaded
hepatocytes. Although the sporozoite traverse of dermis
and Kupffer cells is indispensable for the liver stage
development into EEFs in vivo, the cell traversal itself is(HGF) secretion and activating the MET receptor, resulting
in suppressed hepatocyte apoptosis and facilitating
sporozoite development into EEFs. Although the Leiriao
et al. study strongly suggested that the development of P.
berghei in the liver involves host factors, activation of the
HGF/MET signaling pathway is not essential for liver stage
P. yoelii rodent malaria parasites or for the P. falciparum
human malaria parasite. Thus, little is known about thecommon regulatory circuit that is active during liver-stage
development in Plasmodium species.
In the mosquito vector, Plasmodium sporozoites arerod-shaped. After invading hepatocytes, the rod-shaped
sporozoites undergo bulbous expansion and transform
into spherical EEFs. The morphological transformationof sporozoites into early EEFs can be induced outside ofhepatocytes and is known to require serum and an optimal
temperature of 37°C. Ca2+ is known to regulate temperaturedependent sporozoite development into EEFs under cellfree conditions. However, it is also known that ectopic
morphological transformation of sporozoites outside
hepatocytes is detrimental to the life cycle of Plasmodium
parasites, as P. berghei lacking PUF2, an RNA binding

protein, displays spherical EEF-like parasites in the mosquito
salivary glands, resulting in failure of parasite invasion into
the hepatocytes. Thus, it seems likely that morphologicaltransformation into EEFs may be tightly controlled so thatit only occurs in hepatocytes during the parasite’s life
cycle; however, the host factors that critically regulate themorphological transformation of sporozoites as well as theirdifferentiation into EEFs in the infected hepatocytes are notwell understood.
Here, we show that sporozoite differentiation into EEFs
in the liver involves protein kinase C ζ-mediated NF-κB

activation, which robustly induces the expression of C-X-C
chemokine receptor type 4 (CXCR4) in hepatocytes andsubsequently elevates intracellular Ca 2+ levels, thereby
triggering sporozoite transformation into EEFs. Blocking
CXCR4 expression by genetic or phar macologicalintervention profoundly inhibited the liver stage developmentof the P. berghei rodent malaria parasite and the human P.
falciparum parasite also. Collectively, our experiments showthat CXCR4 is a key host factor for Plasmodium developmentin the liver, and CXCR4 warrants further investigation formalaria prophylaxis.

Immunolo g y

Immunoparasitology

Figure. CXCR4 regulates the liver-stage Plasmodium development by Ca2+ increment in hepatocytes.

Recent Publications
⿎⿎ Bando H, Pradipta A, Iwanaga S, Okamoto T, Okuzaki D, Tanaka S,
Vega-Rodríguez J, Lee Y, Ma JS, Sakaguchi N, Soga A, Fukumoto
S, Sasai M, Matsuura Y, Yuda M, Jacobs-Lorena M, Yamamoto M.
CXCR4 regulates Plasmodium development in mouse and humanhepatocytes. J. Exp. Med. 216:1733-1748 (2019) doi: 10.1084/
jem.20182227.
⿎⿎ Bando H, Lee Y, Sakaguchi N, Pradipta A, Ma JS, Tanaka S, Cai Y,
Liu J, Shen J, Nishikawa Y, Sasai M, Yamamoto M. Inducible nitricoxide synthase is a key host factor for toxoplasma GRA15-dependent
disruption of the gamma interferon-induced antiparasitic human
response. MBio. (2018) doi: 10.1128/mBio.01738-18.
⿎⿎ 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 proteinsin interferon-inducible GTPase-mediated host defense. Nature
Immunology. (2017) 18:899-910. doi: 10.1038/ni.3767.
⿎⿎ Lee Y, Sasai M, Ma J, Sakaguchi N, Ohshima J, Bando H, Saitoh T,
Akira S, Yamamoto M. p62 plays a specific role in interferon-γ-induced
presentation of a Toxoplasma vacuolar antigen. Cell Rep. (2015)
13:223-233. doi: 10.1016/j.celrep.2015.09.005.
⿎⿎ Ohshima J, Sasai M, Liu J, Yamashita K, Ma JS, Lee Y, Bando H,
Howard JC, Ebisu S, Hayashi M, Takeda K, Standley DM, Frickel EM,
Yamamoto M. RabGDIα is a negative regulator of interferon-γ-inducible
GTPase-dependent cell-autonomous immunity to Toxoplasma gondii.
Proc. Natl. Acad. Sci. USA. (2015) 112:E4581-4590. doi: 10.1073/
pnas.1510031112.

37

biological processes. We recently found that Xkr8

secondary necrosis, release cellular components, andcan be activated by phosphorylation (Figure 2). This

activate the immunity, leading to SLE-type autoimmune

phosphorylation-induced activation of Xkr8 was independent

disease. Similarly, a large number of male germ cells

from the caspase-mediated activation. We will study

undergo apoptosis, and are cleared by Sertoli cells. We
found that apoptotic germ cells expose PtdSer in an Xkr8dependent manner, and many dead Xkr8-/- germ cells areleft unengulfed in the testis, causing male infertility.
As mentioned above, PtdSer is exposed in various

which kinase(s) is responsible for activating Xkr8, andin which biological process it is activated. We are alsointerested in the molecular mechanism of how flippases
flip phospholipids, and how scramblases scramble
phospholipids at plasma membranes.

Immunolo g yBiochemistry and Immunology

autoimmunity. It is likely that unengulfed dead cells undergo

Shigekazu Nagata, PhD
Professor

Shigekazu Nagata

Associate Professor
Postdoctoral Fellow
Research Assistant
Visiting Scientist
Support Staff

Katsumori Segawa
2
4
2
1

Scramblase

Flippase

Outer
Inner
Phosphatidylcholine

P h o s p h o l i p i d s a re a s y m m e t r i c a l l y d i s t r i b u t e d
between inner and outer leaflets of plasma membranes.
Phosphatidylserine (PtdSer) is exclusively localized in the
inner leaflet. This asymmetrical distribution of phospholipids
is maintained by ATP-dependent phospholipid flippasesthat translocate PtdSer from outer to inner leaflets (Figure 1).
When cells undergo apoptosis, the asymmetrical distributionof phospholipids is disrupted by a scramblase(s) that
non-specifically scrambles phospholipids between the
two leaflets, leading to PtdSer-exposure. The PtdSer, thus
exposed to the cell surface, works as an “eat me” signal of
apoptotic cells for macrophages. In addition to apoptotic
cells, the PtdSer-exposure is observed in activated platelets,
lymphocytes and mast cells, capacitated sperm, aged
erythrocytes, exosomes, and enveloped virus.
We identified two P4-type ATPases (ATP11A and11C) and their subunit CDC50A as flippases at plasma
membranes. The CDC50A works as a chaperone for ATP11A
and 11C to escort them from ER to plasma membranes, andis required for their flippase activity at plasma membranes.
ATP11A and 11C contain caspase-recognition sites in the
middle of molecules, and their flippase activity is destroyed
during apoptosis. ATP11C-null mice suffer from B cell
deficiency. We found that B cell progenitors express ATP11C
but not ATP11A. Thus, B cells in ATP11C-/- mice have no
flippase at plasma membranes, causing sustained PtdSerexposure, and are engulfed alive by macrophages.
There are two families of membrane proteins carrying
10 transmembrane regions that support non-specific38

scrambling of phospholipids at plasma membranes. Fivemembers (TMEM16C, 16D, 16F, 16G and 16J) of the
TMEM16 family function as Ca2+-dependent scramblases.
Using a microarray system with lipid bilayers in which
phospholipids are asymmetrically distributed, we showedthat a single dimeric molecule of TMEM16F can scramble
phospholipids. TMEM16F is ubiquitously expressed, whileother members are expressed in specific tissues. The
TMEM16F -/- platelets cannot expose PtdSer, leading to
the reduced ability to produce thrombin for blood clotting.
In fact, human patients of Scott syndrome, a congenital
bleeding disorder, were found to carry a loss of function
mutation in the TMEM16F gene.
Three members (Xkr4, Xkr8 and Xkr9) of the Xkr familyenhance the scrambling of phospholipids during apoptosis.
Xkr8 is expressed ubiquitously, while Xkr4 and Xkr9 are
expressed in a tissue-specific manner, the brain or intestine,
respectively. These Xkr members contain a caspaserecognition site in the C-terminal region, and are cleaved
by caspase to function as a scramblase. Thus, in apoptotic
cells, caspase cleaves and irreversibly inactivates ATP11A
and 11C flippases, while it cleaves and activates the Xkr8
scramblase, to quickly and irreversibly expose PtdSer(Figure 2). The PtdSer, thus on the dead cell’s surface, isrecognized by macrophages for engulfment. Lymphocytes,
neutrophils express only Xkr8, and a lack of Xkr8 prevents
the PtdSer-exposure and engulfment by macrophages. We
found that the inefficient engulfment of apoptotic neutrophils
and lymphocytes due to the Xkr8 mutation activates the

Phosphatidylserine

ATP-dependent

Energy independent

ATP11A and ATP11C
Segawa et al. Science 2014

TMEM16 Family
Suzuki et al. Nature 2010
XKR8 Family
Suzuki et al. 2013

Flippase

Scramblase

ATP11A/11C

Xkr8

Figure 1. Flippase and Scramblase.
Plasma membranes in eukaryotic cells are comprisedof outer and inner leaflets, in which phosphatidylserine(PtdSer) and phosphatidylcholine are locatedexclusively at inner or outer leaflets, respectively. The
asymmetrical distribution of PtdSer is maintained by an
ATP dependent flippase. We showed that ATP11A and
11C are flippases at plasma membranes. In various
biological processes, the asymmetrical distribution ofphospholipids is disrupted by the action of scramblase.
We identified two family of proteins (TMEM16 andXkr) as Ca2+- and caspase-dependent scramblases,
respectively.

Apoptotic Cells

Irreversible
inactivation

caspaseIrreversible
activation

Activated Cells?

reversible
inactivation?

phosphatidylserine

KinasePreversible
inactivation

Figure 2. Activation of Xkr8’s scramblase andi n a c t i va t i o n o f f l i p p a s e s by t w o i n d e p e n d e n tmechanisms.
In apoptosis, caspase cleave Xkr8 to activate
scramblase while it cleaves flippases (ATP11A and 11C)
to inactivate, causing irreversible exposure of PtdSer.
Under activated conditions, unidentified kinase(s)phosphorylates Xkr8 to activate scramblase. At thesame time, it seems that flippases are phosphorylated
to inactivate their flippase activity.

Recent Publications
⿎⿎ Yamashita Y, Suzuki C, Uchiyama Y and Nagata S. Infertility caused byinefficient apoptotic germ cell clearance in Xkr8-deficient male mice.
Mol. Cell Biol. 40, e00402-e00419 (2020).

12217 (2018).
⿎⿎ Nagata S and Tanaka M. Programmed cell death and the immune
system. Nat. Rev. Immunol. 17, 333-340 (2017).

⿎⿎ Ryoden Y, Fujii T, Segawa K and Nagata S. Functional expression of
the P2X7 ATP receptor requires Eros. J. Immunol. 204, 559-568 (2020).
⿎⿎ Sakuragi T, Kosako H and Nagata S. Phosphorylation-mediatedactivation of mouse Xkr8 scramblase for phosphatidylserine exposure.
Proc. Natl. Acad. Sci. USA. 116, 2907-2912 (2019).
⿎⿎ Segawa K, Yanagihashi Y, Yamada K, Suzuki C, Uchiyama Y and
Nagata S. Phospholipid flippases enable precursor B cells to flee
engulfment by macrophages. Proc. Natl. Acad. Sci. USA. 115, 12212-

39

Toshihide Yamashita, MD/PhD
Professor

Toshihide Yamashita

Associate Professor

Takahide Itokazu
Yuki Fujita
Hiroshi Nakagawa
Oluwaseun Fatoba
Hiroshi Tsujioka
Yosuke Ohtake
Toru Nakanishi
Toke Jost Isaksen
3
6
2

Assistant Professor

Postdoctoral Fellow
Visiting Scientist
Support Staff

Disorders of the central nervous system, such ascerebrovascular diseases, cerebrospinal trauma, andencephalomyelitis, often cause spatiotemporal changes
in the nervous system and in various biological systems,
such as the immune system and vascular system. We

biological systems control a series of processes (Figure 2).
Particularly, the ultimate goal of this study 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 involvedin the operation of living organisms with neural network
disorders within the central nervous system by observing
such disorders and their functional recovery process withrespect to the dynamics of the entire biological system andby conducting a comprehensive analysis of the association
between each system.

We perceive the central nervous system as a single
organ within a biological system; further, studies from theperspective of the involvement of the entire biologicalsystem in disorders and recovery of neural networks are
scarce. By perceiving disorders in the neural networks andthe 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 well as theirsignificance that may potentially create a new and neverbefore-seen trend in Life Sciences.

Immunolo g y

Molecular Neuroscience

have analyzed disorders of the neural networks in thecentral nervous system and the subsequent restoration
process from the perspective of the functional network of
biological systems (Figure 1). Further, we have analyzed themechanism by which the spatiotemporal dynamics in those

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

Recent Publications

Figure 1. The mechanism of spontaneous functional recovery.
40

⿎⿎ Hamaguchi M, Muramatsu R, Fujimura H, Mochizuki H, Kataoka H
and Yamashita T. Circulating transforming growth factor-β1 facilitates
remyelination in the adult central nervous system. eLife 8, e41869(2019).

⿎⿎ Fujita Y, Masuda K, Nakato R, Katou Y, Tanaka T, Nakayama M, Takao
K, Miyakawa T, Shirahige K and Yamashita T. Cohesin regulates
formation of neuronal networks in the brain. J. Exp. Med. 214, 14311452 (2017).

⿎⿎ Ta n a b e S a n d Ya m a s h i t a T. B - 1 a l y m p h o c y t e s p r o m o t eoligodendrogenesis during brain development. Nat. Neurosci. 21, 506516 (2018).

⿎⿎ Fujitani M, Zhang S, Fujiki R, Fujihara Y and Yamashita T. A
chromosome 16p13.11 microduplication causes hyperactivity throughdysregulation of miR-484/protocadherin-19 signaling. Mol. Psychiatry
22, 364-374 (2017).

⿎⿎ Kuroda M, Muramatsu R, Maedera N, Koyama Y, Hamaguchi M,
Fujishima H, Yoshida M, Konishi M, Itoh N, Mochizuki H and Yamashita
T. Promotion of central nervous system remyelination by peripheral
FGF21. J. Clin. Invest. 127, 3496-3509 (2017).

41

to inhibit their osteoblast differentiation, and to maintainspaces for HSCs (Seike et al. Genes Dev. 2018).
We are studying the molecular regulation of CAR cellproperties and generation and the roles of CXCL12-CXCR4
signaling and CAR cells in the spatiotemporal regulation oflymphohematopoiesis during homeostasis and diseases.

Immunolo g yStem Cell Biology and Developmental Immunology

Takashi Nagasawa, MD/PhD

Special microenvironments known as niches are
essential for the maintenance of hematopoietic stem
cells (HSCs), which give rise to blood cells, and lymphohematopoiesis within bone marrow cavities. We isolated
a chemokine, CXCL12 (SDF-1/PBSF) as a molecule that
stimulates the growth of B cell precursors (Nagasawa etal. PNAS 1994) and found that CXCL12 and its receptor
CXCR4 are essential for colonization of bone marrow by
HSCs during embryogenesis (Nagasawa et al. Nature
1996; Ara et al. Immunity 2003), maintenance of a pool of
HSCs (Sugiyama et al. Immunity 2006), and developmentof immune cells, including B cells, plasmacytoid dendritic
cells (pDCs) and NK cells in bone marrow (Nagasawa.
Nat. Rev. Immunol. 2006) as well as vascular formation andcardiogenesis (Tachibana et al. Nature 1998). Subsequently,
we identified a population of reticular cells expressing
CXCL12 at high levels, termed CXCL12-abundant reticular(CAR) cells, within bone marrow (Tokoyoda et al. Immunity
2004; Sugiyama et al. Immunity 2006) and indicated that
CAR cells are mesenchymal stem cells, which give riseto adipocytes and osteoblasts (Seike et al. Genes Dev.
2018), the major producer of CXCL12 and SCF (Omatsu at
al. Immunity 2010), and the major cellular components ofniches for HSCs and hematopoietic cells, including B cells(Omatsu at al. Immunity 2010; Shimoto et al. Blood 2017).

42

Professor

Takashi Nagasawa

Associate Professor
Assistant Professor
Support Staff

Tatsuki Sugiyama
Yoshiki Omatsu
1

CAR cell-specific transcription factors, Foxc1 andEbf1/Ebf3 are essential for formation and maintenanceof niches for HSCs and immune cells
In 2014, 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 developing and maintaining niches for HSCs andimmune cells (Omatsu et al. Nature 2014).
Bone marrow is the tissue filling the space betweenbone surfaces. CAR cells have potential to differentiate
into osteoblasts in bone marrow. However, it had remained
unclear how osteogenesis was prevented in most CAR cells
to maintain HSC niches and marrow cavities. We foundthat the transcription factor Ebf3 is preferentially expressedin CAR cells 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 bone in aged mice. In mice lacking both
Ebf1 and Ebf3, CAR cells exhibiting a normal morphology
were abundantly present but their niche function wasmarkedly impaired with depleted HSCs at 1 week of age.
Subsequently, the mutants become progressively more
osteosclerotic, leading to the complete occlusion of marrowcavities in early adulthood. Expressions of osteogenic genes
were increased in CAR cells with reduced HSC-niche factor
expression in the absence of Ebf1/Ebf3. Thus, HSC cellularniches express Ebf3 that is required to create HSC niches,

Figure. The development and functions of CAR cells.
The transcription factors, Foxc1 and Ebf1/Ebf3, and HSC niche factors, CXCL12 and SCF, are preferentially and abundantly expressed in
CAR cells and critical for the formation and maintenance of niches for HSCs and hematopoietic cells, including B cells, within the bonemarrow.

Recent Publications
⿎⿎ Matsushita Y, Nagata M, Kozloff KM, Welch JD, Mizuhashi K,
Tokavanich N, Hallett SA, Link DC, Nagasawa T, Ono W and Ono N. A
Wnt-mediated transformation of the bone marrow stromal cell identity
orchestrates skeletal regeneration. Nat Commun. 11(1), 332(2020).
⿎⿎ Agarwal P, Isringhausen S, Li H, Paterson AJ, He J, Gomariz Á,
Nagasawa T, Nombela-Arrieta C and Bhatia R. Mesenchymal nichespecific expression of Cxcl12 controls quiescence of treatmentresistant leukemia stem cells. Cell Stem Cell. 24(5), 769-784(2019).

⿎⿎ Shimoto M, Sugiyama T and Nagasawa T. Numerous niches forhematopoietic stem cells remain empty during homeostasis. Blood
129(15), 2124-2131(2017).
⿎⿎ Omatsu Y, Seike M, Sugiyama T, Kume T, and Nagasawa, T. Foxc1
is a critical regulator of haematopoietic stem/progenitor cell nicheformation. Nature 508, 536-540(2014).

⿎⿎ Seike M, Omatsu Y, Watanabe H, Kondoh G, Nagasawa T. Stem cellnich-specific Ebf3 maintains the bone marrow cavity. Genes Dev. 32(56), 359-372(2018).

43was downregulated in cirrhotic human liver tissues. Thus,
PKD3 plays a protective role in the development of liver
fibrosis.

Novel MR1 ligands that modulate Mucosal-AssociatedInvariant T (MAIT) Cells

Sho Yamasaki, PhD
Professor

Sho Yamasaki

Assistant Professor

Masamichi Nagae
Eri Ishikawa
Chihiro Motozono
2
5
2
3

Postdoctoral Fellow
Research Assistant
Visiting Scientist
Support Staff

Structural basis for the recognition of pathogen-derived
phosphoglycolipids by C-type lectin receptor DCARThe C-type lectin receptors (CLRs) form a familyof pattern recognition receptors (PRRs) that recognize
numerous pathogens, such as bacteria and fungi, andtrigger innate immune responses. The extracellularcarbohydrate recognition domain (CRD) of CLRs forms a
globular structure that can coordinate a Ca2+ ion, allowing
receptor interactions with sugar-containing ligands.
Although well conserved, the CRD fold can also display
differences that directly affect the specificity of the receptors
for their ligands. Here, we report crystal structures at 1.82.3 Å resolutions of the CRD of murine dendritic cell–
immunoactivating receptor (DCAR/Clec4b1), the only
CLR that binds phosphoglycolipids such as acylated
phosphatidyl-myo-inositol mannosides (AcPIMs) of
mycobacteria. Using mutagenesis analysis, we identified
critical residues, Ala136 and Gln198, on the surface
surrounding the ligand-binding site of DCAR, as well as
an atypical Ca2+-binding motif (Glu-Pro-Ser/EPS 168–170). By
chemically synthesizing a water-soluble ligand analog,
inositol-monophosphate di-mannose (IPM2), we confirmed
the direct interaction of DCAR with the polar moiety of
AcPIMs by biolayer interferometry and co-crystallization
approaches. We also observed a hydrophobic groove
extending from the ligand-binding site that is in a suitable
position to interact with the lipid portion of whole AcPIMs.
These results suggest that the hydroxyl group-binding ability
44

Mucosal-associated invariant T (MAIT) cells are a subsetof recently identified innate-like T lymphocytes that play animportant role in many pathologies ranging from viral andbacterial infection, to autoimmune disorders and cancer.
MAIT cells are activated via the presentation of ligands by
MR1 on antigen presenting cells to the MAIT T cell receptor(TCR), however few studies have explored the effects ofsystematic changes to the ligand structure on MR1 binding
and MAIT cell activation. We report on the first study into theeffects of changes to the sugar motif in the known MAIT cellagonists 7-hydroxy-6-methyl-8-d-ribityllumazine (RL-6-Me7-OH) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil(5-OP-RU). Tetramer staining of MAIT cells revealed that theabsence of the 2’-hydroxy group on the sugar backboneof lumazines improved MR1-MAIT TCR binding, which
could be rationalised using computational docking studies.
Although none of the lumazines activated MAIT cells, all
5-OP-RU analogues showed significant MAIT cell activation,with several analogues exhibiting comparable activity to
5-OP-RU. Docking studies with the 5-OP-RU analogues
revealed different interactions between the sugar backbone
and MR1 and the MAIT TCR compared to those observed
for the lumazines and confirmed the importance of the
2’-hydroxy group for ligand binding and activity. Taken
together, this information will assist in the development of
future potent agonists and antagonists of MAIT cells.

Immunolo g y

Molecular Immunology

and hydrophobic groove of DCAR mediate its specificbinding to pathogen-derived phosphoglycolipids such as
mycobacterial AcPIMs.

Pathogenic contribution of Protein Kinase D in liver
fibrosis
Liver fibrosis is a central pathological process that
occurs in most types of chronic liver diseases. Advanced
liver fibrosis causes cirrhosis, hepatocellular carcinoma,
and liver failure. However, the exact molecular mechanisms
underlying the initiation and progression of liver fibrosis
remain largely unknown. This study was designed toinvestigate the role of protein kinase D3 (PKD3, genesymbol Prkd3) in the regulation of liver homeostasis. We
generated global PKD3-deficient (Prkd3 –/– ) mice andmyeloid cell-specific PKD3-deficient (Prkd3 ∆LysM) mice, andFigure. Crystal structure of the DCAR CRD in complex with IPM2.
Overall structure of the ligand-free DCAR CRD (ligand-free, PDB code: 6LKR)(left), the IPM2 complex (middle). Close-up view of the ligand
binding site bound of DCAR to IPM2 (PDB code: 6LFJ). Hydrophobic groove extending from the ligand-binding site is in a suitable position
to interact with the lipid portion of whole AcPIMs. Mannose and inositol residues are shown by Man and Ins respectively (right).

Recent Publications

found that both Prkd3 –/– mice and Prkd3 ∆LysM mice displayed
spontaneous liver fibrosis. PKD3 deficiency also aggravatedcarbon tetrachloride-induced liver fibrosis. PKD3 is highly
expressed in hepatic macrophages, and PKD3 deficiency
skewed macrophage polarization toward a profibrotic
phenotype. The activated profibrotic macrophages
produced TGF-β that in turn activates hepatic stellate
cells (HSCs) to become matrix-producing myofibroblasts.
Moreover, PKD3 deficiency decreased the phosphataseactivity of SHP1, a known PKD3 substrate, resulting insustained STAT6 activation in macrophages. In addition, we
observed that PKD3 expression in hepatic macrophages

⿎⿎ Omahdi Z, Horikawa Y, Nagae M, Toyonaga K, Imamura A, Takato K,
Teramoto T, Ishida H, Kakuta Y, Yamasaki S. Structural insight into therecognition of pathogen-derived phosphoglycolipids by C-type lectin
receptor DCAR. J. Biol. Chem. 295, 5807-5817 (2020).
⿎⿎ Imai T, Matsumura T, Mayer-Lambertz S, Wells CA, Ishikawa E,
Butcher SK, Barnett TC, Walker MJ, Imamura A, Ishida H, Ikebe T,
Miyamoto T, Ato M, Ohga S, Lepenies B, van Sorge NM, Yamasaki S.
Lipoteichoic acid anchor triggers Mincle to drive protective immunity
against invasive group A Streptococcus infection. Proc. Natl. Acad. Sci.
USA. 115, E10662-E10671(2018).
⿎⿎ Nagata M, Izumi Y, Ishikawa E, Kiyotake R, Doi R, Iwai S, Omahdi
Z, 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-E3294 (2017).
⿎⿎ Toyonaga K, Torigoe S, Motomura Y, Kamichi T, Hayashi JM, Morita
YS, Noguchi N, Chuma Y, Kiyohara H, Matsuo K, Tanaka H, Nakagawa
Y, Sakuma T, Ohmuraya M, Yamamoto T, Umemura M, Matsuzaki G,
Yoshikai Y, Yano I, Miyamoto T, Yamasaki S. C-type lectin receptor
DCAR recognizes mycobacterial phosphatidyl-inositol mannosides topromote a Th1 response during infection. Immunity 45, 1245-57 (2016).
⿎⿎ 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).

45

autophagic pathway in senescent cells. These results reveal
the cellular vulnerability of senescent cells, and provide
valuable novel insights into the resistance of senescent cells
to death and open up new possibilities for its control.

Immunolo g yAging Biology

Eiji Hara, PhD

Oncogenic proliferative signals are coupled to a varietyof growth inhibitory processes, such as the inductionof apoptotic cell death and stable cell-cycle arrest, in
phenomena termed cellular senescence. Both apoptosis andcellular senescence are considered to serve as importantsafeguards against neoplasia. However, unlike apoptotic
cells, senescent cells are viable for long periods of time andthereby accumulate with age in various organs and tissues.
Moreover, it has recently become apparent that senescentcells are not merely non-dividing, but eventually develop asecretory profile composed of pro-inflammatory cytokines,
chemokines, and extracellular matrix-degrading proteases,a typical signature termed the senescence-associatedsecretory phenotype (SASP). Although SASP reportedly
plays some beneficial roles, it also exhibits deleterious side
effects such as chronic-inflammation and/or tumorigenesis,
depending on the biological context. Thus, although
cellular senescence primarily acts as a tumor suppression
mechanism, the accumulation of senescent cells in aged
tissues may eventually promote the age-related decline oforgan function and/or associated diseases, such as cancer.
Indeed, the clearance of p16INK4a-positive senescent cells
from aged transgenic mice reportedly delays in the onsetof various age-related dysfunctions, such as sarcopenia,
cataracts, atherosclerosis, loss of adipose tissue, andtumorigenesis, thus extending the healthy lifespan. Alongsimilar lines, the elimination of therapy-induced senescentcells reduced several side-effects of chemotherapy andeven cancer recurrence in mice. Thus, it is anticipated that46

Professor

Eiji Hara

Associate Professor
Assistant Professor
Postdoctoral Fellow
Research Assistant
Visiting Scientist
Support Staff

Sugiko Watanabe
Shimpei Kawamoto
3
3
1
2

the removal of senescent cells could prevent the toxicity
of anticancer treatments and enhance their therapeutic
benefits.
Senolytic drugs, which specifically induce cell death insenescent cells, are likely to represent a new therapeutic
avenue, and several candidate drugs were identified using
a bioinformatics approach. However, the senolytic drugs
identified to date were not discovered by a truly unbiased
high-throughput screening (HTS) method, and thus appear
to have limitations in clinical applications. For example, ina phase II study of ABT263 (a specific inhibitor of the antiapoptotic proteins BCL2 and BCL-xL) for the treatment ofadvanced and recurrent small-cell lung carcinoma patients,
transient thrombocytopenia and neutropenia were reported
as side-effects. Furthermore, the combination of dasatinib
and quercetin (D+Q), another previously reported senolytic
drug, apparently exhibited remarkable cell-type specificity,although its mechanisms of action remain obscure.
Therefore, the identification of more effective senolytic
drugs and the elucidation of their mechanisms of action
are required towards a better strategy for the removal ofsenescent cells in vivo.
Here, we have identified bromodomain and extraterminal domain (BET) family protein inhibitor (BETi) as apromising new class of senolytic drugs. The blockade of
BRD4, a BET family protein, by chemical inhibitors or RNA
interference robustly provokes senolysis. This is due, at leastin part, to the combined effects of the attenuation of nonhomologous end joining (NHEJ) repair and the activation of

Figure. The model distinguishing mechanisms that may operate in the absence (left) or presence (right) of BETi in senescent cells.

Recent Publications
⿎⿎ Wakita M., et al. A BET family protein degrader provokes senolysis bytargeting NHEJ and autophagy in senescent cells. Nat. Commun. 11,1935 (2020).
⿎⿎ Gorgoulis V., et al. Cellular senescence: defining a path forward. Cell
179, 813-827 (2019).

47

dependent regulation of mTORC1 (Figure 2). These findings

on lysosomes. Recent analysis using p18 KO cells that lack

identified the interacting molecular surface as a potential

regulatory components of Rag GTPase, such as GATOR1

therapeutic target in lifestyle diseases, such as diabetes

and FLCN, showed that p18-Ragulator complex provides

mellitus and cancer, both of which are linked to dysfunctiona regulatory platform that is indispensable for amino acid-of the mTORC1 pathway.

Immunolo g y

Oncogene Research

insights into the role of p18-mediated regulation of mTORC1

Masato Okada, PhD
Professor

Masato Okada

Associate Professor
Assistant Professor

Shigeyuki Nada
Kentaro Kajiwara(S.A.) Tetsuya Kimura
1
2
1

Postdoctoral Fellow
Research AssistantSupport Staff

Figure 1. Role of CDCP1 in the Src-mediated growth factor signaling.

Role of Src tyrosine kinase in tumor progression
We have investigated the role of Src tyrosine kinasein tumor progression. Src is the first-identified oncogenic
tyrosine kinase, but no significant mutation of the SRC
gene occurs in any type of human cancer. Nonetheless, thefunction of Src is frequently upregulated in various malignant
cancers, and it is appreciated that upregulated Src plays
a crucial role in tumor progression, particularly in theacquisition of invasive and metastatic features. To elucidate
the molecular mechanisms underlying upregulation of Src,
we investigated the regulatory mechanism of SRC gene
expression and searched for Src-activating factors. We foundthat TGF-β treatment directly induces SRC gene expression
via the Smad pathway coupled with the AP1 transcription
factors, and determined the promoter and enhancer regions
located upstream of the SRC gene. The upregulation of Src
contributes to the progression of TGF-β-induced epithelialmesenchymal transition. We also found that activated Src
is ubiquitinated and promotes its secretion via exosomes tosuppress its oncogenic potential. In addition, we identified
CDCP1 as a Src-activating membrane glycoprotein in lipid
rafts. Upregulation of CDCP1 induces prominent activationof Src and the STAT3 pathway, which promotes the invasive
activity of epithelial cells. We also found that ablation of
CDCP1 inhibits HGF-induced morphological changes andcell growth, and attenuates membrane presentation of MET,
resulting in inhibition of invasive activity induced by HGF.
These findings suggest that CDCP1 is a co-receptor of MET
48(Figure 1). Furthermore, ablation of CDCP1 suppresses thecompensatory renal hypertrophy, indicating that CDCP1 isrequired for the HGF-MET signaling even in vivo. CDCP1 andMET are crucial for promoting cancer cell invasion; therefore,
we expect this study to identify a potential therapeutic target
in some types of cancer.

Role of p18 in the regulation of mTORC1 nutrient
signaling
We previously identified a new Src substrate termed
p18/Lamtor1, which exclusively localizes to lipid rafts oflysosomes. Subsequent analysis revealed that p18 functions
by forming a hetero-heptamer complex (Ragulator),
consisting of p18, p14, MP1, HBXIP, p10, RagA/C, and it isrequired for activation of mTORC1 on lysosomes. Conditional
KO of p18 in the epidermis showed that p18-mTORC1 is
crucial not only for anabolism of bio-materials, but also
for catabolism via autophagy, indicating that p18 is tightly
associated with the regulation of mTORC1 nutrient signalingin vivo. Recent studies in the intestinal tissues revealed thatthe p18-mediated mTORC1 signaling promotes the anabolic
metabolism required for robust production of mucin ingoblet cells. These findings underscore the critical role of
p18 in the regulation of metabolic homeostasis in various
tissues and cells. To further analyze the regulation of the p18
complex at the molecular level, we determined the crystal
structure of Ragulator. This revealed that p18 wraps aroundthe other components of Ragulator and provided significant

Figure 2. Molecular basis for the regulation of mTORC1 on lysosomes.

Recent Publications
⿎⿎ Tanaka K, Ito Y, Kajiwara K, Nada S, Okada M. Ubiquitination of
Src promotes its secretion via small extracellular vesicles. Biochem.
Biophys. Res. Commun. 525, 184-191 (2020).
⿎⿎ Tsukamoto T, Kajiwara K, Nada S, Okada M. Src mediates TGF-β–induced intraocular pressure elevation in glaucoma. J. Cell Physiol.
234, 1730-1744 (2019).
⿎⿎ Kimura T, Kumanogoh A, Okada M. Roles of Lamtor1 in macrophages,
CD4+ T-cells, and regulatory T-cells. Crit. Rev. Immunol. 38, 403-414(2018).

Nakagawa A, Okada M. Structural basis for the assembly of the
Ragulator-Rag GTPase complex. Nat. Commun. 8, 1625 (2017).
⿎⿎ Kimura T, Nada S, Takegahara N, Okuno T, Nojima S, Kang S, Ito D,
Morimoto K, Hosokawa T, Hayama Y, Mitsui Y, Sakurai N, SarashinaKida H, Nishide M, Maeda Y, Takamatsu H, Okuzaki D, Yamada M,
Okada M, Kumanogoh A. Polarization of M2 macrophages requires
Lamtor1 that integrates cytokine and amino-acid signals. Nat.
Commun. 7, 13130 (2016).

⿎⿎ Yonehara R, Nada S, Nakai T, Nakai M, Kitamura A, Ogawa A,
Nakatsumi H, Nakayama KI, Li S, Standley DM, Yamashita E,

49

Immunolo g y

Signal Transduction

Nobuyuki Takakura, MD/PhD
Professor

Nobuyuki Takakura

Associate Professor

Hisamichi Naito
Hiroyasu Kidoya
32
1

Postdoctoral Fellow
Research AssistantSupport Staff

Our research team is involved in studies in terms ofvascular biology and stem cell biology. Among the results ofour research in 2019, two of the interesting ones are shownhere.

modifications (Figure, Naito et al. Nature Protoc. 2020).

1) How integrity of blood vessels is induced after birth,is there stem cell population in endothelial cells?

The cellular and molecular mechanisms of blood vessel
formation in mice and humans is thought to be basicallysimilar. Although gene modification is available to observe
such mechanism in mice, it is not easy to assess in vivo
function of cells or molecules for blood vessel formation inhumans. So far, merely in vitro proliferation, migration, andtube formation analyses have been utilized to evaluate bloodvessel formation by using human ECs from several organ
origins. An important issue that we need to be concern
about is that many different types of cells affect ECs topromote in vivo blood vessel formation and we need toevaluate the 3D structure of blood vessels with time course.
We have recently succeeded in visualizing human bloodvessels spatiotemporally using a patient derived xenograft(PDX) model generated in immunodeficient mice (Tsukada
et al. paper in preparation). Using this PDX model, we willassess the mechanism of human vascular formation.

So far, the existence of tissue specific stem cells has
been elucidated in many organs, such as hematopoietic,
neural, gut, epidermal (including hair) stem cells and soon. In the cardiovascular system, although the cardiac
stem cell population was identified by using cell surface
markers, vascular endothelial stem cells (VESCs) have notbeen identified by their cell surface markers. We previously
reported that endothelial cells (ECs) with highly proliferative
potential were fractioned in side population (SP) cells which
express ABC transporter abundantly. EC-SP cells indeed
differentiate into main population ECs and contribute toECs for long term in the hindlimb ischemia model (Naitoet al, EMBO J. 2012); however, we have not shown thehierarchy of ECs from stem cell population into terminallydifferentiated ECs through progenitor population. Recently,
we identified specific cell surface markers (CD157 andCD200) to identify VESCs and showed abilities of single
cell transplantation and clonal expansion of VESCs in theliver injury model (Wakabayashi et al. Cell Stem Cell 2018).
This year, we reported the details of methods of how CD157
positive VESCs can be isolated from the liver with some
50

Figure. New mechanistic insight of blood vessel formation by endothelial stem cells.

2) How human blood vessel formation can be induced
and visualized in mice?

Recent Publications
⿎⿎ Naito H, Wakabayashi T, Ishida M, Gil CH, Iba T, Rahmawati FN,
Shimizu S, Yoder MC, Takakura N. Isolation of tissue-resident vascularendothelial stem cells from mouse liver. Nat. Protoc. 15, 1066-1081(2020).
⿎⿎ Kidoya H, Muramatsu F, Shimamura T, Jia W, Satoh T, Hayashi Y, Naito
H, Kunisaki Y, Arai F, Seki M, Suzuki Y, Osawa T, Akira S, Takakura
N. Regnase-1-mediated post-transcriptional regulation is essential forhematopoietic stem and progenitor cell homeostasis. Nat. Commun.
10, 1072 (2019).
⿎⿎ Naito H, Iba T, Wakabayashi T, Tai-Nagara I, Suehiro JI, Jia W, Eino
D, Sakimoto S, Muramatsu F, Kidoya H, Sakurai H, Satoh T, Akira
S, Kubota Y, Takakura N. TAK1 prevents endothelial apoptosis andmaintains vascular integrity. Dev. Cell 48, 151-166 (2019).
⿎⿎ Eino D, Tsukada Y, Naito H, Kanemura Y, Iba T, Wakabayashi T,
Muramatsu F, Kidoya H, Arita H, Kagawa N, Fujimoto Y, Takara K,
Kishima H, Takakura N. LPA4-mediated vascular network formation
increases the efficacy of anti-PD-1 therapy against brain tumors.
Cancer Res. 78, 6607-6620 (2018).
⿎⿎ Wakabayashi T, Naito H, Suehiro JI, Lin Y, Kawaji H, Iba T, Kouno
T, Ishikawa-Kato S, Furuno M, Takara K, Muramatsu F, Weizhen J,
Kidoya H, Ishihara K, Hayashizaki Y, Nishida K, Yoder MC, Takakura N.
CD157 marks tissue-resident endothelial stem cells with homeostatic
and regenerative properties. Cell Stem Cell 22, 384-397 (2018).

51

Manabu Fujimoto, MD/PhD

Trim21/IgG heavy chain/HLA-DR complex is expressedon the cell surface, and becomes a target for
autoantibodies (ongoing)
We have found that some misfolded proteins generated
in the ER can be transported to the cell surface by HLAclass II alpha and beta heterodimers. Furthermore, thesemisfolded protein/HLA class II complexes are revealed astargets for autoantibodies. Immunoglobulin heavy chain(IgG HC) is regarded as one of the misfolded proteinsthat can be complexed with HLA class II molecules. Thistime, we have identified that cytosolic SS-A Ro52 protein:
Trim21 associates with IgG HC/HLA-DR complex, andbecomes a target for autoantibodies in several systemic
autoimmune diseases, such as Sjogren’s syndrome, orsome dermatomyositis subtypes. We discovered that acytoplasmic/nuclear protein complexes with HLA class IIand becomes a target for autoantibodies.

Aging alteration of skin T cells is different from that ofblood T cells (ongoing)
Immune function is known to decrease with age,resulting in increased numbers of tumors and infections.
We have assessed T cell density, diversity and function
in individuals of various ages to study the immunologic
effects of aging on human skin. No decline in the densityof T cells was noted in whole skin or dermis with advanced
age. Of note, the frequency of epidermal T cells, particularly
52

Professor

Manabu Fujimoto

Postdoctoral Fellow
Research Assistant1
1

disorder caused by the constitutive activation of mTORC1
as a result of the inactivation of causative gene TSC1 or
TSC2. The disease is characterized by skin disorders
including hamartomas, hypomelanotic macules and central
nervous system abnormalities such as epilepsy and autism.
The mechanisms of hypomelanotic macules, epilepsy andautism remain unknown, although that of hamartomas are
known. To elucidate the common mechanisms describedabove, we made MITF-conditional knock out, Mitf-M-cre
Tsc2 flox-/-, mice, which developed vitiligo and epilepsy.
We elucidated that abnormal ER stress which results inmitochondrial oxidative stress occurred in both neurons
in the brain and melanocytes in the skin of the mice, anddemonstrated that these abnormalities were restored by the
inhibition of mTORC1.

Immunolo g y

Cutaneous Immunology

CD49a+ CD8 resident memory T cells, was higher in elderly
individuals. T cell diversity and antibacterial responses weremaintained in the skin of elderly individuals but declined in
the blood. Our findings suggest that skin T cells maintain
diversity and protective cytokine production in elderly
individuals despite reduced T-cell diversity and function inblood.

Dysregulation of circular and resident T cells targetingmelanocyte in autoimmune vitiligo
Vitiligo is an autoimmune disorder related to melanocyte
loss; however, the exact interplay between antigen-specificautoimmunity and local oxidative stress remains unclear.
We found a reduced suppressive function of activatedTregs on Melan-A-specific CD8+ T cells in the circulating
cells of vitiligo patients compared with healthy controls. This
result suggests that T cell anergy with Tregs dysfunction
in circulation may participate in the immune response tomelanocytes in vitiligo patients. In addition, skin resident
memory CD8+ T cells preferentially isolated from the vitiligo
skin are now processed for single cell transcriptome
analysis to unveil the specific character towards stressedmelanocytes.

Elucidation of common mechanism of both skin andbrain disorders by mTORC1
Tuberous sclerosis complex (TSC) is an inherited

Figure. Difference in T lymphocyte antigen receptor repertoire between the blood and skin from the youth and elderly.
T cells were harvested from the blood and skin from people of different ages, and the repertoire of T cell antigen receptors were analyzed.
Representative results from 26-year-old and 90-year-old subjects. (Koguchi-Yoshioka et al. submitted.)

Recent Publications
⿎⿎ Inoue M, Tanboon J, Hirakawa S, Komaki H, Fukushima T, Awano
H, Tajima T, Yamazaki K, Hayashi R, Mori T, Shibuya K, Yamanoi T,
Yoshimura H, Ogawa T, Katayama A, Sugai F, Nakayama Y, Yamaguchi
S, Hayashi S, Noguchi S, Tachimori H, Okiyama N, Fujimoto M,
Nishino I. Association of dermatomyositis sine dermatitis and with antinuclear matrix protein 2 autoantibodies. JAMA Neurol. (2020) in press.
⿎⿎ Okiyama N, Yamaguchi Y, Kodera M, Hamaguchi Y, Yokozeki H,
Ishiguro N, Fujimoto M. Distinct histopathologic patterns of finger
eruptions in dermatomyositis based on myositis-specific autoantibody
profiles. JAMA Dermatol.155, 1080-1082 (2019).

Takehara K. BAFF inhibition attenuates fibrosis in scleroderma by
modulating the regulatory and effector B cell balance. Sci. Adv.4,
eaas9944 (2018).
⿎⿎ Saito A, Okiyama N, Kubota N, Nakamura Y, Fujisawa Y, Watanabe
R, Ishitsuka Y, Bleackley RC, Fujimoto M. Blockade of granzyme B
remarkably improves mucocutaneous diseases with keratinocyte deathin interface dermatitis. J. Invest. Dermatol. 138, 2079-2083 (2018).

⿎⿎ Matsushita T, Kobayashi T, Mizumaki K, Kano M, Sawada T, Tennichi
M, Okamura A, Hamaguchi Y, Iwakura Y, Hasegawa M, Fujimoto M,

53

found. The ILC2 selectivity and cytotoxicity of these extracts

activator and inhibitor from marine microbe extract library’as well as its effects in vivo will be assessed and further the

project. To date, 480 types of extracts have been screened

effective component of the extract will be defined with thein vitro and some candidates for ILC2 inhibitors have been

aim of developing an ILC2 therapeutic agent (Figure 3).

Immunolo g yInnate Immune Systems

from Nagasaki university as part of the ‘Screening of ILC2

Kazuyo Moro, Ph.D
Professor

Kazuyo Moro

Postdoctoral Fellow
Support Staff

1
1
Figure 1. Invasion of helminth induce the release of IL-33 from
epithelial cells via necrosis. IL-33 induces IL-5 and IL-13 production
from ILC2 to induce strong type 2 immune responses. ILC2derived IL-5 and IL-13 eliminate parasites by inducing eosinophilia
and mucin production, respectively.

Group 2 innate lymphoid cells (ILC2) play crucial roles
in type 2 immune responses.
ILC2 are multifunctional innate lymphocytes that playroles in type 2 immunity and tissue homeostasis at barriersurfaces of the body. ILC2 are localized to the adipose
tissue, lung, colon, skin, bone marrow, and other peripheral
tissues and monitoring pathogen invasion. Upon invasion
with helminth, damaged epithelial cells release IL-33
and IL-25, which activate ILC2 and initiate IL-5 and IL-13
production. IL-5 and IL-13 induce type 2 immune responses
including eosinophilia and mucin production from goblet
cells. Furthermore, there is increasing evidence that ILC2
are strongly associated with allergic diseases such asasthma and atopic dermatitis. Cysteine proteases derived
from mites and pollen strongly induce IL-33 release from
epithelial cells, resulting in the induction of excess ILC2mediated immune responses, and cause allergic symptoms.
Therefore, the treatment of allergic diseases targeting ILC2
is expected as a promising approach (Figure 1) .

ILC2 increase susceptibility to allergy in response toenvironmental factors.
T cell antigens derived from mites and pollen are known
to be a trigger of allergic diseases. On the other hand,allergic diseases can also be caused by environmental
factors such as drugs, stress, exercise and cold weather that
do not act as T cell antigens. However, how environmental
54

factors cause allergy symptoms is unclear. We found that
ILC2 produced IL-4 in response to environmental stress,
and resulted in the secretion of IgE from B1 cells without
antigen stimulation. We also revealed that IgE from B1
cells promoted the survival of FcεRⅠα+ cells such as mast
cells and basophils, leading to the increase of allergic
susceptibility. Consistently, the IgE-mediated accumulationof FcεRⅠα+ cells induces severe allergic symptoms
through the amplification of ILC2-mediated responses
via cysteinyl leukotriene (CysLT) upon IL-33 stimulation.
These findings suggest that ILC2, B1 cells, and FcεRⅠα+cells compose a positive feedback loop. Taken together,
our research demonstrated that ILC2 play a crucial rolein the pathogenesis of allergic diseases in response toenvironmental factors (Figure 2).

Screening of inhibitors for ILC2 function from marine
microbe extract
T h e re i s a c c u m u l a t i n g e v i d e n c e t h a t I L C 2 a reresponsible for the development of allergy symptoms.
Moreover, recent studies demonstrated the involvement of
ILC2 in various diseases such as obesity, rheumatism, andidiopathic pulmonary fibrosis, indicating the possibility that
ILC2 is a therapeutic target for allergic diseases as wellas various diseases. Inhibitors for ILC2 function, however,
have not yet been developed. Therefore, to develop drugs
for these diseases, we screened for an inhibitor for ILC2
using the library of marine microbe extracts received

Figure 2. IL-4 derived from ILC2 in response to environmental
factors promotes IgE production from B1 cells. This IgE enhances
survival of mast cells and basophils and cause severe allergy
upon IL-33 stimulation. Moreover, Cysteinyl leukotriene (CysLT)
released by mast cells and basophils induces IL-4 production from
ILC2, suggesting that these innate immune cells compose positive
feedback machinery to increase susceptibility to allergy.

Figure 3. Inhibitor of ILC2 function was screened for using libraryof marine microbe extracts. ILC2 are cultured with 480 types ofmarine microbe extract under IL-33 stimulation, some extracts
suppressed ILC2 proliferation as well as IL-5 and IL-13 production
from ILC2, suggesting that these extracts have a potential tosuppress ILC2 functions.

Recent Publications
⿎⿎ Kobayashi T, Ricardo-Gonzalez RR, Moro K. Skin-resident innate
lymphoid cells - Cutaneous innate guardians and regulators. Trends
Immunol. 41(2), 100-112, doi:10.1016/j.it.2019.12.004 (2020).
⿎⿎ Sasaki T, Moro K, Kubota T, Kubota N, Kato T, Ohno H, Nakae S, Saito
H, Koyasu S. Innate lymphoid cells in the induction of obesity. Cell
Rep. 28(1), 202-217.e7, doi:10.1016/j.celrep.2019.06.016 (2019).

mesenchymal cells support the differentiation of fetal liver-derived
ILC2. J. Exp. Med. 215 (6), 1609-1626 (2018).
⿎⿎ Moro K, Kabata H, Tanabe M, Koga S, Takeno N, Mochizuki M,
Fukunaga K, Asano K, Betsuyaku T, Koyasu S. Interferon and IL-27
antagonize the function of group 2 innate lymphoid cells and type 2
innate immune responses. Nat. Immunol. 17 (1), 76-86 (2016).

⿎⿎ Kabata H, Moro K, Koyasu S. The group 2 innate lymphoid cell (ILC2)
regulatory network and its underlying mechanisms. Immunol. Rev. 286(1), 37-52 (2018).
⿎⿎ Koga S, Hozumi K, Hirano KI, Yazawa M, Terooatea T, Minoda A,
Nagasawa T, Koyasu S, Moro K. Peripheral PDGFRalpha (+) gp38 (+)

55

Tfr are expandable in culture and can retain their memory
characteristics, indicating that the mass production ofUsing the CyTOF technique, we have recently foundthese cells as a novel cell therapy may be possible. We arethat many Tfr in the blood and tonsils of humans have acontinuing to investigate this point. We believe that furthernaïve phenotype. This unique combination of a memory
like Tfr phenotype with naïve characteristics raises thepossibility that it is possible to take advantage of both ofthese factors since in most cases memory like cells do not
expand easily in culture. However, we find that naïve likeprofiling of these cells in both healthy and autoimmune
donors and improving ways to experimentally manipulatethem will provide both novel insights into any defects in theirfunction in these settings.

Immunolo g y

Human Immunology (Single Cell Immunology)

Application of single cell immunology to Tregs

James Badger Wing, PhD
Associate Professor

James Badger Wing

Support Staff

1

Figure 1. Multiple roles of Tfr cells in the regulation of humoral immunity.

Background
The Human Immunology (single cell immunology) lab
was established in November of 2019 and our work focuseson both single cell biology and its applications to humanimmunology. In particular, we have an established interestin regulatory T cells (Tregs) expressing the transcription
factor Foxp3, which play a critical role in the control of
immune homeostasis. Recently it has become clear that a
specialized subset of Tregs, T-follicular regulatory cells, (Tfr)
have a particular role in the control of T-follicular helper (Tfh)cell-driven germinal center (GC) responses responsible for
antibody production. Recent results strongly suggest thatTfr are critical for the regulation of autoantibody production,
vaccine response and the production of high affinity IgE
responsible for allergies. However, due to their recent
discovery in 2011 our understanding of Tfr formation andfunction still has many gaps. Previously, we and others
have demonstrated that Tfr have a high frequency in humanblood and lymphoid organs, often making up as many as
50% of total Tregs. Partly because of this combination ofhigh frequency and role in the control of antibody production
Tfr are believed to be important to human disease. However,very little is known about changes to their phenotype andfunction in autoimmune disease patients since the firstcomprehensive descriptions of their phenotype in humans
were carried out by us and others in 2017. The status andsubtypes of germinal center resident Tfr in human tonsils ineither non-inflamed tonsillar hypertrophy or IgA nephropathy
56

patients are largely unknown at this time. A clear goal of the
field is to determine how to manipulate a specific subset
of Tregs as for example enhancement of Tfr function would
allow control of autoimmune antibody production without
affecting the role of Tregs in tumor Immunosurveillance.

Mass Cytometry and single cell immunology
Tregs have key roles in multiple sites and recent
advances in single cell biology have made it clear that
Tregs show a great range of heterogeneity of states in
autoimmunity and cancer. We specialize in the use of single
cell technologies in order to better understand the function
and phenotype of Treg cells and other cells in the humanimmune system. In particular mass cytometry (CyTOF) is atechnique that allows us to assess the protein expressionof millions of cells in a single experiment producing large
datasets allowing the detailed comparison of Immune
cells in different donors and tissue sites. Additionally, incollaboration with other groups at both IFReC and abroad
we are developing novel techniques to combine sequencing
based protein analysis with epigenetic analysis with thebroader aim of combining these powerful, but cell limited,
techniques with the collection of larger CyTOF datasets for
multi-omic analysis. This would be greatly beneficial since
it allows an understanding of how epigenetic modifications
affect protein expression on a single cell level.

Figure 2. CyTOF analysis of human immune Tregs.

Recent Publications
⿎⿎ Sakaguchi S, Mikami N, Wing JB, Tanaka A, Ichiyama K, Ohkura N.
Regulatory T cells and human disease. Annu. Rev. Immunol. 38 (2020)
online ahead of print.
⿎⿎ Wing JB, Tanaka A, Sakaguchi S. Human FOXP3+ regulatory T cell
heterogeneity and function in autoimmunity and cancer. Immunity 50(2), 302-316 (2019).

⿎⿎ Wing JB, Tay C, Sakaguchi S. Control of regulatory T cells by co-signal
molecules. Co-signal Molecules in T Cell Activation, 179-210 (2019).
⿎⿎ Wing JB, Tekgüç M, Sakaguchi S. Control of germinal center
responses by T-follicular regulatory cells. Front. Immunol. 9, 1910
(2018).

⿎⿎ Ha D, Tanaka A, Kibayashi T, Tanemura A, Sugiyama D, Wing JB, Lim
EL, Teng KWW, Adeegebe D, Newell EW, Katayama I, Nishikawa H,
Sakaguchi S. Differential control of human Treg and effector T cells intumor immunity by Fc-engineered anti–CTLA-4 antibody. Proc. Natl.
Acad. Sci. USA. 116 (2), 609-618 (2019).

57

analysis as well as the potential benefits of the generated
data to the scientific community. The sequencing data fromour lab can provide not only insight into gene expression
profiles in correlation to human physiology but also intonovel transcriptome findings in bacteria or other nonmodel organisms, studies of non-coding RNAs, and into thepathology roles of post transcript-regulator such as miRNAs.

Immunolo g y

Human Immunology (Single Cell Genomics)

Daisuke Okuzaki, PhD

58

Associate Professor

Daisuke Okuzaki

Visiting Scientist

2

The Laboratory of Human Immunology was establishedin November 2019 for the purpose of accelerating theapplication of single cell sequencing technology to
improve the connection between fundamental science
research and clinical applications. In strategic allianceswith top-level immunology research institutes as well as
the Graduate School of Medicine and Faculty of Medicine,include the data generated from different independent
research groups as well as from publicly available
resources, and will be made available to the collaborating
researchers for conducting integration, annotation, and data
mining as shown in the figure.
In addition, we developed a pipeline called UNAGI or
UNAnnotated Gene Identifier, which processes long-read

Osaka university, the lab also utilizes the facilities of the
Genome Information Research Center of Research Institutefor Microbial Diseases. Last year, multiple equipment
assets including single cell resolution sequencing systems
capable of sequencing molecules from tens of thousandsof cells simultaneously (Chromium (10xGenomics), a BD
Rhapsody Express Single-Cell Analysis System, cuttingedge next generation sequencers (Illumina NovaSeq6000) with the highest throughput currently available in
the industry, and a large-scale supercomputer server
for the storage and processing of experiment data wereintroduced, making IFReC one of the few research centersin the country capable of conducting NGS experiments ontens of thousands of samples. A research paper (Hasegawa
et al. Nat. Immunol. 2019) that was published in 2019was based on such single-cell sequencing experiments.
Furthermore, experiment data for over one hundred samples
was generated in collaboration with the various researchlaboratories in IFReC. We are currently developing a singlecell gene expression profile based system for novel immune
cell type identification as well as the most comprehensive
database of immune cells in Japan. The database willsequencing data obtained from nanopore sequencing, andcompared it with the standard Illumina pipeline by studying
the Saccharomyces cerevisiae transcriptome in full-length
cDNA samples generated from two different biologicalsamples: haploid and diploid cells (Al Kadi et al. Funct.
Integr. Genomics 2020). The result outperformed the Illumina
pipeline and FLAIR in transcript reconstruction and further
contributed to the discovery of 3877 unannotated transcripts
including 1282 intergenic transcripts.
In another study (Okuzaki et al. Cancer Sci. 2020),
we elucidated the process of cell transformation driven
by c-Src, a tyrosine kinase found upregulated in various
human cancers by analyzing the expression profiles ofmiRNAs in a doxycycline-inducible Src expression system.
We found that miRNA (miR)-129-1-3p was downregulated
in the early phase of c-Src-induced cell transformationand that the reexpression of miR-129-1-3p disrupted
c-Src-induced cell transformation. In addition, miR-1291-3p downregulation was tightly associated with tumor
progression in human colon cancer cells/tissues. Resultsof these studies demonstrate the value of our developmentof robust algorithms for high throughput sequencing data

Figure. Overview of the research process.

Recent Publications
⿎⿎ Al Kadi M., et al. UNAGI: an automated pipeline for nanopore fulllength cDNA sequencing uncovers novel transcripts and isoforms
in yeast. Funct. Integr. Genomics, doi: 10.1007/s10142-020-00732-1(2020).
⿎⿎ Okuzaki D., et al. c-Src promotes tumor progression throughdownregulation of microRNA-129-1-3p. Cancer Sci. 111, 418-428, doi:
10.1111/cas.14269 (2020).

with immunoglobulin G1, is a molecular target of intravenous
immunoglobulin therapy for Kawasaki disease. Sci. Rep. 7, 11334, doi:
10.1038/s41598-017-11108-0 (2017).
⿎⿎ Okuzaki D., et al. Microarray and whole-exome sequencing analysis
of familial Behçet’s disease patients. Sci. Rep. 6, 19456, doi: 10.1038/
srep19456 (2016).

⿎⿎ Hasegawa T., et al. Identification of a novel arthritis-associatedosteoclast precursor macrophage regulated by FoxM1. Nat. Immunol.
20, 1631-1643, doi: 10.1038/s41590-019-0526-7 (2019).
⿎⿎ Okuzaki D., et al. FCN1 (M-ficolin), which directly associates59Im aging

Single Molecule Imaging
Toshio Yanagida, PhD
Ben Seymour, MD/PhD

60

Professor

Toshio Yanagida
Ben Seymour

Postdoctoral Fellow
Support Staff

1
2

The central focus of our IFReC research is trying tounravel the symptomatology of inflammatory disease. In
diseases such as inflammatory arthritis, it is well recognisedthat the most troubling symptoms are fatigue and pain.
Fatigue in particular is presumed to be triggered by theacute inflammatory response, such as by blood-brain
communication of cytokines. However, what makes fatigue

measures of fatigue. And in particular, this shows a strong
correlation with serum levels of IL-6. Our current research
now is attempting to localise the effective interaction
between IL-6 and fatigue-related behaviour in the brain,
based on neuroimaging data.
At the same time, we have also been developing
functional biomarkers, using machine learning tools, forsuch a difficult problem is that it becomes treatment
resistant: even when the acute inflammatory process
is controlled with therapy, such as immunologics, thesymptoms persist. Therefore some process in the brain
leads to the maintenance of symptoms, and this severely
limits the clinical effectiveness, in terms of holistic metrics
such as quality of life, of immunologics.
We have been studying the brain in patients with
inflammatory arthritis, and animal models thereof. In humans,
we are able to directly quantify fatigue symptoms, to try andunravel the component neural processes with which it isrelated. We do this by studying a set of behavioural tasks
that tap into these core components, and use sophisticatedcomputational models to decompose neural operations,and then map these in the brain using brain imaging. This
process we term computomic brain mapping.
In our results attained over this last year, we havebeen able to make substantial progress in identifying thecore fatigue computational phenotype. In summary, whenprobing learning and motivational behaviour, patients havea clear enhancement of sensitivity to punishments. Thisenhancement strongly correlated with specific clinicalfatigue (and pain) based on resting-state brain imaging,in both humans and rodent models. These have beensuccessful in finding discriminative classifiers. However,components of these classifiers typically correlate poorly
with specific computational or inflammatory (cytokine)
metrics. This suggests that resting-state brain imaging may
have a limited ability to identify mechanistic components of
disease/symptom specific phenotypes, and emphasises theimportance of using targeting behaviour-based approaches
to pinpoint immune-brain interactions.
Beyond this, our other research has involved looking atother (non-inflammatory) clinical conditions using machinelearning based brain biomarkers. And more broadly, we
have been looking at basic mechanisms of motivational,
pain and learning, to provide the fundamental insights
necessary to probe inflammation-linked psychopathology.

Figure 1. In this experiment, we applied computational models of human motivation and decision-making to deconstruct the brain processes
involved behaviour, with the aim to identify regions that are more active in fatigue in inflammatory arthritis and therefore implicated ingenerating the fatigue phenotype. This figure illustrates pregenual and subgenual anterior cingulate cortex as robustly activated, whichstrongly implicates them in fatigue.

Figure 2. In patients with active inflammatory arthritis, serum IL-6 levels are raised, and strongly modulates the prediction error (which drives
human motivated learning) specifically in the hippocampus - a brain region closely related to human memory and consciousness.

Recent Publications
⿎⿎ Takenaka S, Kan S, Seymour B, Makino T, Sakai Y, Kushioka J,
Tanaka H, Watanabe Y, Shibata M, Yoshikawa H and Kaito T. Restingstate amplitude of low-frequency fluctuation is a potentially usefulprognostic functional biomarker in cervical myelopathy. Clin.Orthop.
Relat.Res. doi: 10.1097/CORR.0000000000001157 (2020).

Koizumi A. An evolutionarily threat-relevant odor strengthens human
fear memory. Frontiers in Neuroscience. doi: 10.3389/fnins.2020.00255(2020).

⿎⿎ Ichikawa N, Lisi G, Yahata N, Okada G, Takamura M, Hashimoto RI,
Yamada T, Yamada M, Suhara T, Moriguchi S, Mimura M, Yoshihara
Y, Takahashi H, Kasai K, Kato N, Yamawaki S, Seymour B, Kawato
M, Morimoto J and Okamoto Y. Primary functional brain connections
associated with melancholic major depressive disorder and modulation
by antidepressants. Scientific Reports 10(1),1-12 (2020).
⿎⿎ Taylor JE, Lau H, Seymour B, Nakae A, Sumioka H, Kawato M and61

Im aging

Immunology and Cell Biology
effective at increasing osteoclast motility and changing the
localization of proton pumps, which led to an inhibition ofbone resorption (JBMR plus 2018). Together, these results
demonstrate that the intravital imaging system is a useful
tool for evaluating the similarities and differences in currently
used anti-bone resorptive drugs.

Masaru Ishii, MD/PhD
Professor

Masaru Ishii

Associate Professor

Keizo Nishikawa
Junichi Kikuta
Takao Sudo
Yutaka Uchida12
3

Assistant Professor
Postdoctoral Fellow
Research AssistantSupport Staff

1. Intravital imaging of bone cell dynamics in vivo
We have originally developed a novel imaging systemfor visualizing inside bones by using intravital multiphoton
microscopy. We have succeeded in visualizing the in vivo
behaviors of bone-resorbing macrophage, osteoclast,i.e., the migration and positioning of their precursor
macrophages (Nature 2009, J. Exp. Med. 2010), their modeof bone-resorbing function in vivo (J. Clin. Invest., 2013)
and the functional and physical coupling with bone-forming
osteoblasts (Nat. Commun. 2018). Our study identified two
distinct mature osteoclast (mOCs) functional states; i.e.,bone-resorbing (R) mOCs firmly adhering to bones anddevouring the bone matrix by secreting acids, and nonresorbing (N) mOCs relatively loosely attached and wrigglingalong the bone surface. In order to further analyze theactual event of bone resorption in vivo, we have developed
a new chemical probe for detecting proton secretion in bone
resorption by mOCs (Nat. Chem. Biol. 2016).
We c o u l d d e t e c t a n i n v i v o m o d e o f d y n a m i ccommunication between mature osteoblasts (mOBs)
and mOCs, and found that the mOBs and mOCs were
distributed mainly in a segregated fashion, althoughsome direct cell-to-cell contact was detected between
mOBs and mOCs in spatiotemporally limited areas. A pHsensing fluorescence probe revealed that mOCs secreted
protons for bone resorption when they were not in contact
with mOBs, whereas mOCs contacting mOBs were nonresorptive, suggesting that mOBs could inhibit the bone62

3. Splitting the “AtoM” in search of a treatment forrheumatoid arthritis
Inside live bones, “osteoclasts” and “osteoblasts” areconstantly generated. The former break down bones while
the latter synthesize them. Under well-balanced bone
destruction and formation processes, bones continuallydestroy and form themselves. In the latest study, wedeveloped a unique technology to collect and analyze cells
from arthritic joints, successfully identifying a new type ofbone-destroying osteoclast that contributes to rheumatoid
arthritis (RA), called Arthritis-associated osteoclastogenic
Macrophages, or “AtoMs” (Nat. Immunol. 2019). We foundthat the arthritis-inducing osteoclasts have properties andorigins that are distinct from normal osteoclasts involved inbone metabolism. By specifically inhibiting the differentiationand functions of arthritis-inducing osteoclasts without
affecting normal osteoclasts, their discovery will aid in theestablishment of breakthrough therapy and the developmentof anti-RA drugs.

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 ofbone homeostasis in vivo.

2. Intravital imaging-based investigation ofpharmacological effects
We have shown that our intravital imaging technology
turned out to be a powerful tool for dissecting in vivo
pharmacological actions of various drugs. Regarding thebiologic agents used in clinics for treating rheumatoid
arthritis, anti-IL-6R and anti-TNFα mAbs affected mOCs andswitched bone-resorbing mOCs to non-resorbing cells while
CTLA4-Ig had no action on mOCs but mobilized osteoclast
precursors, eliminating their firm attachment to bone
surfaces. Intravital imaging revealed that various biologic
DMARDs acted at specific therapeutic time-points duringosteoclastic bone destruction, with different efficacies.
These results enable us to grasp the real modes of action ofdrugs, optimizing the usage of drug regimens (Ann. Rheum.
Dis. 2018).
In terms of bisphosphonate agents used in clinics fortreating osteoporosis, we investigated the short-term effectof three types of bisphosphonates, risedronate, alendronate
and minodronate on controlling the bone resorptive activityof mOCs using intravital multiphoton microscopy with a pHsensing fluorescence probe. Risedronate was the mostFigure. In vivo cellular dynamics in 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 visualizingin vivo behaviors with advanced imaging techniques.

Recent Publications
⿎⿎ Hasegawa T., et al. Identification of a novel arthritis-associatedosteoclast precursor macrophage regulated by FoxM1. Nat. Immunol.
20, 1631-43 (2019).
⿎⿎ Matsuura Y., et al. In vivo visualization of different modes of action of
biologic DMARDs inhibiting osteoclastic bone resorption. Ann. Rheum.
Dis. 77, 1219-25 (2018).

molecular probe. Nat. Chem. Biol.12, 579-85 (2016).
⿎⿎ Nishikawa K., et al. Dnmt3a regulates osteoclast differentiation bycoupling to an S-adenosyl methionine-producing metabolic pathway.
Nat. Med. 21, 281-7 (2015).

⿎⿎ Furuya M., et al. Direct cell-cell contact between mature osteoblasts
and osteoclasts dynamically controls their functions in vivo. Nat.
Commun. 9, 300 (2018).
⿎⿎ Maeda H., et al. Real-time intravital imaging of pH variation associatedwith cell osteoclast activity and motility using designed small

63

Im aging

Nuclear Medicine
These preclinical studies suggested that 211AtPhe has
potential to treat glioblastoma. Further histopathologicalstudies are required before proceeding to the clinical trial.

Jun Hatazawa, MD/PhD
Professor

Jun Hatazawa

Associate Professor
Assistant Professor
Research Assistant

Hiroki Kato
Tadashi Watabe
3
Figure 1. Reaction for the labeling of para-borono-L-phenylalanine with 211At.

Glioblastoma is one of the most intractable malignancies
with a 5-year survival rate of 9.8% even when combination
therapy by surgical removal, chemotherapy, and externalradiation was applied.
Radionuclide therapy with alpha particles targeting
intractable cancers is now being developed in Osaka
University. Alpha particles have a strong cell killing effectbecause of high radiation quality factor. To realize this,the key issues are 1) large amount of production of alphaparticles such as actinium-225 (physical half-life:10 day)and Astatine-211 (physical half-life: 7.2 hour) effective
for clinical use, 2) cancer cell specific delivery of alphaparticle-bound molecules, 3) an evaluation of normal tissue
damage. We are now developing astatine-211 labeledphenylalanine (211AtPhe) as a therapeutic agent because ofa cancer specific nature as L-type amino acid transporter 1(Watabe T, et al. Oncotarget 2020), and fluorine-18 labeledfluoro-phenylalanine (18FPhe) (Hatazawa J. et al. Ann. Nucl.

with the mixed gas flow and passed through a Teflon tube
which was cooled by ice water to trap evaporated 211At. After
collection of 211At on the Teflon trap tube for approximately
20 minutes, the trapped 211At was dissolved in approximately
100μL of distilled water (Figure 1) (Watabe T. et al. J. Nucl.
Med. 2019).
Treatment effect of 211AtPhe on glioblastoma cells wasinvestigated in mice bearing C6 glioma xenografts andGL261 allografts. The tumor sizes (mm3) were monitored
using a caliper. In C6 glioma xenograft model, a delay intumor growth was found depending on radioactivity of theinjected 211AtPhe dose. In controls, tumor volume reached
4 mm3 at 7 days after injection of saline, whereas it reachedMBq, and 1.0 MBq injection of 211AtPhe, respectively (Figure(Shimosegawa E. et al. Ann. Nucl. Med 2016; Beshr R. et al.
Ann. Nucl. Med. 2018; Romanov V. et al. Ann. Nucl. Med.
2020) as diagnostic imaging agents with PET.
Astatine-211 was produced by means of AVF Cyclotron
facilities in the Research Center for Nuclear Physics, Osaka
University, by 209Bi(α,2n) 211At nuclear reaction (Ikeda H.

The whole-body distribution of 211AtPhe was investigated211

At was separated from the bismuth target and purified via

Figure 2. Tumor growth suppression effect by 211AtPhe.

Recent Publications

4 mm3 at 18 days, 25 days, and 30 days after 0.1 MBq, 0.5

Med. 1994; Ogawa T. et al. Eur. J. Nucl. Med. 1996) andfluorine-18 labeled fluoro-borono-phenylalanine (18FBPA)et al. Appl. Radiat. Isot. 2018). The metallic Bi target wasirradiated by accelerated alpha particles. The produced64

dry distillation. The irradiated Bi target was put in a quartz
column and heated up to 850°C using an electric tubular
furnace under mixed helium and oxygen gas flow. The
evaporated 211At was then swept out from the quartz column

2).
in normal male ICR mice (n=6, 9 weeks old, mean bodyweight=37.7g). The major organs (brain, thyroid gland,salivary gland, heart, lung, esophagus, liver, stomach, small
intestine, large intestine, kidney, adrenal gland, pancreas,
spleen, and testes) and blood. Thyroid gland and stomach
showed relatively higher accumulation than other organs.
There was no significant difference in body weight between
controls and 211AtPhe treated mice.

⿎⿎ Watabe T., et al. Targeted alpha therapy using astatine ( At)-labeledphenylalanine: A preclinical study in glioma bearing mice. Oncotarget
11, 1399-1398 (2020).
211

⿎⿎ Liu Y., et al. Preclinical evaluation of radiation-induced toxicity I
targeted alpha therapy using [211At]NaAt in mice: a revisit. Trans.
Oncol. 13(4), 100757 (2020).
⿎⿎ Watabe T., et al. Enhancement of 211At uptake via the sodium iodide
symporter by the addition of ascorbic acid in targeted α-therapy ofthyroid cancer. J. Nucl. Med. 60, 1301-1307 (2019).

by means of [18F]fluoro-borono-phenylalanine PET/CT. Ann. Nucl. Med.
32, 702-708 (2018).
⿎⿎ Ikeda H., et al. Application of astatine-210: Evaluation of astatine
distribution and effect of pre-injected iodide in whole body of normal
rats. Appl. Radiat. Isot. 139, 251-255 (2018).
⿎⿎ Shimosegawa E., et al. Assessment of 10B concentration in boron
neutron capture therapy: potential of image-guided therapy using
18FBPA. Ann. Nucl. Med. 30, 749-755 (2016).

⿎⿎ Beshr R., et al. Preliminary feasibility study on differential diagnosis
between radiation-induced cerebral necrosis and recurrent brain tumor

65

Im aging

Biophotonics

Nicholas Isaac Smith, PhD
Associate Professor

Nicholas Isaac Smith

Assistant Professor

Alison Hobro
Nicolas Pavillon
1

Postdoctoral Fellow

66

The biophotonics laboratory develops tools for labelfree analysis of single cells. Single-cell analysis is a populartarget for a large number of researchers, usually pursued
by labeling surface markers, by introducing fluorescentdyes into the cell, or by invasive, techniques such as single
cell RNA sequencing. In contrast, our tools are based onlabel-free optical methods, which aim to produce some ofsuch as the activation state. We previously showed (Pavillion
et al, PNAS 2018) that in a macrophage cell line, theresponse to LPS could be determined from the Ramanderived compositional features, or from the morphologicalfeatures. The Raman-derived cell composition could ingeneral discern LPS-based activation with a higher degreeof accuracy than could be determined from the morphology.

the same discriminatory capability as the more invasive
methods. Additionally, label-free methods are based on
endogenous contrasts of the cell, and can also find novel
features that can be used to discriminate between cell
phenotypes or cell states.
More specifically, we use a combination of labelfree methods: Raman scattering that provides molecularsignatures from a target cell, and quantitative phaseimaging that provides a full quantitative map of the cellmorphology, and it turns out that this information is quite
related to conditions of interest in the cell. Both of these data
sets can be collected in a few seconds or shorter, from asingle cell, providing paired measurements relating to themolecular components and the shape-based features. The
composition of a cell can vary not only due to the conditionsof interest, such as protein expression in response to a
stimulus, but also due to inherent biological variability.
Similarly, the morphology of cells can be seen to varysignificantly from cell to cell when observing a culture dish.
Amongst all this inherent cell-to-cell variability however, liecertain key features, both in molecular composition andmorphological features that can distinguish cell properties

Since cell morphology can be thought of as a “later”
indicator of cellular change, in the sense that signaling
molecules should be expressed or depleted before theoverall cell structure shows visible change, we believe bothof these techniques are valuable in single-cell analysis.
Additionally, both can be paired to maximize the amount of
information that can be obtained from a single-cell without
modifying it or labeling it.
A recent extension of this approach led to some
intriguing results. One question was whether the above
analysis would function equally well in primary cells rather
than a cell line. We then considered macrophages derived
from the mouse peritoneal cavity. Experimentally, to obtainsufficient numbers of cells for statistically relevant analysis
then requires multiple donor mice, with the complication thatmouse-to-mouse variations could then become important.
Turning this complication into a point of interest, we could
then ask to what degree the experimental parameters suchas mouse-to-mouse variation occur, and how strong suchvariations are in comparison to the changes that occur withimmune activation. This led to the first comprehensive labelfree study of how individual peritoneal cavity macrophages

respond to LPS, how the procedures for obtaining them(i.e. resident or elicted) affect the responses, and how thedifferent experimental parameters affect the robustnessof the analysis. This used single-cell measurements, andspanned more than 30,000 cells in total. The results showedthat the label-free analysis could determine single cell LPS
response, whether it was resident or had been recruited into
the peritoneal cavity, and for cells derived from some of themice, the donor mouse could even be determined (ie could
inherently recognize which donor mice were aberrant).
Even when the cell population is highly heterogenous, themethods we developed can still identify cell phenotypes andactivation.
Finally, we saw the first results in the study of thenature and causes of neutrophil extracellular traps (NETS)
in neutrophils using a combination of fluorescent-labeledimaging flow cytometry, and unlabeled Raman imaging.
While NETs have been known for several years now, there
remain many unknown factors in the generation of NETs
and whether different NET types are formed in different
conditions. To study this, we first concentrated on producing
reliable protocols to determine NET generation rates whenstimulated or inhibited by several distinct endocytotic
pathways, and found that one limitation in the study offormation conditions is the lack of sufficient methods torobustly quantify NET formation, and this turns out to be
essential for our future research. NETs can, however, bequantified by building metrics based on fluorescenceintensities and area in imaging flow cytometry, or by carefulanalysis of Raman scattering intensities and signal areas inRaman NET imaging.

Figure. Label-free single cell analysis. Laser-based Raman analysis of single cells provides features that can be used to identify cell
type, drug response, or even donor-specific characteristics. Using cell morphology derived from label-free imaging can provide additional
sensitivity to single cell variations. Cell phenotypes and pathway-specific functional changes can be determined from the non-invasive nonlabelling methods we developed.

Recent Publications
⿎⿎ Pavillon N and Smith NI. Immune cell type, cell activation, and single
cell heterogeneity revealed by label-free optical methods. Sci. Rep. 9,17054 (2019).
⿎⿎ Lelliott PM, Momota M, Shibahara T, Lee MSJ, Smith NI, Ishii KJ,
Coban C. Heparin induces neutrophil elastase-dependent vital andlytic NET formation. Int. Immunol. 32(5), 359-368 (2020).

Opt. Commun. 422, 75-84 (2018).
⿎⿎ Hobro AJ and Smith NI. An evaluation of fixation methods: spatial andcompositional cellular changes observed by Raman imaging. Vib.
Spectrosc. 91, 31-45 (2017).

⿎⿎ Pavillon N, Hobro AJ, Akira S and Smith NI. Noninvasive detection ofmacrophage activation with single-cell resolution through machinelearning. Proc. Natl. Acad. Sci. USA. 115(12), E2676-E2685 (2018).
⿎⿎ Hobro AJ and Smith NI. Vibrational spectroscopic imaging ofpathogens, microorganisms, and their interactions with host systems.

67

Im agingChemical Imaging Techniques
Moreover, we monitored the dynamics of osteoclast

Kazuya Kikuchi, PhD

In Vivo Imaging with Fluorescent Probes Revealed the
Dynamics and Function of Osteoclast Proton Pumps

68

Professor

Kazuya Kikuchi

Associate Professor
Assistant Professor
Postdoctoral Fellow
Research AssistantSupport Staff

Yuichiro Hori
Masafumi Minoshima
1
2
1

proton pumps with the acidic compartments simultaneously
over a long time. We found that the osteoclast proton pumps
were sparsely distributed at the membrane and intracellularcompartments in the osteoclast where few acidic regions
were detected. Concomitant with the accumulation of theosteoclast proton pumps on the bone surface, the acidic
compartments emerged and spread over the bone surface
covered by the osteoclast. Thereafter, the area of acidic
compartments on the bone surface gradually decreased
without the localization change of the osteoclast proton
pumps, indicating that the osteoclast was in an inactive,
non-resorbing state. The image analysis revealed that bone

acidiﬁcation was associated with proton pump accumulation
to the bone surface.
We further applied our imaging system to the in vivo
quantitative evaluation of drugs targeting osteoclast
proton pumps. Afrer administration of Baﬁlomycin A1, aknown proton pump inhibitor, we found the decrease of thered ﬂuorescent signals in the whole area of view. These
results demonstrated that our imaging approach canspatiotemporally analyze the drug efficacies in intact tissues
and provide a useful tool for the development of drugs
against bone diseases and inhibitors of trafficking pathwaysof proton pumps.

values of Rh-1−3 ranged from 4.9 to 5.9, which is suitablefor imaging acidic areas created by mature osteoclasts.
Next, the ring-opening reactions (fluorescence activation) of
the spirolactam were monitored by assessing the absorption
transition of the dyes upon an immediate pH jump from pH
8.0 to 4.0. Remarkably, Rh-2 quickly turned into fluorescentform when the solution acidiﬁed. Because of the suitable pκa

In vivo two-photon ﬂuorescence imaging is a powerfulmodality to monitor cell dynamics deep within the tissues
and organs with high temporal and subcellular resolutions.
Small-molecule-based ﬂuorescent probes are promisingtools as their ﬂuorescence-sensing properties can be tuned
for the optimization of their biomolecular functions and facile
administration by injection. For longitudinal monitoring of theprotein functions in real-time with high signal/backgroundcontrast, the ﬂuorescent probes should have a quick
response and be photostable at the targeted tissues invivo. In this study, we aimed to develop a pH-activatablesmall-molecular probe for multicolor intravital imaging toreveal the functions of osteoclast proton pumps duringosteoclastic bone resorption. Osteoclast proton pumps,
which are vacuolar H+-ATPases (V-type H+-ATPases), areand the rapid ﬂuorescence activation response, Rh-2 wasselected as a dye structure for detecting the area of bone
acidiﬁcation in vivo. Then we synthesized a small-molecular
probe Red-pHocas (red pH-activatable ﬂuorescent probe forosteoclast activity sensing) by the conjugation of Rh-2 with
two alendronates containing bone-targeting bisphosphonate
groups (Figure, left).
Then, Red-pHocas was administrated subcutaneously
to ﬂuorescent reporter mice, where green ﬂuorescentprotein (GFP) is expressed under the promoter of the V-type
H+ATPase α3 subunit (α3-GFP mice) for 3 days then the

Figure. (left) Chemical structure of Red-pHocas. (middle) Two-photon ﬂuorescence image of osteoclasts in bone tissues. Green, GFP fused
proton pumps; red, Red-pHocas; blue, bone tissue. Scale bar: 20 μm. (right) Schematic illustrations of proton pump dynamics and acidic
regions in osteoclasts.

extremely involved in the secretion of several protons todissolve bone minerals. So far, the mechanism by whichmature osteoclasts regulate the localization of the proton
pumps upon bone resorption is still unclear due to the lackof spatiotemporal information on acidic compartments.
We initially synthesized and evaluated pH-activatableﬂ u o re s c e n c e p ro p e r t i e s o f a s e r i e s o f r h o d a m i n e
spirolactams Rh-1−3 based on a rhodamine-B dye.
The ﬂuorescence spectra of Rh-1−3 at different pH
regions revealed that all rhodamine derivatives showed aﬂuorescence increase at the lowered pH regions. The pκaintravital imaging of calvaria bone tissue was performed
using a two-photon excitation microscopy under anesthesia.
Fluorescence images were acquired after processing
the spectral unmixing algorithms that separate distinct
ﬂuorescence signals derived from GFP, Red-pHocas, andsecond-harmonic generation (SHG) of collagen ﬁbers inbones. We found that the ﬂuorescence signals of RedpHocas in the local area on the bone surface overlappedwith osteoclast localizations, indicating it selectively
detected the lowered pH regions upon bone resorption by
activated osteoclasts (Figure, middle).

⿎⿎ Kumar N, Hori Y, Nishiura M & Kikuchi K. Rapid no-wash labelingof PYP-tag proteins with reactive fluorogenic ligands affords stable
fluorescent protein conjugates for long-term cell imaging studies.
Chemical Science 11, 3694-3701 (2020).

⿎⿎ Gao J, Hori Y, Shimomura T, Bordy M, Hasserodt J & Kikuchi K.
Development of fluorogenic probes for rapid high-contrast imaging oftransient nuclear localization of sirtuin 3. ChemBioChem 21, 656-662(2020).

⿎⿎ Gao J, Hori Y, Takeuchi O & Kikuchi K. Live-cell imaging of protein
degradation utilizing designed protein-tag mutant and fluorescentprobe with turn-off switch. Bioconjugate Chem. 31, 577-583 (2020).

⿎⿎ Minoshima M, Kikuta J, Omori Y, Seno S, Suehara R, Maeda H,
Matsuda H, Ishii M & Kikuchi K. In vivo multicolor imaging withfluorescent probes revealed the dynamics and function of osteoclast
proton pumps. ACS Cent. Sci. 5, 1059-1066 (2019).

Recent Publications

⿎⿎ Imoto T, Kawase A, Minoshima M, Yokoyama T, Bito H & Kikuchi K.
Photolytic release of a caged inhibitor of an endogenous transcription
factor enables optochemical control of CREB-mediated gene
expression. Org. Lett., 22, 22-25 (2020).

69

Kazuhiro Suzuki, MD/PhD
Kazuhiro Suzuki

Assistant Professor
Postdoctoral Fellow
Research AssistantSupport Staff

Akiko Nakai
12
2

We have been studying the interactions between the
nervous and immune systems with a special focus on
the roles of adrenergic nerves in the control of adaptive
immune responses. Our study revealed a mechanism bywhich adrenergic nerves control lymphocyte trafficking
through lymph nodes. Inputs from adrenergic nerves tothe β2-adrenergic receptor expressed on lymphocytes

translocated to the plasma membrane after stimulation of
CXCR4. The COMMD3/8 complex interacted with not only
CXCR4 but also other G protein-coupled chemoattractant
receptors, including CXCR5, CCR7, and the oxysterol
receptor EBI2, after activation of the receptors. Interestingly,
COMMD3 and COMMD8 were degraded by the proteasomein the absence of the other, and deficiency of either protein

enhance the responsiveness of a specific set of chemokine
receptors and inhibit lymphocyte exit from lymph nodes(Nakai et al. J. Exp. Med. 2014). This mechanism was foundto generate diurnal variations in lymphocyte numbers inlymph nodes and consequently the magnitude of adaptive
immune responses in synchronization with the circadianoscillation of adrenergic nerve activity (Suzuki et al. J. Exp.
Med. 2016). In search of factors that mediate the crosstalkof signaling between the two different types of G proteincoupled receptors (GPCRs), the β2-adrenergic receptor

produced the same phenotypes, indicating that both
COMMD3 and COMMD8 are required for the stability andfunctions of their complex. Deficiency of COMMD3 or
COMMD8 in B cells reduced their chemotactic responses
through the receptors to which the COMMD3/8 complex
was recruited. Thus, the COMMD3/8 complex is a positive
regulator of chemoattractant receptor signaling.
Agonist binding to GPCRs activates trimeric G proteinsto regulate the generation of second messengers thatmodulate downstream signaling. Agonist-occupied
GPCRs are phosphorylated by GPCR kinases (GRKs) andsubsequently recruit β-arrestins that serve as scaffolds toactivate signaling molecules, including mitogen-activatedprotein kinases (MAPKs). The GRK family consists ofseven mammalian members, among which GRK2, GRK3,
GRK5, and GRK6 are expressed ubiquitously. Different
GRKs phosphorylate distinct sites on the C-terminal tailof the receptor, establishing a barcode that dictates thefunctional consequences of β-arrestin engagement. Thus,
specific targeting of GRKs to activated GPCRs is crucial
for signal transduction. Our mechanistic analysis for theaction of the COMMD3/8 complex demonstrated that thisand chemokine receptors, we identified a protein complex
consisting of copper metabolism MURR1 domain-containing(COMMD) 3 and COMMD8 (COMMD3/8 complex), thefunctions of which have been totally unclear (Figure A). This
year, we demonstrated that the COMMD3/8 complex plays
an important role in chemoattractant receptor signaling andhumoral immune responses (Nakai et al. J. Exp. Med. 2019).
We first identified COMMD8 as a protein that binds to
the C-terminal tail of a representative chemokine receptor
CXCR4. Additional screening revealed the interaction of
COMMD8 with COMMD3. We found that these proteins
constitutively formed a complex in the cytosol, but were70

Professor

showed multiple defects in their migration in vivo (Figure C).

recruits GRK6 to chemoattractant receptors in a GRK2/3-

Additionally, deficiency of COMMD3 or COMMD8 in B cells

dependent manner, which promotes MAPK activation andseverely impaired humoral immune responses (Figure D andconsequently lymphocyte chemotaxis (Figure B). It has
been suggested that the specificity of GRK recruitment to
GPCRs is determined by the relative expression levels of
individual GRKs, which vary among cell type, and distinct
receptor conformations induced by ligand binding. Our
study identifies a GRK-recruiting adaptor, the COMMD3/8
complex, as an additional determinant of GRK specificity for
GPCRs.
Consistent with the reduced chemotactic responses of
COMMD3- and COMMD8-deficient B cells, the mutant B cells

Figure E). Therefore, the COMMD3/8 complex is essential
for proper functioning of the immune system. By exploiting
the unique property of the COMMD3/8 complex, it would
be possible to degrade and disable the protein complex
by pharmacological disruption of the interaction between
COMMD3 and COMMD8. Development of such drugs would
provide a novel approach for immune regulation, which may
be applicable to the treatment of immune disorders.

Im aging

Immune Response Dynamics

protein complex functions as an adaptor that selectively

Figure. Role of the COMMD3/8 complex in GPCR signaling and immune responses.
(A) In silico modeling for the structure of the COMMD3/8 complex. COMMD3, orange; COMMD8, green. (B) Proposed role of the
COMMD3/8 complex in GRK6 recruitment to GPCRs. (C) COMMD8-deficient (Commd8Δ) B cells show a defect in migration toward theouter follicle at 3 h after immunization. (D and E) B cell-specific deficiency of COMMD8 severely impairs the antibody response (D) anddevelopment of germinal centers (E). Scale bars, 200 μm.

Recent Publications
⿎⿎ Nakai A, Fujimoto J, Miyata H, Stumm R, Narazaki M, Schulz S,
Baba Y, Kumanogoh A and Suzuki K. The COMMD3/8 complex is adeterminant of GRK6 specificity for chemoattractant receptors. J. Exp.
Med. 216, 1630-1647 (2019).

⿎⿎ Nakai A, Hayano Y, Furuta F, Noda M and Suzuki K. Control oflymphocyte egress from lymph nodes through β2-adrenergic receptors.
J. Exp. Med. 211, 2583-2598 (2014).

⿎⿎ Nakai A and Suzuki K. Adrenergic control of lymphocyte trafficking andadaptive immune responses. Neurochem. Int. 130, 104320 (2019).
⿎⿎ Suzuki K and Nakai A. Immune modulation by neuronal electric shockwaves. J. Allergy Clin. Immunol. 141, 2022-2023 (2018).
⿎⿎ Suzuki K, Hayano Y, Nakai A, Furuta F and Noda M. Adrenergic controlof the adaptive immune response by diurnal lymphocyte recirculation
through lymph nodes. J. Exp. Med. 213, 2567-2574 (2016).

71

paratope and epitope residues along with high-scoring
docked poses. A flowchart of Adapt is shown in Figure 3.

Figure 1. Distribution of nucleotides around homologs ofRegnase-1 as revealed by MAFFT-DASH.

Figure 2. InterClone: Lymphocyte receptor functional clustering.

Bioin for m a t i c s

Systems Immunology

A web server has been prepared, Adapt (https://sysimm.
org/adapt/), that accepts antibody- antigen pairs in the formof either sequences or structures and returns predicted

Daron M. Standley, PhD
Professor

Daron Standley

Associate Professor

Kazutaka Katoh
Shunsuke Teraguchi
Li Songling
2
4
1
1

Assistant Professor
Postdoctoral Fellow
Research Assistant
Visiting Scientist
Support Staff

72

In 2019 our lab reached several important milestones in
the analysis of adaptive immune receptors, especially in thecontext of single cell sequencing of B and T cells. We also
had a generally productive year in collaborative projects(Metwally H. et al. Sci. Signal 2020; Lee Y. et al. Life Sci.
Alliance 2020; Fukushima K. et al. Immunity 2020; Yamasoba
D. et al. Nat. Microbiol. 2019; Takeda K. et al. J. Allergy Clin.

achieved a specificity (1- False Positive rate) of over 99%
with a sensitivity (True Positive rate) of 62%. A flowchart ofInterClone is shown in Figure 2.
The release of Repertoire Builder and InterClone were
followed by tools that construct models of TCRs withpeptide-MHC complexes and antibodies with their cognate
antigens. ImmuneScape (Li S. et al. Meth. Mol. Biol. 2019)

Immunol. 2019). For general protein functional analysis,
we developed a pipeline for predicting protein-nucleotide
binding sites in 3D using MAFFT and DASH (Rozewicki J. et
al. Nucleic Acids Res. 2019). As shown in Figure 1, the new
pipeline dramatically reveals the conservation of nucleotide
binding sites on homologs of Regnase-1 (Rozewicki. J. et al.
Meth. Mol. Biol. in press).
With regard to repertoire analysis, the first major
milestone was to release a state-of-the-ar t tool for
lymphocyte receptor modeling (Repertoire Builder; Schritt,
D. et al. Mol. Sys. Des. Eng. 2019). What is different about
Repertoire Builder in comparison to other available tools
is that it constructs 3D models of B cell receptors or T cell
receptors in a high-throughput manner, without loss of
accuracy. This makes it possible to model every BCR or TCR
in a 10x Chromium VDJ experiment in under 30 minutes.
What the resulting structures then allow you to do, ismeasure similarities across clones and even across donors.
InterClone (Xu Z. et al. Mol. Sys. Des. Eng. 2019) carries
out such comparisons using machine learning algorithms
that cluster receptors likely to target the same antigen
and epitope. Preliminary tests on anti-HIV antibodies

makes use of known TCR-peptide-MHC complex structuresand models a given TCR-peptide-MHC sequence triplet. It
is fast and accurate, and, to our knowledge the first tool of
its kind.
Adapt (Davila et al. in prep) is a tool for building
antibody-antigen complex models. Because antibodies
can bind antigens on any exposed surface, we can not use
structural templates as we did with ImmuneScape. Instead,
we first predict the possible antibody-antigen interface
residues (paratope and epitope, respectively) using machinelearning algorithms and then dock the antibody antigen 3Dmodels using the predicted paratopes and epitopes as a
guide. Starting from realistic models of unbound antibodies
and antigens with known structure, we carried out leaveone-out cross validation. We found that both the samplingand scoring of the Adapt docking pipeline were significantly
improved over simply using docking alone. Furthermore, we
obtained a 4% improvement in the average area under thereceiver-operating characteristic curve for the final epitope
prediction (0.80) as compared with the initial prediction(0.76), suggesting that the Adapt epitope predictions do
indeed reflect antibody binding specificity.

Figure 3. Adapt: a new way to predict antibody-antigen complex structures.

Recent Publications
⿎⿎ Xu Z., et al. Functional clustering of B cell receptors using sequenceand structural features. Mol. Syst. Des. Eng. 4, 769-778 (2019).
⿎⿎ Takeda K., et al. Allergic conversion of protective mucosal immunity
against nasal bacteria in patients with chronic rhinosinusitis with nasal
polyposis. J. Allergy Clin. Immunol. 143, 1163-1175 e1115, doi: 10.1016/
j.jaci.2018.07.006 (2019).

Res. 47, W5-W10, doi: 10.1093/nar/gkz342 (2019).
⿎⿎ Li S., et al. Structural modeling of lymphocyte receptors and theirantigens. Meth. Mol. Biol. 2048, 207-229, doi: 10.1007/978-1-49399728-2_17 (2019).

⿎⿎ Schritt D., et al. Repertoire Builder: high-throughput structuralmodeling of B and T cell receptors. Mol. Syst. Des. Eng. 4, 761-768(2019).
⿎⿎ Rozewicki J, Li S, Amada KM, Standley DM & Katoh K. MAFFT-DASH:
integrated protein sequence and structural alignment. Nucleic Acids73

Trans-biobank polygenic risk score analysis identified
causal biomarkers for lifespan

Yukinori Okada, MD/PhD

Goal of our laboratory
Genetic backgrounds of individuals have substantial
impacts on risk of a wide range of immune-related diseases.
Statistical immunology is a research field that evaluatescausality of human genetic variations on immune-relateddiseases, using statistical and bioinformatics approaches.
Recent developments of genome sequencing technologies
have provided human genome data from millions of subjects,
and successfully identified comprehensive catalogues ofgenetic risk loci of immune-related diseases. However, little
is known regarding how to develop methodology to integrate
large-scale human genome data with diverse biological andimmunological resources. The goal of our laboratory is todevelop such methods and apply them to the latest largescale disease genome and multi-layer omics data.

Metagenome-wide association study of gut microbiome
revealed disease-specific feature of rheumatoid arthritis
Microbiomes play substantial roles in homeostasis andbiology of a variety of human diseases through interaction
with the host. Metagenome-wide association studies (MWAS)
utilizing whole-genome shotgun sequencing is a promising
tool to elucidate microbiome etiology. We conducted an
initial MWAS of rheumatoid arthritis in Japan. Phylogenetic
case–control association tests showed high abundance ofmultiple species belonging to the genus Prevotella (e.g.,
Prevotella salivae) in the RA case metagenome (Figure 1).
74

Professor

Yukinori Okada

Research AssistantSupport Staff

5
1

Identification of modifiable biomarkers related to lifespanis one of the ultimate goals of human genome studies.
Polygenic risk score (PRS) is a genetic risk of an individual
which aggregates genotype risk of genome-wide SNPs.

We developed a novel statistical genetics method which
utilizes PRS as an instrumental variable to infer causality
on the target phenotype. Through trans-ethnic biobank
collaboration collecting ~700,000 worldwide individuals,
we identified high blood pressure and obesity as causal
biomarkers responsible for lifespan (Sakaue S. et al. Nat.
Med. 2020).

Figure 1. Metagenome-wide association study (MWAS) of gut
microbiome of rheumatoid arthritis in the Japanese population.

Figure 2. Machine learning-based deconvolution of the genomewide association study (GWAS) data of the Japanese population.

Bioin for m a t i c s

Statistical Immunology

Gene functional assessments showed that the abundance of
one redox reaction–related gene (R6FCZ7) was significantly
decreased in the RA metagenome compared to controls.
Our study also identified a population-specific link of themolecular pathways between the metagenome and hostgenome (Kishikawa T. et al. Ann. Rheum. Dis. 2020).

Characterizing natural selection pressure and evolutionof the Japanese population
The history of human beings, namely natural selection
pressure and evolution, is embedded in the genome dataof current human populations. We conducted a genomewide scan of natural selection pressure in the Japanese
population by using large-scale genome-wide association
study (GWAS) data of >170,000 Japanese individuals.
We identified 29 loci with significant selection signatures.
Phenome-wide enrichment analysis identified alcohol
consumption and obesity as key traits for evolution,
while bread consumption and hand grip strength wereidentified for Europeans (Yasumizu Y. et al. Mol Biol Evol2020). Dimension reduction of the Japanese GWAS data
enlightened the population structures, which were clusteredinto “mainland (Hondo)”, and “non-mainland (Ryukyu andHokkaido-Ainu)” (Figure 2). This study demonstrated thevalue of the combinatory approach to applying both linear
and non-linear machine-learning methods (Sakaue S. et al.
Nat. Commun. 2020).

Recent Publications
⿎⿎ Sakaue S., et al. Trans-biobank analysis with 676,000 individuals
elucidates the association of polygenic risk scores of complex traits
with human lifespan. Nat. Med. 26, 542-548 (2020).
⿎⿎ Sakaue S., et al. Dimensionality reduction reveals fine-scale structurein the Japanese population with consequences for polygenic risk
prediction. Nat. Commun.11, 1569 (2020).

population. Mol. Biol. Evol. 37, 1306-1316 (2020).
⿎⿎ Kishikawa T., et al. A metagenome-wide association study of gut
microbiome revealed novel etiology of rheumatoid arthritis in theJapanese population. Ann. Rheum. Dis. 79,103-111 (2020).

⿎⿎ Matoba N., et al. GWAS of 165,084 Japanese individuals identified
nine loci associated with dietary habits. Nat. Hum. Behav. 4, 308-316(2020).
⿎⿎ Yasumizu Y., et al. Genome-wide natural selection signatures
are linked to genetic risk of modern phenotypes in the Japanese75

Associate Professor

Our team integrates computational and single cell
genomics techniques to understand the immune system.
We develop computational methods to analyze and extract
biologically interesting information from single cell data.
We integrate experimental data with publicly available
information into network models of immune regulation. We
apply this framework to study gene regulatory networks

technologies. We are developing methods that leveragetechniques from engineering control to estimate and correctbatch effects. A key focus of our method is on preserving as
much as possible the original cell population structure.

controlling immune cells development, and collaborate withother groups at IFReC to address other immune system
questions.

The large number of datasets accumulating fromsingle cell genomics experiments opens the door toapproaches that study the immune system from a moretheoretical perspective. Transcriptional regulatory networks
are important determinants of cell identity and function.
These networks consist of transcriptional activators andrepressors, including transcription factors and the target
genes they regulate. We use linear regression to model
immune transcriptional regulatory networks. We assume thatthe expression level of each gene depends on the activityof a small number of regulators. Furthermore, regulators
contribute in an additive way to the expression level of theirtarget genes. Using the expression level of the regulators
as a proxy for their activities we apply these methods tostudy the differentiation of immune cells. We use informationfrom trajectory and RNA velocity to infer the differentiation
pathway along which to estimate transcriptional regulatory
networks.

Development of computational methods
Single cell genomics technologies have changed
how we understand cell heterogeneity and identity but
extracting information from these multi-dimensional datasets
remains challenging. One specific area of interest is theidentification of cell specific markers. In collaboration with
Alexis Vandenbon at Kyoto University, we have developed
a method to identify groups of cells with altered expression
patterns from multi-dimensional representations of data (e.g.
UMAP or PCA plot). Our method, called haystack, does
not require any previous clustering of the data, providing
an alternative approach to identify markers associatedwith groups of cells. This method is implemented in the R
package singleCellHaystack, and is available from https://github.com/alexisvdb/singleCellHaystack.
Another important issue is how to combine different
datasets produced by different laboratories using different
76

Diego Diez

regulatory networks control immune cell differentiation.
We collaborate with the Host Defense laboratory to study
the role of the RNase Regnase 1 in T cell development.
Using targeted single cell transcriptomics from the BD
Rhapsody platform, we analyze the expression of hundredsof genes in tens of thousands of single cell transcriptomes
from spleen and thymus in different experimental conditions.
We also study the role of Regnase 1 in NK cell development.
Here we use 10x genomics whole transcriptomic and featurebarcoding to quantify the expression level of a selected
number of proteins from murine splenocytes.
We collaborate with the Experimental Immunology
laboratory and the Systems Immunology laboratory to study
T cell development in SKG mice. In this project we use datafrom 10x genomics single cell transcriptomics and TCR

immune repertoire to investigate how TCR signaling impacts
T cell development. We use computational methods toidentify T cell signaling pathways driving the development of
autoimmunity.
We collaborate with the Immune Regulation laboratory
to study the role of CD4 T cells in human Eosinophilic
Chronic Rhinosinusitis. Here we combine single cell RNA
sequencing with TCR immune repertoire profiling to identify
pathogenic T cells involved in chronic inflammation that may
be responding to bacterial antigens.
Our group is using single cell genomics to study
an increasing number of topics, including NKT celldifferentiation in mice and IgA nephropathy in humans,
using a combination of single cells transcriptomics, featurebarcoding, and (TCR and BCR) immune profiling.

C ombin e d Re s e ar ch F i elds

Quantitative Immunology

Mathematical modeling

Applications to immunology
We are interested in studying how transcriptional

Figure. Single cell RNA-seq of thymocytes gives insight into T cell development: A) clustering, B) RNA velocity C-E) expression ofdifferentiation markers, F) differential expression.

Recent Publications
⿎⿎ Vandenbon A, Diez D. singleCellHaystack: A clustering-independent
method for finding differentially expressed genes in single-celltranscriptome data. bioRxiv 557967; doi: https://doi.org/10.1101/557967(2019).

⿎⿎ Diez D, Agusti A. & Wheelock CE. Network analysis in the investigationof chronic respiratory diseases. From basics to application. Am. J.
Respir. Crit. Care Med. 190, 981-988, doi: 10.1164/rccm.2014030421PP (2014).

⿎⿎ Nakai W, Yoshida T, Diez D, Miyatake Y, Nishibu T, Imawaka N, Naruse
K, Sadamura Y, Hanayama R. A novel affinity-based method for theisolation of highly purified extracellular vesicles. Sci. Rep. 6(1), 33935.
doi: 10.1038/srep33935 (2016).
⿎⿎ Bahrini I, Song JH, Diez D. & Hanayama R. Neuronal exosomes
facilitate synaptic pruning by up-regulating complement factors inmicroglia. Sci. Rep. 5, 7989, doi: 10.1038/srep07989 (2015).

77

Events & Outreach Activities

Date
January 20-24, 2020
Venue	Awaji Yumebutai International Conference Center, Hyogo / Grand
Cube Osaka – Osaka International Convention Center, Osaka(The 11th International Symposium of IFReC)

Lecturer
Nobuyuki Takakura(IFReC, Osaka University, Japan)

Eran Elinav(The Weizmann Institute of Science, Israel)

The program comprised a four-day lecture course on Awaji Island anda one-day international symposium at the Osaka International Convention
Center. Fifty-four young researchers from around the world, selected from
261 applicants of 58 nationalities, learned cutting-edge immunology from
fifteen world-renowned senior researchers at the course and from ten up-andcoming and senior researchers at the symposium. Three young researchers
from companies collaborating with IFReC also attended and joined in thediscussions. The Winter School was highly evaluated by both participants andlecturers as an excellent opportunity for networking with young peers throughactive discussions and communications as well as for its high level of science.
Four participants were awarded the best presentation awards and had an
opportunity to give a presentation at the International Symposium of IFReC.
Dr. Chris Schiering, who was a participant at a previous winter school in 2014,
was invited to the symposium as a speaker and as a role model for the young
researchers. The NIF Winter School on Advanced Immunology has beenorganized jointly with the Singapore Immunology Network (SIgN) since FY2011.
However, this was the last Winter School jointly organized with SIgN due toreform of SIgN, while IFReC will continue to hold the Winter School.

Kiyoshi Takeda(IFReC, Osaka University, Japan)

Olaf Rötzschke(SIgN, Singapore)

Paul Kubes(University of Calgary, Canada)

Florent Ginhoux(SIgN, Singapore)

Tatiana Kisseleva(University of California San Diego, USA)

Sidonia Fagarasan(Riken IMS, Japan)

Kazuyo Moro
(IFReC, Osaka University, Japan)

Joachim Schultze(University of Bonn, Germany)

Lisa Ng(SIgN, Singapore)

Laurent Rénia(SIgN, Singapore)

Oliver Bannard(University of Oxford, UK)

Akihiko Yoshimura(Keio University, Japan)

Shimon Sakaguchi(IFReC, Osaka University, Japan)

80

Title
Endothelial stem cell population in health and diseases
Host micro biome interactions in health and disease
Regulation of intestinal homeostasis
Dissection of allergic reactions in an allergen-saturated environment

Even t s & Ou tr e ach A c t i v i t i e s

The 9th NIF Winter School on Advanced Immunology

Imaging the innate immune system in sterile injury and infections
Macrophage and dendritic cell biology: From development to functions
IL-17 signaling in steatotic hepatocytes and macrophages promotes
alcoholic liver disease-induced hepatocellular carcinoma
Impact of PD-1 deficiency on microbiome and brain
The role of group 2 innate lymphoid cells in idiopathic interstitial
pneumonias
Plasticity within the myeloid cell system
Immune mechanisms of arbovirus pathogenesis: Strategies for
immunotherapies
Rosetting of Plasmodium-infected red blood cells: severity factor or escapemechanisms?
Selection events in germinal centers
Neural damage and repair after ischemic brain injury by innate andadaptive immunity
Treg-Up or Treg-Down to control immune responses

81Speaker

Date
January 24, 2020
Venue	Osaka International Convention Center (Grand Cube Osaka),
Osaka, Japan

Title

Chair: James Badger Wing (IFReC, Osaka University, Japan)
Art Weiss(University of California San Francisco, USA)

Motoko Y. KimuraThis symposium provided an excellent opportunity to share the hottest
academic research achievements in the world with researchers in Japanand to contribute to advancing immunology research at IFReC. Talented
young scientists, who had recently published remarkable research results,
in addition to several well-established senior researchers were invited to thesymposium as speakers. The symposium attracted 187 participants of which
104 were from overseas. The participants of the 9th Winter School on Advanced
Immunology also attended the symposium and joined in the active discussion.
The successful interactions among researchers at the symposium laid thegroundwork for future international collaborations.

New Insights into TCR Ligand Discrimination
CD69 Biology and Pathology(Chiba University, Japan)

Microbial Metabolites Control the Thymic Development ofMucosal Associated Invariant T cells

François Legoux(Institut Curie, France)

Chair: Masahiro Yamamoto (IFReC, Osaka University, Japan)
Thirumala-Devi Kanneganti(St. Jude Children's Research Hospital, USA)

Cevayir Coban(The University of Tokyo / IFReC, Osaka University, Japan)

Olivia Majer(Free University of Berlin, Germany)

Even t s & Ou tr e ach A c t i v i t i e s

Immunology at the Forefront - The 11th International
Symposium of IFReC

Targeting the Inflammasome for the Treatment of Inflammatory
and Infectious Diseases
Host-Plasmodium Interactions and the Sterile Immunity Against
Malaria: Where Are We?
Cellular Mechanisms of Self Versus Non-Self Discrimination by
Nucleic Acid-Sensing TLRs

Chair: Katsumori Segawa (IFReC, Osaka University, Japan)
Marc Beyer(German Center for Neurodegenerative Diseases (DZNE),
Germany)

Justin Taylor(University of Washington, USA)

Chris Schiering(Imperial College London, UK)

Ken Murphy(Washington University in St. Louis, USA)

82

Role of Special-AT-Rich Binding Protein 1 for the Differentiation
and Function of CD4+ T-Cells
B Cells Engineered to Express Pathogen-Specific Antibodies
Protect Against Infection
Aryl Hydrocarbon Receptor in Immunity and Metabolism
High IRF8 Levels Engage AICE-dependent Enhancers for cDC1
Identity

83

Date
Venue

Day2
Speaker

Title
Chair: Katsumori Segawa (IFReC, Osaka University, Japan)

June 27-28, 2019
Taniguchi Memorial Hall, Suita Campus, Osaka University

Shizuo Akira(IFReC, Osaka University, Japan)

Masahiro Yamamoto

The UCL-OU Joint Symposium was organized and held jointly with University
College London (UCL), which is one of the Global Knowledge Partners of Osaka
University. This partnership is expected to promote a collaboration in the fieldof immunology. Such collaboration with UCL will lead to advancing research at
IFReC into acquired immunology. Presentations by 10 UCL researchers and 11
IFReC researchers were made in two days for an audience of 158 participants.
After the symposium, UCL researchers visited IFReC laboratories to discuss
further collaboration. Director Kiyoshi Takeda of IFReC and Dr. Hans Stauss of
UCL agreed that the next joint symposium would be held in London in 2020.
After the joint symposium, Osaka University and UCL concluded an Academic
Exchange Agreement in October 2019.

(IFReC, Osaka University, Japan)

Mahdad Noursadeghi(University College London, UK)

The role of endoribonuclease Regnase-1 in inflammation and immune
response
Cell-intrinsic host defense against intracellular pathogens and its
inflammation
Systems level analysis of immunopathology in tuberculosis
Chair: Eiji Umemoto (IFReC, Osaka University, Japan)

Kiyoshi Takeda(IFReC, Osaka University, Japan)

Lizzy Rosser(University College London, UK)

Mala Maini(University College London, UK)

Regulation of intestinal immune responses by microbiota-derived
metabolites

Even t s & Ou tr e ach A c t i v i t i e s

The 1st UCL-OU Joint Symposium on Immunology

Regulation of arthritic B cell responses by gut microbes and metabolites
Living in the Liver: constraints on adaptive immunity
Chair: James Wing (IFReC, Osaka University, Japan)

Ronjon Chakraverty(University College London, UK)

Day1
Speaker

Title
Chair: Masako Kohyama (IFReC, Osaka University, Japan)

Tomohiro Kurosaki(IFReC, Osaka University, Japan)

Claudia Mauri(University College London, UK)

Hisashi Arase(IFReC, Osaka University, Japan)

Kazuhiro Suzuki(IFReC, Osaka University, Japan)

Function of Tet Proteins in B cell tolerance

Shimon Sakaguchi(IFReC, Osaka University, Japan)

Hans Stauss(University College London, UK)

Autoimmunity in graft-versus-host disease
Conversion of conventional T cells into Treg cells
Genetic engineering of T cell specificity and function

Is rheumatoid arthritis a disease of the gut?
MHC class II-induced misfolded proteins are targets for autoimmune
diseases
GPCR-mediated control of humoral immune responses
Chair: Wataru Ise (IFReC, Osaka University, Japan)

Lucy Walker(University College London, UK)

David Sansom(University College London, UK)

Takashi Nagasawa(IFReC, Osaka University, Japan)

Benedict Seddon(University College London, UK)

Identifying targets of CTLA-4 regulation in vivo
Understanding CTLA-4 interactions with CD80 and CD86
Bone marrow microenvironmental niches for hematopoietic stem cells
and immune cells
Novel functions for inflammatory signalling in T cell development
Chair: Takeshi Inoue (IFReC, Osaka University, Japan)

Masaru Ishii(IFReC, Osaka University, Japan)

Siobhan Burns(University College London, UK)

Shigekazu Nagata(IFReC, Osaka University, Japan)

Sho Yamasaki(IFReC, Osaka University, Japan)
84

Dynamics of immune cell migration and interaction visualized byintravital microscopy
Human Immunity: Lessons from Primary Immunodeficiency
Phosphatidylserine-dependent engulfment of apoptotic cells
Sensing environmental lipids and metabolites via immune receptors
85

IFReC holds seminars throughout the year with speakers from a variety
of disciplines including immunology, imaging, and informatics with the aim ofpromoting collaborative research, as well as to inspire and educate the next
generation of scientists. Since its establishment, IFReC has held more thana hundred seminars, which have served as a forum for effective interaction
between researchers beyond academic disciplines. In FY2019, we held anumber of seminars by guest speakers including world-class scientists such as
Gabriel Núñez and Frederic Geissmann.

86

Date

Talk Title

Speaker

August 19. 2019

Regulation of body cavity
immunity

Yasutaka Okabe(Institute for Frontier Life and Medical Sciences, Kyoto
University, Japan)

September 9, 2019

Pyroptosis: from innate immunity
to cancer

Feng Shao(Deputy Director for Academic Affairs, National Institute of
Biological Sciences, Beijing, China)

November 26, 2019

Host-microbiota interactions inhealth and disease

Gabriel Núñez(Paul de Kruif Endowed Professor, Department of Pathology,
University of Michigan, USA)

November 29, 2019

Peripheral immune control oflatent virus infection

Norifumi Iijima(National Institutes of Biomedical Innovation, Health andNutrition, Japan)

December 16, 2019

The macrophage biology

Frederic Geissmann(William E. Snee Chair of Immunology, Memorial Sloan Kettering
Cancer Center, USA)

January 31, 2020

Introduction for RNA-Seq data
analysis

Hidemasa Bono
(Database Center for Life Science, Japan)

February 6, 2020

Phosphoinositides as key
regulators of membraneorganization

Fubito Nakatsu(Graduate School of Medical and Dental Sciences, Niigata
University, Japan)

Even t s & Ou tr e ach A c t i v i t i e s

IFReC Seminars

87

“IFReC Colloquia” is a series of discussion meetings for IFReC members
held once every other month. At each colloquium, speakers from three 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 exchanges among IFReC members in an informal
setting. These events serve as a platform to promote interdisciplinary researchand deeper understanding of the many research activities conducted in IFReC.
In FY2019, IFReC Colloquia were held in odd-numbered months except March
2020 due to the impact of COVID-19.

Date

Title
Involvement of type I Interferon in the pathogenesis ofconnective tissue diseases.

Hyota Takamatsu(Immunopathology, Kumanogoh Lab)

Tetsuo Hasegawa
41st

May 15,2019

Identification of osteoclast precursor macrophage in arthritis.

Development of pH-activatable fluorescent probes to reveal
dynamics and function of osteoclast proton pumps.
Germinal-center mediated memory B cell development isdriven by cooperation of metabolic fitness and survival.
42nd

43rd

July 24,2019

September 18,
2019

44th

45th

November 20,
2019

January 29,
2020(Immunology and Cell Biology, Masaru
Ishii Lab)

Masafumi Minoshima(Chemical Imaging Techniques, Kikuchi
Lab)

Takeshi Inoue(Lymphocyte Differentiation, Kurosaki
Lab)

Misfolded protein / MHC class II complex in autoimmune
diseases.

Tadahiro Suenaga

Paroxysmal nocturnal hemoglobinuria and Inherited GPI
deficiency caused by the mutations in PIGB gene.

Yoshiko Murakami

B cell-specific MyD88 is involved in the adjuvanticity of a
TLR-independent particulate adjuvant.

Michelle Sue Jann Lee

Negative regulation of T cell activation throughphosphatases.

Takashi Saito

IL-17-induced phosphorylation of Regnase-1 is critical incontrolling target mRNA stability and inflammatory response.

Hiroki Tanaka

Single cell RNA-seq analysis of regulatory T cells for drug
discovery.
B cells in allergic and autoimmune diseases.

(Immunochemistry, Arase Lab)

(Immunoglycobiology, Kinoshita Lab)

(Malaria Immunology, Coban Lab)

(Cell Signaling, Saito Lab)

(Host Defense, Akira Lab)

Naganari Ohkura(Experimental Immunology, Sakaguchi
Lab)

Manabu Fujimoto(Cutaneous Immunology, Fujimoto Lab)TRPM5 negatively modulates innate-like response of LPSstimulated B lymphocytes.

Taiki Sakaguchi

STAT1 displays functionally distinct phosphorylation upon
LPS stimulation.

Hozaifa Saad Hassan Metwally

ILC2 a therapeutic target for type 2 immune diseases.
Initial phospholipid-dependent Irgb6 targeting to Toxoplasma
gondii vacuoles mediates host defense.

88

Speakers

Even t s & Ou tr e ach A c t i v i t i e s

IFReC Colloquia(Mucosal Immunology, Takeda Lab)

(Immune Regulation, Kishimoto Lab)

Kazuyo Moro
(Innate Immune Systems, Moro Lab)

Youngae Lee(Immunoparasitology, Yamamoto Lab)89

“Science Café on the Edge,” organized by IFReC in arelaxing café-like atmosphere, provides the general publicwith an opportunity to participate in informal talks with IFReC
researchers about science, from basic knowledge to advanced
research achievements. We hope to promote communications
between scientists and the general public as well as promote agreater understanding of science technology through the directconversations with IFReC researchers. In FY2019, IFReC held theevent four times.

VisitsSenior high school at Komaba, University of Tsukuba “Research on Kansai Area”
Date	22 May, 2019
Visited Lab
Oncogene Research, IFReC (Prof. Masato OKADA)
Stem Cell Biology and Developmental Immunology, IFReC (Prof. Takashi NAGASAWA)

Even t s & Ou tr e ach A c t i v i t i e s

Science Café on the Edge

Forefront of “Car-T cell therapy,” a novel type of cancer immunotherapy
Guest speaker	Dr. Naoki HOSEN (Departmentof Cancer Stem Cell Biology,
Graduate School of Medicine,
Osaka University)
Date	2 May, 2019
Venue	1st floor, Techno-Alliance Building, Suita
Campus, Osaka University

Nara SSH (Super Science High School) science tour
1 August, 2019
Date
LecturerDr. Masako KOHYAMA (Immunochemistry, IFReC)
Laboratory training Host Defense, Experimental Immunology, Live Immuno-Imaging Facility

The exquisite harmony within you – cross-talk between intestinal bacteria and immune cells
Guest speaker	Dr. Eiji UMEMOTO (Immune
Regulation, Graduate Schoolof Medicine, Osaka University)
Date	16 October, 2019
Venue	Ar t Area B1 (Naniwabashi station,
Keihan Line)

School visit
Date	18 November, 2019
Visited school Hiroshima Municipal Motomachi Senior High School
Lecturer
Prof. Kiyoshi TAKEDA (Mucosal Immunology, IFReC)

Resistant bacteria spreading around the world
Guest speaker	Dr. Yukihiro AKEDA (Section of Bacterial Drug Resistance Research, Research Collaboration Centeron Emerging and Re-emerging Infections, Osaka University)
6 November, 2019
Date
Venue	Art Area B1 (Naniwabashi station, Keihan Line)

What is nuclear medicine? – from image diagnosis to cancer therapy –Guest speaker	Dr. Tadashi WATABE (Department of Nuclear Medicine and Tracer Kinetics, Graduate School of
Medicine, Osaka University)
Date	8 December, 2019
Venue	A lecture room, 1st floor, Lecture Building, School of Medicine, Suita Campus, Osaka University
90

Winter School 2019 for high school faculties
Date	26 December, 2019
Lecturer
Prof. Shizuo AKIRA (Host Defense, IFReC)
Prof. Hisashi ARASE (Immunochemistry, IFReC)
Prof. Sho YAMASAKI (Molecular Immunology, IFReC)91

● Knowledge Capital CHO-Gakko (Super-School) SeriesThis is a program organized by Knowledge Capital under the theme of “Beyond schools and even beyond oneself” forlearning about inspiration in research and its process and also for thinking and talking together with participants. It allows
persons without expert knowledge to attend and provides an opportunity to connect the general public with researchers
belonging to universities, companies and research institutions. In FY2019, a total of seven Knowledge Capital Super-School
events were held and jointly organized by the Public Relations Office (Graduate School of Medicine at Osaka University) andthe Institute for Integrated Cell-Material Sciences (iCeMS at Kyoto University).

「Osaka University × Knowledge Capital」Cutting edge medical research at Osaka University

School 1: Early detection of pancreatic cancer
Guest speaker	Dr. Masamitsu KONNO (Frontier Science for Cancer and Chemotherapy, Graduate School of
Medicine, Osaka University)
11 April, 2019
Date
Venue CAFE Lab., 1st floor, North building, Grand Front Osaka

School 2: Manufacturing antibodies
Guest speaker	Dr. Takeshi INOUE (Lymphocyte Differentiation, IFReC, Osaka university)
Date
15 May, 2019
Venue CAFE Lab., 1st floor, North building, Grand Front Osaka「WPI × Knowledge Capital」KANSAI WPI series

School 1: T he progression of an illness caused by functional and structuralabnormality of capillaries
Guest speaker	Dr. Nobuyuki TAKAKURA (Signal Transduction, IFReC, Osaka university)
Date
25 January, 2020
Venue CAFE Lab., 1st floor, North building, Grand Front Osaka

School 2: Crazing: printing without using ink!

Even t s & Ou tr e ach A c t i v i t i e sCollaboration with KNOWLEDGE CAPITAL

Guest speaker	Dr. Masateru ITO (Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University)
Date
1 February, 2020
Venue CAFE Lab., 1st floor, North building, Grand Front Osaka

School 3: Revealing immune and inflammatory dynamics with the latest intravital
imaging technology
Guest speaker	Dr. Masaru ISHII (Immunology and Cell Biology, IFReC, Osaka university)
Date
8 February, 2020
Venue CAFE Lab., 1st floor, North Building, Grand Front Osaka● Workshop Fes.
As a third type of venue intended for education in addition to schools and homes, Knowledge Capital regularly organizes
workshops in collaboration with universities and private companies.
We hope to offer a foundation upon which children can develop their creativity based on their own experiences gained
through their participation in workshops of various themes.
In FY2019, IFReC participated in Workshop Fes. twice, once in summer and once in autumn.

IFReC Immunology Lab – Learning through games about the mechanism protecting our bodies

School 3: Sports medicine for the Tokyo 2020 Olympic and Paralympic Games
Guest speaker	Dr. Ken NAKATA (Medicine for Sports and Performing Arts, Graduate School of Medicine, Osaka
University)
19 June, 2019
Date
Venue CAFE Lab., 1st floor, North building, Grand Front Osaka

Date
Venue
Date
Venue

3-4 August, 2019
CAFE Lab., 1st floor, North building, Grand Front Osaka
23-24 November, 2019
Event space, 4th floor, North Building, Grand Front Osaka

School 4: Toxoplasmosis and immunity
Guest speaker	Dr. Miwa SASAI (Immunoparasitology, IFReC, Osaka university)
Date
17 July, 2019
Venue CAFE Lab., 1st floor, North building, Grand Front Osaka

92

93

Date
November 11, 2019
Venue	LaLaport EXPOCITY

Osaka University held Osaka University Co-Creation Day@EXPOCITY on Saturday, 30 November, 2019 to expand anddeepen interactions among private companies, local governments, and the local community.
About 5,000 visitors such as families with kids, married couples, and groups attended and the event ended successfully.
Under the theme of “IFReC Immunology Lab,” visitors to IFReC’s booth participated in a fill-in-the-blank quiz regarding
cancer therapy and diagnostic methods shown on thedisplayed posters and a game using models of blood
cells, and viewed slides of normal and colorectal
cancer tissues under a microscope.

“Kagaku Zanmai” in Aichi
Date
Venue
Host

December 27, 2019NINS Okazaki Conference Center, Aichi
Okazaki High-school, Aichi prefecture

Super Science High School Student Fair
The Super Science High School (SSH) Student Fair 2019 washeld on August 7-8 at Kobe International Exhibition Hall. IFReC
together with other WPI institutes operated collaborative booths at
the event. This event is an important opportunity for presenting WPI
information to SSH students from around the country.

Even t s & Ou tr e ach A c t i v i t i e s

Osaka University Co-Creation Day

WPI Science Symposium
The 8th WPI Science Symposium was held at the Yasuda
Auditorium on the Hongo Campus of The University of Tokyo on
January 12, 2020. The theme was “The Power of Mathematics:
Bridging Worlds Together.” IFReC and other WPI institutes used
this event to publicize their research achievements at each booth.

“Kagaku Zanmai (addiction to science)” is an advanced initiative called “Aichi model”. In this program, the high schools in
Aichi prefecture that are actively engaged in science education, collaborate with universities and research institutes to conduct
research and presentation. At the annual meeting of Kagaku Zanmai 2019, WPI institutes organized the presentation booth fordemonstrating the top level researches in Japan.

94

95

AAAS 2020 Annual Meeting

The Nature journal (© 2020 Springer Nature) has been hosting

Osaka University including IFReC participated in the annualinternational career fairs to connect talented researchers to job
opportunities and provide them with career information andadvice. IFReC had a booth exhibition at the Nature Careers Livein London (October 3) and in New York (October 19-20). At bothbooths, IFReC introduced their research activities and recruited
outstanding young researchers for the Advanced Postdoc Programwith the aim of advancing research and improving the level ofresearch in Japan by promoting the global circulation of talented
researchers.

meeting of the American Association for the Advancement of
Science (AAAS 2020) on February 13-16, 2020 in Seattle, USA.
The AAAS is the largest international non-profit organization in
the world for the advancement of science. The annual meetingof the AAAS is a widely recognized scientific event, with various
networking opportunities for researchers, journalists, government
officials, and citizens. The main theme for AAAS 2020 was“ENVISIONING TOMORROW’S EARTH.” In his plenary address, Bill
Gates (Bill & Melinda Gates Foundation/Microsoft founder) talked
about the obligations of the wealthy, including himself, to the global
environment.
At AAAS 2020, the staff from Osaka University including IFReC
organized an exhibition booth introducing the university’s research
projects, research environments, recruitment information (e.g.,advanced postdoc at IFReC), etc. IFReC also exhibited a booth at“Family Science Days.” The event featured a number of hands-on
activities and demonstrations for families and kids. IFReC’s booth

Even t s & Ou tr e ach A c t i v i t i e s

Nature Jobs Career Expo in London & NY

focused on observing cancer tissues and classifying immune cells,
and was enthusiastically received by visitors all day.

96

97

Japanese Language Class
Japanese language classes are held for overseas
researchers / students to alleviate any stress andinconvenience in their research and in their daily lives
that may be caused by the language barrier.
IFReC has continued holding the classes since2012, and to date, the cumulative total is over 300
participants.
We offer two lecture-style classes, one is for anelementary to a pre-intermediate level, and anotheris for an intermediate to an advanced level. Class
members learn verb and adjective conjugations,basic Japanese grammar, vocabulary of each level,
Kanji, etc., through provided class materials andgroup work.

Voice from IFReC Japanese class participant
Thanks to IFReC for challenging me to learn the Japanese
language. The class taught me so much about Japanese culture
as well as language and helped me to grow a lot. Teachers teachus from the heart, and staffs are very kind and willing to help us
adjust to life in Japan. I was happy with the class and had very
nice experiences. Thanks again.
- Youngae, who had participatedin the class for a decade.

98

Information

Shimon SAKAGUCHIKen J. ISHII▪ The Order of Cultural Merit of Japan▪ The Paul Elrich and Ludwig Darmstaedter Prize, Germany▪ The German Immunoogy Prize▪ Honorary Doctorate from University of Birmingham▪ JSI Award

In for m a t ion

Major Awards“Mechanism of vaccine adjuvant and its clinical application”

Each prize was given for his outstanding achievements on the discovery of theRegulatory T cell and elucidating its roles in autoimmune diseases, allergies, andcancers.

Tadamitsu KISHIMOTO

Takeshi INOUE▪ The 2019 Keio Medical Science Prize▪ JSI Young Investigator Award

The reason for selection and the major achievement is “IL-6: from molecule tomedicine”.

“Elucidation of molecules that control B cell differentiation and activation”(Photo: Keio University)

Shigekazu NAGATA

Katsumori SEGAWA▪ The 2019 ECDO Honorary Award▪ Osaka University Prize

The research on “Phosphatidylserine-dependent efferocytosis and entosis” ishighly appreciated.

100

Kiyoshi TAKEDA

Kazutaka KATOH & Daron M. STANDLEY▪ The Mochida Memorial Academic Awardth▪ The 8 in “The Most Cited Articles of the Heisei Period” ranking in all the research fields“Discovering the mechanism of maintenance of intestinal homeostasis”“MAFFT multiple sequence alignment software version 7: improvements in performance and usability” Mol.Biol.Evol.(2013).

101

In for m a t ion

Common Facilities (IFReC, RIMD, Animal Resource Center)

Composition & Finance

IFReC and its parent institution, the Research Institute for Microbial Diseases (RIMD) are located on thesame site, constituting a large research complex. The complex contains the Core Instrumentation Facility, theAnimal Resource Center and 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 and skilled
technicians provide in-house services to IFReC and RIMD researchers. The Animal Resource Center consistsof three buildings for specific pathogen-free (SPF) animals and the live immuno-imaging facility. With a largecapacity animal-breeding facility in IFReC, researchers are able to choose animal rooms suitable for theirexperiment purpose.
Using these common facilities, IFReC researchers are able to effectively and smoothly carry out theirexperiments to promote their world-leading research at IFReC.

Composition
Number of IFReC Staff
350

Researchers (JPN)
Researchers (Overseas)

300

Administrative and Support Staff

250
102

117

92

106
90

70

200

89
86

105
52

150
6

4

2

50
1

16
8

93

119

117

128

138

44

47

43

33

137

65

44

102

42

40

24
5

55

42

38

100

3

54

54

48

87

82

2016

2017

129

124

117

2018

2019 Feb. 2020

0
2008

2009

2010

2011

2012

2013

2014

2015

Year
❶ IFReC Research Building❷ Integrated Life Science Building❸ Main Building, Research Institute for Microbial Diseases, RIMD
❹ South Building, Research Institute for Microbial Diseases, RIMD
❺ Cutting-edge Research Building for Infectious Diseases❻ Animal Resource Center for Infectious Diseases

Animal Resource Center for Infectious Diseases⃝ Specific pathogen-free (SPF) animal facility⃝ Sperm/ embryo freezing and preservation⃝ In vitro fertilization and embryo transplantation⃝ Intracytoplasmic sperm injection⃝ Transgenic and knock-out animals⃝ Genome editing in experimental animals

Live immuno-imaging facility⃝ SPF animal experiment facility with 11.7T MRI, in vivo
imager & two-photon microscope.

102

Finance
Sources

From the
University
10.9%

Network Administration Office⃝ Provision and maintenance of network infrastructure:
LAN system and servers (web, mail, mailing lists,
etc.)

Core Instrumentation Facility⃝ Basic and advanced instruments⃝ In-house service⃝ DNA sequencing, cell sorting, electron microscopy,
mass spectrometry and next-generation sequencing
analysis⃝ Radio isotope facility

Expenditure

Main Grant
32.6%

JPN
3,242,579,814

Grant-in-Aid
for Scientific
Research
11.4%

Management
Expenses Grant
19.6%

Donation
11.2%
Contract
Research
14.3%

Individual
research project
29.2%

Personnel
30.0%

JPN
3,242,579,814

Equipment
18.8%

Project Activities
21.5%

Travel0.5%103Unique Support Programs for Young Researchers

Around the world, leading research institutions are in fierce competition to discover excellent young

To strengthen our international research network and our basis for international collaborative research,

researchers. IFReC has, therefore, established the Advanced Postdoc system, which offers outstanding young
researchers opportunities to work with field-leading researchers in IFReC as well as to conduct their own
research under their own merit. Under this system, IFReC has employed seven excellent postdoctoral fellows
in 2018 and 2019. They were assigned to laboratories in IFReC with an internationally competitive salary andresearch funds (three million JPY per year) to conduct original research.

IFReC has established two kinds of financial support programs for researchers. The first one is the “IFReC
Kishimoto Foundation Fellowship,” which has been used to invite international researchers to Osaka. The
second one is the “Program for International Circulation of Young Talented Researchers” for those who wish
to participate in overseas research activities. These programs aim to develop the practical skills and abilitiesof young researchers in international collaborative research and to develop their network with researchers
overseas. Since 2009, over 120 researchers have received the grants.

In for m a t ion

Advanced Postdoc Program

IFReC Advanced Postdoctoral fellows

Grant for Next Generation Principal Investigators
This program aims to foster the next generation of principal investigators at IFReC. In particular, challenging
research that will contribute to the development of immunology or basic research that has the potential to create
a new field of study in immunology is selected. IFReC has selected three PIs for FY2018-2020 and two PIs for
FY2019-2021, and provides continuous research funding for a period of three fiscal years.
The grant is expected to generate excellent research achievements, raise the international recognition of
the next generation of PIs, and contribute to the acquisition of external research funding required to operate thelaboratories.

104

105

Regnase-1-mediated post-transcriptional regulation is essential for hematopoietic
stem and progenitor cell homeostasis.
Hiroyasu Kidoya, Fumitaka Muramatsu, Teppei Shimamura, Weizhen Jia, Takashi Satoh, Yumiko Hayashi, Hisamichi Naito, Yuya
Kunisaki, Fumio Arai, Masahide Seki, Yutaka Suzuki, Tsuyoshi Osawa, Shizuo Akira, and Nobuyuki Takakura.

Akiko Nakai, Jun Fujimoto, Haruhiko Miyata, Ralf Stumm, Masashi Narazaki, Stefan Schulz, Yoshihiro Baba, Atsushi Kumanogoh,
and Kazuhiro Suzuki.

Nature Communications 10: 1072 (2019)

J Exp Med 216: 1630-1647 (2019)

The balance between self-renewal and differentiation of hematopoietic
stem and progenitor cells (HSPCs) maintains hematopoietic homeostasis,
failure of which can lead to hematopoietic disorder. HSPC fate is controlled
by signals from the bone marrow niche resulting in alteration of the stem
cell transcription network. Regnase-1, a member of the CCCH zinc finger
protein family possessing RNAse activity, mediates post-transcriptional
regulatory activity through degradation of target mRNAs. The precise
function of Regnase-1 has been explored in inflammation-related cytokine
expression but its function in hematopoiesis has not been elucidated.
Nobuyuki Takakura (Signal Transduction, IFReC/RIMD) and his researchgroup show that Regnase-1 regulates self-renewal of HSPCs throughmodulating the stability of Gata2 and Tal1 mRNA. In addition, they foundthat dysfunction of Regnase-1 leads to the rapid onset of abnormal
hematopoiesis. Thus, the data reveal that Regnase-1-mediated posttranscriptional regulation is required for HSPC maintenance and suggestthat it represents a leukemia tumor suppressor.

Lymphocyte migration is mediated by G proteincoupled receptors (GPCRs) that respond to chemoattractive
molecules, represented by chemokines. Agonist binding

Phosphorylation-dependent Regnase-1 release from endoplasmic reticulum is
critical in IL-17 response.
Hiroki Tanaka, Yasunobu Arima, Daisuke Kamimura, Yuki Tanaka, Noriyuki Takahashi, Takuya Uehata, Kazuhiko Maeda, Takashi
Satoh, Masaaki Murakami, and Shizuo Akira.

J Exp Med 216: 1431-1449 (2019)Regnase-1 (also known as Zc3h12a or MCPIP-1) isan endoribonuclease involved in mRNA degradation of
inflammation-associated genes. Regnase-1 is inactivatedin response to external stimuli through post-translational
modifications including phosphorylation, yet the preciserole of phosphorylation remains unknown. Hiroki Tanaka,
Shizuo Akira (Host Defense, IFReC) and their researchgroup demonstrated that interleukin (IL)-17 induces
phosphorylation of Regnase-1 in an Act1-TBK1/IKKi–
dependent manner, especially in nonhematopoietic
cells. Phosphorylated Regnase-1 is released from theendoplasmic reticulum (ER) into the cytosol, thereby losing
its mRNA degradation function, which leads to expression of
IL-17 target genes. By using CRISPR/Cas-9 technology, theygenerated Regnase-1 mutant mice, in which IL-17–induced
Regnase-1 phosphorylation is completely blocked. Mutant
mice (Regnase-1AA/AA and Regnase-1ΔCTD/ΔCTD) were resistantto the IL-17–mediated inflammation caused by T helper 17(Th17) cells in vivo. Thus, Regnase-1 plays a critical role in
106

The COMMD3/8 complex determines GRK6 specificity for chemoattractant
receptors.

the development of IL-17–mediated inflammatory diseases
via the Act1-TBK1-IKKi axis, and blockade of Regnase-1
phosphorylation sites may be promising for treatment of
Th17-associated diseases.

to GPCRs activates heteromeric G proteins to regulate
the generation of second messengers that modulate
downstream signaling. Agonist-occupied GPCRs arephosphorylated by GPCR kinases (GRKs) and subsequently
recruit β-arrestins that serve as scaffolds to activate
signaling molecules, including mitogen-activatedprotein kinases (MAPKs). The GRK family consistsof seven mammalian members, among which GRK2,
GRK3, GRK5, and GRK6 are expressed ubiquitously.
Different GRKs phosphorylate distinct sites on
the C-terminal tail of the receptor, establishing abarcode that dictates the functional consequencesof β-arrestin engagement. Thus, specific targetingof GRKs to activated GPCRs is crucial for signal

In for m a t ion

Selected Articles

transduction. However, the molecular mechanism that
determines the specificity of GRK targeting is poorly
understood. Kazuhiro Suzuki (Immune Response Dynamics,
IFReC) and his group identified a protein complex consistingof copper metabolism MURR1 domain-containing (COMMD)
3 and COMMD8 (COMMD3/8 complex) as an adaptor
that selectively recruits a specific GRK to chemoattractant
receptors and promotes lymphocyte migration.

CXCR4 regulates Plasmodium development in mouse and human hepatocytes.
Hironori Bando, Ariel Pradipta, Shiroh Iwanaga, Toru Okamoto, Daisuke Okuzaki, Shun Tanaka, Joel Vega-Rodríguez, Youngae
Lee, Ji Su Ma, Naoya Sakaguchi, Akira Soga, Shinya Fukumoto, Miwa Sasai, Yoshiharu Matsuura, Masao Yuda, Marcelo JacobsLorena, and Masahiro Yamamoto.

J Exp Med 216: 1733-1748 (2019)
The liver stage of the etiological agent of malaria,
Plasmodium, is obligatory for successful infection of its
various mammalian hosts. Differentiation of the rod-shaped
sporozoites of Plasmodium into spherical exoerythrocytic
forms (EEFs) via bulbous expansion is essential for parasite
development in the liver. However, little is known about thehost factors regulating the morphological transformation of
Plasmodium sporozoites in this organ. The research groupof Masahiro Yamamoto (Immunoparasitology, IFReC/RIMD)
showed that sporozoite differentiation into EEFs in the liver
involves protein kinase C ζ-mediated NF-κB activation,
which robustly induces the expression of C-X-C chemokine
receptor type 4 (CXCR4) in hepatocytes and subsequently
elevates intracellular Ca 2+ levels, thereby triggeringparasite also. Collectively, the experiments show that CXCR4
is a key host factor for Plasmodium development in theliver, and CXCR4 warrants further investigation for malaria
prophylaxis.

sporozoite transformation into EEFs. Blocking CXCR4
expression by genetic or pharmacological intervention
profoundly inhibited the liver stage development of the P.
berghei rodent malaria parasite and the human P. falciparum107Tetsuo Hasegawa, Junichi Kikuta, Takao Sudo, Yoshinobu Matsuura, Takahiro Matsui, Szandor Simmons, Kosuke Ebina, Makoto
Hirao, Daisuke Okuzaki, Yuichi Yoshida, Atsushi Hirao, Vladimir V. Kalinichenko, Kunihiro Yamaoka, Tsutomu Takeuchi, andMasaru Ishii.

Kiyoharu Fukushima, Takashi Satoh, Fuminori Sugihara, Yuki Sato, Toru Okamoto, Yuichi Mitsui, Sachiyo Yoshio, Songling Li,
Satoshi Nojima, Daisuke Motooka, Shota Nakamura, Hiroshi Kida, Daron M. Standley, Eiichi Morii, Tatsuya Kanto, Motoko
Yanagita, Yoshiharu Matsuura, Takashi Nagasawa, Atsushi Kumanogoh, and Shizuo Akira.

Nature Immunology 20: 1631-1643 (2019)

Immunity 52: 542-556 (2020)

Masaru Ishii (Immunology and Cell Biology, IFReC/
Graduate School of Medicine) show osteoclasts in pannus
originate exclusively from circulating bone marrow-derived
cells and not from locally resident macrophages. They
identify murine CX3CR1hiLy6CintF4/80+I-A+/I-E+ macrophages(termed here “arthritis-associated osteoclastogenic
macrophages [AtoMs]”) as the osteoclast precursor (OP)containing population in the inflamed synovium, comprisinga subset distinct from conventional OPs in homeostatic bone
remodeling. Tamoxifen-inducible Foxm1 deletion suppressed
the capacity of AtoMs to differentiate into osteoclasts in vitro
and in vivo. Furthermore, synovial samples from humanrheumatoid arthritis (RA) patients contained CX3CR1+HLA-

FoxM1 inhibitor, thiostrepton, constituting a potential target
for RA treatment.

AtoMs, and human osteoclastogenesis was inhibited by the

Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid
biosynthesis and ER-associated degradation.
Yicheng Wang, Yusuke Maeda, Yi-Shi Liu, Yoko Takada, Akinori Ninomiya, Tetsuya Hirata, Morihisa Fujita, Yoshiko Murakami, andTaroh Kinoshita.

Nature Communications 11: 860 (2020)
Glycosylphosphatidylinositol (GPI) is a glycolipid
for post-translational modification of many cell-surface
proteins in eukaryotic cells. The structure of the coreb a c k b o n e o f G P I i s c o n s e r v e d w h e re a s t h e
structural variation of GPI anchors is introducedby side-chain modifications. In some mammalian
GPI-APs, the N-acetylgalactosamine side-chain
linked to the first mannose is further modified withgalactose by an unknown galactosyltransferase(GPI-Gal-T). The research group of Taroh Kinoshita(Immunoglycobiology, IFReC/RIMD) performed
genome-scale CRISPR-Cas9 knockout screening
for GPI-Gal-T and found that B3GALT4, known as
GM1 synthase, is GPI-Gal-T. They also demonstrated
the requirement of lactosylceramide for efficientgalactosylation of GPI side chain. In addition, we showthat GPI biosynthesis in the endoplasmic reticulum(ER) is regulated by ER-associated degradation
system to prevent GPI accumulation. Thus, ourFibrosis is an incurable disorder of unknown etiology.
Segregated-nucleus-containing atypical monocytes (SatMs)
discovered by the authors are critical for the development offibrosis. The research group of Satoh and Shizuo Akira (HostDefense, IFReC) examined the mechanisms that recruit
SatMs to pre-fibrotic areas. A screen based on cytokine
expression in the fibrotic lung revealed that the chemokine
Cxcl12, which is produced by apoptotic nonhematopoietic

triggering of apoptosis. Thus, Rbm7 in epithelial cells playsa critical role in the development of fibrosis by regulating
ncRNA decay and thereby the production of chemokinesthat recruit SatMs.

cells, was essential for SatM recruitment. Analyses of lungtissues at fibrosis onset showed increased expression of
Rbm7, a component of the nuclear exosome targetingcomplex. Rbm7 deletion suppressed bleomycin-induced
fibrosis and at a cellular level, suppressed apoptosis of
nonhematopoietic cells. Mechanistically, Rbm7 boundto noncoding (nc) RNAs that form subnuclear bodies,including Neat1 speckles. Dysregulated expression of Rbm7
resulted in the nuclear degradation of Neat1 speckles,
the dispersion of the DNA repair protein BRCA1, and the

DRhiCD11c+CD80−CD86+ cells that corresponded to mouse108

Dysregulated expression of the nuclear exosome targeting complex component
RBM7 in non-hematopoietic cells licenses the development of fibrosis.

In for m a t ion

Identification of a novel arthritis-associated osteoclast precursor macrophage
regulated by FoxM1.

work demonstrates cross-talks of GPI biosynthesis withglycosphingolipid biosynthesis and the ER quality control
system.

Noncanonical STAT1 phosphorylation expands its transcriptional activity into
promoting LPS-induced IL-6 and IL-12p40 production.
Hozaifa Metwally, Toshio Tanaka, Songling Li, Gyanu Parajuli, Sujin Kang, Hamza Hanieh, Shigeru Hashimoto, Jaya P. Chalise,
Yohannes Gemechu, Daron M. Standley, and Tadamitsu Kishimoto.

Science Signaling 13: eaay0574 (2020)
The lipopolysaccharide (LPS)–induced endocytosisof Toll-like receptor 4 (TLR4) is an essential step inthe production of interferon-β (IFN-β), which activates
the transcription of antiviral response genes by STAT1
phosphorylated at Tyr701. Hozaifa Saad Hassan Metwally,
Tadamitsu Kishimoto (Immune Regulation, IFReC) and theresearch group showed that noncanonical phosphorylationin response to LPS confers STAT1 with distinct DNA binding
and gene-regulatory properties that promote both IL12B
expression and IL6 mRNA stabilization. Their study provides
a potential mechanism for how TLR4 endocytosis regulate
proinflammatory cytokine production.

109

1

2

3

4

5

6

7

8

9

10

110

Adachi, Y; Tonouchi, K; Nithichanon, A; Kuraoka, M; Watanabe,
A; Shinnakasu, R; Asanuma, H; Ainai, A; Ohmi, Y; Yamamoto,
T; Ishii, KJ; Hasegawa, H; Takeyama, H; Lertmemongkolchai,G; Kurosaki, T; Ato, M; Kelsoe, G; Takahashi, Y. Exposure of an
occluded hemagglutinin epitope drives selection of a class ofcross-protective influenza antibodies. Nature Communications
10, 3883 (2019).
Akamatsu, M; Mikami, N; Ohkura, N; Kawakami, R; Kitagawa,
Y; Sugimoto, A; Hirota, K; Nakamura, N; Ujihara, S; Kurosaki,
T; Hamaguchi, H; Harada, H; Xia, GL; Morita, Y; Aramori, I;
Narumiya, S; Sakaguchi, S. Conversion of antigen-specificeffector/memory T cells into Foxp3-expressing T-reg cells
by inhibition of CDK8/19. Science Immunology 4, eaaw2707(2019).
Akiyama, M; Ishigaki, K; Sakaue, S; Momozawa, Y; Horikoshi,
M; Hirata, M; Matsuda, K; Ikegawa, S; Takahashi, A; Kanai, M;
Suzuki, S; Matsui, D; Naito, M; Yamaji, T; Iwasaki, M; Sawada,
N; Tanno, K; Sasaki, M; Hozawa, A; Minegishi, N; Wakai, K;
Tsugane, S; Shimizu, A; Yamamoto, M; Okada, Y; Murakami, Y;
Kubo, M; Kamatani, Y. Characterizing rare and low-frequency
height-associated variants in the Japanese population. Nature
Communications 10, 4393 (2019).
Akizuki, S; Ishigaki, K; Kochi, Y; Law, SM; Matsuo, K; Ohmura, K;
Suzuki, A; Nakayama, M; Iizuka, Y; Koseki, H; Ohara, O; Hirata,
J; Kamatani, Y; Matsuda, F; Sumida, T; Yamamoto, K; Okada, Y;
Mimori, T; Terao, C. PLD4 is a genetic determinant to systemic
lupus erythematosus and involved in murine autoimmune
phenotypes. Annals of the Rheumatic Diseases 78, 509-518(2019).
Amariuta, T; Luo, Y; Gazal, S; Davenport, EE; van de Geijn, B;
Ishigaki, K; Westra, HJ; Teslovich, N; Okada, Y; Yamamoto,
K; Price, AL; Raychaudhuri, S. IMPACT: Genomic annotationof cell-state-specific regulatory elements inferred from theepigenome of bound transcription factors. American Journal of
Human Genetics 104, 879-895 (2019).
Aoki, M; Watabe, T; Nagamori, S; Naka, S; Ikeda, H;
Kongpracha, P; Horitsugi, G; Kanai, Y; Shimosegawa, E; Kanai,
Y; Hatazawa, J. Distribution of LAT1-targeting PET tracer was
independent of the tumor blood flow in rat xenograft models of
C6 glioma and MIA PaCa-2. Annals of Nuclear Medicine 33,
394-403 (2019).
Arase, N; Tanemura, A; Jin, H; Nishioka, M; Aoyama, Y; Oiso, N;
Matsunaga, K; Suzuki, T; Nishigori, C; Kawamura, T; Shimizu,
T; Ito, A; Fukai, K; Abe, Y; Yang, LL; Tsuruta, D; Takeoka, K;
Iwatani, Y; Hidaka, Y; Nishida, M; Yamauchi-Takihara, K; Arase,
H; Fujimoto, M; Katayama, I. Autoantibodies detected inpatients with vitiligo vulgaris but not in those with rhododendrolinduced leukoderma. Journal of Dermatological Science 95,
80-83 (2019).
Argilaguet, J; Pedragosa, M; Esteve-Codina, A; Riera, G; Vidal,
E; Peligero-Cruz, C; Casella, V; Andreu, D; Kaisho, T; Bocharov,
G; Ludewig, B; Heath, S; Meyerhans, A. Systems analysis
reveals complex biological processes during virus infection fatedecisions. Genome Research 29, 907-919 (2019).
Bando, H; Lee, Y; Sakaguchi, N; Pradipta, A; Sakamoto, R;
Tanaka, S; Ma, JS; Sasai, M; Yamamoto, M. Toxoplasma effector
GRA15-dependent suppression of IFN-gamma-induced
antiparasitic response in human neurons. Frontiers In Cellularand Infection Microbiology 9, 140 (2019).
Bando, H; Pradipta, A; Iwanaga, S; Okamoto, T; Okuzaki, D;
Tanaka, S; Vega-Rodriguez, J; Lee, Y; Ma, JS; Sakaguchi, N;
Soga, A; Fukumoto, S; Sasai, M; Matsuura, Y; Yuda, M; JacobsLorena, M; Yamamoto, M. CXCR4 regulates Plasmodium
development in mouse and human hepatocytes. Journal of
Experimental Medicine 216, 1733-1748 (2019).

11

12

Boocock, J; Leask, M; Okada, Y; Matsuo, H; Kawamura, Y;
Shi, YY; Li, CG; Mount, DB; Mandal, AK; Wang, WQ; Cadzow,
M; Gosling, AL; Major, TJ; Horsfield, JA; Choi, HK; Fadason,
T; O’Sullivan, J; Stahl, EA; Merriman, TR. Genomic dissectionof 43 serum urate-associated loci provides multiple insights
into molecular mechanisms of urate control. Human Molecular
Genetics 29, 923-943 (2020).
Braganza, CD; Shibata, K; Fujiwara, A; Motozono, C; Sonoda,
KH; Yamasaki, S; Stocker, BL; Timmer, MSM. The effect of MR1
ligand glyco-analogues on mucosal-associated invariant T(MAIT) cell activation. Organic & Biomolecular Chemistry 17,8992-9000 (2019).

13

Canete, PF; Sweet, RA; Gonzalez-Figueroa, P; Papa, I; Ohkura,
N; Bolton, H; Roco, JA; Cuenca, M; Bassett, KJ; Sayin, I; Barry,
E; Lopez, A; Canaday, DH; Meyer-Hermann, M; Doglioni, C; deSt Groth, BF; Sakaguchi, S; Cook, MC; Vinuesa, CG. Regulatory
roles of IL-10-producing human follicular T cells. Journal of
Experimental Medicine 216, 1843-1856 (2019).

14

Chalise, JP; Hashimoto, S; Parajuli, G; Kang, SJ; Singh, SK;
Gemechu, Y; Metwally, H; Nyati, KK; Dubey, PK; Zaman, MMU;
Nagahama, Y; Hamza, H; Masuda, K; Kishimoto, T. Feedback
regulation of Arid5a and Ppar-gamma 2 maintains adipose
tissue homeostasis. Proceedings of the National Academy of
Sciences USA 116, 15128-15133 (2019).

15

Clark, DW; Okada, Y; Moore, KHS; Mason, D et al. Associationsof autozygosity with a broad range of human phenotypes.
Nature Communications 10, 4957 (2019).

16

Cossarizza, A; Chang, HD; Radbruch, A; Acs, A; Adam, D etal. Guidelines for the use of flow cytometry and cell sorting in
immunological studies (second edition). European Journal of
Immunology 49, 1457-1973 (2019).

17

DeVorkin, L; Pavey, N; Carleton, G; Comber, A; Ho, C; Lim, J;
McNamara, E; Huang, HC; Kim, P; Zacharias, LG; Mizushima, N;
Saitoh, T; Akira, S; Beckham, W; Lorzadeh, A; Moksa, M; Cao,
Q; Murthy, A; Hirst, M; DeBerardinis, RJ; Lum, JJ. Autophagy
regulation of metabolism is required for CD8+ T cell anti-tumor
immunity. Cell Reports 27, 502 (2019).

18

19

20

21

Endo, T; Mikedis, MM; Nicholls, PK; Page, DC; de Rooij, DG.
Retinoic acid and germ cell development in the ovary andtestis. Biomolecules 9, 775 (2019).
Fatoba, O; Ohtake, Y; Itokazu, T; Yamashita, T. Immunotherapies
in Huntington’s disease and alpha-Synucleinopathies. Frontiersin Immunology 11, 337 (2020).

24

Gao, J; Hori, Y; Nishiura, M; Bordy, M; Hasserodt, J; Kikuchi,
K. Engineered protein-tag for rapid live-cell fluorogenic
visualization of proteins by anionic probes. Chemistry Letters49, 232-235 (2020).

25

Gao, JC; Hori, Y; Shimomura, T; Bordy, M; Hasserodt, J;
Kikuchi, K. Development of fluorogenic probes for rapid highcontrast imaging of transient nuclear localization of sirtuin 3.
Chembiochem 21, 656-662 (2020).

26

Hamaguchi, M; Muramatsu, R; Fujimura, H; Mochizuki, H;
Kataoka, H; Yamashita, T. Circulating transforming grow factorbeta 1 facilitates remyelination in the adult central nervous
system. Elife 8, e41869 (2019).

27

Hasegawa, T; Ishii, M. Visualizing bone tissue in homeostaticand pathological conditions. Proceedings of the Japan
Academy Series B-Physical and Biological Sciences 96, 43-49(2020).

28

Hasegawa, T; Kikuta, J; Sudo, T; Matsuura, Y; Matsui, T;
Simmons, S; Ebina, K; Hirao, M; Okuzaki, D; Yoshida, Y;
Hirao, A; Kalinichenko, VV; Yamaoka, K; Takeuchi, T; Ishii, M.
Identification of a novel arthritis-associated osteoclast precursor
macrophage regulated by FoxM1. Nature Immunology 20, 1631
(2019).

29

Hashimoto, S; Furukawa, S; Hashimoto, A; Tsutaho, A; Fukao,
A; Sakamura, Y; Parajuli, G; Onodera, Y; Otsuka, Y; Handa,
H; Oikawa, T; Hata, S; Nishikawa, Y; Mizukami, Y; Kodama, Y;
Murakami, M; Fujiwara, T; Hirano, S; Sabe, H. ARF6 and AMAP1
are major targets of KRAS and TP53 mutations to promoteinvasion, PD-L1 dynamics, and immune evasion of pancreaticcancer. Proceedings of the National Academy of Sciences USA
116, 17450-17459 (2019).

30

Hayashi, Y; Jia, WZ; Kidoya, H; Muramatsu, F; Tsukada, Y;
Takakura, N. Galectin-3 inhibits cancer metastasis by negatively
regulating integrin beta 3 expression. American Journal of
Pathology 189, 900-910 (2019).

31

Herppich, S; Toker, A; Pietzsch, B; Kitagawa, Y; Ohkura, N;
Miyao, T; Floess, S; Hori, S; Sakaguchi, S; Huehn, J. Dynamic
imprinting of the Treg cell-specific epigenetic signature indeveloping thymic regulatory T cells. Frontiers in Immunology
10, 2382 (2019).

32

Hochsmann, B; Murakami, Y; Osato, M; Knaus, A; Kawamoto,
M; Inoue, N; Hirata, T; Murata, S; Anliker, M; Eggermann,
T; Jager, M; Floettmann, R; Hollein, A; Murase, S; Ueda, Y;
Nishimura, JI; Kanakura, Y; Kohara, N; Schrezenmeier, H;
Krawitz, PM; Kinoshita, T. Complement and inflammasome
overactivation mediates paroxysmal nocturnal hemoglobinuria
with autoinflammation. Journal of Clinical Investigation 129,5123-5136 (2019).

33

Hori, Y; Kikuchi, K. Chemical tools with fluorescence switches
for verifying epigenetic modifications. Accounts of Chemical
Research 52, 2849-2857 (2019).

34

Hossain, MM; Tsuzuki, T; Sakakibara, K; Imaizumi, F; Ikegaya,
A; Inagaki, M; Takahashi, I; Ito, T; Takamatsu, H; Kumanogoh,
A; Negishi, T; Yukawa, K. PlexinA1 is crucial for the midline
crossing of callosal axons during corpus callosum development
in BALB/cAJ mice. Plos One 14, e0221440 (2019).

Fisch, D; Bando, H; Clough, B; Hornung, V; Yamamoto, M;
Shenoy, AR; Frickel, EM. Human GBP1 is a microbe-specificgatekeeper of macrophage apoptosis and pyroptosis. Embo
Journal 38, e100926 (2019).
Fujimoto, K; Kawaguchi, Y; Shimohigoshi, M; Gotoh, Y; Nakano,
Y; Usui, Y; Hayashi, T; Kimura, Y; Uematsu, M; Yamamoto, T;
Akeda, Y; Rhee, JH; Yuki, Y; Ishii, KJ; Crowe, SE; Ernst, PB;
Kiyono, H; Uematsu, S. Antigen-specific mucosal immunity
regulates development of intestinal bacteria-mediateddiseases. Gastroenterology 157, 1530 (2019).

22

Fujita, Y; Yamashita, T. The effects of leptin on glial cells inneurological diseases. Frontiers in Neuroscience 13, 828(2019).

23

Fukushima, K; Satoh, T; Sugihara, F; Sato, Y; Okamoto, T; Mitsui,
Y; Yoshio, S; Li, SL; Nojima, S; Motooka, D; Nakamura, S; Kida,
H; Standley, DM; Morii, E; Kanto, T; Yanagita, M; Matsuura, Y;
Nagasawa, T; Kumanogoh, A; Akira, S. Dysregulated expressionof the nuclear exosome targeting complex component Rbm7
in nonhematopoietic cells licenses the development of fibrosis.
Immunity 52, 542 (2020).

35

Ikeda-Matsuo, Y; Miyata, H; Mizoguchi, T; Ohama, E; Naito,
Y; Uematsu, S; Akira, S; Sasaki, Y; Tanabe, M. Microsomal
prostaglandin E synthase-1 is a critical factor in dopaminergic
neurodegeneration in Parkinson’s disease. Neurobiology of
Disease 124, 81-92 (2019).

36

Ikumi, K; Odanaka, M; Shime, H; Imai, M; Osaga, S; Taguchi,
O; Nishida, E; Hemmi, H; Kaisho, T; Morita, A; Yamazaki, S.
Hyperglycemia is associated with psoriatic inflammation in bothhumans and mice. Journal of Investigative Dermatology 139,
1329 (2019).

37

Imanishi, T; Saito, T. T cell co-stimulation and functional
modulation by innate signals. Trends in Immunology 41, 200212 (2020).

38

Imoto, T; Kawase, A; Minoshima, M; Yokoyama, T; Bito, H;
Kikuchi, K. Photolytic release of a caged inhibitor of an
endogenous transcription factor enables optochemical controlof CREB-mediated gene expression. Organic Letters 22, 22-25(2020).

39

Imoto, T; Muramatsu, M; Miyasaka, H; Mizukami, S; Kikuchi,
K. Improvement in photostability of fluorescein by lanthanide
Ions based on energy transfer-based triplet state quenching.
Chemistry Letters 48, 1181-1184 (2019).

40

Ishifune, C; Tsukumo, S; Maekawa, Y; Hozumi, K; Chung, DH;
Motozono, C; Yamasaki, S; Nakano, H; Yasutomo, K. Regulationof membrane phospholipid asymmetry by Notch-mediatedflippase expression controls the number of intraepithelialTCRαβ+CD8αα+ T cells. Plos Biology 17, e3000262 (2019).

41

Kamada, T; Togashi, Y; Tay, C; Ha, D; Sasaki, A; Nakamura, Y;
Sato, E; Fukuoka, S; Tada, Y; Tanaka, A; Morikawa, H; Kawazoe,
A; Kinoshita, T; Shitara, K; Sakaguchi, S; Nishikawa, H. PD-1+
regulatory T cells amplified by PD-1 blockade promote
hyperprogression of cancer. Proceedings of the National
Academy of Sciences USA 116, 9999-10008 (2019).

42

Kang, SJ; Tanaka, T; Narazaki, M; Kishimoto, T. Targetinginterleukin-6 signaling in clinic. Immunity 50, 1007-1023 (2019).

43

Kashiyama, N; Miyagawa, S; Fukushima, S; Kawamura, T;
Kawamura, A; Yoshida, S; Eiraku, S; Harada, A; Matsunaga, K;
Watabe, T; Toda, K; Hatazawa, J; Sawa, Y. MHC-mismatchedallotransplantation of induced pluripotent stem cell-derived
cardiomyocyte sheets to improve cardiac function in a primate
ischemic cardiomyopathy model. Transplantation 103, 15821590 (2019).

44

Kawamura, Y; Nakaoka, H; Nakayama, A; Okada, Y; Yamamoto,
K et al. Genome-wide association study revealed novel lociwhich aggravate asymptomatic hyperuricaemia into gout.
Annals of the Rheumatic Diseases 78, 1430-1437 (2019).

45

Kayama, H; Tani, H; Kitada, S; Opasawatchai, A; Okumura, R;
Motooka, D; Nakamura, S; Takeda, K. BATF2 prevents T-cellmediated intestinal inflammation through regulation of the IL-23/
IL-17 pathway. International Immunology 31, 371-383 (2019).

46

Kishikawa, T; Maeda, Y; Nii, T; Motooka, D; Matsumoto, Y;
Matsushita, M; Matsuoka, H; Yoshimura, M; Kawada, S;
Teshigawara, S; Oguro, E; Okita, Y; Kawamoto, K; Higa, S;
Hirano, T; Narazaki, M; Ogata, A; Saeki, Y; Nakamura, S;
Inohara, H; Kumanogoh, A; Takeda, K; Okada, Y. Metagenomewide association study of gut microbiome revealed novel
aetiology of rheumatoid arthritis in the Japanese population.
Annals of the Rheumatic Diseases 79, 103-111 (2020).

47

Kitazume-Taneike, R; Taneike, M; Omiya, S; Misaka, T; Nishida,
K; Yamaguchi, O; Akira, S; Shattock, MJ; Sakata, Y; Otsu, K.
Ablation of Toll-like receptor 9 attenuates myocardial ischemia/
reperfusion injury in mice. Biochemical and BiophysicalResearch Communications 515, 442-447 (2019).

48

Kiyozumi, D; Nakano, I; Sato-Nishiuchi, R; Tanaka, S; Sekiguchi,
K. Laminin is the ECM niche for trophoblast stem cells. Life
Science Alliance 3, e201900515 (2020).

In for m a t ion

Publications

111

50

51

Kubagawa, H; Honjo, K; Ohkura, N; Sakaguchi, S; Radbruch, A;
Melchers, F; Jani, PK. Functional roles of the IgM Fc receptor in
the immune system. Frontiers in Immunology 10, 945 (2019).

52

Kumar, A; Kumar, H. Long noncoding RNA: TRIMming the viralload. Cellular & Molecular Immunology 16, 843-845 (2019).

53

54

55

56

Kumar, N; Hori, Y; Kikuchi, K. Photoactive yellow protein andits chemical probes: an approach to protein labelling in living
cells. Journal of Biochemistry 166, 121-127 (2019).
Leach, S; Shinnakasu, R; Adachi, Y; Momota, M; MakinoOkamura, C; Yamamoto, T; Ishii, KJ; Fukuyama, H; Takahashi,
Y; Kurosaki, T. Requirement for memory B-cell activationin protection from heterologous influenza virus reinfection.
International Immunology 31, 771-779 (2019).
Lee, MSJ; Natsume-Kitatani, Y; Temizoz, B; Fujita, Y; Konishi,
A; Matsuda, K; Igari, Y; Tsukui, T; Kobiyama, K; Kuroda, E;
Onishi, M; Marichal, T; Ise, W; Inoue, T; Kurosaki, T; Mizuguchi,
K; Akira, S; Ishii, KJ; Coban, C. B cell-intrinsic MyD88 signaling
controls IFN-gamma-mediated early IgG2c class switching inmice in response to a particulate adjuvant. European Journal of
Immunology 49, 1433-1440 (2019).
Lee, Y; Yamada, H; Pradipta, A; Ma, JS; Okamoto, M; Nagaoka,
H; Takashima, E; Standley, DM; Sasai, M; Takei, K; Yamamoto, M.
Initial phospholipid-dependent Irgb6 targeting to Toxoplasma
gondii vacuoles mediates host defense. Life Science Alliance 3,
e201900549 (2020).

57

Lelliott, PM; Momota, M; Lee, MSJ; Kuroda, E; Iijima, N; Ishii,
KJ; Coban, C. Rapid quantification of NETs in vitro and in whole
blood samples by imaging flow cytometry. Cytometry Part A
95A, 565-578 (2019).

58

Leu, C; Stevelink, R; Smith, AW; Goleva, SB; Kanai, M;
Ferguson, L; Campbell, C; Kamatani, Y; Okada, Y; Sisodiya,
SM; Cavalleri, GL; Koeleman, BPC; Lerche, H; Jehi, L; Davis,
LK; Najm, IM; Palotie, A; Daly, MJ; Busch, RM; Lal, D. Polygenic
burden in focal and generalized epilepsies. Brain 142, 34733481 (2019).

59

Lim, EL; Okkenhaug, K. Phosphoinositide 3-kinase delta is aregulatory T-cell target in cancer immunotherapy. Immunology
157, 210-218 (2019).

60

Lu, YG; Oura, S; Matsumura, T; Oji, A; Sakurai, N; Fujihara,
Y; Shimada, K; Miyata, H; Tobita, T; Noda, T; Castaneda, JM;
Kiyozumi, D; Zhang, Q; Larasati, T; Young, SAM; Kodani, M;
Huddleston, CA; Robertson, MJ; Coarfa, C; Isotani, A; Aitken,
RJ; Okabe, M; Matzuk, MM; Garcia, TX; Ikawa, M. CRISPR/
Cas9-mediated genome editing reveals 30 testisenrichedgenes dispensable for male fertility in mice. Biology of
Reproduction 101, 501-511 (2019).

61

62

112

Kobari, S; Kusakabe, T; Momota, M; Shibahara, T; Hayashi, T;
Ozasa, K; Morita, H; Matsumoto, K; Saito, H; Ito, S; Kuroda, E;
Ishii, KJ. IL-33 is essential for adjuvant effect of hydroxypropylbeta-cyclodexrin on the protective intranasal influenza
vaccination. Frontiers in Immunology 11, 360 (2020).

63

Maekawa, A; Arase, N; Tamai, K; Nomura, T; Kiyohara, E;
Wataya-Kaneda, M; Arase, H; Katayama, I; Fujimoto, M. Caseof epidermolytic ichthyosis with impairment of pulmonary
function and exacerbated skin manifestations in a late middleaged adult. Journal of Dermatology 46, e480e482 (2019).

64

Martin, AR; Kanai, M; Kamatani, Y; Okada, Y; Neale, BM;
Daly, MJ. Clinical use of current polygenic risk scores may
exacerbate health disparities. Nature Genetics 51, 584-591(2019).

65

Mastellos, DC; Blom, AM; Connolly, ES; Daha, MR; Geisbrecht,
BV; Ghebrehiwet, B; Gros, P; Hajishengallis, G; Holers, VM;
Huber-Lang, M; Kinoshita, T; Mollnes, TE; Montgomery, RA;
Morgan, BP; Nilsson, B; Pio, R; Ricklin, D; Risitano, AM;
Taylor, RP; Mantovani, A; Ioannidis, JPA; Lambris, JD. ‘Stealth’
corporate innovation: an emerging threat for therapeutic drug
development. Nature Immunology 20, 1409-1413 (2019).

78

Minoshima, M; Kikuta, J; Omori, Y; Seno, S; Suehara, R; Maeda,
H; Matsuda, H; Ishii, M; Kikuchi, K. In vivo multicolor imaging
with fluorescent probes revealed the dynamics and function of
osteoclast proton pumps. Acs Central Science 5, 1059-1066(2019).

79

Mizuno, A; Okada, Y. Biological characterization of expression
quantitative trait loci (eQTLs) showing tissue-specific opposite
directional effects. European Journal of Human Genetics 27,
1745-1756 (2019).

80

Molineros, JE; Looger, LL; Kim, K; Okada, Y; Terao, C et al.
Amino acid signatures of HLA Class-I and II molecules arestrongly associated with SLE susceptibility and autoantibody
production in Eastern Asians. Plos Genetics 15, e1008092(2019).

81

Morimoto, A; Kikuta, J; Ishii, M. Intravital multiphoton
microscopy as a novel tool in the field of immunopharmacology.
Pharmacology & Therapeutics 206, UNSP 107429 (2020).

82

Motomura, Y; Kobayashi, T; Moro, K. The Neuropeptide CGRP
induces bipolar syndrome in group 2 innate lymphoid cells.
Immunity 51, 598-600 (2019).

83

Murakami, M; Tognini, P. The Circadian Clock as an Essential
Molecular Link Between Host Physiology and Microorganisms.
Frontiers in Cellular and Infection Microbiology 9, 469 (2020).

84

Nagata, S; Sakuragi, T; Segawa, K. Flippase and scramblase
for phosphatidylserine exposure. Current Opinion in
Immunology 62, 31-38 (2020).

Matsuda, A; Asada, Y; Suita, N; Iwamoto, S; Hirakata, T; Yokoi, N;
Ohkawa, Y; Okada, Y; Yokomizo, T; Ebihara, N. Transcriptome
profiling of refractory atopic keratoconjunctivitis by RNA
sequencing. Journal of Allergy and Clinical Immunology 143,
1610 (2019).

85

Nagatake, T; Hirata, S; Koga, T; Kuroda, E; Kobari, S; Suzuki, H;
Hosomi, K; Matsumoto, N; Yanrismet, Y; Shimojou, M; Morimoto,
S; Sasaki, F; Ishii, KJ; Yokomizo, T; Kunisawa, J. BLT1 mediates
commensal bacteria-dependent innate immune signals toenhance antigen-specific intestinal IgA responses. Mucosal
Immunology 12, 1082-1091 (2019).

Matsumura, T; Endo, T; Isotani, A; Ogawa, M; Ikawa, M. An
azoospermic factor gene, Ddx3y and its paralog, Ddx3x
are dispensable in germ cells for male fertility. Journal of
Reproduction and Development 65, 121-128 (2019).

86

Naito, H; Wakabayashi, T; Ishida, M; Gil, CH; Iba, T; Rahmawati,
FN; Shimizu, S; Yoder, MC; Takakura, N. Isolation of tissueresident vascular endothelial stem cells from mouse liver.
Nature Protocols 15, 1066-1081 (2020).

Matsumura, T; Ikebe, T; Arikawa, K; Hosokawa, M; Aiko, M;
Iguchi, A; Togashi, I; Kai, S; Ohara, S; Ohara, N; Ohnishi,
M; Watanabe, H; Kobayashi, K; Takeyama, H; Yamasaki, S;
Takahashi, Y; Ato, M. Sequential sensing by TLR2 and mincle
directs immature myeloid cells to protect against invasive group
A streptococcal infection in mice. Cell Reports 27, 561 (2019).

87

Nakai, A; Fujimoto, J; Miyata, H; Stumm, R; Narazaki, M; Schulz,
S; Baba, Y; Kumanogoh, A; Suzuki, K. The COMMD3/8 complex
determines GRK6 specificity for chemoattractant receptors.
Journal of Experimental Medicine 216, 1630-1647 (2019).

88

Nakai, A; Suzuki, K. Adrenergic control of lymphocyte trafficking
and adaptive immune responses. Neurochemistry International
130, 104320 (2019).

89

Nakanishi, T; Fujita, Y; Tanaka, T; Yamashita, T. Anti-repulsive
guidance molecule-a antibody treatment and repetitivetranscranial magnetic stimulation have synergistic effects on
motor recovery after spinal cord injury. Neuroscience Letters709, 134329 (2019).

66

67

Matoba, N; Akiyama, M; Ishigaki, K; Kanai, M; Takahashi,
A; Momozawa, Y; Ikegawa, S; Ikeda, M; Iwata, N; Hirata, M;
Matsuda, K; Kubo, M; Okada, Y; Kamatani, Y. GWAS of smoking
behaviour in 165,436 Japanese people reveals seven new lociand shared genetic architecture. Nature Human Behaviour 3,
471-477 (2019).

69

70

71

72

73

Maeda, Y; Takeda, K. Host-microbiota interactions inrheumatoid arthritis. Experimental and Molecular Medicine 51,
150 (2019).
Maeda, Y; Tsuda, T; Takeda, Y; Koyama, S; Hayama, Y; Nojima,
S; Kimura, T; Ito, D; Takamatsu, H; Kang, SJ; Nishide, M;
Morimoto, K; Hosokawa, T; Kinehara, Y; Kato, Y; Nakatani,
T; Takeda, K; Hayama, M; Obata, S; Akazawa, H; Shikina, T;
Inohara, H; Kumanogoh, A. SEMA4A promotes eosinophil
survival and contributes to eosinophil-mediated allergic
diseases. Allergology International 68, 274-276 (2019).

77

Metwally, H; Tanaka, T; Li, SL; Parajuli, G; Kang, SJ; Hanieh,
H; Hashimoto, S; Chalise, JP; Gemechu, Y; Standley, DM;
Kishimoto, T. Noncanonical STAT1 phosphorylation expands its
transcriptional activity into promoting LPS-induced IL-6 and IL12p40 production. Science Signaling 13, eaay0574 (2020).

Masuda, T; Low, SK; Akiyama, M; Hirata, M; Ueda, Y; Matsuda,
K; Kimura, T; Murakami, Y; Kubo, M; Kamatani, Y; Okada, Y.
GWAS of five gynecologic diseases and cross-trait analysis inJapanese. European Journal of Human Genetics 28, 95-107(2020).

68

76

Mesin, L; Schiepers, A; Ersching, J; Barbulescu, A; Cavazzoni,
CB; Angelini, A; Okada, T; Kurosaki, T; Victora, GD. Restrictedclonality and limited germinal center reentry characterize
memory B cell reactivation by boosting. Cell 180, 92 (2020).

Matoba, N; Akiyama, M; Ishigaki, K; Kanai, M; Takahashi,
A; Momozawa, Y; Ikegawa, S; Ikeda, M; Iwata, N; Hirata, M;
Matsuda, K; Murakami, Y; Kubo, M; Kamatani, Y; Okada, Y.
GWAS of 165,084 Japanese individuals identified nine lociassociated with dietary habits. Nature Human Behaviour 4,308-316 (2020).
Matoba, T; Imai, M; Ohkura, N; Kawakita, D; Ijichi, K; Toyama, T;
Morita, A; Murakami, S; Sakaguchi, S; Yamazaki, S. Regulatory
T cells expressing abundant CTLA-4 on the cell surface with aproliferative gene profile are key features of human head andneck cancer. International Journal of Cancer 144, 2811-2822(2019).

Matsuo, T; Hashimoto, M; Sakaguchi, S; Sakaguchi, N; Ito,
Y; Hikida, M; Tsuruyama, T; Sakai, K; Yokoi, H; Shirakashi, M;
Tanaka, M; Ito, H; Yoshifuji, H; Ohmura, K; Fujii, T; Mimori, T.
Strain-specific manifestation of lupus-like systemic autoimmunity
caused by Zap70 mutation. Journal of Immunology 202, 31613172 (2019).

74

Matsuoka, S; Hashimoto, D; Kadowaki, M; Ohigashi, H; Hayase,
E; Yokoyama, E; Hasegawa, Y; Tateno, T; Chen, XZ; Aoyama, K;
Oka, H; Onozawa, M; Takeda, K; Akashi, K; Teshima, T. Myeloid
differentiation factor 88 signaling in donor T cells accelerates
graft-versus-host disease. Haematologica 105, 226-234 (2020).

75

Matsushita, K; Tanaka, H; Yasuda, K; Adachi, T; Fukuoka,
A; Akasaki, S; Koida, A; Kuroda, E; Akira, S; Yoshimoto, T.
Regnase-1 degradation is crucial for IL-33-and IL-25-mediated
ILC2 activation. JCI Insight 5, e131480 (2020).

90

Nakatochi, M; Kanai, M; Nakayama, A; Hishida, A; Kawamura,
Y et al. Genome-wide meta-analysis identifies multiple novelloci associated with serum uric acid levels in Japaneseindividuals. Communications Biology 2, 115 (2019).

91

Nakatsukasa, H; Oda, M; Yin, J; Chikuma, S; Ito, M; KogaIizuka, M; Someya, K; Kitagawa, Y; Ohkura, N; Sakaguchi, S;
Koya, I; Sanosaka, T; Kohyama, J; Tsukadar, Y; Yamanaka, S;
Takamuraenya, T; Lu, QJ; Yoshimura, A. Loss of TET proteinsin regulatory T cells promotes abnormal proliferation, Foxp3
destabilization and IL-17 expression. International Immunology
31, 335-347 (2019).

92

Nishi, C; Yanagihashi, Y; Segawa, K; Nagata, S. MERTK tyrosine
kinase receptor together with TIM4 phosphatidylserine receptor
mediates distinct signal transduction pathways for efferocytosis
and cell proliferation. Journal of Biological Chemistry 294,
7221-7230 (2019).

93

Nishida, S; Tsuboi, A; Tanemura, A; Ito, T; Nakajima,
H; Shirakata, T; Morimoto, S; Fujiki, F; Hosen, N; Oji, Y;
Kumanogoh, A; Kawase, I; Oka, Y; Azuma, I; Morita, S;
Sugiyama, H. Immune adjuvant therapy using Bacillus
Calmette-Guerin cell wall skeleton (BCG-CWS) in advanced
malignancies: A phase 1 study of safety and immunogenicity
assessments. Medicine 98, e16771 (2019).

94

Noguchi, E; Akiyama, M; Yagami, A; Hirota, T; Okada, Y; Kato,
Z; Kishikawa, R et al. HLA-DQ and RBFOX1 as susceptibility
genes for an outbreak of hydrolyzed wheat allergy. Journal ofAllergy and Clinical Immunology 144, 1354-1363 (2019).

95

Nyati, KK; Agarwal, RG; Sharma, P; Kishimoto, T. Arid5a
regulation and the roles of Arid5a in the inflammatory Response
and Disease. Frontiers In Immunology 10, 2790 (2019).

96

Nyati, KK; Zaman, MMU; Sharma, P; Kishimoto, T. Arid5a,
an RNA-binding protein in immune regulation: RNA stability,
inflammation, and autoimmunity. Trends in Immunology 41, 255268 (2020).

97

Ogawa, K; Okuno, T; Hosomichi, K; Hosokawa, A; Hirata, J;
Suzuki, K; Sakaue, S; Kinoshita, M; Asano, Y; Miyamoto, K;
Inoue, I; Kusunoki, S; Okada, Y; Mochizuki, H. Next-generation
sequencing identifies contribution of both class I and II HLAgenes on susceptibility of multiple sclerosis in Japanese.
Journal of Neuroinflammation 16, 162 (2019).

98

Ogawa, K; Stuart, PE; Tsoi, LC; Suzuki, K; Nair, RP; Mochizuki,
H; Elder, JT; Okada, Y. A transethnic mendelian randomization
study identifies causality of obesity on risk of psoriasis. Journalof Investigative Dermatology 139, 1397-1400 (2019).

99

Oh, JE; Iijima, N; Song, E; Lu, PW; Klein, J; Jiang, RY;
Kleinstein, SH; Iwasaki, A. Migrant memory B cells secreteluminal antibody in the vagina. Nature 571, 122 (2019).

100

Oh-hora, M; Lu, XY; Shiokawa, M; Takayanagi, H; Yamasaki,
S. Stromal interaction molecule deficiency in T cells promotes
spontaneous follicular helper T cell development and causes
type 2 immune disorders. Journal of Immunology 202, 26162627 (2019).

101

Ohshima, K; Nojima, S; Tahara, S; Kurashige, M; Kawasaki, K;
Hori, Y; Taniguchi, M; Umakoshi, Y; Okuzaki, D; Wada, N; Ikeda,
J; Fukusaki, E; Morii, E. Serine racemase enhances growth ofcolorectal cancer by producing pyruvate from serine. Nature
Metabolism 2, 81 (2020).

102

Okumura, R; Kodama, T; Hsu, CC; Sahlgren, BH; Hamano, S;
Kurakawa, T; Iida, T; Takeda, K. Lypd8 inhibits attachment ofpathogenic bacteria to colonic epithelia. Mucosal Immunology
13, 75-85 (2020).

103

Orimo, T; Sasaki, I; Hemmi, H; Ozasa, T; Fukuda-Ohta, Y;
Ohta, T; Morinaka, M; Kitauchi, M; Yamaguchi, T; Sato, Y;
Tanaka, T; Hoshino, K; Katayama, K; Fukuda, S; Miyake, K;
Yamamoto, M; Satoh, T; Furukawa, K; Kuroda, E; Ishii, KJ;
Takeda, K; Kaisho, T. Cholera toxin B induces interleukin-1
beta production from resident peritoneal macrophages throughthe pyrin inflammasome as well as the NLRP3 inflammasome.
International Immunology 31, 657-668 (2019).

In for m a t ion

49

Knaus, A; Kortrum, F; Kleefstra, T; Stray-Pedersen, A; Dukic, D;
Murakami, Y; Gerstner, T; van Bokhoven, H; Iqbal, Z; Horn, D;
Kinoshita, T; Hempe, MA; Krawitz, PM. Mutations in PIGU impair
the function of the GPI transamidase complex, causing severe
intellectual disability, epilepsy, and brain anomalies. American
Journal of Human Genetics 105, 395-402 (2019).

113

105

Osada-Oka, M; Goda, N; Saiga, H; Yamamoto, M; Takeda, K;
Ozeki, Y; Yamaguchi, T; Soga, T; Tateishi, Y; Miura, K; Okuzaki,
D; Kobayashi, K; Matsumoto, S. Metabolic adaptation toglycolysis is a basic defense mechanism of macrophages for
Mycobacterium tuberculosis infection. International Immunology
31, 781-793 (2019).

106

Ozaki, T; Muramatsu, R; Nakamura, H; Kinoshita, M; Kishima, H;
Yamashita, T. Proteomic analysis of protein changes in plasma
by balloon test occlusion. Journal of Clinical Neuroscience 72,
397-401 (2020).

107

Ozasa, K; Temizoz, B; Kusakabe, T; Kobari, S; Momota, M;
Coban, C; Ito, S; Kobiyama, K; Kuroda, E; Ishii, KJ. Cyclic
GMP-AMP triggers asthma in an IL-33-dependent mannerthat is blocked by amlexanox, a TBK1 Inhibitor. Frontiers in
Immunology 10, 2212 (2019).

108

109

Pavillon, N; Smith, NI. Immune cell type, cell activation,and single cell heterogeneity revealed by label-free opticalmethods. Scientific Reports 9, 17054 (2019).

110

Reinink, P; Buter, J; Mishra, VK; Ishikawa, E; Cheng, TY;
Willemsen, PTJ; Porwollik, S; Brennan, PJ; Heinz, E; Mayfield,
JA; Dougan, G; van Els, CA; Cerundolo, V; Napolitani, G;
Yamasaki, S; Minnaard, AJ; McCeland, M; Moody, DB; Van
Rhijn, I. Discovery of Salmonella trehalose phospholipids
reveals functional convergence with mycobacteria. Journal of
Experimental Medicine 216, 757-771 (2019).

111

Reja, SI; Minoshima, M; Hori, Y; Kikuchi, K. Development of aneffective protein-labeling system based on smart fluorogenic
probes. Journal of Biological Inorganic Chemistry 24, 443-455(2019).

112

Romanov, V; Isohashi, K; Alobthani, G; Beshr, R; Horitsugi,
G; Kanai, Y; Naka, S; Watabe, T; Shimosegawa, E; Hatazawa,J. Evaluation of the total distribution volume of F-18-FBPA in
normal tissues of healthy volunteers by non-compartmental
kinetic modeling. Annals of Nuclear Medicine 34, 155-162(2020).

113

Rozewicki, J; Li, SL; Amada, KM; Standley, DM; Katoh, K.
MAFFT-DASH: integrated protein sequence and structuralalignment. Nucleic Acids Research 47, W5-W10 (2019).

114

Ryoden, Y; Fujii, T; Segawa, K; Nagata, S. Functional expressionof the P2X7 ATP receptor requires Eros. Journal of Immunology
204, 559-568 (2020).

115

Sakaue, S; Akiyama, M; Hirata, M; Matsuda, K; Murakami, Y;
Kubo, M; Kamatani, Y; Okada, Y. Functional variants in ADH1B
and ALDH2 are non-additively associated with all-cause
mortality in Japanese population. European Journal of Human
Genetics 28, 378-382 (2020).

116

114

Pareek, S; Kurakawa, T; Das, B; Motooka, D; Nakaya, S;
Rongsen-Chandola, T; Goyal, N; Kayama, H; Dodd, D;
Okumura, R; Maeda, Y; Fujimoto, K; Nii, T; Ogawa, T; Iida,
T; Bhandari, N; Kida, T; Nakamura, S; Nair, GB; Takeda, K.
Comparison of Japanese and Indian intestinal microbiota
shows diet-dependent interaction between bacteria and fungi.
Npj Biofilms and Microbiomes 5, 37 (2019).

Sakaue, S; Hirata, J; Kanai, M; Suzuki, K; Akiyama, M; Too,
CL; Arayssi, T; Hammoudeh, M; Al Emadi, S; Masri, BK;Halabi, H; Badsha, H; Uthman, IW; Saxena, R; Padyukov, L;
Hirata, M; Matsuda, K; Murakami, Y; Kamatani, Y; Okada, Y.
Dimensionality reduction reveals fine-scale structure in theJapanese population with consequences for polygenic risk
prediction. Nature Communications 11, 1569 (2020).

117

Sakaue, S; Kanai, M; Karjalainen, J; Akiyama, M; Kurki, M;
Matoba, N; Takahashi, A; Hirata, M; Kubo, M; Matsuda, K;
Murakami, Y; Daly, MJ; Kamatani, Y; Okada, Y. Trans-biobank
analysis with 676,000 individuals elucidates the association ofpolygenic risk scores of complex traits with human lifespan.
Nature Medicine 26, 542-548 (2020).

118

Sakaue, S; Okada, Y. GREP: genome for REPositioning drugs.
Bioinformatics 35, 3821-3823 (2019).

119

Sanjo, H; Nakayama, J; Yoshizawa, T; Fehling, HJ; Akira, S;
Taki, S. Cutting edge: TAK1 safeguards macrophages against
proinflammatory cell death. Journal of Immunology 203, 783788 (2019).

120

Sasai, M; Yamamoto, M. Decision by injection without infection.
Journal of Experimental Medicine 217, e20192145 (2020).

121

Sasai, M; Yamamoto, M. Innate, adaptive, and cell-autonomous
immunity against Toxoplasma gondii infection. Experimental
and Molecular Medicine 51, 156 (2019).

122

Sato, A; Ono, C; Tamura, T; Mori, H; Izumi, T; Torii, S; Fauzyah, Y;
Yamamoto, T; Morioka, Y; Okuzaki, D; Fukuhara, T; Matsuura, Y.
Rimonabant suppresses RNA transcription of hepatitis B virus
by inhibiting hepatocyte nuclear factor 4 alpha. Microbiology
and Immunology, 345-355 (2020).

123

Schmidleithner, L; Thabet, Y; Schonfeld, E; Kohne, M; Sommer,
D et al. Enzymatic activity of HPGD in Treg cells suppresses
Tconv cells to maintain adipose tissue homeostasis and prevent
metabolic dysfunction. Immunity 50, 1232 (2019).

133

Tanabe, S; Yamashita, T. Function of lymphocytes inoligodendrocyte development. Neuroscientist 26, 74-86 (2020).

134

Tanaka, A; Nishikawa, H; Noguchi, S; Sugiyama, D; Morikawa,
H; Takeuchi, Y; Ha, D; Shigeta, N; Kitawaki, T; Maeda, Y; Saito,
T; Shinohara, Y; Kameoka, Y; Iwaisako, K; Monma, F; Ohishi,
K; Karbach, J; Jager, E; Sawada, K; Katayama, N; Takahashi,
N; Sakaguchi, S. Tyrosine kinase inhibitor imatinib augments
tumor immunity by depleting effector regulatory T cells. Journalof Experimental Medicine 217, e20191009 (2020).

135

Tanaka, A; Sakaguchi, S. Targeting Treg cells in cancer
immunotherapy. European Journal of Immunology 49, 11401146 (2019).

136

Tanuma, M; Kasai, A; Bando, K; Kotoku, N; Harada, K;
Minoshima, M; Higashino, K; Kimishima, A; Arai, M; Ago,
Y; Seiriki, K; Kikuchi, K; Kawata, S; Fujita, K; Hashimoto,H. Direct visualization of an antidepressant analog using
surfaceenhanced Raman scattering in the brain. JCI Insight 5,
e133348 (2020).

137

Tobita, T; Kiyozumi, D; Muto, M; Noda, T; Ikawa, M. Lvrn
expression is not critical for mouse placentation. Journal of
Reproduction and Development 65, 239-244 (2019).

138

Tsuda, T; Nishide, M; Maeda, Y; Hayama, Y; Koyama, S; Nojima,
S; Takamatsu, H; Okuzaki, D; Morita, T; Nakatani, T; Kato, Y;
Nakanishi, Y; Futami, Y; Suga, Y; Naito, Y; Konaka, H; Satoh, S;
Naito, M; Izumi, M; Obata, S; Nakatani, A; Shikina, T; Takeda,
K; Hayama, M; Inohara, H; Kumanogoh, A. Pathological andtherapeutic implications of eosinophil-derived semaphorin 4D
in eosinophilic chronic rhinosinusitis. Journal of Allergy andClinical Immunology 145, 843 (2020).
Tsuji, T; Cheng, J; Tatematsu, T; Ebata, A; Kamikawa, H;
Fujita, A; Gyobu, S; Segawa, K; Arai, H; Taguchi, T; Nagata, S;
Fujimoto, T. Predominant localization of phosphatidylserine at
the cytoplasmic leaflet of the ER, and its TMEM16K-dependent
redistribution. Proceedings of the National Academy of
Sciences USA 116, 13368-13373 (2019).

124

Schritt, D; Li, SL; Rozewicki, J; Katoh, K; Yamashita, K;
Volkmuth, W; Cavet, G; Standley, DM. Repertoire builder: highthroughput structural modeling of B and T cell receptors.
Molecular Systems Design & Engineering 4, 761-768 (2019).

139

125

Shimizu, Y; Kohyama, M; Yorifuji, H; Jin, H; Arase, N; Suenaga,
T; Arase, H. Fc gamma RIIIA-mediated activation of NK cells
by IgG heavy chain complexed with MHC class II molecules.
International Immunology 31, 303-314 (2019).

140

126

Sidwell, T; Liao, Y; Garnham, AL; Vasanthakumar, A; Gloury,
R; Blume, J; Teh, PP; Chisanga, D; Thelemann, C; Rivera,
FD; Engwerda, CR; Corcoran, L; Kometani, K; Kurosaki, T;
Smyth, GK; Shi, W; Kallies, A. Attenuation of TCR-induced
transcription by Bach2 controls regulatory T cell differentiationand homeostasis. Nature Communications 11, 252 (2020).

Tsujioka, H; Yamashita, T. Comparison of gene expression
profile of the spinal cord of sprouting-capable neonatal andsprouting-incapable adult mice. Bmc Genomics 20, 619 (2019).

141

127

Simmons, S; Sasaki, N; Umemoto, E; Uchida, Y; Fukuhara,
S; Kitazawa, Y; Okudaira, M; Inoue, A; Tohya, K; Aoi, K; Aoki,
J; Mochizuki, N; Matsuno, K; Takeda, K; Miyasaka, M; Ishii,
M. Highendothelial cell-derived S1P regulates dendritic cell
localization and vascular integrity in the lymph node. Elife 8,
e41239 (2019).

Usui, R; Yabe, D; Fauzi, M; Goto, H; Botagarova, A; Tokumoto,
S; Tatsuoka, H; Tahara, Y; Kobayashi, S; Manabe, T; Baba, Y;
Kurosaki, T; Herrera, PL; Nagashima, K; Ogura, M; Nagashima,
K; Inagaki, N. GPR40 activation initiates store-operated Ca2+
entry and potentiates insulin secretion via the IP3R1/STIM1/
Orai1 pathway in pancreatic beta-cells. Scientific Reports 9,15562 (2019).

142

van der Peet, PL; Gunawan, C; Watanabe, M; Yamasaki, S;
Williams, SJ. Synthetic beta-1,2-mannosyloxymannitol glycolipid
from the fungus Malassezia pachydermatis signals throughhuman mincle. Journal of Organic Chemistry 84, 6788-6797(2019).

143

Wagner, M; Ealey, KN; Tetsu, H; Kiniwa, T; Motomura, Y; Moro, K;
Koyasu, S. Tumor-derived lactic acid contributes to the paucity
of intratumoral ILC2s. Cell Reports 30, 2743 (2020).

144

Wang, YC; Maeda, Y; Liu, YS; Takada, Y; Ninomiya, A;
Hirata, T; Fujita, M; Murakami, Y; Kinoshita, T. Crosstalks of glycosylphosphatidylinositol biosynthesis withglycosphingolipid biosynthesis and ER-associated degradation.
Nature Communications 11, 860 (2020).

145

Weber, M; Yamada, N; Tian, X; Bull, SD; Minoshima, M; Kikuchi,
K; Mackenzie, AB; James, TD. Sensing peroxynitrite in different
organelles of murine RAW264.7 macrophages with coumarinbased fluorescent probes. Frontiers in Chemistry 8, 39 (2020).

128

Spiliopoulou, A; Colombo, M; Plant, D; Nair, N; Cui, J et al.
Association of response to TNF inhibitors in rheumatoid arthritis
with quantitative trait loci for CD40 and CD39. Annals of theRheumatic Diseases 78, 1055-1061 (2019).

129

Suematsu, R; Miyamoto, T; Saijo, S; Yamasaki, S; Tada, Y;
Yoshida, H; Miyake, Y. Identification of lipophilic ligands ofSiglec5 and-14 that modulate innate immune responses.
Journal of Biological Chemistry 294, 16776-16788 (2019).

130

Takahashi, R; Amano, H; Ito, Y; Eshima, K; Satoh, T; Iwamura,
M; Nakamura, M; Kitasato, H; Uematsu, S; Raouf, J;
Jakobsson, PJ; Akira, S; Majima, M. Microsomal prostaglandin
E synthase-1 promotes lung metastasis via SDF-1/CXCR4mediated recruitment of CD11b+Gr1+MDSCs from bone marrow.
Biomedicine & Pharmacotherapy 121, 109581 (2020).

131

Takeuchi, Y; Hirota, K; Sakaguchi, S. Impaired T cell receptor
signaling and development of T cell-mediated autoimmune
arthritis. Immunological Reviews 294, 164-176 (2020).

132

Takeuchi, Y; Hirota, K; Sakaguchi, S. Synovial tissue
inflammation mediated by autoimmune T cells. Frontiers in
Immunology 10, 1989 (2019).

146

Xu, ZC; Li, SL; Rozewicki, J; Yamashita, K; Teraguchi, S; Inoue, T;
Shinnakasu, R; Leach, S; Kurosaki, T; Standley, DM. Functional
clustering of B cell receptors using sequence and structuralfeatures. Molecular Systems Design & Engineering 4, 769-778(2019).

147

Yamada, M; Fujita, Y; Hayano, Y; Hayakawa, H; Baba,
K; Mochizuki, H; Yamashita, T. Increased expression offibronectin leucine-rich transmembrane protein 3 in the dorsal
root ganglion induces neuropathic pain in rats. Journal of
Neuroscience 39, 7615-7627 (2019).

148

Yamaguchi, T; Teraguchi, S; Furusawa, C; Machiyama, H;
Watanabe, TM; Fujita, H; Sakaguchi, S; Yanagida, T. Theoreticalmodeling reveals that regulatory T cells increase T-cellinteraction with antigen-presenting cells for stable immune
tolerance. International Immunology 31, 743-753 (2019).

149

Yamamoto, K; Sakaue, S; Matsuda, K; Murakami, Y; Kamatani,
Y; Ozono, K; Momozawa, Y; Okada, Y. Genetic and phenotypiclandscape of the mitochondrial genome in the Japanese
population. Communications Biology 3, 104 (2020).

150

Yamamoto, T; Kanuma, T; Takahama, S; Okamura, T; Moriishi,
E; Ishii, KJ; Terahara, K; Yasutomi, Y. STING agonists activate
latently infected cells and enhance SIV-specific responses
ex vivo in naturally SIV controlled cynomolgus macaques.
Scientific Reports 9, 5917 (2019).

151

Yamashita, Y; Suzuki, C; Uchiyama, Y; Nagata, S. Infertility
caused by inefficient apoptotic germ cell clearance in Xkr8deficient male mice. Molecular and Cellular Biology 40, e0040219 (2020).

152

Yorifuji, H; Arase, N; Kohyama, M; Hirano, T; Suenaga, T;
Kumanogoh, A; Arase, H. Transport of cellular misfolded
proteins to the cell surface by HLA-B27 free heavy chain.
Biochemical and Biophysical Research Communications 511,
862-868 (2019).

153

Zhu, C; Luo, X; Espulgar, WV; Koyama, S; Kumanogoh, A; Saito,
M; Takamatsu, H; Tamiya, E. Real-time monitoring and detectionof single-cell level cytokine secretion using LSPR technology.
Micromachines 11, 107 (2020).

In for m a t ion

104

Osa, A; Uenami, T; Naito, Y; Hirata, H; Koyama, S; Takimoto,T; Shiroyama, T; Futami, S; Nakatsubo, S; Sawa, N; Yano, Y;
Nagatomo, I; Takeda, Y; Mori, M; Kida, H; Kumanogoh, A.
Monitoring antibody binding to T cells in a pembrolizumabtreated patient with lung adenocarcinoma on hemodialysis.
Thoracic Cancer 10, 2183-2187 (2019).

The articles were published between April, 2019 and March, 2020. The data were acquired using Web of Science Core
CollectionTM on May 15, 2020, and sorted by alphabetical order of the first authors.
115

By Hankyu Railway
15 min. walk east from Kita-Senri Station on the Hankyu Senri Line.
By Osaka Monorail
25 min. walk from Handai-byoin-mae Station on the Osaka Monorail.
By Hankyu Bus
Rout 1: Get on the bus bound for “Handai Honbu Mae” or “Ibaraki Mihogaoka” from SenriChuo Station. Get off at Handai Honbu Mae, walk 15 min.
Rout 2: Get on the bus bound for “Onohara Higashi, Fuji Kasai” from Senri-Chuo Station.
Get off at “Handai-guchi”, walk 5 min.
By Kintetsu Bus
Get on the bus bound for “Handai Honbu Mae” or “Ibaraki Mihogaoka” from Hankyu
Ibarakishi Station (via JR Ibaraki Station). Get off at “Handai Honbu Mae”, walk 15 min.

SUITA CAMPUS Osaka University

IncforAc e smsa M
t ion
ap

Access from the Nearest Station

Access Map

Handai-guchi
Handai-guchi
Ibaraki
IbarakiMihogaoka
Mihogaoka

Graduate
Graduate
SchoolSchoolof Medicineof Medicine

Senri
Senri
Gate
Gate

Graduate
Graduate
SchoolSchoolof Engineeringof Engineering

Osaka
Osaka
Monorail
Monorail
Handai-byoin-mae
Handai-byoin-mae
StationStationHandaiHandai
Honbu
Honbu
Mae
Mae

HankyuHankyuKita-Senri
Kita-Senri
StationStationGraduate
Graduate
SchoolSchoolof of
Frontier
Frontier
Bioscience
Bioscience

ADDRESSTEL

FAX
E-mail116

IFReC Research Building,
Osaka University 3-1 Yamadaoka, Suita,
565-0871, Osaka, Japan
General Affairs: +81-6-6879-4275
Accounting: +81-6-6879-4917
Research Planning & Management Office:+81-6-6879-4777+81-6-6879-4272
ifrec-office@ifrec.osaka-u.ac.jp

Main
Main
Gate
Gate
KitaKita
FireFire
StationStation
Ha Ha
nkynkyu Ru R
ailwailway ay117