WPI Immunology Frontier Research Center 2020-2021

World PremierInternational
Research Center

Os a k a Un i ver si ty

Immunology
Frontier
Research
Cent er
Annual Reportof IFReC
2020-2021

Edit: Research Planning & Management Office, IFReC
Published in June, 2021

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

Contents

Message from the Director……………………………………………………………………………………… 1
Organization………………………………………………………………………………………………………… 5
Kiyoshi TAKEDA, MD/PhD
Director

Laboratories……………………………………………………………………………………………………… 11■ Immunology Groups……………………………………………………………………………………

14■ Imaging Groups……………………………………………………………………………………………

66■ Bioinformatics Groups…………………………………………………………………………………

78■ Units for Combined Research Fields………………………………………………………

82

Events & Outreach Activities…………………………………………………………………………… 85
Information………………………………………………………………………………………………………… 91
Access Map…………………………………………………………………………………………………… 108

WPI Immunology Frontier Research Center

As the Director of the Immunology Frontier ResearchCenter (WPI-IFReC) at Osaka University, I am very
pleased to present the IFReC annual report for the year
2020-2021.
Since its inception in 2007, IFReC has establisheditself as a high-profile international research center for
immunology with the broad support of many people.
Furthermore, since the establishment of the WPI
Academy in 2017, IFReC has continued efforts to create
history as a research center through a novel academicindustry partnership agreement.
I assumed the directorship of IFReC from Prof.
Shizuo Akira in 2019. Soon after that time from aroundthe end of 2019, an infectious disease (COVID-19)
caused by a novel coronavirus called SARS-CoV-2
has been spreading throughout the world and has
developed into a pandemic.
COVID-19 still runs rampant across the globe, andas of March 2021, travel between countries remains
severely restricted. Although the traditional role ofscientists involves directly discussing their findings
across national borders, the COVID-19 crisis has forced
the cancellation or postponement of many academic
events, including international symposia and seminars
with invited guest speakers. Similarly, many outreachactivities such as science cafés or visits by high school
students have also been put on hold.
However, using remote conferencing systems, wehave gradually started to hold alternative events. In

March 2021, we started an online seminar called the“Immuno-Seminar Series” featuring internationallyrenowned researchers. Of course, we encounteredsome constraints, such as time zone differences, but
we will continue our international collaborations as
a WPI research center without interruption. Looking
ahead toward the day when the COVID-19 situation is
resolved, we are already planning to resume holding
international symposia and our annual “Winter School,”an international educational program that has continued
for ten years.
As a research center for immunology receiving
government support, all members of IFReC recognize
the necessity of conducting basic research as ourcontribution to society to overcome this fundamental
problem of COVID-19. Therefore, we launched the “TeamOsaka University Research on COVID-19” consortium
through which we are promoting a team-based
approach to research that surpasses the framework ofindividual research.
In FY2020, we welcomed two principal investigators.
One is Dr. Naoki Hosen, who is a world-renowned
researcher in the field of cancer immunology, and theother is Dr. Yasutaka Okabe who is joining our center
under the Young Lead Researcher program.
Even in these difficult times, IFReC will continue
to contribute to the further advancement of sciencethrough research and education.

1

Efforts of Osaka University and IFReC in the
COVID-19 Epidemic
Jun SAKANOUE

Osaka University’s response to COVID-19
The number of people infected with the new coronavirus (SARS-CoV-2) has been increasing worldwide since the end of2019 and has reached 120 million (44,000 in Japan) while the number of deaths has exceeded 2.6 million (8,300 in Japan) (WHO
as of March 10, 2021).
Osaka University has planned its response to the “New Era of COVID-19” by establishing the “Osaka University
Coronavirus Infectious Disease Task Force” in March 2020, which consists of the university president, department directors,
and infectious disease experts. The task force established “Osaka University’s COVID-19 Response Levels” and preparedguidelines for infection prevention measures. Telecommuting was introduced for the first time and the administrative office in
IFReC has been promoting it more actively than what the university’s response levels specify. However, for now, users must
provide their own internet environment for telecommuting and telecommuting is expected decline once the coronavirus crisis
passes. For students of the university (including international students), tuition reduction and exemption and extension of
payment deadlines were offered. The university also provides an environment for online classes and lends out wireless routers.
For telecommuters, a consultation service was set up at the Health and Counseling Center to provide them with support. In
addition to the above, IFReC also recommends and sponsors twice-monthly PCR tests for the faculty and staff. Fortunately,
there have been no COVID-19 cases so far among IFReC members and their close relatives.
Despite the preventive measures taken by Osaka University, the cumulative total number of infected people is 110 (asof March 10, 2021), which is about 0.35% of the total number of its members and does not differ much from the national
percentage as a whole. This number includes COVID-19 patients at the university hospital and also the clusters of infections
that have occurred among some university students (Osaka University website).

Research Promotion in the New Era of Corona by the “Center for Infectious Disease Education andResearch”
Some departments at Osaka University, including IFReC, the Graduate School of Medicine, the University Hospital,
the Graduate School of Science, the Research Institute for Microbial Diseases, and the Institute of Industrial Science, arecollaborating with organizations outside the university to promote comprehensive research directly related to emerging
infectious diseases, including pathological analysis, the development of drugs, preventive vaccines and treatment strategies,
and the analysis of infection spread. Furthermore, as a ten-year plan, the university decided to establish a comprehensive
research and development center that will work on basic research, clinical research, clinical trials, and practical application,by combining the university’s outstanding knowledge in protein science, pathogenic microbiology, genome science,nanotechnology, and information science. Fortunately, the research organizations including IFReC are concentrated on the
Suita Campus, which is an ideal environment for a center for the comprehensive research and development on infectious
diseases. IFReC is also a member of this “Center for Infectious Disease Education and Research” and is expected to play aleadership role in the center.

Dr. Wani, a mascot of Osaka University appeals the university’s policy.

COVID-19 related research at Osaka University

2

Osaka University is one of the centers of excellence in the field of infectious diseases and immunology. However, its
COVID-19 related research has been unable to produce substantial results and only a few papers on basic research in thisfield appear on the Osaka University’s website after 2020. One of them is the finding by the Laboratory of Immune Regulation
at IFReC, which reveals cytokine IL-6 increases in the blood during early COVID-19, and promotes thrombus formation via PAI(plasminogen activator inhibitor) -1, a promoter of blood clotting from blood vessels (Kang et al. PNAS 2020). Osaka University
supports the “Sustainable Development Goals (SDGs)” promoted by the United Nations. The aforementioned PNAS paper andanother one by the Laboratory of Lymphocyte Differentiation that revealed the differentiation mechanism of memory B cells(Inoue et al. JEM 2020) were selected as good examples for one of the SDGs, “Health for All” (cf. “Selected Articles”).

Establishing Center for Infectious Disease Education and Research (CIDER)

3

Organization

Osaka University

MEXT

International Advanced
Research Institute (IARI)WPI Initiative Program Committee

As of March, 2021

Organization

Organization Chart

JSPS

Joint Research Chairs with the Pharmaceutical Companies
International
Scientific
Advisory
Board

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

Informatics Group
Unit for Combined Research Fields

Core Instrumentation Facility

Common Facilities

Director

Management
Committee

Network Administration Office

Board of Representatives

Administrative Office

DeputyDirectors

Administrative
Director

Animal Resource Center for Infectious Diseases

General Affairs Section
Administrative
Department• General Affairs Section• Accounting Section

Research Planning and Management Office• Employment and acceptance procedures 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

Research Planning and Management Office
Cooperative Institutions

6• Institute for Frontier Life and Medical Sciences, Kyoto University, Japan• RIKEN Center for Integrative Medical Sciences, Japan• University College London, UK• ImmunoSensation2, Clusters of Excellence, the Rheinische Friedrich-Wilhelms-University of Bonn,
Germany• 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.)7World Premier International Research Center Initiative (WPI)● Program Director
Akira UKAWA

As of October, 2020
Director, Center for WPI Center, JSPS, Japan● Deputy Program Director
Minoru YOSHIDA

8Group Director, Chemical Genomics Research Group, RIKEN Center for Sustainable
Resource Science, JapanWPI Academy
In 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

● Program Committee Members

Toshio KUROKI

Rita COLWELL

Distinguished University Professor, University of Maryland, USA● Program 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)Maki KAWAI

Director General, Institute for Molecular Science (IMS)/National Institutes of Natural
Sciences (NINS), Japan

Klaus von KLITZING

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

Kiyoshi KUROKAWA

Professor Emeritus, National Graduate Institute for Policy Studies Former president,
Science Council of Japan

Chuan Poh LIM

Chairman, Singapore Food Agency (SFA)

Hiroshi MATSUMOTO

President, RIKEN, Japan

Ryozo NAGAI

President, Jichi Medical University, Japan

Ryoji NOYORI (Chair)Director-General of Center for Research and Development Strategy (CRDS)/Japan
Science and Technology Agency (JST)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, Institute of Research into the Fundamental Laws of the Universe (IRFU/
CEA), France

Organization

Committees & Advisory Board for IFReC

As of October, 2020
Special Advisor, Research Center for Science Systems, JSPS, Japan

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

International Scientific Advisory Board for IFReC

As of March, 2021

Lewis LANIER

University of California, San Francisco

Immunology

Anne O’GARRAThe Francis Crick InstituteImmunology

Jeffrey RAVETCH

Rockefeller University

Immunology

Art WEISS

University of California, San Francisco/Howard Hughes Medical
InstituteImmunology

Joachim SCHULTZ

DZNE/LIMES Institute, University of Bonn

Imaging9

Laboratories
As of March, 2021

Host Defense

Laboratories of IFReC

Immunoglycobiology

Immunopathology

Stem Cell Biology andDevelopmental Immunology

Aging Biology

Oncogene Research

Shizuo Akira

Takashi Nagasawa

Eiji Hara

Masato Okada#innateimmunity#pathogenrecognition#macrophage #regnase#carcell #stemcell#niche#aging #sasp#cancer#mtor #src

Cutaneous Immunology

Innate Immune Systems

Immunochemistry

Signal Transduction

Taroh Kinoshita
Yoshiko Murakami

Atsushi Kumanogoh

Hisashi Arase

Nobuyuki Takakura

Manabu Fujimoto

Kazuyo Moro#gpi-anchor#paroxysmalnocturnalhemoglobinuria#immunesemaphorin#autoimmunediseases#t-cellactivation#mhc #neo-self#misfoldedprotein#malaria #covid-19#bloodvessels #stemcell#cancer#intractableskindiseases#allergy#ilc2#autoimmunediseases

Human Immunology(Single Cell Immunology)

Human Immunology(Single Cell Genomics)

Immune Homeostasis

Immune Regulation

Immune Regulation

Mucosal Immunology

Tadamitsu Kishimoto

Hitoshi Kikutani

Kiyoshi Takeda

James Badger Wing

Daisuke Okuzaki

Yasutaka Okabe#rheumatism #il-6#th17differentiation#sle#anti-nuclearantibody(ana)#gutImmunity#inflammatoryboweldisease(ibd)#microbiota#humandisease #singlecell#treg#humandisease #singlecell#genomics#macrophage#immunehomeostasis

Experimental Immunology

Cell Signaling

Lymphocyte Differentiation

Cellular Immunotherapy

Single Molecule Imaging

Immunology andCell Biology

Shimon Sakaguchi

Takashi Saito

Tomohiro Kurosaki

Naoki Hosen

Toshio Yanagida
Ben Seymour

Masaru Ishii#treg #immunetolerance#cancerimmunology#t-cellactivation#tcrsignal#memoryb-cell#antibodyproduction#cancer#cartcell#singlemoleculeimaging#membraneprotein#osteoclast #liveimaging#cancermetastasis

Vaccine Science

Cevayir Coban

Ken J. Ishii

Masahiro Yamamoto

Jun Hatazawa

Kazuya Kikuchi

Kazuhiro Suzuki#malariaparasite#vaccine#vaccine#adjuvant#parasite #toxoplasma#immuneevasion#pet/mri #fbpa#cancertherapy#chemicalbiology#fluorescentprobe#adrenergicrceptor#lymphocytetrafficking#commd38

Biochemistry &Immunology

Molecular Neuroscience

Immunoparasitology

Chemical Imaging
Techniques

Malaria Immunology

Molecular Immunology

Nuclear Medicine

Biophotonics

Immune Response Dynamics

Systems Immunology

Statistical Immunology

Shigekazu Nagata

Toshihide Yamashita

Sho Yamasaki

Nicholas Isaac Smith

Daron Standley

Yukinori Okada#macrophage#celldeathsignal#apoptosis#centralnervoussystem#encephalomyelitis#lectin#novelimmunereceptor#labelfree #ramanscattering#intracellimaging#immunerepertoire#receptormodeling#statisticalgenetics #bigdata#diseaseriskgenes

12

13

Shizuo Akira, MD/PhD
Professor

Shizuo Akira

Associate Professor

Kazuhiko Maeda
Takashi Satoh (-July, 2020)
Hiroki Tanaka
Kanako Kuniyoshi
2655Assistant Professor
Postdoctoral Fellows
Research Assistants
Visiting Scientists
Support Staff

Our research focuses on the innate immune system,
which is an evolutionally 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.

Molecular mechanism of endoribonuclease Regnase-1
in inflammation

14

Regnase-1 is a member of CCCH-type zinc finger
proteins and 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 IL6 and IL12 p40 in macrophages and c-Rel,
Ox40, and IL2 in CD4+ T cells. The Regnase-1 protein iscleaved by MALT1 protease activity after T cell receptor
stimulation, resulting in the enhancement of T cell activation.
Dynamic control of Regnase-1 expression is critical formodulation of T cell activation. Based on these findings, andthrough the use of tissue-specific Regnase-1-deficient miceand mutant mice, we promote the understanding of the
precious roles of Regnase-1 in immune and non-immune
cells.
Regnase-1 is inactivated in response to external stimuli
through posttranslational modifications, yet the precise role

pathogenesis is poorly understood and there are few

prevention and/or blocking of intestinal inflammatory

effective treatments. The development of fibrosis isdisorders.

associated with activation of monocytes and macrophages.

We are further studying to achieve the goal of a

We identified a new macrophage subset wherecomprehensive understanding of the innate immune system

Ceacam1+Msr1+Ly6C-F4/80-Mac1+ monocytes, which weand to develop an effective treatment for immune-related
inflammatory diseases.

Macrophages consist of at least two subsets. 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 the homeostasisof peripheral organs, key immunological processes anddevelopment of various diseases. Among these diseases,
fibrosis is a life-threatening disease of unknown aetiology. Its

termed SatM (segregated-nucleus-containing atypicalmonocytes), share granulocyte characteristics, are regulated
by C/EBPβ(CCAAT/enhancer binding protein beta), and are
critical for fibrosis. To investigate the physiological role of
SatM and related subsets, we recently identified an RNAbinding protein RBM7 that is a component of the NEXT
(nuclear exosome targeting) complex. We found that theexpression of RBM7 increased in the fibrotic phase, RBM7deletion in nonhematopoietic cells suppresses fibrosis, anddysregulated expression RBM7 triggers apoptosis via
nuclear degradation of noncoding RNA Neat1. RBM7 inepithelial cells plays a critical role in the development offibrosis by regulating ncRNA decay, thereby producing
chemokines that recruit SatMs. Therefore, we believe theinhibition of RBM7 could lead to an effective treatment offibrosis in patients.

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

Figure 2. Dysregulated expression of the nuclear exosome
targeting complex component Rbm7 in non-hematopoietic cells
licenses the development of fibrosis.

Role of SatM, an atypical monocyte and committedprogenitor involved in fibrosisof phosphorylation largely remains unknown. We showedthat IL-17 induces the phosphorylation of Regnase-1 in an
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 the treatment of T helper 17-associateddiseases.
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-1ΔIEC
mice, which are Regnase-1-deficient in IECs, were resistantto body 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
predominantly 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.

Immunology

Host Defense

Regnase-1-deficiency in IECs has beneficial effects on the

Recent Publications
⿎⿎ Akira S and Maeda K. Control of RNA stability in immunity. Annu. Rev.
Immunol. 39: 481-509 (2021).

⿎⿎ Maeda K, Caldez MJ, Akira S. Innate immunity in allergy. Allergy 74:
1660-1674 (2019).

⿎⿎ Fukushima K, Satoh T, Sugihara F, Sato Y, Okamoto T, Mitsui Y, Yoshio
S, Li S, 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 expression of the nuclear exosome targetingcomplex component Rbm7 in non-hematopoietic cells licenses thedevelopment of fibrosis. Immunity 52: 542-556 (2020).

⿎⿎ Nagahama Y, Shimoda M, Mao G, Singh SK, Kozakai Y, Sun X,
Motooka D, Nakamura S, Tanaka H, Satoh T, Maeda K, Akira S.
Regnase-1 controls colon epithelial regeneration via regulation ofmTOR and purine metabolism. Proc. Natl. Acad. Sci. USA. 115: 1103611041 (2018).

⿎⿎ Tanaka H, Arima Y, Kamimura D, Tanaka Y, Takahashi N, Uehata T,
Maeda K, Satoh T, Murakami M, Akira S. Phosphorylation-dependent
Regnase-1 release from endoplasmic reticulum is critical in IL-17
response. J. Exp. Med. 216: 1431-1449 (2019).

15

working with Hrd1), and Dahlin2 (a component of Hrd1

cellular GPI levels, we found that GPI biosynthesis is undercomplex) have high GPI biosynthesis levels (6- to 7-foldthe control of the ER-associated degradation (ERAD)

increase) (Wang et al. Nat Commun. 2020). We continue

system. Particularly, cells defective in Hrd1 (a major E3 inworking to understand the molecular mechanisms of thethe ERAD system), UBE2J1 and UBE2G2 (E2 enzymes

upregulation of GPI biosynthesis.

Immunology

Immunoglycobiology

have been unclear. Using a convenient method to determine

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

16

Professor

Taroh Kinoshita
Yoshiko Murakami

Postdoctoral Fellows
Research Assistants
Support Staff

1
1
3

PNH is a hematopoietic stem cell (HSC) disordercharacterized by complement-mediated hemolysis andthrombosis, and bone marrow failure. In patients with PNH,
the affected blood cells are defective inglycosylphosphatidylinositol-anchored proteins (GPI-APs),
including the complement regulatory proteins CD55 andCD59. Deficiency of these complement regulatory proteinsrenders erythrocytes sensitive to complement-mediateddestruction and plays a fundamental role in thrombosis. The
classic form of PNH (PIGA-PNH) is caused by a somatic
mutation of the PIGA gene in the HSC and subsequentclonal expansion, in which bone marrow failure is implicated.
PIGA is involved in the first step of GPI biosynthesis and is
the only X-linked gene among 27 genes involved in GPI-AP
biosynthesis. A somatic mutation in one allele of PIGA issufficient to generate GPI-AP deficient cells in females aswell as males. Recently, in collaboration with Dr. Muus, Dr.
Langemeijer and their colleagues in the Netherlands, wereported on PNH caused by a homozygous mutation of the
PIGB gene in chromosome 15q (PIGB-PNH) (Langemeijer S
et al, Blood Adv 2020). We demonstrated that the patient isheterozygous for a loss-of-function variant of PIGB and a
copy-number neutral loss-of-heterozygosity (CN-LOH) eventin an HSC caused homozygosity of the PIGB mutation.
Almost all blood cells including erythrocytes andgranulocytes were GPI-AP deficient, indicating predominanthematopoiesis by the PIGB mutant clone. We also foundthat a 70-kbp deletion previously implicated in clonal
hematopoiesis was present in the same chromosome 15q
as the PIGB mutation, perhaps accounting for dominance of
the PIGB mutant clone. The patient’s mother who isheterozygous for the same PIGB mutation and 70-kbp
deletion had at least four PNH clones. All of them had CNLOH of various lengths spanning PIGB and a region of the
70-kbp deletion, therefore, characterizing familial PNH. Like
patients with PIGT-PNH, the PIGB-PNH patient had recurrent
autoinflammatory symptoms such as joint pains andurticaria. We suspect accumulated GPI biosynthetic
intermediate plays a role in the inflammatory symptoms.

Studies on inherited GPI deficiencies (IGD)
We have been contributing to the global collaboration
studies led by Dr. Campeau in Canada aiming to discoverand characterize the first cases of IGD caused by mutations
in genes, for which association with IGD has not been
established. This year, we published a paper on PIGF-IGD
and PIGK-IGD (Nguyen et al. Am J Hum Genet, 2020; Salian
et al. Hum Genet, 2021).

Studies on GPI biosynthesis.
Side-chains of some GPI-APs, such as prion proteins,are modified by sialic acid. How sialic acid modification of
GPI side-chain occurs has been unknown. In collaboration
with Dr. Kobayashi of Hokkaido University and Dr. Nishikazeof the Shimadzu Corporation, we analyzed a human prion
protein and found that its GPI side-chain is modified by α2,3linked N-acetylneuraminic acid (Kobayashi et al. J Biol
Chem. 2020). This will allow us to identify an enzyme thatmediates the sialylation.
Mechanisms regulating the levels of GPI biosynthesis

Figure 1. Germline PIGB mutation and 70kb deletion became homozygous by CNLOH.
Upper: Proband’s and mother’s blood granulocytes contained PNH cells.
Lower: One and four PNH clones with CN-LOH regions of different lengths were found in proband and mother,respectively. (Adopted from Langemeijer, S. et al, Blood Adv., 2020, 4(22): 5755-5761)

Recent Publications
⿎⿎ Langemeijer S, Schaap C, Preijers F, Jansen JH, Blijlevens N, Inoue N,
Muus P, Kinoshita T and Murakami Y. Paroxysmal nocturnal
hemoglobinuria caused by CN-LOH of constitutional PIGB mutation
and 70-kb microdeletion on 15q. Blood Adv., 4: 5755-5761 (2020).
⿎⿎ Wang Y, Maeda Y, Liu YS, Takada Y, Ninomiya A, Hirata T, Fujita M,
Murakami M & 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, et al. 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 & Kinoshita T. Free,
unlinked glycosylphosphatidylinositols on mammalian cell surfaces
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, et al. Identification of a Golgi GPIN-acetylgalactosamine transferase with tandem transmembraneregions in the catalytic domain. Nat. Commun. 9: 405- (2018).

17

Immunology

Immunopathology
Atsushi Kumanogoh, MD/PhD
Professor

Atsushi Kumanogoh

Assistant Professor
Research Assistants
Support Staff

Jun Fujimoto
5
5

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’ such as
semaphorins and their receptors. Semaphorins were originallyidentified as axon-guidance molecules that function during
neuronal development. However, cumulative evidence
indicates that semaphorins also participate in immune
responses, both physiological and pathological, and they
are now considered to be potential diagnostic and/or
therapeutic targets for a range of diseases. Beyond suchbasic implications, we are trying to apply the findings fromthis proposed study to diagnosis/therapy for humanimmunological disorders, such as autoimmunity, allergy,
immune deficiency, cancer/metastasis, and neurodegenerative
diseases. We recently focus on the cross-talk among neuronal,
immune and metabolic systems since some of thesemaphorins’ expression are regulated by a metabolic sensor,
mTOR, in which we investigated the biological and pathologicalsignificance of Lamtor1/p18, an amino acid sensor localized18

at the lysosome.
Lysosomes are involved in nutrient sensing via themechanistic target of rapamycin complex 1 (mTORC1).
mTORC1 is tethered to lysosomes by the regulator complex,a heteropentamer in which Lamtor1 wraps around Lamtor.
Although the Ragulator complex is required for cell
migration, the mechanisms by which it participates in cell
motility remain unknown. Here, we show that lysosomes
move to the uropod in motile cells, providing a platform
where Lamtor1 interacts with the myosin phosphatase Rhointeracting protein (MPRIP) independently of mTORC1 andinterferes with the interaction between MPRIP and MYPT1, a
subunit of myosin light chain phosphatase (MLCP), thereby
increasing myosin II–mediated actomyosin contraction.
Additionally, formation of the complete Ragulator complex isrequired for leukocyte migration and pathophysiologicalimmune responses. Together, our findings demonstrate thatthe lysosomal Ragulator complex plays an essential role inleukocyte migration by activating myosin II through interactingwith MPRIP.

Figure. Schematic of the mechanism of regulation of myosin II activity by the Ragulator complex
In immotile cells (upper), the Ragulator complex localized to lysosomes is preferentially distributed in the perinuclear region (left), and
MPRIP anchors MLCP on myosin–actin bundles by binding MYPT1, a subunit of MLCP, resulting in suppression of MLC phosphorylation(right). In motile cells exposed to chemokines (lower), the lysosomes bearing the Ragulator complex moves to the uropod (left), where theRagulator complex interacts with MPRIP. This interaction interferes with the interaction between MPRIP and MYPT1 and decreases MLCP
activity, thereby increasing MLC phosphorylation. Consequently, cell motility is facilitated (right). MLC, myosin regulatory light chain; MLCP,
myosin light chain phosphatase; MPRIP, myosin phosphatase Rho-interaction protein; MYPT1, myosin phosphatase-targeting subunit 1;
PP1δ, a catalytic subunit of the type I protein serine/threonine phosphatase family; M20, 20-kDa small subunit; p14, Lamtor2; MP1,
Lamtor3; p10, Lamtor4; HBXIP, Lamtor5.

Recent Publications
⿎⿎ Nakatani T, Tsujimoto K, Park J, Jo T, Kimura T, Hayama Y, Konaka H,
Morita T, Kato Y, Nishide M, Koyama S, Nada S, Okada M, Takamatsu
H, Kumanogoh A. The lysosomal Ragulator complex plays an essential
role in leukocyte trafficking by activating myosin II. Nat Commun. inpress.

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, et al The activated conformation of integrin β.
Nat Med 23: 1436-1443 (2017).

⿎⿎ Tsuda T, Nishide M, Maeda Y, Hayama Y, Koyama S, et al. Pathologicaland Therapeutic Implications of Eosinophil-Derived 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, et al. Semaphorin 6D reverse signaling controls macrophagelipid metabolism and anti-inflammatory polarization. Nature Immunol.
19: 561-570 (2018).

19

Immunology

Immunochemistry
Hisashi Arase, MD/PhD
Professor

Hisashi Arase

Associate Professor
Assistant Professor
Postdoctoral Fellows
Research Assistants
Visiting Scientists
Support Staff

Masako Kohyama
Wataru Nakai
3515A) Host pathogen interaction mediated by paired
receptorB) Misfolded proteins complexed with MHC class II
molecules are targets for autoimmune diseases.

Paired receptors are composed of activating andinhibitory receptors. We found that PILRα, one of paired
receptors, plays an important role in the regulation of
immune response (Nat. Immunol. 2012; Int. Immunol. 2015;
Eur. J. Immunol. 2016) as well as HSV-1 infection (Cell 2008;
J. Virol. 2009). Similarly, Siglec-4 (myelin-associatedglycoprotein, MAG), one of paired receptors, is involved in
VZV infection (PNAS 2010; J. Biol. Chem. 2015). Thesefindings suggested that paired receptors are involved in
immune regulation as well as viral infection.

MHC class II allelic polymorphisms are associated withsusceptibility to many autoimmune diseases. However, it hasremained unclear how MHC class II molecules are involvedin autoimmune disease susceptibility. We found that cellular
misfolded autoantigens are rescued from protein
degradation by MHC class II molecules (Int. Immunol.
2013). Furthermore, we found that misfolded proteinscomplexed with MHC class II molecules are targets for
autoantibodies in autoimmune diseases such as rheumatoid
arthritis, antiphospholipid syndrome, and ANCA-associatedvasculitis (PNAS 2014; Blood. 2015; Int, Immunol. 2019; Br.
J. Dermatol. 2017; Arthritis. Rheumatol. 2017; Arthritis.
Rheumatol. 2021). 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 are involved in the pathogenicity of
autoimmune diseases (Figure 2).

LILR is another type of paired receptor family. We foundthat activating LILRA2 recognizes abnormal immunoglobulins
cleaved by microbial proteases but not normal
immunoglobulins. LILRA2 seems to be a sensor to detect
immunoglobulin abnormalities in microbial infection (Nature
Microbiology 2016). On the other hand, we found that
RIFINs, products of a multigene family of Plasmodium
falciparum, are similar to MHC class I molecules, bind to
inhibitory LILRB1 and LILRB2, and downregulate immune
response. These findings suggest that binding of RIFIN to
LILRB1 plays an important role in immune evasion by
Plasmodium falciparum (Figure 1. Nature 2017; Nature 2020;
BBRC 2021).
20

Figure 1. Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors
Plasmodium falciparum evades host immune response by engaging inhibitory
receptors using RIFINs (Nature 2017; Nature 2020; BBRC 2021).

Figure 2. Misfolded proteins transported to the cell surface by MHC class II molecules aretargets for autoantibodies.
Cellular misfolded proteins are generally degraded in the cells and are not transported tooutside the cells. Therefore, misfolded proteins transported to the cell surface by MHC class II
molecules may be recognized as ‘neo-self’ antigens by the immune system, which might initiateaberrant immune responses to the self-antigens (Int. Immunol. 2013; PNAS 2014; Blood 2015,
Br. J. Dermatol. 2017; Arthritis Rheumatol. 2017, Arthritis Rheumatol. 2020).

Recent Publications
⿎⿎ Sakoguchi A, Saito F, Hirayasu K, Shida K, Matsuoka S, Itagaki S,
Nakai W, Kohyama M, Suenaga T, Iwanaga S, Horii T, Arase H.
Plasmodium falciparum RIFIN is a novel ligand for inhibitory immune
receptor LILRB2. Biochem Biophys Res Commun. 2021 548: 167-173(2021).
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 by Autoantibodies
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 microbiallycleaved immunoglobulins. Nature Microbiology 1: 1-7 (2016).
⿎⿎ Jin H, Arase N, Hirayasu K, Kohyama M, Suenaga T, Saito F, et al.
Autoantibodies to IgG/HLA-DR complexes are associated withrheumatoid arthritis susceptibility. Proc. Natl. Acad. Sci. USA. 111:
3787-3792 (2014).

21

Tadamitsu Kishimoto, MD/PhD
Professor

Tadamitsu Kishimoto

Associate Professor

Shigeru Hashimoto
Sujin Kang
42
2

Postdoctoral Fellows
Research Assistants
Support Staff

endocytosis independently of IFN-β and NF-κB signaling. In

lnc-AU021063 enhances the levels of a tribble homolog 3(Trib3) protein in response to IL-6 by protecting thehuman macrophages, lipopolysaccharide LPS-bound TLR4

degradation of Trib3 and activates a MAPK pathway which

endocytosis activated noncanonical phosphorylation of

result in exacerbation of invasion and metastasis of BC in

STAT1 at Thr749, which altered its DNA target motif. Thr749–

vitro and in vivo. Taken together, our study provides evidence

phosphorylated STAT1 promoted the expression of the geneencoding AT-rich interactive domain-containing protein 5A(ARID5A), which stabilizes interleukin 6 (IL6) mRNA.
Moreover, Thr749–phosphorylated STAT1 directly enhancedof the existence of an aberrant IL-6/Arid5a/lnc-AU021063
signaling that leads to BC invasion and metastasis throughTrib3 induction, which could be novel therapeutic targets in
BC malignancies.

the transcription of the gene encoding IL12B (Metwallyet.al., Sci Signal, 2020). In order to understand the biologicalsignificance of Thr749 (Thr748 in mice) phosphorylation of
STAT1, we generated mice with a specific non-phosphomimic (T748A) mutation in STAT1 (KI mice). Intriguingly,
STAT1 KI mice showed enhanced survival against LPSinduced sepsis compared to WT littermates indicating anovel fundamental role of Thr748 phosphorylation of STAT1 inshaping the host immune response against sepsis.
Currently, we are investigating the detailed mechanisms of
how Thr748/9 phosphorylation of STAT1 regulates the hostimmune response in the biological contexts of infection andinflammation.

Arid5a regulation of invasion and metastasis of breast
cancer through a novel noncoding RNA under IL-6
signaling

22

Role of endothelial homeostasis in cytokine storm andrelated diseases

Distinct STAT1 phosphorylation confers distinct DNA
binding and gene-regulatory properties

Cytokine release syndrome (CRS) is a life-threatening
complication induced by systemic inflammatory responses
to infections, including bacteria and SARS-CoV-2, andchimeric antigen receptor (CAR) T-cell therapy. There are
currently no immunotherapies with proven clinical efficacy
and knowledge of the molecular details of CRS
pathogenesis is limited. We found that interleukin-6 (IL-6)
signaling induced pro-inflammatory cytokine production andactivated the coagulation cascade during CRS. Patientsdiagnosed with CRS, including those with sepsis, acuterespiratory distress syndrome, or burns, had common
manifestations: strikingly elevated levels of four proinflammatory cytokines, IL-6, IL-8, MCP-1, and IL-10, and a
coagulation cascade activator, plasminogen activator
inhibitor-1 (PAI-1). Endothelial IL-6 trans-signaling formed an
inflammation circuit for robust IL-6, IL-8, and MCP-1
production and promoted PAI-1 production, and blockade
by a humanized monoclonal antibody, tocilizumab, blunted
activation of endothelial cells in vitro. Severe SARS-CoV-2
patient plasma also exhibited increased levels of the four
cytokines and PAI-1, which were restored by tocilizumab
treatment. Thus, a unique pattern of immune dysregulationin CRS was characterized by excessive IL-6-mediatedcytokines and PAI-1 production, associated with hyperinflammation (Kang et al, PNAS, 2020 and Figure 1).

Lipopolysaccharide (LPS)–induced toll-like receptor 4(TLR4) endocytosis has emerged as a key step for the
production of interferon (IFN)-β, which activates thetranscription of antiviral response genes through Janus
kinase (JAK)-activated Tyr701 phosphorylation of signaltransducer and activator 1 (STAT1) signaling. TLR4 endocytosis
also promotes proinflammatory cytokines production, atleast in part through mediating a late-phase activation of
nuclear factor (NF)-κB. Our research showed that STAT1

Breast cancer (BC) metastasis is the major cause of
mortality in women worldwide. Arid5a functions as an RNAbinding protein and a transcription factor, and plays crucial
roles in the development of inflammatory and autoimmune
diseases via the augmentation of IL-6 signaling. However,
the role of Arid5a in hallmark of cancer such as invasion andmetastasis remain elusive. We found IL-6-Arid5a axis
transcriptionally upregulates a novel long noncoding RNA(lncRNA)-AU021063 which contributes to the promotion ofinvasion and metastasis of BC cells. Mechanistically, thisImmunology

Immune Regulation

serves as a proinflammatory effector downstream of TLR4

Arid5a acts as a dual regulator in mesenchymal tumors
to generate an immunosuppressive microenvironment
The acquisition of mesenchymal traits leads to immune
evasion in various cancers, but the molecular mechanisms
involved remain unclear. We identified Arid5a as a regulatorof both immune-evasion and mesenchymal phenotypes ofpancreatic ductal adenocarcinoma (PDAC) and colorectal
cancer (CRC). Arid5a expression was increased in both celllines. Arid5a expression was increased upon TGF-β-induced
EMT. Arid5a promotes in vivo tumor growth in an extrinsic
immune cell-dependent manner by disrupting the balanceof anti-tumor and suppressive tumor-infiltrating leukocytes inthe tumor microenvironment (TME). In Arid5a-deficient celltumors, gMDSC and Treg infiltration was suppressed, but
cytotoxic T-lymphocyte recruitment was promoted. Arid5a
augments Ido1 and Ccl2 expression by the posttranscriptional stabilization of their mRNAs by binding totheir 3’-UTRs. Our results indicate that Arid5a acts as a dual
regulator in mesenchymal malignant tumors. Arid5a induces
the suppressive effects of Ido1 on T-cells by reducing
intratumoral Trp concentrations, and promotes Treg
differentiation/activation. Arid5a induces Ccl2 expression in
the TME and recruits immunosuppressive cells (Tregs and
gMDSCs) to the TME. Our findings indicates that Arid5a is apromising and possible drug target for tumor immunotherapy.

Recent Publications
⿎⿎ Kishimoto T, Interleukin-6: From arthritis to CAR-T cell therapy and
COVID-19. Int Immunol. dxab011. doi: 10.1093/intimm/dxab011 (2021).
⿎⿎ Kang S et al, IL-6 trans-signaling induces plasminogen activator
inhibitor-1 from vascular endothelial cells in cytokine release
syndrome. Proc Natl Acad Sci U S A. 117(36): 22351-22356 (2020).
⿎⿎ Kang S et al, Historical overview of the interleukin-6 family cytokine. J
Exp Med. 217(5): e20190347 (2020).

Figure 1. How Actemra© suppresses inflammation; IL-6 in blood
promotes thrombus formation via PAI-1. By suppressing IL-6,
Actemra prevents reduces the severity of the pneumonia
caused by the cytokine storm.

⿎⿎ Metwally H et al. A Non-canonical STAT1 Phosphorylation Expands its
Transcriptional Activity into Promoting LPS–induced IL-6 and IL-12p40
Expression. Sci. Signal. eaay0574 (2020).
⿎⿎ Parajuli G et al. Arid5a augments tryptophan metabolism andchemokine expression to promote the immune evasion ofmesenchymal subtypes of pancreatic and colorectal cancer. Cancer
Immunol Res 2021 (in press).

23

function, particularly the inconsistency in phenotypes of two

differentiation but suppress plasma cell differentiation in GC.

different RUBCN KO mice, but also posit that focusing

Our findings may not only explain the controversy of RUBCN

vaccine design efforts on enhancing autophagy in B cells.

Immunology

Immune Regulation

deficiency enhances autophagy to promote Bmem cell

Hitoshi Kikutani, MD/PhD
Professor

Hitoshi Kikutani

Associate Professor
Assistant Professor
Research Assistants
Support Staff

Shuhei Sakakibara
Chao-Yuan Tsai
1
1Figure 1. Our mouse model for studying germline precursors of pathogenic anti-dsDNA antibodies
derived from SLE patients.

Characterization of low-affinity progenitor B cells forpathogenic anti-dsDNA antibody-producing cells derived
from SLE
Our previous study has demonstrated that low-affinity
anti-ssDNA B cells can acquire high-affinity to both ss- anddsDNA after only one or two mutations (Sakakibara S et al.,2017). This raises the questions of whether low-affinity antissDNA precursor B cells are subjected to immunologicaltolerance and in what conditions they undergo clonal
expansion and affinity mutation to differentiate into highaffinity anti-dsDNA antibody-producing cells. To addressthese questions, we generated a site-directed knock-in (KI)mouse line, G9gl, which carries unmutated IgH and L chainsderived from one of the SLE anti-DNA antibody clones(Figure 1). We confirmed that the G9gl BCR-expressing B
cells react to ssDNA, but not dsDNA. These low-affinity antissDNA precursor B cells exhibited anergic phenotype suchas lowered BCR expression and shorter half-life in vitro,
which caused their exclusion from GC reactions induced inseveral experimental settings. When the KI mice were
immunized with bacterial DNA-conjugated methylated BSA,
G9gl B cells were found in the memory B cell compartment,
but not GC B. These G9gl-expressing IgG+ memory B cells

24

did not acquire somatic mutations to augment their anti-DNA
reactivity. Therefore, clonal anergy tightly regulates lowaffinity anti-DNA precursor B cells not to evolve into highaffinity anti-dsDNA antibody-producing cells.

The role of enhanced autophagy in memory B celldifferentiation
Autophagy is required for a variety of cellular functions
including the differentiation of pre-B and B-1a cells andsurvival of long-lived memory and plasma B cells. Since
RUBCN (A RUN domain and cysteine-rich domain
containing, Beclin 1-interacting protein), a negative regulatorof canonical autophagy, was strongly induced in germinal
center B (BGC) cells and activated B cells, B cell-specific
RUBCN knockout (RUBCN B-KO) mice were generated to
study the function of RUBCN in B cells. The comparative
study of RUBCN B-KO and wild-type (WT) mice revealed
that, in addition to originally reported 130-kD RUBCN(RUBCN130), B cells also expressed a 100-kD isoform(RUBCN100) that appeared to be translated from analternative start-codon of the same messenger RNA.
Interestingly, B cells of RUBCN B-KO mice lacked theexpression of RUBCN130 as expected but still expressed
100

130

RUBCN . Although selective RUBCN deficiency did notaffect the GC function, the number of antigen-specific IgG1+
memory B (Bmem) cells was higher in RUBCN B-KO than WT
mice. In contrast, the number of ASCs in the bone marrows
of RUBCN B-KO mice was lower than that of WT mice. Suchincrease of Bmem cells also contribute to the increased recall

Figure 2. Alteration of B cell fate by RUBCN isoforms through regulation of mTOR activity and autophagy.

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).

responses in RUBCN B-KO mice. Additionally, the mTOR
activity was down-regulated in the BGC cells of RUBCN B-KO
mice compared to that of WT mice. Although atg7 deletionin B cells did not affect BGC and Bmem cell differentiations, theincrease of Bmem cells was cancelled in RUBCN/ATG7
B-DKO mice. These results indicate that selective RUBCN130

25

the intestine, and mice lacking these molecules are sensitive

ATP-hydrolyzing ectoenzymes, such as E-NPP3, E-NTPD1,

to allergic or chemically-induced colitis. Thus, regulation ofand E-NTPD2, are expressed in innate/adaptive immune

extracellular ATP by mucosal ATP-hydrolyzing ectoenzymes

cells (mast cells/basophils/regulatory T cells/intraepithelial

is required for appropriate immune responses in thelymphocytes/macrophages), fibroblasts, and glial cells in

intestine.

Immunology

Mucosal Immunology

of intestinal immune homeostasis. Other than epithelial cells,

Kiyoshi Takeda, MD/PhD
Professor

Kiyoshi Takeda

Associate Professor
Assistant Professor

Hisako Kayama
Ryu Okumura
Mari Murakami
Taiki Sakaguchi
2
102

Postdoctoral Fellows
Research Assistants
Support Staff

We previously demonstrated that extracellular adenosine
triphosphate (ATP) released by microbes contributes to
induction of Th17 cells by activating CD70+ dendritic cells in
the intestinal lamina propria through the P2X and P2Y
receptors. To avoid inappropriate immune reactions in theintestine, luminal ATP is strictly controlled by epithelial ATPhydrolyzing ectoenzymes, such as ectonucleotide
pyrophosphatase/phosphodiesterases (E-NPPs) andectonucleoside triphosphate diphosphohydrolases(E-NTPDases). E-NTPDases convert ATP to ADP as well as
ADP to AMP, while E-NPPs hydrolyze ATP to AMP. We havefound that E-NTPD7, which highly expressed in small
intestinal epithelial cells, inhibits excessive Th17 responses
through hydrolysis of luminal ATP. In addition, E-NPP3 on theintestinal epithelial cells depresses the apoptosis ofplasmacytoid dendritic cells (DCs) in the small intestine andPeyer’s patches.
Although intestinal phagocytes, which express P2X/P2Y
receptors, have some protective effects, these cells can alsofunction in pathological conditions. Thus, the number ofintestinal phagocytes and their functions are finely tuned toprevent intestinal inflammation.

26

However, the molecular mechanism underlying theregulation of the luminal ATP concentration in the large
intestine remains poorly understood. Among E-NTPD and
E-NPP family members, E-NTPD8 was highly expressed incolonic epithelial cells. Therefore, we generated Entpd8 -/mice to assess its physiological roles. A higher level ofluminal ATP in the colon was observed for Entpd8 -/- micethan wild-type mice. Entpd8 -/- mice developed more severe
DSS-induced colitis accompanied by increased numbers of
IL-17-producing CD4+ T cells and neutrophils in the lamina
propria of the colon compared with those in wild-type mice.
In this context, the ablation of neutrophils by treatment with
anti-Gr-1 antibodies, but not T cells, mitigated the severity ofcolitis in Entpd8 -/- mice. In addition, the introduction of P2rx4
deficiency into Entpd8 -/- mice ameliorated DSS-induced
colitis with lower levels of neutrophils accumulation.
Extracellular ATP promoted glycolysis in neutrophils througha P2X4 receptor-dependent Ca2+ influx, which is linked toprolonged survival and elevated ROS production in thesecells. Thus, E-NTPD8 limits intestinal inflammation bycontrolling metabolic alteration toward glycolysis via theP2X4 receptor in neutrophils (Figure 1).

E-NTPD8 prevents neutrophil-mediated intestinal
pathology

Immunomodulatory effects of ATP-hydrolyzing
ectoenzymes

In the small intestine, E-NTPD7 and E-NPP3 control
luminal ATP concentration, which contributes to theregulation of innate and adaptive immune responses.

A series of studies on ATP-hydrolyzing ectoenzymes
expressing in intestinal epithelial cells has shown that control
of luminal ATP concentration is essential for the maintenance

Figure 1. E-NTPD8 prevents neutrophil-mediated colitis by hydrolyzing luminal ATP.
E-NTPD8 in colonic epithelial cells maintains gut homeostasis through hydrolysis
of luminal ATP produced by commensal bacteria (left). An increased level ofluminal ATP caused by lack of E-NTPD8 modulates neutrophil physiology andleads to prolonged survival and enhancement of ROS production, through P2X4mediated promotion of glycolysis, which links to aggravation of colitis (right).

Recent Publications
⿎⿎ Sakaguchi T, Okumura R, Ono C, Okuzaki D, Kawai D, Okochi Y,
Tanimura N, Murakami M, Kayama H, Umemoto E, Kioka H, Ohtani T,
Sakata Y, Miyake K, Okamura Y, Baba Y, Takeda K: TRPM5 negatively
regulates calcium-dependent responses in lipopolysaccharidestimulated B lymphocytes. Cell Reports 10: 107755 (2020).
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 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. GPR31-dependent
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).

27

Shimon Sakaguchi, MD/PhD

28

Professor

Shimon Sakaguchi

Associate Professor
Assistant Professor
Postdoctoral Fellows
Research Assistants
Visiting Scientists
Support Staff

Kenji Ichiyama
Yusuke Takeshima
4
2
13
7

Our laboratory studies: (i) the cellular and molecularbasis of immunologic self-tolerance, in particular the roles ofregulatory T cells; (ii) the strategy for eliciting effective
immune responses to autologous tumor cells, or inducing
immunologic tolerance to organ transplants, by manipulating
the mechanism of immunologic self-tolerance; and (iii) the
cause and pathogenetic mechanism of systemic
autoimmune diseases, such as rheumatoid arthritis, byutilizing an animal model that we developed.
Treg cells, which specifically express the transcription
factor Foxp3, are actively engaged in the maintenance of
immunological self-tolerance and homeostasis. Furthermore,
the establishment of the Treg-specific epigenetic landscapeand the transcription factor network involving FoxP3 coordinate
Treg development and function. While FoxP3 functionallyacts as a strong repressor of Il2, Ifng, and other genes upon
Treg activation, the Treg epigenome encompassing Tregspecific CpG hypomethylation, chromatin accessibility andhistone modifications, reinforces activation of enhancers,
especially super-enhancers, associated with Treg signature
genes such as Foxp3, Cd25 and Ctla4, and stabilizes theirexpression. Notably, the establishment of such Treg-specificepigenome is FoxP3-independent and a large portion of
Treg-specific super-enhancers are concurrently activated at
the precursor stage, prior to FoxP3 expression (Kitagawa etal., 2017). These findings suggest that genetic and epigenetic
alterations at Treg function-associated genes may becausative of autoimmune and other inflammatory diseases
by affecting Treg development and function (reviewed in
Sakaguchi 2021).
Mutations of Treg signature genes, such as FOXP3,

IL2RA (CD25) and CTLA4, indeed impair Treg development
and function and consequently produce in humans severe
autoimmune diseases reminiscent of murine FoxP3
deficiency. FOXP3 mutations, in particular, cause profoundTreg-specific deficiency/dysfunction, resulting in IPEX
(Immune dysregulation, Poly-endocrinopathy, Enteropathy,
X-linked) syndrome accompanying a variety of autoimmune
diseases (e.g., type 1 diabetes and autoimmune thyroiditis),
inflammatory bowel disease, and allergy in infants. In
contrast to such monogenic “Tregopathies” with distinct Treg
deficiency or dysfunction, functional Treg anomalies foundin common polygenic autoimmune diseases (such as type 1
diabetes and rheumatoid arthritis), which afflict ~10% of the
population worldwide, have been tantalizingly equivocal andcontroversial, in particular, over whether a detected anomaly/
variation is a “cause” or a “consequence” of autoimmunity.
Meanwhile, genome-wide association studies (GWAS) of
common autoimmune diseases have revealed that ~60% of
autoimmune-causal single nucleotide polymorphisms (SNPs)
are mapped to non-coding enhancer regions of immune
cells, especially CD4+ T cells. This raises a critical questionof whether such autoimmune SNPs, for example, found at
the CD25 or CTLA4 locus in type 1 diabetes, should affectCD4+ helper T cells or CD4+ Tregs as a gain-of- or loss-offunction variant, respectively.
This year, we addressed the issue by characterizing
genome-wide epigenetic profiles of human Tregs and CD4+
Tconvs in naïve and activated states. We found the presenceof Treg-specific distinct CpG hypomethylated regions at
Treg signature gene loci (e.g., FOXP3, CD25, and CTLA4),
which are largely included in Treg-specific super-enhancers

acting as expression quantitative trait loci (eQTLs)

and other epigenetic changes (Ohkura et al., 2020). Notably,

controlling endogenous Treg development and function. The

autoimmune SNPs are highly (~9-fold) enriched in Treg-

results suggest the altered function or development ofspecific DNA demethylated regions present in naïve Tregs,

natural Treg cells holds the key to susceptibility to common

compared with activation-induced demethylated regions.

autoimmune diseases (Figure 1). Further attempts to link

The findings indicate that many causative autoimmune SNPs
are present at enhancer regions of Treg function-associatedgenes such as CD25 and CTLA4 (Ohkura et al., 2020),

GWAS and Treg immunobiology may elucidate a key
contribution of Tregs in common autoimmune diseases.

Immunology

Experimental Immunology

and closely associated with Treg-specific gene transcription

Figure 1. Autoimmune SNPs are enriched in naïve Treg cell-specific CpG hypomethylated regions.
Single-nucleotide polymorphisms (SNPs) associated with common autoimmune diseases were predominantly
enriched in CpG demethylated regions (DRs) specifically present in naïve Treg cells but much less enriched in
activation-induced DRs common in Tconv and Treg cells.
Adapted from Ohkura, Yasumizu et al., Immunity, 2020.

Recent Publications
⿎⿎ Sakaguchi S. Taking regulatory T cells into medicine. J. Exp. Med.
218(6): e20210831 (2021).
⿎⿎ Kawakami R, Kitagawa Y, Chen KY, Arai M,Ohara D, Nakamura Y,
Yasuda K, Osaki M, Mikami N, Lareau CA, Watanabe H, Kondo G,
Hirota K, Ohkura N, and Sakaguchi S. Coordinated activation of
distinct Foxp3 enhancer elements for Treg development, maintenance,
and immunological self-tolerance. Immunity 54(5): 947-961 (2021).
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 52(6): 1119-1132 (2020).

⿎⿎ Mikami N, Kawakami R, Chen KY, Sugimoto A, Ohkura N, Sakaguchi
S. Epigenetic conversion of conventional T cells into regulatory T cellsby CD28 signal deprivation. Proc Natl Acad Sci U S A. 117(22): 1225812268 (2020).
⿎⿎ 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, 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. 217(2): e20191009 (2020).

29

Immunology

Cell Signaling
Takashi Saito, PhD
Professor

Takashi Saito

Support Staff

1

The objective of our team is to determine the molecularmechanisms of T cell activation, differentiation and function.
Ultimately, we wish to elucidate the onset of and to modulate
T cell function/activation to prevent immune diseases suchas autoimmunity and allergic inflammation. For this purpose,
we analyzed the regulation of T cell activation/function froma signaling perspective.

1. Regulation of T cell activation through inhibitory
checkpoint receptors
Our initial finding that TCR-microclusters (MC) are
dynamically generated and initiate T cell activation led us toanalyze the dynamics of signaling molecules at the immune
synapse.
Similar to our previous studies of CTLA4 and PD-1, we
analyzed the dynamic regulation of another inhibitory costimulation receptor, LAG3. LAG3 was also colocalized with
the TCR-MC to mediate an inhibitory signal for T cell
activation. The structural requirement of LAG3 for cluster
formation and inhibitory function was analyzed. Various
forms of anti-LAG3 together with anti-PD1 Abs were analyzed
for the capacity to augment T cell activation for the purposeof checkpoint therapy. Our analyses provide a dynamic viewof negative regulation and define inhibitory mechanisms of
LAG3.
30

2. Inhibitory mechanism of T cell activation throughphosphatases
We analyzed the negative regulation of T cell activation
not only by checkpoint inhibitory receptors but also by
receptor-associated phosphatases, particularly by
autoimmune-related PTPN22. Its deficiency resulted inenhanced activation and an increase in effector/memory T
cells. PTPN22 generates clusters which are associated with
TCR-MC. A PTPN22 mutant causing susceptibility to
autoimmune diseases was found to be defective in recruitment
to the TCR-MC. Analysis of the associated proteins revealed
that PTPN22 was recruited to the TCR-MC to comprise an“inhibitory complex” with other inhibitory molecules to inhibit
T cell activation. These studies help define the autoimmune
susceptibility caused by the mutation.

3. Regulation of T cell function by innate signaling
We also analyzed the modulation of T cell activation andfunction by innate signals. We previously found that T cells
were activated by co-stimulation of TCR and TLRs. TLR2, inparticular, activated effector T cells (as Th1) but not naïve T
cells in the absence of TCR stimulation. We then analyzed
the reason why naïve T cells fail to respond to TLR2. We
found that naïve T cells failed to respond to TLR2 stimulation
due to the defective expression of TIRAP. TIRAP is expressed
upon stimulation through mTORC1 activation via TCR or IL-2
signaling (Figure).

Figure. TLR2-Mediated T-Cell Activation is dependent on TIRAP Expression in T cells and regulated by
mTORC1 signaling.
Effector T cells are activated by TLR2 signals in the absence of TCR signals. TLR2-mediated activation ismediated through TIRAP, whose expression is induced by mTOR signals through TCR or IL-2 signals,
whereas naïve T cells are not activated by TLR2 due to the lack of TIRAP expression.

Recent Publications
⿎⿎ Imanishi T, Unno M, Kobayashi W, Yoneda N, Akira S and Saito T.
mTORC1 signaling controls TLR2-Mediated T-cell activation by
inducing TIRAP expression. Cell Reports. 32: 107911-107911 (2020).
⿎⿎ Imanishi T and Saito T. T Cell co-stimulation and functional modulation
by innate signals. Trends lmmunol. 41: 200-212 (2020).

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 Mei S, et al. Inhibition of T cell activation and function by theadaptor protein CIN85. Sci Signal. 12: eaav4373 (2019).

⿎⿎ Sasaki T, Yajima T, Shimaoka T, Ogawa S, Saito T, Yamaoka K,
Takeuchi T and Kubo M. Synergistic effect of lgG4 antibody and CTLs
causes tissue inflammation in lgG4-related disease. Int. lmmunol. 32;
163-174 (2020).

31islands in the IRF4 gene locus that were demethylated

immunization. In vitro experiments showed that Tet double

specifically in plasma cells in a Tet2/Tet3-dependent manner.

knockout B cells proliferated normally and were capable ofThus, Tet-dependent demethylation of CpG islands isgenerating cells with IRF4int, but not with IRF4hi. IRF4

essential for maintenance of high IRF4 expression, but notoverexpression rescued the defects of Tet double knockout

for initiation of IRF4 expression.

B cells in plasma cell differentiation. We also identified CpG

Immunology

Lymphocyte differentiation

failed to differentiate into plasma cells upon TD and TI-II

Tomohiro Kurosaki, MD/PhD
Professor

Tomohiro Kurosaki

Associate Professor

Wataru Ise
Takeshi Inoue
Ryo Shinnakasu
112
4Assistant Professor
Postdoctoral Fellows
Research Assistants
Visiting Scientists
Support Staff

Generation mechanisms of GC-dependent memory B
cells

32

Memory B cells and long-lived plasma cells (LLPCs) areresponsible for effective long-term immunity against
pathogens. The majority of these cells responding to T celldependent antigens are generated from the germinal center(GC) reaction. Indeed, memory B cells emerge from the GC
as recirculating cells, and upon secondary antigen
challenge, they are primed to elicit rapid antibody
responses.
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 tobe primarily composed of specificities possessing thehighest affinity for the primary antigen. On the other hand,
we and others have recently demonstrated that GC B cellswith weak T cell help are preferentially recruited into thememory B cell compartment. Since GC B cells receiving
weak T cell help are generally thought to undergo apoptosis,
our model has raised the question of how precursor cells tomature memory B cells are prevented from dying and able
to differentiate into memory B cells.
To address this question, after identifying a memoryprone population (pro-memory and pre-memory B cells), we
have identified key features for these precursor cells todevelop to the mature memory B cells. We found that promemory B cells were starting upregulation of Bcl2 and cell-surface BCR, contributing to development of mature
memory B cells. Furthermore, we provide evidence that
downregulation of Bcl6 in pro-memory B cells could be oneof the potential regulatory mechanisms to increase Bcl2 andBCR. Together, we propose a model in which weak help
from T cells together with provision of an increased survival
signal are key for GC B cells to adopt a memory B cell fate,
and that, in regard to the increased survival signal, stepwise
decreases in Bcl6 expression (pro-memory, pre-memory,
mature memory B cells) play a key role.

Figure. During germinal center (GC) reaction, light zone B cells are selected to differentiate intomemory B cells, plasma cells, or to re-enter the dark zone. We have identified and characterizeda small GC population of precursors for memory B cells, and found that the GC B cells withlower mTORC1 activity resulting from receiving weak T cell help and those with a higher
survival signal from surface B cell receptors favor a memory B cell fate.

Epigenetic regulation on B cell differentiation
Differentiation from B cells into plasma cells is a lineageswitch, thus being accompanied by dramatic changes ingene expression, which extinguish B-cell identity but provide
the ability to constantly secrete large amounts of antibodies.
The key transcription factors, IRF4, Blimp-1, and Xbp-1, forsuch lineage-decision are well known and their targets havebeen intensively characterized. In contrast, epigenetic
modification during this transition has been relatively uncharacterized. A recent study has revealed that genomic
DNA isolated from plasma cells is overall hypo-methylated,compared to that from naïve B cells. However, the precise
molecular mechanisms by which DNA methylation regulates
plasma cell differentiation are not understood. Hence, we
examined the effects of B cell-intrinsic deficiency of DNA
demethylases, Tet2 and Tet3. Tet double knockout cells

Recent Publications
⿎⿎ Inoue T, Shinnakasu R, Kawai C, Ise W, Kawakami E, Sax N, Oki T,
Kitamura T, Yamashita K, Fukuyama H, Kurosaki T. Exit from germinal
center to become quiescent memory B cells depends on metabolic
reprograming and provision of a survival signal. J Exp Med. 218(1):e20200866 (2021).
⿎⿎ Fujii K, Tanaka S, Hasegawa T, Narazaki M, Kumanogoh A, Koseki H,
Kurosaki T, Ise W. Tet DNA demethylase is required for plasma celldifferentiation by controlling expression levels of IRF4. Int Immunol.
32(10): 683-690 (2020).
⿎⿎ Tanaka S, Ise W, Inoue T, Ito A, Ono C, Shima Y, Sakakibara S,
Nakayama M, Fujii K, et al. Tet2 and Tet3 in B cells are required torepress CD86 and prevent autoimmunity. Nat. Immunol. 21(8): 950961 (2020).

⿎⿎ Fukuyama H, Shinnakasu R & Kurosaki T. Influenza vaccination
strategies targeting the hemagglutinin stem region. Immunol. Rev.
296(1): 132-141 (2020).
⿎⿎ Mesin L, Schiepers A, Ersching J, Barbulescu A, Cavazzoni CB,
Angelini A, Okada T, Kurosaki T & Victora GD. Restricted Clonality and
Limited Germinal Center Reentry Characterize Memory B Cell
Reactivation by Boosting. Cell 180: 92-106 (2020).

33

Immunology

Malaria Immunology
Cevayir Coban, MD
Professor

Cevayir Coban

Malaria, caused by Plasmodium parasites, often leads tosevere complications such as cerebral malaria and death.
The immune pathology causing these complications ispoorly defined. In our lab, we elucidate the mechanisms
involved in the host-Plasmodium parasites interactions (Nature
Reviews Immunology, 2018). Our final goal is to develop
vaccines and drugs against malaria and other infectious
diseases.
In 2020, the treatment of COVID-19 patients byhydroxychloroquine alone or in combination with other drugs
has captured a great deal of attention and triggeredconsiderable debate. Although, the quinoline based-drugs
has led to a spectacular reduction in death from malaria in
the world, however, scientists have been forced to seek
alternative drugs to treat malaria due to the emergence ofchloroquine-resistant parasites in the 1960s (Figure). The

repurposing of hydroxychloroquine against viral infections,various types of cancer and autoimmune diseases has beenongoing for more than 70 years with no clear understandingof its mechanism of action. We have proposed in our recent
review article that further investigation and better
understanding of how chloroquine targets the host's cellularand immune responses is needed (Current Opinion in
Immunology, 2020).
Heparin is a common anticoagulant; however, itsbiological effects are not limited to coagulation and remain
incompletely understood. For example, heparin is used asanti-malarial drug due to its ability to inhibit Plasmodium
parasite invasion of erythrocytes. We have demonstrated an
unexpected finding that heparin has ability to induce neutrophil
extracellular traps (NETs) (International Immunology, 2020).

Figure. Possible mechanism (s) of action of chloroquine during blood stage malaria infection. After invasionof erythrocytes, Plasmodium parasites form their own digestive vacuole (DV), a lysosome-like acidic
compartment important for parasite metabolism and survival. In acidic DVs, the host-hemoglobin isdegraded by parasite proteases for the vital needs, such as amino acids and the free-heme is detoxified byconverting it into insoluble crystals hemozoin. A weak base chloroquine accumulates in DVs, increases DV
pH and binds heme and crystal surfaces, thereby blocks every step of hemozoin formation which eventuallyleads heme toxicity and parasite death. In the absence of hemoglobin degrading proteases hemoglobin
remains undigested and free heme is significantly diminished and the effect of chloroquine on parasites
does not occur. Ineffective presence of chloroquine, on the other hand, may create the chloroquine-resistantparasites via a mutation in P. falciparum chloroquine resistance transporter (PfCRT) (modified from Current
Opinion in Immunology, 2020).

Recent Publications
⿎⿎ Coban C. The host targeting effect of chloroquine in malaria Current
Opinion Immunology 66: 98-107 (2020).

⿎⿎ Coban C, Lee MSJ, Ishii KJ. Tissue-specific immunopathology duringmalaria infection. Nature Reviews Immunology 18(4): 266-278 (2018).

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 32(5): 359-368 (2020).

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 2(12): eaam8093 (2017).

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-mediated early
IgG2c class switching in response to a particulate adjuvant. European
J Immunology 49(9): 1433-1440 (2019).

34

35

Ken J. Ishii, MD/PhD
Professor

Ken J. Ishii

Associate Professor
Assistant Professor
Postdoctoral Fellows
Research Assistants
Visiting Scientists
Support Staff

Kouji Kobiyama
Burcu Temizoz
1
3
2
2

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 biological responses toadjuvants. Such knowledge will enable us to develop novel
concepts, modalities and next generation immuno-preventive
and/or therapeutic agents against infectious diseases, cancer
and allergy as well as other non-communicable diseases.

1) Researches and development of a mRNA vaccine andmonoclonal antibodies against SARS-CoV-2

36

In 2020, the coronavirus disease (COVID-19) pandemic
caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) led to the successful development of two
mRNA-based vaccines, encoding the full length of the viralsurface spike protein, with high efficacy and reasonablesafety. However, reactogenicity, such as fever, caused byinnate immune responses to the vaccine formulation remains
to be improved. To overcome this potential issue, wedeveloped a lipid nanoparticle (LNP)-based mRNA vaccine,
encoding the SARS-CoV-2 spike protein receptor-binding
domain (LNP-mRNA-RBD), that improved immunogenicity
by removing reactogenic materials from the vaccine
formulation in mice and conferred protection against SARSCoV-2 infection. The vaccine is filed for patent, finished forpre-clinical development, and is underway to first in humanclinical trial by the end of March 2021.
In addition to vaccine development, we started togenerate monoclonal antibody against SARS-CoV-2 obtained
from the volunteer recovered from COVID-19. We havefor the adjuvant effect of HP-β-CD when it is administeredcases it is unclear which specific molecules contribute to

intranasally. In contrast, the subcutaneous injection of HP-β-their stimulatory activity. We previously reported that both

CD and the intranasal administration of alum, as a damage-subcutaneous and intranasal administration ofhydroxypropyl-β-cyclodextrin (HP-β-CD), a pharmaceuticalassociated molecular patterns (DAMPs)-inducing adjuvant,or cholera toxin, as a mucosal adjuvant, enhanced humoral

excipient widely used to improve solubility, can act as aneffective adjuvant for an influenza vaccine. However, themechanisms by which mucosal immune pathway is critical
for the intranasal adjuvant activity of HP-β-CD have not been
fully delineated. Here, we show that intranasally administeredHP-β-CD elicits a temporary release of IL-33 from alveolar
epithelial type 2 cells in the lung; notably, IL-33 expression
in these cells is not stimulated following the use of othervaccine adjuvants. The experiments using gene deficient
mice suggested that IL-33/ST2 signaling is solely responsible

immunity in an IL-33-independent manner, suggesting that
the IL-33/ST2 pathway is unique to the adjuvanticity of
intranasally administered HP-β-CD. Furthermore, the releaseof IL-33 was involved in the protective immunity against
influenza virus infection which is induced by the intranasal
administration of HP-β-CD-adjuvanted influenza split
vaccine. In conclusion, our results suggest that anunderstanding of administration route- and tissue-specificimmune responses is crucial for the design of unique
vaccine adjuvants.

Immunology

Vaccine Science

modulate the immunogenicity of vaccines, although in manyobtained several clones with very high affinity, filed for patent,
and now is underway for its efficacy in the animal model.

2) Discovery of Self-Assembling Small Molecules as
Vaccine Adjuvants
Immune potentiators, termed adjuvants, trigger early
innate immune responses to ensure the generation of robustand long-lasting adaptive immune responses of vaccines.
Presented here is a study that takes advantage of a selfassembling small-molecule library for the development of anovel vaccine adjuvant. Cell-based screening of the libraryand subsequent structural optimization led to the discovery
of a simple, chemically tractable deoxycholate derivative(molecule 6, also named cholicamide) whose well-definednanoassembly potently elicits innate immune responses inmacrophages and dendritic cells. Functional andmechanistic analyses indicate that the virus-like assembly
enters the cells and stimulates the innate immune response
through Toll-like receptor 7 (TLR7), an endosomal TLR that
detects single-stranded viral RNA. As an influenza vaccine
adjuvant in mice, molecule 6 was as potent as Alum, aclinically used adjuvant. The studies described here pave
the way for a new approach to discovering and designingself-assembling small-molecule adjuvants against
pathogens, including emerging viruses.

3) IL-33 Is Essential for Adjuvant Effect of Hydroxypropyl-βCyclodexrin on the Protective Intranasal Influenza Vaccination
Vaccine adjuvants are traditionally used to augment andFigure. Summary for the animal experiments.
mRNA vaccine encoding purified LNP-mRNA RBD SARS-CoV2

Recent Publications
⿎⿎ Rizaldy D, Toriyama M, Kato H, Fukui R, Fujita F, Nakamura M, Okada
F, Morita A, Ishii KJ. Increase in primary cilia in the epidermis ofpatients with atopic dermatitis and psoriasis. Exp Dermatol. 30(6):792-803 (2021).

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 inanti-influenza virus immunity. Int Immunol. 32(3): 203-212 (2020).

⿎⿎ Jin S, Vu HT, Hioki K, Noda N, Yoshida H, Shimane T, Ishizuka S,
Takashima I, Mizuhata Y, Beverly Pe K, Ogawa T, Nishimura N,
Packwood D, Tokitoh N, Kurata H, Yamasaki S, Ishii KJ, Uesugi M.
Discovery of Self-Assembling Small Molecules as Vaccine Adjuvants.
Angew Chem Int Ed Engl. 60(2): 961-969 (2021).

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).

37

the PV. This suggests that Irgb6 has a role as a pioneer inthat Irgb6 is capable of binding a specific phospholipid on

the process by which multiple IRG proteins access the PV.

the PV membrane. Furthermore, the absence of Irgb6

Irgb6-deficient mice are highly susceptible to infection by acauses reduced targeting of other effector IRG proteins tostrain of T. gondii avirulent in wild-type mice.

Immunology

Immunoparasitology

involves vacuole ubiquitination and breakdown. We show

Masahiro Yamamoto, PhD
Professor

Masahiro Yamamoto

Associate Professor
Postdoctoral Fellows
Research Assistants
Visiting Scientists
Support Staff

Miwa Sasai
12
1
1

Healthy mammalian hosts activate immune responses
against pathogenic infections. The innate immune response
firstly induces interleukin-12 (IL-12) production by antigenpresenting cells (APCs) such as macrophages and dendritic
cells. This is done via recognition of pathogen-derived
components by microbe pattern recognition receptors suchas Toll-like receptors (TLRs). IL-12 subsequently stimulates
the anti-pathogen type I immune response, wherein naïve
CD4+ or CD8+ T cells become antigen-specific Th1 cells andcytotoxic T cells, respectively, with the help of APCs. Th1
cells, cytotoxic T lymphocytes, and natural killer cells produce
interferon-γ (IFN-γ) to activate the various cell-autonomous
programs targeting vacuolar pathogens.
One of the IFN-γ-induced cell-autonomous programs is
associated with IFN-inducible GTPases, such as p47
immunity-related GTPases (IRGs) and p65 guanylatebinding proteins (GBPs). IRGs and GBPs belong to the
dynamin GTPase superfamily, and can target wide ranges ofbacterial, fungal, and protozoan vacuolar pathogens. In
mice, the IRG protein family consists of three regulator IRG
proteins (Irgm1, Irgm2, and Irgm3) and over 20 effector IRG
proteins and decoys. There are four effector IRG proteins
known to be expressed in mice: Irga6, Irgb6, Irgb10, andIrgd. Regulator IRG proteins harboring GX4GMS in the first38

nucleotide-binding motif (G1) are mainly associated withhost endomembranes, such as the Golgi apparatus andendoplasmic reticulum (ER). Effector IRG proteins possessa universally conserved GX4GKS sequence in the G1 motif,
enabling binding to both GTP and GDP. GTPase activity has

been demonstrated for Irga6 and Irgm3. Regulator IRG
proteins can maintain effector IRG proteins in an inactive
GDP-bound state, potentially preventing the latter frominappropriate activation on host cell membrane-boundedvesicular systems. In their absence, effector IRG proteins
likely form GTP-bound aggregates, and are unable tointeract with the T. gondii PV. There are 11 members of themouse GBP family, all of which have the conserved GTP
binding motifs. GBP mutants lacking GTPase activity areincapable of accumulating at the T. gondii PV membrane(PVM). When these IFN-inducible GTPases are recruited to
the PVM, it becomes vesiculated and disrupted, resulting indeath of the vacuolar pathogen. Thus, GTPase activitydependent IRG and GBP accumulation is well establishedas important for cell-autonomous immunity to vacuolar
pathogens.
The mechanism by which IRG proteins access the T.
gondii PV from the cytosolic compartments can be passive.
This process depends on diffusion from the cytoplasmic
pools rather than active transport involving TLR-mediatedsignaling pathways or microtubule networks. Although IRG
proteins are localized on the PVM within a few minutes of T.
gondii infection, little is known about the mechanism bywhich IRG proteins recognize and destroy the PVM thus far.
This process is important for IFN-γ-induced cell-autonomous
immunity. Among the effector IRG proteins, Irgb6 and Irgb10
are loaded first and most efficiently onto the T. gondii PVM.
Here, we characterized the role of IRG protein Irgb6 inthe cell-autonomous response against T. gondii, which

Figure. Initial phospholipid-dependent Irgb6 targeting to Toxoplasma gondii vacuoles mediates host defense

Recent Publications
⿎⿎ 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 Sci. Alliance. 3 (2020).

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. 13: 223-233(2015).

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 human hepatocytes.
J Exp Med. 216: 1733-1748 (2019).

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 U S A. 112: E4581-4590 (2015).

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 proteins ininterferon-inducible GTPase-mediated host defense. Nature Immunology.
18: 899-910 (2017).

39

Shigekazu Nagata, PhD

40

Professor

Shigekazu Nagata

Associate Professor
Postdoctoral Fellows
Research Assistants
Visiting Scientists
Support Staff

Katsumori Segawa
142
1

Phospholipids are asymmetrically distributed 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 flippases,
which translocate PtdSer from outer to inner leaflets (Figure1). When cells undergo apoptosis, the asymmetrical
distribution of 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 viruses.
We identified two P4-type ATPases (ATP11A and 11C)
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

As mentioned above, PtdSer is exposed in various

apoptotic neutrophils and lymphocytes due to the Xkr8

biological processes. We recently found that Xkr8 can bemutation activates autoimmunity. It is likely that unengulfed
dead cells undergo secondary necrosis, release cellular

activated by phosphorylation (Figure 2). This phosphorylationinduced activation of Xkr8 was independent from thecomponents, and activate the immunity, leading to SLE-type

caspase-mediated activation. We will study which kinase(s)autoimmune disease. Similarly, a large number of male germ
cells undergo apoptosis, and are cleared by Sertoli cells.
We found that apoptotic germ cells expose PtdSer in an
Xkr8-dependent manner, and many dead Xkr8 -/- germ cells

is responsible for activating Xkr8, and which biologicalprocess it activates. We are also interested in the molecularmechanism of how flippases flip phospholipids, and how
scramblases scramble phospholipids at the plasma
membranes.

are left unengulfed in the testis, causing male infertility.

transmembrane regions that support non-specificscrambling 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 phospholipids during apoptosis.
Xkr8 is expressed ubiquitously, while Xkr4 and Xkr9 are
expressed, in a tissue-specific manner, in the brain andintestine, respectively. These Xkr members contain a
caspase-recognition site in the C-terminal region, and arecleaved by caspase to function as a scramblase. Thus, inapoptotic 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’ssurface, is recognized by macrophages for engulfment.
Lymphocytes and neutrophils express only Xkr8, and a lackof Xkr8 prevents the PtdSer-exposure and engulfment by

Figure 1. Flippase and Scramblase.
Plasma membranes in eukaryotic cells are comprised of outerand inner leaflets, and phosphatidylserine (PtdSer) andphosphatidylcholine are located exclusively at the inner and outerleaflets, respectively. The asymmetrical distribution of PtdSer ismaintained 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 and Xkr, as Ca2+- andcaspase-dependent scramblases, respectively.

Immunology

Biochemistry & Immunology

macrophages. We found that the inefficient engulfment of

Figure 2. Activation of Xkr8’s scramblase and inactivation offlippases by two independent mechanisms.
In apoptosis, caspase cleaves 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 the same time, it seems that flippases arephosphorylated to inactivate their flippase activity.

Recent Publications
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. Nat. Acad. Sci. USA 116: 2907-2912 (2019).

⿎⿎ Nagata S. Apoptosis and clearance of apoptotic cells. Annu. Rev.
Immunol. 36: 489-517 (2018).
⿎⿎ Nagata S, and Tanaka M. Programmed cell death and the immune
system. Nat. Rev. Immunol. 17: 333-340 (2017).

⿎⿎ Segawa K, Yanagihashi Y, Yamada K, Suzuki C, Uchiyama Y, andNagata S. Phospholipid flippases enable precursor B cells to flee
engulfment by macrophages. Proc. Nat. Acad. Sci. USA 115: 1221212217 (2018).

41

Immunology

Molecular Neuroscience
Toshihide Yamashita, MD/PhD
Professor

Toshihide Yamashita

Associate Professor

Takahide Itokazu
Yuki Fujita
Hiroshi Nakagawa
Oluwaseun Fatoba
Hiroshi Tsujioka
Yosuke Ohtake
Toru Nakanishi
Shinichiro Hiraga
Nao Shibuya
12
62

Assistant Professor

Postdoctoral Fellows
Research Assistants
Visiting Scientists
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, suchas the immune system and vascular system. We haveanalyzed disorders of the neural networks in the central
nervous system and the subsequent restoration processfrom the perspective of the functional network of biologicalsystems (Fig. 1). Further, we have analyzed the mechanism
by which the spatiotemporal dynamics in those biologicalsystems control a series of processes (Fig. 2). Particularly,
the ultimate goal of this study is to elucidate the manner inwhich the control mechanism is affected by the associations
among the nervous system, immune system, and vascularsystem. Additionally, we aim to elucidate the processes
involved in the functioning of living organisms with neural

Figure 1. The mechanism of spontaneous functional recovery

network disorders within the central nervous system byobserving such disorders and their functional recovery
process with respect to the dynamics of the entire biologicalsystem and by conducting a comprehensive analysis of theassociation between each system.
We observe the central nervous system as a single
organ within a biological system. Further, studies from theperspective of how the entire biological system is involved indisorders and recovery of neural networks are scarce. By
observing disorders in neural networks and the biologicalreactions during the subsequent recovery process as a“scrap-and-build” strategy, we aim to elucidate themechanisms behind a series of reactions as well as theirsignificance that may potentially lead to a new and original
trend in Life Sciences.

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

Recent Publications
⿎⿎ Ito M, Muramatsu R, Kato Y, Sharma B, Uyeda A, Tanabe S, Fujimura
H, Kidoya H, Takakura N, Kawahara Y, Takao M, Mochizuki H, Fukamizu
A & Yamashita T. Age-dependent decline in myelination capacity is
mediated by apelin-APJ signaling. Nat. Aging 1: 284-294 (2021).

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

⿎⿎ Fujita Y, Nakanishi T, Ueno M, Itohara S & Yamashita T. Netrin-G1
regulates microglial accumulation along axons and supports thesurvival of layer V neurons in the postnatal mouse brain. Cell Rep. 10:107580 (2020).

⿎⿎ Fujita Y, Masuda K, Nakato R, Katou Y, Tanaka T, Nakayama M, Takao
K, Miyakawa T, Shirahige K & Yamashita T. Cohesin regulates formationof neuronal networks in the brain. J. Exp. Med. 214: 1431-1452 (2017).

⿎⿎ Tanabe S & Yamashita T. B-1a lymphocytes promote oligodendrogenesis
during brain development. Nat. Neurosci. 21: 506-516 (2018).

42

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

43

Immunology

Molecular Immunology
Sho Yamasaki, PhD
Professor

Sho Yamasaki

Assistant Professor

Masamichi Nagae
Eri Ishikawa
2
7
45

Postdoctoral Fellows
Research Assistants
Visiting Scientists
Support Staff

Helicobacter pylori metabolites exacerbate gastritis
through C-type lectin receptors
Helicobacter pylori causes gastritis, which has been
attributed to the development of H. pylori-specific T cells
during infection. However, the mechanism underlying innate
immune detection leading to the priming of T cells is not fully
understood, as H. pylori evades TLR detection. Here, wereport that H. pylori metabolites modified from host cholesterol
exacerbate gastritis through the interaction with C-type
lectin receptors. Cholesteryl acyl α-glucoside (αCAG) andcholesteryl phosphatidyl α-glucoside (αCPG) were identified
as noncanonical ligands for Mincle (Clec4e) and DCAR
(Clec4b1). During chronic infection, H. pylori-specific T cell
responses and gastritis were ameliorated in Mincle-deficient
mice, although bacterial burdens remained unchanged.
Furthermore, a mutant H. pylori strain lacking αCAG andαCPG exhibited an impaired ability to cause gastritis. Thus
H. pylori-specific modification of host cholesterol plays apathophysiological role that exacerbates gastricinflammation by triggering C-type lectin receptors.

44

Figure 1. Pathogenic conversion of host lipids by Helicobacter exacerbate gastritis through C-type lectin receptors

Identification, crystallization and epitope determinationof public Tfh TCR shared and expanded in COVID-19
patients
T cells play pivotal roles in protective immunity against
SARS-CoV-2 infection. Follicular helper T (Tfh) cells mediate
the production of antigen-specific antibodies; however, T
cell receptor (TCR) clonotypes used by SARS-CoV-2specific Tfh cells have not been well characterized. Here,
we first identified and crystallized public TCR of Tfhclonotypes that are shared and expanded in unhospitalized
COVID-19-recovered patients. These clonotypes
preferentially recognized SARS-CoV-2 spike (S) protein
epitopes which are conserved among emerging SARSCoV-2 variants. Among SARS-CoV-2 S epitopes, S864-882,
presented by multiple frequent HLA alleles, could activate
multiple public Tfh clonotypes in COVID-19-recovered
patients. Furthermore, HLA tetramer loaded with this epitope
preferentially bound to CD4+ T cells expressing CXCR5.
Highly public Tfh clonotypes cross-reacted with symbiotic
bacteria, but not common cold human coronaviruses. In thisstudy, we identified and crystallized public TCR for SARSCoV-2 that may contribute to the prevention of COVID-19
aggravation.

Figure 2. Public Tfh clonotypes cross-reacting with conserved SARS-CoV-2 spike
epitopes and commensal bacteria were expanded in recovered COVID-19 patients

Recent Publications
⿎⿎ Nagata M, Toyonaga K, Ishikawa E, Haji S, Okahashi N, Takahashi M,
Izumi Y, et al. Helicobacter pylori metabolites exacerbate gastritis
through C-type lectin receptors. J Exp Med. 218: e20200815 (2021).
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, et al. Lipoteichoic acid anchor triggers Mincle todrive protective immunity against invasive group A Streptococcus
infection. Proc Natl Acad Sci U S A. 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, et al.
C-Type Lectin Receptor DCAR Recognizes Mycobacterial
Phosphatidyl-Inositol Mannosides to Promote a Th1 Response during
Infection. Immunity 45: 1245-57 (2016).

45

Perichondrial cells

Takashi Nagasawa, MD/PhD
Professor

Takashi Nagasawa

Associate Professor
Support Staff

Yoshiki Omatsu
1

Immunology

Stem Cell Biology and Developmental Immunology

Bone Marrow
Osteoblasts

P-Erythroid,
p-Myeloid

Ebf3/1
Foxc1

CAR cells

46

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 achemokine, CXCL12 (SDF-1/PBSF) as a molecule that
stimulates the growth of B cell precursors (Nagasawa et al.
PNAS 1994). We found that CXCL12 and its receptor CXCR4
are essential for colonization of bone marrow by HSCs
during embryogenesis (Nagasawa et al. Nature 1996; Ara etal. Immunity 2003), maintenance of a pool of HSCs (Sugiyama
et al. Immunity 2006), and development of immune cells,
including B cells, plasmacytoid dendritic cells (pDCs) andNK cells in bone marrow (Nagasawa. Nat. Rev. Immunol.
2006) as well as vascular formation and cardiogenesis(Tachibana et al. Nature 1998). Based on these findings, we
identified a population of reticular cells expressing CXCL12
at high levels, termed CXCL12-abundant reticular (CAR)
cells within murine bone marrow (Tokoyoda et al. Immunity
2004; Sugiyama et al. Immunity 2006). We found that CAR
cells are the major producer of CXCL12 and SCF (Omatsuat al. Immunity 2010), and the major cellular components ofniches for HSCs and immune cells (Omatsu at al. Immunity
2010; Shimoto et al. Blood 2017). In addition, we determined
the nature of CAR cells, showing that CAR cells aremesenchymal stem cells, which give rise to adipocytes andosteoblasts (Seike et al. Genes Dev. 2018) and thattranscription factors, Foxc1 and Ebf3, are preferentiallyexpressed in CAR cells and play a critical role in theformation and maintenance of niches for HSCs and immune
cells, inhibiting differentiation of CAR cells into adipocytes

and osteoblasts, respectively (Omatsu et al. Nature 2014;
Seike et al. Genes Dev. 2018). These studies clarified thenature and functions of CAR cells in murine bone marrow.
However, the human counterpart of CAR cells has not been
fully described.
To address this issue, we analyzed human bone marrowusing a flow cytometry-based in-situ technique that enables
high-throughput detection of mRNA at single-cell resolution
and identified cells expressing much higher CXCL12 thanother cells. Like murine CAR cells, CXCL12hi cells had the(HSPC)

Foxc1hi Ebf3hi

Lymphoid progenitor

adipocytes

CXCL12hiSCFhi
Figure 1. The development and functions of CAR cells.
CAR cells are the major cellular components of non-hematopoietic cells in bone marrow characterized by several salientfeatures in both mice and humans. The transcription factors, Foxc1 and Ebf1/Ebf3, and cytokines, CXCL12 and SCF, arepreferentially expressed in CAR cells and critical for the formation and maintenance of niches for HSCs and immune cells,
within the bone marrow.

potential to differentiate into adipocytes and osteoblasts,
and most CXCL12hi cells expressed much higher levels of
SCF, Foxc1, and Ebf3 than any other bone marrow cells andsynovial mesenchymal cells, which had the potential todifferentiate into adipocytes and osteoblasts. Histologically,
the nuclei of CXCL12hi cells were identified and quantified
by Ebf3 expression in fixed marrow sections. CXCL12hi cells
sorted from residual bone marrow aspirates of chronic
myeloid leukemia (CML) patients expressed reduced levelsof HSC niche factors, CXCL12, SCF, Foxc1, and Ebf3 incorrelation with increased leukemic burden. Together, we
identified the human counterpart of CAR cells and enabled
the evaluation of their alterations in various hematologicaldisorders by flow cytometric and histological analyses.
We are studying the molecular regulation of CAR cellproperties and development, and the roles of CXCL12CXCR4 signaling and CAR cells in the spatiotemporal
regulation of lymphohematopoiesis during homeostasis anddiseases.

Recent Publications
⿎⿎ Aoki K, Kurashige M, Ichii M, Higaki K, Sugiyama T, Kaito T, Ando W,
Sugano N, Sakai T, Shibayama H, HANDAI Clinical Blood Club,
Takaori-Kondo A, Morii E, Kanakura Y, and Nagasawa T. Identificationof CXCL12-abundant reticular cells in human adult bone marrow. Br J
Haematol. 193: 659-668 (2021).
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: 769-784 (2019).
⿎⿎ Seike M, Omatsu Y, Watanabe H, Kondoh G, Nagasawa T. Stem cellniche-specific Ebf3 maintains the bone marrow cavity. Genes Dev. 32:
359-372 (2018).

⿎⿎ Shimoto M, Sugiyama T, and Nagasawa T. Numerous niches forhematopoietic stem cells remain empty during homeostasis. Blood
129: 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).

47

Eiji Hara, PhD
Professor

Eiji Hara

Assistant Professor

Shimpei Kawamoto
Tomonori Matsumoto
Masahiro Wakita
22
2

Postdoctoral Fellows
Research Assistants
Support Staff

Cellular senescence is the state of irreversible cell cycle
arrest that can be induced by a variety of potentially
oncogenic stimuli and has therefore long been consideredto suppress tumorigenesis, acting as a guardian of
homeostasis. Emerging evidence, however, reveals thatsenescent cells also promote secretion of various proinflammatory and pro-proliferative factors. This newly
identified senescence-associated secretory phenotype
termed SASP is likely to be associated with homeostatic
disorders including cancer. It is therefore quite possible that
accumulation of senescent cells during aging in vivo may
contribute to aging-associated cancers. By conducting thefollowing studies, we aim to clarify the molecularmechanisms underlying aging-associated cancer.

48

Oncogenic proliferative signals are coupled to a varietyof growth inhibitory processes, such as the induction of
apoptotic cell death and stable cell-cycle arrest, in aphenomenon termed cellular senescence. Both apoptosis
and cellular senescence are considered to serve asimportant safeguards against neoplasia. However, unlike
apoptotic cells, senescent cells are viable for long periodsof time and thereby accumulate with age in various organs
and tissues. Moreover, it has recently become apparent thatsenescent cells are not merely non-dividing, but eventuallydevelop a secretory profile composed of pro-inflammatory
cytokines, chemokines, and extracellular matrix-degrading
proteases, a typical signature of 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 senescentcells in aged tissues may eventually promote the age-related
decline of organ function and/or associated diseases, suchas cancer (Figure 1). Indeed, the clearance of p16INK4apositive senescent cells from aged transgenic micereportedly delays the onset of various age-related
dysfunctions, such as sarcopenia, cataracts, atherosclerosis,
loss of adipose tissue, and tumorigenesis, thus extending
the healthy lifespan. Along similar lines, the elimination oftherapy-induced senescent cells reduced several sideeffects of chemotherapy and even cancer recurrence inmice. Thus, it is anticipated that the removal of senescentcells could prevent the toxicity of anticancer treatments andenhance its 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, in aphase 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

promising new class of senolytic drugs. The blockade ofdrug, apparently exhibited remarkable cell-type specificity,

BRD4, a BET family protein, by chemical inhibitors or RNA

although its mechanisms of action remain obscure.

interference robustly provokes senolysis. This is due, at least

Therefore, the identification of more effective senolytic drugs

in part, to the combined effects of the attenuation of non-and the elucidation of their mechanisms of action arehomologous end joining (NHEJ) repair and the activation ofrequired 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 a

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.

Immunology

Aging Biology

and quercetin (D+Q), another previously reported senolytic

Figure 1. Cellular senescence initially inhibits proliferation of damaged cells, thereby
acting as a fail-safe mechanism. However, in the long term, senescent cells eventuallypromote tumorigenesis via SASPs.

Recent Publications
⿎⿎ Wakita M, Takahashi A, Sano O, Loo TM, Imai Y, Narukawa M, Iwata H,
Matsudaira T, Kawamoto S, Ohtani N, Yoshimori T, Hara E. A BET
family protein degrader provokes senolysis by targeting NHEJ andautophagy in senescent cells. Nat. Commun. 11: 1935 (2020).
⿎⿎ Takahashi A, Loo TM, Okada R, Kamachi F, et al. Downregulation ofcytoplasmic DNases is implicated in cytoplasmic DNA accumulationand SASP in senescent cells. Nat. Commun. 9: 1249 (2018).

⿎⿎ Takasugi M, Okada R, Takahashi A, Chen DV, Watanabe S, Hara E.
Small extracellular vesicles secreted from senescent cells promote
cancer cell proliferation through EphA2. Nat. Commun. 8: 15728 (2017).
⿎⿎ Takahashi A, Okada R, Nagao K, Kawamata Y, Hanyu A, Yoshimoto S,
Takasugi M, Watanabe S, Kanemaki MT, Obuse C, Hara E. Exosomes
maintain cellular homeostasis by excreting harmful DNA from cells.
Nat. Commun 8: 15287 (2017).

and p21
⿎⿎ Okuma A, Hanyu A, Watanabe S, Hara E. p16
promote tumour growth by enhancing myeloid-derived suppressor
cells chemotaxis. Nat. Commun. 8: 2050 (2017).
INK4a

Cip1/Waf1

49

molecular surface as a potential therapeutic target in

Ragulator complex provides a regulatory platform that islifestyle diseases, such as diabetes mellitus and cancer,

indispensable for amino acid-dependent regulation ofmTORC1 (Fig. 2). These findings identified the interacting

both of which are linked to dysfunction of the mTORC1
pathway.

Immunology

Oncogene Research

GTPase, such as GATOR1 and FLCN, showed that p18-

Masato Okada, PhD
Professor

Masato Okada

Associate Professor
Assistant Professor

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

Postdoctoral Fellows
Research Assistants
Support Staff

Figure 1. Role of the CDCP1-Met-Src-STAT3 axis in regenerative renal growth.

Role of Src tyrosine kinase in tumor progression

50

We investigated the role of Src tyrosine kinase in tumor
progression. Src is the first oncogenic tyrosine kinase tohave been identified and 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 acrucial role in tumor progression, particularly in the acquisitionof invasive and metastatic features. To elucidate themolecular mechanisms underlying upregulation of Src, weinvestigated the regulatory mechanism of SRC gene
expression and searched for Src-activating factors. We
found that TGF-βtreatment directly induces SRC gene
expression via the Smad pathway coupled with additionaltranscription factors, and determined the promoter andenhancer regions located upstream of the SRC gene. The
upregulation of Src contributes to the progression of TGF-βinduced epithelial-mesenchymal transition. We also foundthat activated Src is ubiquitinated and promotes its secretion
via exosomes to suppress its oncogenic potential. In
addition, we identified CDCP1 as a Src-activating membrane
glycoprotein in lipid rafts. Upregulation of CDCP1 induces
prominent activation of 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 and cell growth, and attenuates
membrane presentation of MET, resulting in inhibition ofinvasive activity induced by HGF. These findings suggestthat CDCP1 is a co-receptor of MET (Fig. 1). Furthermore,ablation of CDCP1 suppresses the compensatory renal

hypertrophy, indicating that CDCP1 is required for the HGFMET signaling even in vivo. CDCP1 and MET are crucial for
promoting cancer cell invasion; therefore, we expect thisstudy to identify a potential therapeutic target in some typesof cancer.

Role of p18 in the regulation of mTORC1 nutrient
signaling
We previously identified a new Src substrate termed
p18/Lamtor1, which exclusively localizes into lipid rafts oflysosomes. Subsequent analysis revealed that p18 functions
by forming a hetero-heptamer complex (Ragulator),
consisting of p18, p14, MP1, HBXIP, p10, and RagA/C, andis required for activation of mTORC1 on lysosomes.
Conditional KO of p18 in the epidermis showed that p18mTORC1 is crucial not only for anabolism of bio-materials
but also for catabolism via autophagy, indicating that p18 istightly associated with the regulation of mTORC1 nutrient
signaling in vivo. Recent studies in intestinal tissues revealed
that the p18-mediated mTORC1 signaling promotes the
anabolic metabolism required for robust production of mucin
in goblet 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 previously determinedthe crystal structure of Ragulator. This revealed that p18
wraps around the other components of Ragulator andprovided significant insights into the role of p18-mediatedregulation of mTORC1 on lysosomes. Recent analysis using
p18 KO cells that lack regulatory components of Rag

Figure 2. Function and regulation of the mTORC1 signaling on lysosomal membrane.

Recent Publications
⿎⿎ Kajiwara K, Yamano S, Aoki K, Okuzaki D, Matsumoto K, Okada M.
CDCP1 promotes compensatory renal growth by integrating Src andMet signaling. Life Sci Alliance. 2021 4: e202000832 (2021).

⿎⿎ Nada S, Okada M. Genetic dissection of Ragulator structure andfunction in amino acid-dependent regulation of mTORC1. J Biochem.
168: 621-632 (2020).

⿎⿎ Chee W-Y, Kurahashi Y, Kim J, Miura K, Okuzaki D, Ishitani T, Kajiwara
K, Nada S, Okano H, Okada M. β-catenin-promoted cholesterol
metabolism protects against cellular senescence in naked mole-rat
cells. Commun Biol. 4: 357 (2021).

⿎⿎ Ito S, Nada S, Yamazaki D, Kimura T, Kajiwara K, Miki H, Okada M.
p18/Lamtor1-mTORC1 Signaling Controls Development of Mucinproducing Goblet Cells in the Intestine. Cell Struct Funct. 45: 93-105(2020).

⿎⿎ Yao Y, Hong S, Ikeda T, Mori H, MacDougald OA, Nada S, Okada M,
Inoki K. Amino acids enhance polyubiquitination of Rheb and its
binding to mTORC1 by blocking lysosomal ATXN3 deubiquitinase
activity. Mol Cell. 80: 437-451.e6. (2020).

51

Immunology

Signal Transduction
Nobuyuki Takakura, MD/PhD
Professor

Nobuyuki Takakura

Associate Professor

Hisamichi Naito
Hiroyasu Kidoya
43
2

Postdoctoral Fellows
Research Assistants
Support Staff

Our research team is involved in the research of vascularbiology and stem cell biology. In 2020, we obtained several
achievements and among them, interesting results on selfrenewal in hematopoietic stem cells are shown here.
Hematopoietic stem cells (HSCs) give rise to the blood
supply and excessive division of HSCs will cause cell
exhaustion. Therefore, it is very important to maintain abalance between self-replication and differentiation of HSCs.
However, the cellular and molecular mechanisms
coordinating the balance between HSC quiescence anddifferentiation are not fully understood.
As a consequence of analyzing the molecularmechanism for HSC quiescence, we found that galectin-3(Gal-3) is highly expressed in quiescent HSCs. Gal-3 is amember of the galactose binding lectin family and plays arole in various biological processes such as development,
differentiation, cell cycle, and apoptosis. Our research foundthat the percentage of long-term HSCs (LT-HSC) in the G0
phase decreased in BM from Gal-3 KO relative to wild-typemice and that the reconstruction ability of LT-HSCs in Gal-3

52

KO mice was attenuated. These results illustrate that Gal-3
regulates the cell-cycle of HSCs and plays a critical role inmaintaining the quiescent state.
To elucidate how Gal-3 maintains HSC quiescence, we
analyzed the regulatory mechanism and function of Gal-3 asfollows. It is well known that Angiopoietin-1 or Thrombopoietin,secreted from bone marrow niche cells, binds to thereceptor Tie2 or Mpl on the surface of HSCs. We found that
nuclear translocation of NF-κB mediated by the PI3K/AKT
pathway following stimulation by Tie2 or Mpl expressed in
HSCs promotes the production of Gal-3. Moreover, we foundthat Gal-3 upregulated in HSCs binds to Sp1 and induces
p21 transcription, resulting in the inhibition of cell cycle
progression and maintenance of LT-HSC quiescence.
This research clarified a novel mechanism of bonemarrow niche for the maintenance of HSCs and may
contribute to stem cell expansion in culture and bonemarrow transplantation, as well as promoting leukemia
cancer stem cell-targeted therapy and other clinicalapplications.

Figure. Proposed model showing the molecular mechanisms essential for maintaining (long term-hematopoietic stem cell)
LT-HSC quiescence.
Angiopoietin-1 (Ang-1) or Thrombopoietin (Thpo) secreted from bone marrow niche cells, binds to the receptor Tie2 or
Mpl on the surface of LT-HSCs. Nuclear translocation of NF-κB mediated by activated AKT following stimulation by Tie2
or Mpl expressed in LT-HSCs promotes the production of Gal-3, which then binds to Sp1 and induces p21 transcription,
resulting in the inhibition of cell cycle progression and maintenance of LT-HSC quiescence.

Recent Publications
⿎⿎ Jia W, Kong L, Kidoya H, Naito H, Muramatsu F, Hayashi Y, Hsieh HY,
Yamakawa D, Hsu DK, Liu F-T, and Takakura N. Indispensable role of
Galectin-3 in promoting quiescence of hematopoietic stem cells.
Nature Commun. 12: 2118 (2021).

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 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 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).

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 isessential for hematopoietic stem and progenitor cell homeostasis. Nat
Commun. 10: 1072 (2019).

53

the skin of Japanese infants and showed that infants whodeveloped AD early in life were more likely to have cheekskin colonized by S. aureus. However, infants harboring S.
aureus with acquired spontaneous mutations in Agr wereManabu Fujimoto, MD/PhD
Professor

Manabu Fujimoto

Associate Professor
Research Assistants
Visiting Scientists
Support Staff

Yumi Matsuoka-Nakamura
1
1
1

Our research team is interested in understanding thepathogenesis of autoimmune diseases, inflammatory skindiseases, and cancer, and translating novel researchknowledge into clinical applications.

The local T-cell immunity in the skin is maintained more
efficiently in advanced age than circulating T-cell memory
Recent studies have highlighted that human resident
memory T cells (TRM) are functionally distinct fromcirculating T cells. Thus, it can be postulated that skin T
cells age differently from blood-circulating T cells. We
assessed T-cell density, diversity, and function in skinsamples of various ages. No decline in the density of T cells
was noted with advancing age, and the frequency of
epidermal CD49a+ CD8 TRM was increased in elderly
individuals regardless of ethnicity. T-cell diversity andantipathogen responses were maintained in the skin ofelderly individuals but declined in the blood. Our findings
demonstrate that in elderly individuals, skin T cells maintaintheir density, diversity, and protective cytokine production
despite the reduced T-cell diversity and function in blood.

Ganglioside GD3 suppress the functional activities of
TRM in cutaneous T-cell lymphoma

54

In cutaneous T-cell lymphoma (CTCL), which arises fromskin-tropic memory T cells, malignant T cells and benign T
cells are confined in the same skin lesions. It is thus difficult
to evaluate the phenotypic characteristics and functional
activities of benign T cells in CTCL. Disialoganglioside withthree glycosyl groups (GD3) is increasingly expressed on
the surface of solid malignant tumor cells and takes part intumor progression and suppression of tumor immunity.
However, the role of GD3 in CTCL is not well-understood.
We found that the benign T cells included limited TRM in
CTCL skin lesions and the expression of GD3 in themalignant T cells inversely correlated with IL-17A production
from the benign CD4 T cells. Revealing the role of GD3 ininhibiting the production of IL-17A in CTCL would aid theunderstanding of the suppressive mechanism of theantitumor activity by malignant tumor cells.

Atopic dermatitis (AD) is commonly associated withmore likely to remain healthy, despite the presence of thiscolonization by Staphylococcus aureus in the affected skin.
Inhibition of Staphylococcus aureus Agr–mediated quorum
sensing is known to protect against AD. We performed
whole-genome sequencing of S. aureus strains isolated frombacterium on their skin. This work suggests that S. aureus
and associated functional quorum sensing may play a rolein the onset of AD in children.

Figure 1. The density of T cells, both CD4 and CD8 fractions,increased in the epidermis of elderly individuals.

Figure 2. Whole-genome sequencing of S. aureus strains isolated
from the skin of Japanese infants.

Immunology

Cutaneous Immunology

Staphylococcus Agr expression is critical for epidermalcolonization and associates with atopic dermatitis
development.

Imbalance of follicular helper CD4+ T cell subsets induces
B cell alteration and contributes to the pathogenesis ofsystemic sclerosis.
Systemic sclerosis (SSc) is a connective tissue diseaseof unknown aetiology characterized by features of
autoimmunity, vasculopathy, inflammation and fibrosis on
skin and multiple internal organs, as well as themusculoskeletal system. Until now, the full pathogenesis of
SSc remains unknown. We assessed circulating follicularhelper CD4+ T cells (cTFH) and B cells, and found that thefrequency of TFH1 cells was increased and correlated withcytokine concentrations of IL-21 and IL-6 that induce B celldifferentiation in SSc. cTFH cells from SSc showed activatedphenotype with expressing higher cytokine levels comparedwith healthy controls. These results suggest that an
imbalance of cTFH toward TFH 1 may induce B cell alteration
through IL-21 and IL-6 pathways and promote inflammation,
contributing to the pathogenesis of SSc.

Recent Publications
⿎⿎ Kume M, Kiyohara E, Matsumura Y, Koguchi-Yoshioka H, Tanemura A,
Hanaoka Y, Taminato M, Tashima H, Tomita K, Kubo T, Watanabe R,
Fujimoto M. Ganglioside GD3 May Suppress the Functional Activities
of Benign Skin T Cells in Cutaneous T-Cell Lymphoma. Front Immunol.
12: 651048 (2021).
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 anti-nuclear
matrix protein 2 autoantibodies. JAMA Neurol. 77(7): 872-877 2020.
⿎⿎ Koguchi-Yoshioka H, Hoffer E, Cheuk S, Matsumura Y, Vo S, Kjellman
P, Grema L, Ishitsuka Y, Nakamura Y, Okiyama N, Fujisawa Y, Fujimoto
M, Eidsmo L, Clark RA, Watanabe R. Skin T cells maintain theirdiversity and functionality in the elderly. Commun Biol. 4(1): 13 (2021).

⿎⿎ Koguchi-Yoshioka H, Watanabe R, Matsumura Y, Okiyama N, Ishitsuka
Y, Nakamura Y, Fujisawa Y, Fujimoto M. The possible linkage of
granzyme B-producing skin T cells with the disease prognosis ofalopecia areata. J Invest Dermatol. 141(2): 427-429 (2021).
⿎⿎ Nakamura Y, Takahashi H, Takaya A, Inoue Y, Katayama Y, Kusuya Y,
Shoji T, Takada S, Nakagawa S, Oguma R, Saito N, Ozawa N, Nakano
T, Yamaide F, Dissanayake E, Suzuki S, Villaruz A, Varadarajan S,
Matsumoto M, Kobayashi T, Kono M, Sato Y, Akiyama M, Otto M,
Matsue H, Núñez G, Shimojo N. Staphylococcus Agr virulence iscritical for epidermal colonization and associates with atopic dermatitis
development. Sci Transl Med. 12(551): eaay4068 (2020).

55

currently trying to further fractionate the components tothree extracts still contain a variety of components, we are

identify the effective compound.

Immunology

Innate Immune Systems

ILC2 and only suppress cytokine production. Since theseKazuyo Moro, PhD
Professor

Kazuyo Moro

Assistant Professor
Postdoctoral Fellows
Research Assistants
Visiting Scientists
Support Staff

Eriko Kudo
11
1
1

Figure 1. ILC2-derived IL-4 regulates allergic susceptibility through innate IgE production by B1 cells

Our team has been focused on group 2 innate lymphoidcells (ILC2), an innate lymphocyte lineage that we identified
in 2010. ILC2 localize in a variety of tissues such as fat,lung, intestine, liver, and skin, and mediate immune
responses to helminth and fungal infections via strong type2 cytokine production, including IL-5, IL-13, IL-9, GM-CSF.
Unlike T and B lymphocytes, ILC2 lack antigen-specificreceptors and are activated by cytokines produced byepithelial cell-derived cytokines such as IL-33. Because
ILC2 are also known to produce type 2 cytokines in many
diseases including allergic disorders and pulmonary fibrosis(PF), we wish to establish new therapies for these diseases
through regulating ILC2.

56

IL-4, a key cytokine in allergy, is involved in multiple
immune reactions including Th2 cell development, IgG1 andIgE production by B cells and M2 macrophage differentiation.
While Th2 cells produce IL-4 together with IL-5 and IL-13
under antigen-induced TCR stimulation, ILC2 fail to produce
IL-4 under IL-33 stimulation, which induces a large amountof IL-5 and IL-13. Based on this fact, ILC2 are not thought tocontribute to IL-4-mediated immune responses, even though
they express high levels of the IL-4 gene. However, wedemonstrated the mechanism underlying interleukin (IL)-4
production by ILC2 that depends on IL-2, IL-33 andcysteinyl leukotriene (CysLT)-mediated Ca2+ signaling andinduces ‘innate immunoglobulin E (IgE)’ production by B1
cells. Innate IgE prolonged the survival of FcεR+ cells andaccelerated ILC2-mediated allergic responses. The CysLT
inhibitor blocked innate IgE-mediated augmentation ofasthma symptoms, suggesting that innate IgE-primed FcεR+

cells positively regulate the activation of ILC2 via CysLTs.
Overall, these data demonstrated that ILC2, B1 cells, andFcεR+ cells generate an amplification circuit that defines
allergic susceptibility (Fig.1).
Pulmonary fibrosis (PF) is a disease that causes collagendeposition in the lungs. Despite numerous studies on thepathology of PF, lack of animal models has significantly
hampered advancement of our understanding of thedisease. We found that 100% of Ifngr1 -/-Rag2 -/- mice lacking
suppression mechanism for ILC2 and ILC3 spontaneously
developed PF. Through studying of Ifngr1 -/-Rag2 -/- mice, wehave unveiled an indispensable contribution of ILC2 andILC3 to the fibrosis formation. Single-cell RNA-sequence
analysis indicated that ILC3 activation occurred during thedisease-onset phase in Ifngr1 -/-Rag2 -/- mice, which triggered
subsequent activation of ILC2 subpopulation. By dissecting
pathomechanisms of disease initiation in vitro and in vivo,
we have delineated complex networking among ILC2, ILC3
and fibroblasts, which will lead us to develop novel
therapeutic options that prevent the progression of thedisease (Fig.2).
The development of new drugs is required to suppress
ILC2, because ILC2 induces various diseases such asallergies, pulmonary fibrosis, and metabolic diseases. In
collaboration with Nagasaki University, we searched for an
ILC2 inhibitor from the marine microbe extract library.
Among 520 marine microbe extracts, 3 extracts contained acomponent that suppressed cytokine production by ILC2.
We confirmed that these extracts do not affect the survival of

Figure 2. Ifngr1-/-Rag2-/- mouse lacking suppression mechanism for ILC2 and ILC3 spontaneously developed pulmonary fibrosis.

Recent Publications
⿎⿎ Kobayashi T, Ricardo-Gonzalez RR & Moro K. Skin-Resident Innate
Lymphoid Cells - Cutaneous Innate Guardians and Regulators. Trends
in immunology 41(2): 100-112 (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
reports 28(1): 202-217. e207 (2019).

⿎⿎ 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(8): 2743-2757. e2745 (2020).

⿎⿎ Motomura Y, Kobayashi T & Moro K. The Neuropeptide CGRP Induces
Bipolar Syndrome in Group 2 Innate Lymphoid Cells. Immunity 51(4):598-600 (2019).

⿎⿎ Miyajima Y, Ealey KN, Motomura Y, Mochizuki M, Takeno N, Yanagita
M, Economides AN, Nakayama M, Koseki H & Moro K. Effects of
BMP7 produced by group 2 innate lymphoid cells on adipogenesis.
International immunology 32(6): 407-419 (2020).

57

Immunology

Human Immunology (Single Cell Immunology)
James Badger Wing, PhD
Associate Professor

James Badger Wing

Postdoctoral Fellows
Research Assistants

2
1

Figure 1. Main Treg Subgroups Present in Human Blood and tumors (Wing et al. Immunity 2019)

The Human Immunology (single cell immunology)
focuses on single cell biology and its applications to humanimmunology. Recent advances in single cell technologies,
such as mass cytometry (also known as CyTOF) haverevealed high levels of heterogeneity among immune cells.
The mass cytometry technique can measure single cell
proteome in great details of up to 50 protein markers on
millions of single cells (Hartman and Bendall, Nat Rev
Rheumatol, 2020). This makes it uniquely well placed toobtain a wide-ranging picture of the human immune system.
Primarily we use mass cytometry to explore two areas,
autoimmunity and infectious diseases.
We have an established interest in regulatory T cells(Tregs) expressing the transcription factor Foxp3, which playa critical role in the control of immune homeostasis. Recently
it has become clear that Tregs present in humans harbor agreat deal of heterogeneity in both blood and tumors (Figure1). Recently it has become clear that a specialized subset ofTregs, T-follicular regulatory cells, (Tfr) have a particular rolein the control of T-follicular helper (Tfh) cell-driven germinal
center (GC) responses responsible for antibody production.
Recent results strongly suggest that Tfr are critical for the
regulation of autoantibody production, vaccine response
and the production of high affinity IgE responsible for allergy.
However, very little is known about changes to theirphenotype and function in autoimmune disease patients
since the first comprehensive descriptions of theirphenotype in humans was carried out by us and others in
2017. A clear goal of the field is to determine how tomanipulate specific subset of Tregs as for example
enhancement of Tfr function would allow control of
autoimmune antibody production without affecting the roleof Tregs in tumor Immunosurveillance.

Recent Publications
⿎⿎ Wing JB & Sakaguchi S. Using mass cytometry to address Tfh andTfr heterogeneity. Methods in Molecular Biology. Edition “T follicularhelper cells” (2021 in press).

⿎⿎ Sakaguchi S, Mikami N, Wing JB, Tanaka A, Ichiyama K, Ohkura N.
Regulatory T cells and human disease. Annual Review of Immunology
38: 541-566 (2020).

⿎⿎ Mimitou EP et al. Scalable, multimodal profiling of chromatin accessibility
and protein levels in single cells. bioRxiv DOI: 10.1101/2020.09.08.
286914. Nature Biotechnology (in press).

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 Yang et al. CTLA-4 expression by B-1a B cells is essential for immune
tolerance. Nature Communications 12: 525 (2021).

58

59

of Medicine, and analyzing the peripheral blood samples

the proper treatments for prior and post intubation patients.

taken from the patients sent to Osaka University Hospitalduring all four waves of COVID-19 in Japan. Currently, weOn the other hand, the importance of evaluating

Daisuke Okuzaki, PhD
Associate Professor

Daisuke Okuzaki

Postdoctoral Fellows
Visiting Scientists
Support Staff

3
2
1

The Laboratory of Human Immunology was establishedin November 2019 for the purpose of accelerating theapplication of single cell sequencing technology to improvethe connection between fundamental science research andclinical applications. One of the major research projects weare currently focused on is a genome project in collaboration
with the Trauma and Acute Critical Care Center in the Osaka
University Graduate School of Medicine. Many of thepatients sent to the Trauma and Acute Critical Care Center
have acute inflammation and suffer from systemic
inflammatory response syndrome (SIRS). The severity of
SIRS usually has to be clinically classified within several
hours upon patient arrival. Classification is based on clinicalcriteria such as fever, hypothermia, tachycardia, tachypnea
or leucopoenia. Although treatment of the clinical syndrome
is a top priority in the Trauma and Acute Critical Care Center,
progress in the research of its pathological classification
and associated molecular mechanism has not advanced inJapan as it has in Europe and America.

60

We hereby kick started a collaboration research project
involving the measurement and collection of biomolecular
data such as DNA, RNA and protein from patented
biomaterials, while integrated with cutting edge single cell
sequencing technology. As a prospective observational
study, we collected ELISA, RNA sequencing, Olink or single
cell sequencing data from patients three days and sevendays post arrival. The resulting multiomics data, combinedwith clinical pathology and bioinformatic multi-dimensional
analysis, can be utilized to classify SIRS patients based onmolecular properties into pathological classifications suchare analyzing multiple aspects of the molecular properties ofvaccination efficacy is gradually increasing given the current

immune cells from dozens of patients.

nationwide vaccination efforts. We intend to elucidate thepathological mechanism of the novel coronavirus as well as
develop potential treatments through the use of single cell
RNA sequencing of peripheral blood samples taken fromhealthy individuals prior and post vaccination. In connection
with the Joint Research Coronavirus Task Force and serving
as a member of the All Handai research team, we are
currently joining forces with the young researchers of IFReC
as well as the Graduate School of Medicine and the Faculty

We have co-authored two papers in which we apply
single cell RNA sequencing. They include a study of PTHinduced bone formation conducted by Morimoto et al.,published in Nat Commun. 2021 and a successful
observation of hematopoietic recovery supported by innate
lymphoid cells under stress conditions performed by Sudo T,
et al., published on J Exp Med. 2021.

Immunology

Human Immunology (Single Cell Genomics)

Especially for the given circumstance, it is urgent to selectas sepsis, ARDS, PCAS, trauma, heat stroke or burn.
Through these classifications, proper treatment canpotentially be selected in a timely manner upon arrival.
Furthermore, with COVID-19 raging worldwide, it is alsoclassified as a SIRS and the course of treatment for patientsbrought to Osaka University Hospital should be preferably
decided prior to arrival. Our proposal to search forappropriate biomarkers using proteome and transcriptome
profiling techniques and to apply them clinically was
selected as a project for the AMED " Research Program on
Emerging and Re-emerging Infectious Diseases. This
project is being conducted in cooperation with the Trauma
and Acute Critical Care Center at Osaka University and the
Osaka Prefectural Nakakawachi Emergency and Critical
Care Center.
Currently there are no conventionally establishedbiomarkers or drug treatments for COVID 19. In order tosearch for the appropriate biomarkers to reflect thepathological classification for COVID-19, cohorts weredivided into the three groups of research, development andverification. Applying the same strategy as the aforementioned
SIRS studies, we plan to collect experiment data using RNA
sequencing, mass spectrometry, Olink proteomics, andsingle cell RNA sequencing. Through the integration withpanomics data a set of “pathological molecular scores” and“pathological correlated clusters” can be defined. The novel“pathological classification” is determined by the definedclusters and scores, and enables selective therapeuticintervention depending on the COVID-19 symptoms.

Figure. Overview of the research process.

Recent Publications
⿎⿎ Morimoto A, et al. SLPI is a critical mediator that controls PTH-inducedbone formation. Nat Commun. 12(1): 2136 (2021).

⿎⿎ Okuzaki D, et al. c-Src promotes tumor progression throughdownregulation of microRNA-129-1-3p. Cancer Sci. 111: 418-428 (2020).

⿎⿎ Sudo T, et al. Group 2 innate lymphoid cells support hematopoietic
recovery under stress conditions. J Exp Med. 218(5): e20200817 (2021).

⿎⿎ Hasegawa T, et al. Identification of a novel arthritis-associated osteoclast
precursor macrophage regulated by FoxM1. Nat. Immunol. 20: 16311643 (2019).

⿎⿎ Al Kadi M, et al. UNAGI: an automated pipeline for nanopore fulllength cDNA sequencing uncovers novel transcripts and isoforms inyeast. Funct. Integr. Genomics. doi:10.1007/s10142-020-00732-1(2020).

61

A deficiency in mice caused the reduction of GATA6 gene

receptors (RARs) bind to the promoter of the GATA6 gene in

expression. Taken together, these results provide insight into

a retinoic acid-dependent manner. In addition, the treatment

the mechanism of generation of tissue specialization ofof RAR inverse agonist (BMS493) abolished the expression

resident macrophages.

of GATA6 gene in peritoneal macrophages. Lastly, a vitamin

Immunology

Immune Homeostasis

peritoneal macrophages. We found nuclear retinoic acid

Yasutaka Okabe, PhD
Associate Professor

Yasutaka Okabe

Postdoctoral Fellows
Research Assistants
Support Staff

1
2
1

Figure 1. Functional specialization of tissue-resident macrophages is an essential component of tissue homeostasis.

The laboratory of Immune Homeostasis was establishedat IFReC in September 2020. The focus of the laboratory is
to understand tissue homeostasis, which is an essential
mechanism for maintaining tissue in its optimal functioning
state. The principle of tissue homeostasis can be describedas the maintenance of the level of regulated variables (e.g.,
tissue size, interstitial fluid volume) within an acceptable
range. This is regulated by the orchestration of parenchymal
cells that perform the primary function of tissues with othersupportive cell types under the control of tissue
environmental cues.

62

In this perspective, we study tissue-resident
macrophages. Although macrophages have been
historically studied in the context of their functions in hostdefense and the clearance of dead cells, they are
increasingly appreciated to serve many important roles intissue development, normal physiology, and repair. Tissueresident macrophages are present in virtually every
mammalian tissue and they function as essential
components for the maintenance of tissue homeostasis.
They perform tissue-specific functions that are critical fornormal tissue physiology (Figure 1). Accordingly,
abnormalities of tissue-resident macrophage functions often
link to various pathologies including osteopetrosis, type 2
diabetes, immune deficiency and neurodevelopmental
diseases. These evidences suggest that understanding
tissue macrophage functions could provide a novel
therapeutic approach for these diseases.

The heterogeneity of tissue-resident macrophage
phenotypes is considered to be a functional specializationof macrophages as a consequence of their adaptation to
local tissue environments (Figure 2). Tissue-resident
macrophages, in response to tissue environmental cues,
activate the corresponding functional polarization programs
which accompany the induction of specific gene-expression
programs. We have characterized tissue-specifictranscriptional programs of resident macrophages andidentified hundreds of genes that are selectively expressedin tissue macrophages. Among these genes, we foundtranscription factor GATA6 is uniquely expressed inmacrophages that reside in the peritoneal cavity. We
reported that GATA6 acts as a master transcriptional
regulator for functional specialization of peritoneal
macrophages, as the deletion of the GATA6 gene inperitoneal macrophages resulted in a defect in theperitoneal-specific gene expression program. Additionally,
we and other groups found the GATA6-mediating program
controls macrophage positioning within the peritoneal cavity,
local proliferation, and peritoneal-specific immune
responses.
Although functional specialization of tissue-resident
macrophages is thought to be induced under the influenceof tissue micro-environments, the identity of tissue-derived
signals was largely unknown. We reported that retinoic acid,
which is a lipophilic molecule derived from vitamin A, plays
an essential role in the induction of the GATA6 gene in

Figure 2. Macrophages are essential components for the maintenance of tissue homeostasis.

Recent Publications
⿎⿎ Okabe Y. Molecular Control of the Identity of Tissue-Resident
Macrophages. International Immunology 30: 485-491 (2018).

⿎⿎ Okabe Y., & Medzhitov R. Tissue-specific signals control reversible
program of localization and functional polarization of macrophages.
Cell 157: 832-844 (2014).

⿎⿎ Okabe Y, & Medzhitov R. Tissue Biology Perspective on Macrophages.
Nature Immunology 17: 9-17 (2016).

63

development of effective CAR-T cell therapy, the mostestablished huge numbers of mAbs reacting with AML cells,

critical problem is lack of appropriate cancer-specific target

and are now selecting ones recognizing antigen structures

antigens. We have started looking for cell surface antigen

highly specific for AML cells. In the case of solid tumors,

structures for various types of cancer in collaboration withwhile inefficient trafficking of CAR-T cells to tumor sites andseveral departments treating cancer in Osaka University

immune-suppressive tumor microenvironment hamper

Hospital.

Immunology

Cellular Immunotherapy

cancer-specific cell surface antigen. We have alreadyNaoki Hosen, MD/PhD
Professor

Naoki Hosen

Associate Professor
Assistant Professor

Takafumi Yokota
Kana Hasegawa
Kentaro Fukushima
Jiro Fujita
Hisashi Kato
Michiko Ichii
Yasutaka Ueda
3

Support Staff

We are focusing on cellular immunotherapy, especiallychimeric antigen receptor (CAR)-T cell therapy for cancer.
CAR is generated by fusing the antigen recognition domainof a tumor-specific monoclonal antibody (mAb) with CD3ζand a costimulatory molecule such as CD28 or 4-1BB, andtransduced into T cells using retroviruses or lentiviruses tomake CAR-T cells. CAR-T cells specifically recognize
cancer cells using the cancer-specific mAb-derived antigenrecognition domain, and are activated. Activated CAR-T
cells kill tumor cells and also proliferate extensively. CAR-T
cells share the advantages of both mAbs and cytotoxic T
cells, and have a high affinity and specificity for tumor cells,
as well as high cytotoxicity and proliferation potential. CD19
CAR-T cells showed surprisingly high effectiveness against
acute lymphocytic leukemia and malignant lymphoma.
Currently, many researchers are trying to develop CAR-T
cells for various types of cancer. However, cell surface
target antigens have to be identified for each kind of cancer
to develop CAR-T cells.

Development of a new CAR-T cell therapy for multiple
myeloma64

Cancer-specific cell surface antigens are ideal targets
for CAR-T cell therapy. However, such antigens are not likely
to remain unidentified following extensive searching bytranscriptome or proteome analysis. We discovered that theactive conformer of an integrin could serve as a specifictherapeutic target for multiple myeloma (MM), which is anincurable hematological cancer characterized by theaccumulation of neoplastic plasma cells in the bone marrow(BM). We first identified MMG49 as an MM-specific mAbafter screening more than 10,000 anti-MM mAb clones, andfound that it specifically recognized a subset of integrin β7
molecules. The MMG49 epitope is located in the N-terminal
region of the β7 chain, which is predicted to be inaccessible

Figure. MMG49 CAR T-cell therapy targeting the active conformation of integrin β7 is promising for MM

in the resting integrin conformer, but exposed in the active
conformation. Elevated expression and constitutive
activation of integrin β7 conferred high MMG49 reactivity on
MM cells, whereas MMG49 binding was barely detectable inother types of cells, including normal integrin β7+lymphocytes. Furthermore, T cells transduced with MMG49derived CAR exerted anti-MM effects without damaging
normal hematopoietic cells. A clinical trial of MMG49 CAR
T-cell for MM is now on-going.

Development of CAR-T cell therapy targeting antigen
structures formed as results of post-translational eventsin various types of cancer
The above finding suggests that cancer
immunotherapeutic targets may yet be identified in manycell-surface proteins that undergo post-translationalchanges, even if the expression of the proteins themselves
is not cancer-specific. Thus, we applied the same strategy
to various types of cancer. For hematological cancers suchas acute myeloid leukemia (AML), the only hurdle fordeveloping CAR-T cell therapy is lack of an appropriate

Recent Publications
⿎⿎ Shingai Y, Yokota T, Okuzaki D, Sudo T, et al. Autonomous TGFbeta
signaling induces phenotypic variation in human acute myeloid
leukemia. Stem Cells (2021), in press.

⿎⿎ Hosen N, Matsunaga Y, Hasegawa K, et al. The activated conformationof integrin beta7 is a novel multiple myeloma-specific target for CAR T
cell therapy. Nat Med 23: 1436-1443 (2017).

⿎⿎ Toda J, Ichii M, Oritani K, et al. Signal-transducing adapter protein-1 isrequired for maintenance of leukemic stem cells in CML. Oncogene
39: 5601-5615 (2020).

65

Imaging

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

Toshio Yanagida
Ben Seymour

Figure 1. Behavioural data panel based in inferring the underlying computation parameters that may be modulated as a function of
inflammatory disease. These can be used as a biomarker of immune-brain interactions.

The lab continues to study what happens in the brain
during injury and inflammation. We focus on how these elicit
a suite of behavioural responses that are appropriate to
injury: pain, fatigue, and lowered mood. That is, thesebehaviours are adaptive in normal circumstances, in thatthey allow the individual to protect tissue that may bevulnerable as a result of existing damage, conserve energy,
and reduce unnecessary reward seeking behaviour thatwould consume resources not critical for survival. In
particular, we have been developing behavioural tasks thatallow us estimate and quantify these effects in any
individual, which therefore provide a biomarker of the impactof an inflammatory disease on cognitive and affectivefunction. Our analysis is based on sophisticatedcomputational models of behaviour, and yields a set ofinferred parameters that capture the underlying mechanistic
basis of immune-brain modulation. Practically, this is a panelof results that reflects an immune-behavioural signature in
any individual, as shown in Fig 1. We have been validating
this methodology in patients with rheumatoid arthritis. In thecontext of COVID-19, this testing ability has particular
relevance to the evaluation of patients with long COVID
syndrome. This refers to the persistent symptoms of fatigue
and cognitive blunting that effects a high proportion of postCOVID sufferers (up to 87%), and represents a major
challenge to healthcare systems in 2021 onwards. We have66

therefore been developing our methodology to be used in
an online setting (as a mobile-phone or tablet application)that would potentially allow us to gather large-scale data in
any country.
In parallel, we continue to develop theoretical andcomputational models of injury-related behaviour, using
mathematical modelling and simulation based approaches.
In particular, we’ve been studying how the brain uses pain
signals to shape conservative (i.e. non-risky / safe)
behaviour in dangerous environments. The broader context
for this work is that we want to understand how changes inhomeostatic status, such as that which occurs after injury or
inflammation, can change our behaviour. Ultimately we want
to be identify how and where immune or neural information
can signal to the brain that behaviour needs to change.
Using our modelling approach, we have proposed
algorithms that capture the trade-off between risky andconservative behaviour in the context of dangerous
environments, and shown how they predict behaviour in aset of artificial intelligence test-bed scenarios: we do this insimple environments (termed tabular reinforcement
learning), and complex environments (termed deep
reinforcement learning) – see figure 2. These simulations
provide predictions that can now been tested in real humanexperiments – both in healthy people, and those with injuries
or inflammatory disease.

Figure 2. Some examples of simulations of an artificial agent that inhabits a dangerous world, and needs to navigate to avoid states thatcause injury, in simple and complex (deep) environments.

Recent Publications
⿎⿎ Zhang S, Yoshida W, Mano H, Yanagisawa T, Mancini F, Shibata K,
Kawato M and Seymour B. Pain control by co-adaptive learning in abrain-machine interface. Current Biology 30(20): 3935-3944 (2020).
⿎⿎ Becker S, Loffler M, Seymour B. Reward enhances pain discriminationin humans. Psychological Science 31(11) (2020).

67

Masaru Ishii, MD/PhD
Professor

Masaru Ishii

Associate Professor
Assistant Professor

Junichi Kikuta
Takao Sudo
Yutaka Uchida
3
2
1

Postdoctoral Fellows
Research Assistants
Support Staff

1. Intravital bone imaging revealing osteoclast andosteoblast dynamics in vivo
We developed a novel imaging system to visualize
inside bones using intravital multiphoton microscopy. Using
this methodology, we succeeded in visualizing the in vivo
behaviors of osteoclast and osteoblast as well as the activityof bone-resorbing by osteoclasts. In addition to developmentof methodology, we are also updating various materials forbone imaging such as mouse models to visualize osteoclast
and osteoblast and probes to visualize the bone resorption
activity of osteoclasts. Our major contributions to the field ofbone biology research are as follows:• Identifying S1P (Sphingosine-1-phosphate) as a key
factor in controlling the migratory behavior of osteoclast
precursors (Nature 2009, J. Exp. Med. 2010).
• Showing the substantial contribution of S1P-mediated68

migration of bone cells by generating S1P transporter
deficient mice (J. Clin. Invest. 2012).
• Identifying Vitamin D as significantly suppressing bone
destruction by modulating S1P-mediated migration of
osteoclast precursor (Proc. Natl. Acad. Sci. USA 2013).
• Proposing two distinct mature osteoclast functional
states: bone-resorbing mature osteoclasts and nonresorbing mature osteoclasts (J. Clin. Invest. 2013).
• Showing the importance of epigenetic processes ofosteoclast in the regulation of cellular metabolism anddifferentiation (Nat. Med. 2015).
• Developing a new fluorescence probe for detecting

osteoclast that contributes to RA (rheumatoid arthritis),
called Arthritis-associated osteoclastogenic Macrophages,
or “AtoMs” (Nat. Immunol. 2019). We found that the arthritis-

Inside live bones, osteoclasts and osteoblasts are

inducing abnormal osteoclasts have distinct properties andconstantly generated. Under well-controlled bone

origins from normal osteoclasts involved in bone

destruction and formation processes, bone constantly
undergoes destruction and formation to maintainhomeostasis. In our previous study, we developed a unique
technology to collect and analyze cells from arthritic joints,
successfully identifying a new type of bone-destroying

metabolism. By targeting the differentiation and functions ofarthritis-inducing abnormal osteoclasts, we will be able toestablish innovative therapeutic strategies and nextgeneration of anti-RA drugs that do not affect normal
osteoclasts and normal bone homeostasis.

Imaging

Immunology and Cell Biology

2. Identification of origin and function of abnormal
osteoclasts: “AtoM”

3. A new mechanism for bone marrow recovery fromchemotherapy treatment: Emergency switch by innate
lymphoid cells

bone surface pH to visualize bone resorption by mature
osteoclasts (Nat. Chem. Biol. 2016).
• Applying intravital bone imaging to drug efficacy
evaluation and elucidation of mechanism of action (Ann.
Rheum. Dis, 2018; JBMR plus, 2018).
• Showing the importance of cell to cell communication
between mature osteoblasts and mature osteoclasts forbone homeostasis (Nat. Commun. 2018).
PTH (parathyroid hormone) is an important hormone that
controls bone and calcium metabolism and is currently used
as a therapeutic agent for osteoporosis. In a previous study(Nat. Commun. 2018), we demonstrate that PTH suppresses
bone resorption through promoting interaction of osteoblasts
and osteoclasts but could not clarify the detailed molecularmechanism. In the follow-up study (Nat. Commun. 2021), we
identify SLPI (secretory leukocyte protease inhibitor) as a
key mediator for PTH-induced bone formation. We showed
SLPI up-regulation in osteoblasts from PTH-treated mice.
Furthermore, Slpi mutant mice analysis exhibited that PTHinduced bone mass increase was suppressed in Slpi mutant
mice. Through this study, we demonstrate that two distinct
functions of SLPI for PHT-induced bone formation; 1)
intracellular SLPI effects on osteoblasts to promote osteoblast
differentiation and proliferation, 2) extracellular SLPI effects
on the interaction with osteoblasts and osteoclasts andsuppresses bone resorption by osteoclasts. Our results
suggest that SLPI regulates both bone formation and bone
resorption.

Figure. In vivo cellular dynamics in various immune systems.
Immune cells are highly dynamic and interconnect various tissues
and organs, by forming a ‘soft-wired’ network. We are elucidating
the basic principle controlling the dynamic nature of immune cellsby visualizing in vivo behaviors using advanced imaging
techniques.

We discovered that iLC2 (type 2 innate lymphoid cells)in the bone marrow detect a critical state in bone marrowcells after chemotherapy treatment and promote recovery
from blood cell loss through GM-CSF (Granulocyte
Macrophage colony-stimulating Factor) secretion (J. Exp.
Med 2021). We developed an intravital imaging system for
HSPCs (hematopoietic stem/progenitor cells) and found theunusual behavior of HSPCs transplanted into the bonemarrow with chemotherapy treatment. We performed gene
expression analysis using RNAseq for the HSPCs showing
the abnormal behavior and found that the HSPCs
proliferated under stimulation of GM-CSF from the bonemarrow environment after chemotherapy treatment. Further
single-cell RNAseq analysis identified that iLC2 was
responsible for GM-CSF secretion in the injured bonemarrow. Transplantation of iLC2 into chemotherapy treatedmice accelerated the recovery from blood cell loss. We
expect GMCSF and iLC2 can be applied in new therapeuticstrategies for recovery from blood cell loss after
chemotherapy treatment.

Recent Publications
⿎⿎ Morimoto A, et al. SLPI is a critical mediator that controls PTH-inducedbone formation. Nat. Commun. 12: 2136 (2021).
⿎⿎ Sudo T, et al. Group 2 innate lymphoid cells support hematopoietic
recovery under stress conditions. J. Exp. Med. 218: e20200817 (2021).
⿎⿎ Matsui T, et al. Nonlinear optics with near-infrared excitation enablereal-time quantitative diagnosis of human cervical cancers. Cancer
Res. 80: 3745-3754 (2020).

⿎⿎ Hasegawa T, et al. Identification of a novel arthritis-associated osteoclast
precursor macrophage regulated by FoxM1. Nat. Immunol. 20: 1631-43(2019).
⿎⿎ Simmons S, et al. High-endothelial cell-derived S1P regulates dendritic
cell localization and vascular integrity in the lymph node. eLife. 8:
e41239 (2019).

69

Jun Hatazawa, MD/PhD
Professor

Jun Hatazawa

Associate Professor
Assistant Professor
Postdoctoral Fellows
Visiting Scientists
Support Staff

Hiroki Kato
Tadashi Watabe
1
1
1

The Nuclear Medicine Laboratory has been working on
a cancer therapy by means of alpha-emitting radioisotopes
and on chronic neuro-inflammation imaging by means ofpositron emission tomography (PET) in patients with multiple
sclerosis and in those with child-onset epilepsy.
Targeted Alpha Therapy (TAT) is an application of theradiation emitted from alpha-particles (= 4He nucleus) totreat locally advanced and/or metastatic cancers. Alphaparticles have a robust cell-killing effect because of its highradiation quality factor. To develop TAT, the key issues are1) producing large amounts of alpha particles such as
Actinium-225 (225Ac, physical half-life: 9.9 day) andRadium-223 (223Ra, physical half-life: 11.4 day) effective forclinical use, 2) delivering alpha particle-bound molecules totarget specific cancer cells, and 3) evaluating damage tonormal tissue. In a clinical setting, 223Ra dichloride is used totreat castration-resistant prostate carcinoma patients withbone metastasis. These alpha emitting radio-isotopes areproduced in foreign-based nuclear reactors and not inJapan.
The element 85, Astatine-211 (211At, physical half-life: 7.2hour), is another alpha-emitting radio-isotope and is produced
by a particle accelerator in the Research Center for Nuclear
Physics at Osaka University. We succeeded in the
production and purification of 211At for preclinical studies
70(Watabe T, et al., J. Nucl. Med. 2019). 211At belongs to the
halogen family in the periodic table and its distribution issimilar to 131I. After administering 0.1 MBq or 1.0 MBq of
211

At-NaAt, elevated amounts accumulated in the thyroid

inflammation. In patients with multiple sclerosis, 11C-acetate
was utilized to evaluate the metabolism of reactive

TAT agents to target various entities of human carcinoma.

astrocytes in the brains of patients with multiple sclerosis.

Another potential TAT agent is fibroblast activation

Kinetic analysis revealed that the rate constant K2,

protein inhibitor (FAPI) labeled with 225Ac. FAPI targets

representing efflux of acetate from glial cells to systemic

cancer stroma cells developed by the Heidelberg University
Hospital group (Giesel FL, et al., J. Nucl. Med. 2019). We
confirmed a tumor growth suppression effect of 225Ac-FAPI

circulation, was higher than normal in MS patients both inwhite matter and gray matter (Kato H, et al., J Cerebr. Blood.
Flow. Metab. 2020). In patients with child-onset epilepsy,
translocator protein targeting PET tracer 11C-DPA713

in the tumor xenograft model of mice (Watabe T, et al., J.
Nucl. Med. 2020).
Neuro-inflammation is associated with a variety ofchronic neurological diseases. We developed methods thatuse PET to evaluate the severity and extent of neuro-

gland, stomach, bladder, heart, lungs, spleen, kidneys, andtestis in rats, similar to the results after 131I-NaI administration.
The 0.1 MBq group showed no abnormalities while the 1.0
MBq group showed decreased body weight and foodintake. Histological analysis showed atrophy and fibrosis in
the thyroid gland. The administration of 1.0 MBq of 211AtNaAt resulted in transient toxicity in the white blood cells
and testis without severe hematological or renal toxicity. The
total cholesterol, total albumin, and total protein increased
with no signs of recovery probably due to hypothyroidism(Liu Y, et al., Trans. Oncol. 2020). Based on these results, aphase I/II clinical trial of 211Na-NaAt for the treatment of
intractable thyroid carcinoma is planned with support from
AMED (Principal Investigator: Watabe T).
We are developing a new TAT molecule, α-Methyl-ltyrosine (AMT). AMT has a high affinity for LAT1 which is
predominantly expressed on the membrane of human major
cancers (Kaneda-Nakashima K, et al., Cancer Science2020). 211At-labeled α-methyl-l-tyrosine (211At-AAMT) is apotential carrier of 211At into tumors. We evaluated theaccumulation of211

At-AAMT in vivo in mice by means of
SPECT (E-cam, Siemens, Munich, Germany). The SPECT
image showed an accumulation of 211At-AAMT in thetransplanted tumor. In an experiment of tumor growth
suppression, the PANC-1 cells tumor-bearing mice wereintravenously injected with 211At-AAMT at 0.4 MBq/mouse.
This treatment suppressed tumor growth for 40 days without
significant loss of body weight. The 211At-AAMT of 0.1MBq/
mouse and 1 MBq/mouse reduced metastatic lesions in the

Imaging

Nuclear Medicine

lungs of the B16F10 metastasis model in C7BL/65 mice. Our
results suggests that 211At-AAMT can be a candidate for theshowed increased accumulation in the epileptogenic fociindicating neuro-inflammation in the epileptic foci (KagitaniShimono K, et al., J. Neuroinflam. 2020).

Figure. Treatment effect (A) and change in body weight (B) in PANC-1 xenograft mice model after injection of 225Ac-FAPI-04(Watabe T, et al. J Nucl Med. 61:563-569, 2020)

Recent Publications
⿎⿎ Liu Y, Watabe T, aneda-Nakashima K, et al. Preclinical evaluation ofradiation-induced toxicity in targeted alpha therapy using [211At] NaAt
in mice: a revisit. Translational Oncology 13: 100757 (2020).

⿎⿎ Kagitani-Shimono K, et al. Clinical evaluation of neuroinflammation inchild-onset focal epilepsy: a translocator protein PET study J
Neuroinflammation. 18: 8 (2021).

⿎⿎ Kaneda-Nakashima K, Zhang Z, Manabe Y, et al. K-emitting cancer
therapy using 211At-AAMT targeting LAT1. Cancer Science (2020). doi:
10.1111/cas.14761

⿎⿎ Kato H, et al. Astrocyte metabolism in multiple sclerosis investigatedby 1-C-11 acetate PET. J Cerebr Blood Flow Metab. 41(2): 369-379(2021).

⿎⿎ Watabe T, et al. Theranostics Targeting Fibroblast Activation Protein in
the Tumor Stroma: 64Cu-and 225Ac-Labeled FAPI-04 in Pancreatic
Cancer Xenograft Mouse Models. J Nucl Med. 61(4): 563-569 (2020).

71

Kazuya Kikuchi, PhD
Professor

Kazuya Kikuchi

Associate Professor
Assistant Professor
Postdoctoral Fellows
Research Assistants
Support Staff

Yuichiro Hori
Masafumi Minoshima
1
2
1

An Acid Activatable Fluorescence Probe for Imaging
Osteocytic Bone Resorption Activity in Deep Bone
Cavities

72

Long lived osteocytes constitute around 90-95% of allbone cells and exist within disk-shaped cavities called
osteocytic lacunae that are present throughout themineralized bone matrix. Osteocytes project their longdendrites into these canals, which allows them to detect andmodulate shear stress through communication with surface
bound osteoblasts and osteoclasts that are responsible formodifying bone structures. Osteocytes are known to directly
remodel the bone walls of their lacuna-canalicular systems
in response to hormones produced by lactating or
hibernating animals, with lacunae bone wall mineralisation
producing larger cavities in a process known as osteocytic
osteolysis. However, the physiological role of osteocytes in
direct bone mineralisation is still open to debate, so theavailability of a high-resolution imaging method that would
enable osteocytic osteolysis to be imaged in real time would
be extremely useful. We have previously developed areversible OFF/ON pH-sensing probe (pHocas-3) containing
bisphosphonate derived alendronate fragments that bindstrongly to the bone matrix enabling visualisation of
osteoclast mediated bone resorption processes.
Unfortunately, attempts to employ pHocas-3 for the intravital
imaging of low-pH regions in osteocytic lacunae within thebones of living mice proved unsuccessful.
Thus, we developed a new probe (pHocas-RIS) that
could be used to visualize the low pH environments ofosteocytic lacunae that contained acid secreting osteocytes(Figure 1, top). Conjugation of the moderate bone binding
drug risedronate to a pH-activatable BODIPY fluorophoreafforded pHocas-RIS in order to penetrate osteocytic
lacunae cavities embedded deep within the bone matrix.
The fluorescence quantum yields (QY) of the probes atdifferent pH values were acquired by measuring theirabsorption and fluorescence emission spectra in a citratephosphate buffer. The fluorescence intensity of the pHocasRIS probe increased approximately 15-fold as the pH
decreased from 8.0 to 4.0, with changes in its QY at different
pH levels used to calculate the pKa value of its aniline moiety
as 6.8.
Imaging studies were then carried out using transgenic
TRAP-tdTomato mice containing osteoclasts expressing a
red fluorescent protein. Daily subcutaneous administrationof both probes to these genetically modified mice over a 3
day period was followed by their anaesthetisation and twophoton imaging of the medullary cavities of their thin calvariaparietal bones. Processing of the images were carried out
using spectroscopic unmixing algorithms that enabled
fluorescence signals from the probe (green), tdTomato (red)and second-harmonic generation (SHG) emissions of bone
collagen fibres (blue) to be distinguished. Imaging studies
revealed the presence of green fluorescence signals fromprotonated pH-activatable pHocas-RIS produced from theaction of stationary acid secreting osteoclasts in directcontact with bone surfaces within the bone marrow cavity.
Further imaging studies revealed the presence of small
numbers of ring-shaped green fluorescence signals of the
protonated pHocas-RIS that were present in bone obtained
from deep cortical regions (Figure 1, bottom). It is proposed

and no fluorescence for a bone section treated at pH 10.0.

to the walls of osteocytic lacunae containing bone resorptive

This clearly demonstrated that the intensity of the pH-

osteocytes that were actively secreting acid to create a low

activatable fluorescent response of the pHocas-RIS probe

pH environment. The pH responsiveness of the pHocas-RIS

can be used to effectively image the pH of osteocytic

probe within the lacunae of the bone matrix was confirmed

lacunae in living bone. Our pH responsive probe can beby carrying out ex vivo fluorescent imaging of cryogenically
prepared deep bone slices at different pH levels. Thesestudies revealed the presence of strong fluorescent signals
for osteocytic lacunae in a bone section treated at pH 4.0,
reduction in fluorescent intensity of a bone section at pH 7.0,

used to visualize the bone mineralizing activities of acidproducing osteocytes in real time, thus providing a valuable
imaging method to explore their central role in remodelingbone-matrix in health and disease.

Imaging

Chemical Imaging Techniquesthat these fluorescent signals originate from probes boundFigure 1. (top) Chemical structure of risedronate containing pH-activatable pHocas-RIS probe.
	(bottom) Two-photon excitation imaging of calvaria parietal bones after administration of
pHocas-RIS. White arrows indicate protonated pHocas-RIS bound to the walls of acidic
osteocytic lacunae.

Recent Publications
⿎⿎ Kowada T, Arai K, Yoshimura A, Matsui T, Kikuchi K & Mizukami S.
Optical Manipulation of Subcellular Protein Translocation Using a
Photoactivatable Covalent Labeling System. Angew. Chem. Int. Ed. inpress (2021).
⿎⿎ Hori Y, Nishiura M, Tao T, Baba R, Bull SD & Kikuchi K. Fluorogenic
Probes for Detecting Deacylase and Demethylase Activity towards
Post-translationally-modified Lysine Residues. Chem. Sci. 12: 29482503 (2021).
⿎⿎ Hashimoto R, Minoshima M, Kikuta J, Yari S, Bull SD, Ishii M, &Kikuchi K. An Acid Activatable Fluorescence Probe for Imaging
Osteocytic Bone Resorption Activity in Deep Bone Cavities. Angew.
Chem. Int. Ed. 59: 20996-21000 (2020).

⿎⿎ Imoto T, Minoshima M, Yokoyama T, Emery BP, Bull SD, Bito H, &Kikuchi K. A Photoactivatable Antagonist for Controlling CREB
Dependent Gene Expression. ACS Cent. Sci. 6: 1813-1818 (2020).
⿎⿎ Torii K, Hori Y, Watabe K, & Kikuchi K. Development of Photoswitchable
Fluorescent Molecules Using Arylazopyrazole. Bull. Chem. Soc. Jpn.
93: 821-824 (2020).

73

Kazuhiro Suzuki, MD/PhD
Professor

Kazuhiro Suzuki

Assistant Professor
Postdoctoral Fellows
Research Assistants
Support Staff

Akiko Nakai
1
2
2

Our laboratory has been studying the interactions
between the nervous and immune systems with a special
focus on the roles of adrenergic nerves, which constitute theefferent arc of the sympathetic nervous system, in thecontrol of adaptive immune responses. Our study revealed amechanism by which adrenergic nerves control lymphocyte
trafficking through lymph nodes. Inputs from adrenergic
nerves to the β2-adrenergic receptor expressed on
lymphocytes enhance the responsiveness of a specific setof chemokine receptors and inhibit lymphocyte exit fromlymph nodes (Nakai et al., J. Exp. Med. 2014). This mechanism
was found to generate diurnal variations in lymphocyte
numbers in lymph nodes and consequently the magnitudeof adaptive immune responses in phase 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 and74

chemokine receptors, we identified a protein complex
consisting of copper metabolism MURR1 domain-containing(COMMD) 3 and COMMD8 (COMMD3/8 complex), of which
the functions had been totally unclear (Fig. A).
We first identified COMMD8 as a protein that binds to
the C-terminal tail of a representative chemokine receptor
CXCR4. Additional proteomic screening revealed theinteraction of COMMD8 with COMMD3. We found that these
proteins constitutively formed a complex in the cytosol, butwere translocated to the plasma membrane after stimulationof 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 proteasome in theabsence of the other, and deficiency of either protein produced
the same phenotypes, indicating that both COMMD3 andCOMMD8 are required for the stability and functions of theircomplex. Deficiency of COMMD3 or COMMD8 in B cells
reduced their chemotactic responses mediated by thereceptors to which the COMMD3/8 complex was recruited.
Thus, the COMMD3/8 complex is a positive regulator ofchemoattractant receptor signaling (Nakai et al., J. Exp.
Med. 2019).
Agonist binding to GPCRs activates trimeric G proteins
to induce 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-activated protein
kinases (MAPKs). The GRK family consists of seven
mammalian members, among which GRK2, GRK3, GRK5,
and GRK6 are expressed ubiquitously. Different GRKs
phosphorylate distinct sites on the C-terminal tail of thereceptor, establishing a barcode that dictates the functional
consequences of β-arrestin engagement. Thus, specifictargeting of GRKs to activated GPCRs is crucial for signaltransduction. Our mechanistic analysis of the action of the
COMMD3/8 complex demonstrated that this protein complex
functions as an adaptor that selectively recruits GRK6 tochemoattractant receptors, which promotes MAPK activationand consequently lymphocyte chemotaxis (Fig. B). It has
been suggested that the specificity of GRK recruitment to
GPCRs is determined by the relative expression levels of

COMMD3/8 complex plays essential roles in the control of B

receptor conformations induced by ligand binding. Our
study identifies a GRK-recruiting adaptor, the COMMD3/8

cell migration and induction of humoral immune responses(Nakai et al., J. Exp. Med. 2019). To test the involvement ofcomplex, as an additional determinant of GRK specificity for

the COMMD3/8 complex in autoimmune diseases, we

GPCRs (Nakai et al., J. Exp. Med. 2019).
Consistent with the reduced chemotactic responses of
COMMD3- and COMMD8-deficient B cells, the mutant B
cells showed multiple defects in their migration in vivo (Fig.
C). Additionally, deficiency of COMMD3 or COMMD8 in B
cells severely impaired production of antigen-specificantibodies (Fig. D), which was accompanied with reduced
formation of germinal centers (Fig. E). Therefore, theestablished an experimental system that allowed inducible
deletion of the COMMD3/8 complex in moue models of
autoimmunity. Notably, deletion of the COMMD3/8 complex
in the course of the diseases blocked their clinical andpathological progression with suppressed production of
autoantibodies. These findings suggest that the COMMD3/8
complex is a potential therapeutic target for autoimmune
diseases.

Imaging

Immune Response Dynamics

individual GRKs, which vary among cell type, and distinct

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) Defective outer follicular migration of COMMD8-deficient (Commd8Δ) B cells at an early time point (3 h) after immuniza tion.
(D and E) Impaired antibody production (D) and germinal center formation (E) in B cell-specific COMMD8-deficient mice. Scale bars, 200 μm.

Recent Publications
⿎⿎ Nakai A, Leach S and Suzuki K. Control of immune cell trafficking
through inter-organ communication. Int. Immunol. 2021 in press.
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 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).
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 Suzuki K and Nakai A. Immune modulation by neuronal electric shockwaves. J. Allergy Clin. Immunol. 141: 2022-2023 (2018).

75

Nicholas Isaac Smith, PhD
Associate Professor

Nicholas Isaac Smith

using a combination of fluorescent-labeled imaging flow

therefore used Raman imaging which can non-invasively

cytometry, and unlabeled Raman imaging. NETS are

track these changes, finding that the uptake, processing

important both in pathogen defense and also in numerous

and recovery from exposure to fatty acids varies depending

disease pathology, for example, vasculitis, sepsis, andon the chain length of the fatty acid and the degree ofothers. It is thought that different types of NETs form insaturation. Toxicity was also clarified at higher levels of lipid
exposure (Sugiyama et. al. Analyst, 2020). Images of theprocess are shown in Figure 1.
We continued our ongoing study of the nature andcauses of neutrophil extracellular traps (NETS) in neutrophils

different circumstances, however, there are very limited
methods for easily evaluating the NETs as they form aroundthe neutrophils. We are now combining quantitative phaseimaging, Raman analysis, and deep-learning to evaluate thisspecific question.

Imaging

Biophotonics

changes associated different fatty acids in live cells. We

Associate Professor (Lecturer) Nicolas Pavillon
Assistant Professor
Alison Hobro
Postdoctoral Fellows 1
Support Staff
1

76

The biophotonics laboratory develops tools for label-free
analysis of single cells. Single-cell analysis is a populartarget for a large number of researchers, usually pursued bylabeling surface markers, by introducing fluorescent dyes
into the cell, or by invasive, yet comprehensive, techniquessuch as single cell RNA sequencing. In contrast, our tools
are based on label-free optical methods, which aim to
produce some of the same discriminatory capability as themore invasive methods. Additionally, label-free methods are
based on endogenous contrasts of the cell, and can alsofind novel features that can be used to discriminate between
cell phenotypes or cell states.
We develop both fundamental techniques for label-free
analysis, and in collaboration with other groups in IFReC,
also develop applications in immunological cellular imaging
and analysis. We use a variety of label-free approaches,
including Raman scattering, which provides molecularsignatures from a target cell, as well as quantitative phaseimaging, which provides a complete quantitative map of thecell morphology. It is fortunate that both of these sets of
information can be linked to cellular functions of interest,and can be used as classifiers of cell type or cell behavior.
When looking at a culture dish, the morphology of cells canbe seen to differ dramatically from cell to cell. Similarly, thebiomolecular composition of each cell varies, though it isless trivial to observe. It may seem obvious that thecomposition of a cell reflects important aspects of its
biological role, such as the protein expression in response
to a stimulus, but each cell’s composition also varies due toinherent and somewhat random biological variability. This is
one reason why a multimodal approach can be ideal for

teasing out subtle cellular biomolecular signals that are
related to biological questions, which can otherwise be
hidden by cell-to-cell differences. It also that to ensure thecellular information we measure is significant, a large
number of single cell measurements is required.
We then continued to push towards higher throughput
single-cell analysis. With larger numbers of samples, andsubtle but important variations between classes of cells, weevaluated how well different machine-learning tools could
assist the data analysis. There are many types of machinelearning tools available. Some of the more “deep-learning”
style of tools can classify aspects of samples, but are lesshelpful in terms of interpreting the differences in a biologicalsense. We therefore choose the method based on the
problem we are trying to solve. Our recent study publishedthis year (Pavillon et. al. Analyst, in press, 2021) showed that
if we use linear methods and suppress any features that do
not contribute to classification, the ability to discriminate
between subtle features across single cells can become
very robust. The remaining features can also be easier tointerpret than similar information in more traditional methods.
We demonstrated this in a well-defined case where cellular
protein synthesis was inhibited, measurements were madein inhibited and control cells, and the resulting discrimination
model showed us features consistent with mRNA
accumulation in the cell.
In applications more directly related to human disease,
we completed a study of fatty-acid induced changes inmacrophage cells. Non-alcoholic fatty liver disease (NAFLD)
is related to lipid-induced transitions of cells. There are very
few ways to label and track the uptake and downstream

Figure 1. Uptake of palmitic acid observed in Raw264.7 macrophage cells. We compared the uptake of four different fatty acids (Palmitic,
Steric, Oleic and Linoleic), and evaluate the transitions occurring in the macrophage. Changing the type of fatty acid, by varying the chain
length and degree of saturation, affects the amount of uptake, the uptake timeline and the later recovery from exposure. Raman images wereacquired at different concentrations, fatty acid type, and different time points. Panel (a) shows the Raman intensity (area under the curve
between 2876 - 2891 cm-1) of five macrophage cells exposed to different concentrations of palmitic acid for 24 hours, (b) shows the average
Raman spectra extracted from a 10 x 10 pixel block from all cells for each concentration of palmitic acid used and a reference Raman
spectrum of palmitic acid, (c) PCA scores images for PC1 and PC2 for cells exposed to palmitic acid and (d) the reference spectra for
purified palmitic acid aids the interpretation of the signals found in the macrophage cell. The five cells shown here are representative of thelarger datasets collected for all cells per condition. From: Sugiyama et al. Analyst, 2020.

Recent Publications
⿎⿎ Pavillon N and Smith NI. Deriving accurate molecular indicators ofprotein synthesis through Raman-based sparse classification. Analyst.
in press (2021).

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): 2676-2685 (2018).

⿎⿎ Sugiyama T, Hobro AJ, Pavillon N, Umakoshi T, Verma P, and Smith N.
Label-free Raman mapping of saturated and unsaturated fatty aciduptake, storage, and return toward baseline levels in macrophages.
Analyst 146 (4): 1268-1280 (2020).

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 Lelliott PM, Momota M, Shibahara T, Lee MSJ, Smith NI, Ishii KJ, andCoban C. Heparin induces neutrophil elastase-dependent vital andlytic NET formation. Int. Immunol. 32 (5): 359-368 (2020).

77

Bioinformatics

Systems Immunology
Daron Standley, PhD
Professor

Daron Standley

Associate Professor

Kazutaka Katoh
Shunsuke Teraguchi
Li Songling
Llamas Covarrubias Mara Anais
2
4
1

Assistant Professor
Postdoctoral Fellows
Research Assistants
Support Staff

A. Software for sequence and structural analysis of Abs
and TCRs
MAFFT is a very widely-used multiple sequence alignment(MSA) package. Aside from the obvious importance of MSAs
in evolutionary biology, MSAs are becoming more and more
important in structural bioinformatics as well. We utilize
MAFFT MSAs for antibody and TCR structure prediction
using Repertoire Builder. Repertoire Builder, in turn, is usedby our antibody docking package, AbAdapt. Repertoire
Builder is also used in our TCR-peptide-MHC modelingpackage, ImmuneScape, as well as for our BCR/TCR
clustering software, InterClone. Moreover, protein structures
can be used to improve protein MSA accuracy. To this end,
we have developed MAFFT-DASH for structure-informed
multiple alignment (Rosewicki, J. et al. 2019).

B. Structural modeling SARS-CoV-2 infection-enhancing
Abs
During the COVID-19 pandemic, we analyzed
betacoronaviruses that have infected non-human hosts, andidentified a group of potentially important residues for virus
evolution (Saputri, D. S. et al 2020), a subset of which turned
out to characterize SARS-CoV-2 variants with enhanced
infectivity (Katoh, K. and Standley, D.M. 2021). We also

78

worked closely with Prof. Arase and several other labs tounderstand the structure and mechanism of a subset of
antibodies that enhance SARS-CoV-2 infection (Liu, Y. et al,2021). A key finding is that these antibodies appear tobridge neighboring spikes, and, by ding so, alter theconformation of the host receptor binding domain. We arecontinuing to study these antibodies using molecular
dynamics and repertoire analysis in order to clarify both theirrole in disease progression and their prevalance in humanpupulations.

C. Structural modeling of CAR T cell receptors
One of the applications of protein structural modeling is
to develop more effective chimeric antigen receptor T cells(CAR T cells), most of which utilize a single-chain antibody
heavy and light chain (scFv) linked by a short peptide
fragment. In collaboration with a U. Penn team using antiCD22 CAR T cells to treat acute lymphoblastic leukemia(ALL), we predicted the oligomerization state of the scFVand helped show that dimeric scFVs were significantly more
effective than monomeric scFVs at tumor killing (Singh, N. et
al. 2021). This research, in turn, has renewed efforts in our
lab to understand the mechanistic role of TCR oligomerizationin T cell activation and inactivation.

Figure 1. A, in-house tools that can be used to model antibody structure and function; B, enhancing antibodies (pink, cyan) bridge SARSCoV-2 spike trimers (green) on the virus surface (grey); C, the length of the scFv linker affects oligomerization, which, in turn, has significant
effects on CAR T cell efficacy.

Recent Publications
⿎⿎ Singh N et al. Antigen-independent activation enhances the efficacy of
41BB co-stimulated CD22 CAR T cells. Nat. Med. in press (2021).

⿎⿎ Schritt D et al. Repertoire Builder: High-throughput structural modelingof B and T cell receptors. Mol. Syst. Des. Eng. 4: 761-768 (2019).

⿎⿎ Liu Y et al. An infectivity-enhancing site on the SARS-CoV-2 spike
protein is targeted by COVID-19 antibodies. Cell in press (2021).

⿎⿎ Rozewicki J, Li S, Amada KM, Standley DM & Katoh K. MAFFT-DASH:
integrated protein sequence and structural alignment. Nucleic Acids
Res 47: W5-W10 (2019).

⿎⿎ Xu Z et al. Functional clustering of B cell receptors using sequenceand structural features. Mol. Syst. Des. Eng. 4: 769-778 (2019).

79

Yukinori Okada, MD/PhD
Professor

Yukinori Okada

Research Assistants
Support Staff

4
2

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 of millions of thesubjects, and successfully identified comprehensive
catalogues of genetic risk loci of immune-related diseases.
However, little is known regarding how to develop
methodology to integrate large-scale human genome datawith diverse biological and immunological resources. The
goal of our laboratory is to develop such methods and apply
to the latest large-scale disease genome and multi-layeromics data.

Metagenome-wide association study of gut microbiome
revealed disease-specific feature of multiple sclerosis

80

Microbiomes play substantial roles in biology of a varietyof human immune-related diseases. We conducted
metagenome-wide association studies (MWAS) of gut
microbiome of multiple sclerosis (MS) utilizing wholegenome shotgun sequencing, a promising tool to elucidate
microbiome etiology. Phylogenetic MS case-control association
tests showed discrepancies of clades, including those

implicated in immune system (e.g., Erysipelatoclostridium_sp.
and Gemella morbillorum). Gene association tests found anincreased abundance of one putative dehydrogenase gene(Clo1100_2356) and one ABC transporter related gene
(Mahau_1952) in the MS metagenome. We further
demonstrated interaction between the MS metagenome andhost genome pathways (i.e., MWAS and genome-wide
association study (GWAS) interaction; Kishikawa T et al.
Front Cell Infect Microbiol 2020).

Genetic studies of human immune-related phenotypes
We conducted GWAS of human immune-related

Consideration of biological dynamics in disease status

diseases and clinical subtypes, to identify genetic risk lociprovides directional dosage effect on drug targets, which

and responsible genes. An GWAS of rheumatoid arthritis-

facilitates efficient high-throughput screening of chemicalcompounds from library. To this end, we have developed anovel in silico drug discovery pipeline named Trans-Phar(Figure 2). Trans-Phar screens potential chemicalcompounds by assessing negative correlation between
genetically-regulated case-control gene expression profiles
and compound-regulated gene expression profiles.
Application of Trans-Phar successfully identified the drugs
currently indicated (or under clinical trial) for the input GWAS
diseases themselves. Trans-Phar is computationally simple
and only requests publicly available data, such as GWAS
summary statistics and compound-perturbed gene
expression data (Konuma T et al. Hum Mol Genet 2021).

interstitial lung disease (RA-ILD) in the Japanese population
identified one novel RA-ILD risk locus of RPA3-UMAD1 at
7p21. The RA-ILD risk of the identified variant was relatively
high in the CT image patterns related to fibrosis. Our study
should contribute to elucidation of the complicated aetiology
of RA-ILD. A GWAS of autoimmune pulmonary alveolar
proteinosis (aPAP) of Japanese, which identified that one
common HLA class II allele, HLA-DRB1*08:03, strongly
drove risk of aPAP. The HLA-DRB1*08:03 allele was also
associated with an increased level of anti-GM-CSF antibody,a key driver of the disease (Shirai Y et al. Ann Rheum Dis
2020, Sakaue S et al. Nat Commun 2021).

Figure 1. DEEP*HLA, a convolutional deep learning method for
imputing HLA genotypes.

Figure 2. Trans-Phar, integration of Transcriptome-wide association
study and pharmacological database for in silico drug screening.

Bioinformatics

Statistical Immunology

Novel drug discovery by in silico screening utilizing
human genome and gene expression databases

Application of machine-learning technologies to humangenomics
Dynamic application of machine learning (ML),
especially deep learning (DL), is warranted in the field of
human immunology. As an initial introductory application of
DL to the large-scale population genomes, we developed a
new HLA imputation method named DEEP*HLA (Figure 1).
By converting population genomes into digital images,
DEEP*HLA can computationally estimate HLA gene variantsfrom the surrounding SNPs. DEEP*HLA imputation is lessbiased from distant-dependent linkage disequilibrium (LD)decay, which enabled more accurate imputation of rare HLA
alleles when compared with previous ML methods for HLA
imputation (Naito T et al. Nat Commun 2021).

Recent Publications
⿎⿎ Naito T. et al. A deep learning method for HLA imputation and transethnic MHC fine-mapping of type 1 diabetes. Nat Commun 12: 1639
(2020).

⿎⿎ Kishikawa T. et al. A Metagenome-Wide Association Study of Gut
Microbiome in Patients with Multiple Sclerosis Revealed Novel
Disease Pathology. Front Cell Infect Microbiol 10: 585973 (2021).

⿎⿎ Konuma T. et al. Integration of genetically regulated gene expression
and pharmacological library provides therapeutic drug candidates.
Hum Mol Genet 30: 294-304 (2021).

⿎⿎ Shirai Y. et al. Association of the RPA3-UMAD1 locus with interstitiallung diseases complicated with rheumatoid arthritis in Japanese. Ann
Rheum Dis 79: 1305-1309 (2021).

⿎⿎ Sakaue S. et al. Genetic determinants of risk in autoimmune pulmonary
alveolar proteinosis. Nat Commun 12: 1032 (2021).

81

Associate Professor

Diego Diez

Postdoctoral Fellows

1

Our group integrates computational and single cell
genomics techniques to understand the immune system. We
develop computational methods to analyze single cell data.
We integrate experimental data (including transcriptome,
chromatin accessibility, protein expression, immune repertoireand spatial transcriptomics) with publicly available information
into network models of immune regulation. We apply thisframework to study gene regulatory networks controlling
immune cell development and function.

critical determinants of cell identity and function. We use
linear regression and machine learning to model immune
transcriptional regulatory networks. Using the expression
level of the regulators as a proxy for their activities we apply
these methods to study how these networks change during
immune cell differentiation and under different experimental
conditions.

Development of computational methods

We study the development of NKT cells. SKG mice havea mutation in ZAP70 molecule that causes weakened TCR
signaling resulting in a bias towards development of NKT1
compared to NKT2 in the wild type. Using single celltranscriptomics, protein expression, immune repertoire andchromatin accessibility we study the differentiation of NKT
cells in the thymus and spleen of SKG and WT mice. In a
more clinical setting, we apply single cell genomics to getinsight into IgA nephritis onset and therapies. We analyze
the transcriptome and protein expression of T and B cells in
the PBMC from IgAN patients before and after treatment
with e.g., tonsillectomy and/or steroid immunosuppression.
We combine these datasets with spatial transcriptomics
from tonsils to identify factors that lead to successful
therapy.
We collaborate with other groups at IFReC to study
diverse aspects of immune responses in a basic and clinicalsetting. With the Experimental Immunology laboratory andthe Systems Immunology laboratories, we study how TCR
signaling impacts T cell repertoire in SKG mice. With theAn important problem in single cell genomics is how to
combine different datasets while correcting for batch effects.
A key focus is on preserving the original cell population
structure while not introducing bias. We have developed
Canek, a method that leverages a fuzzy logic framework thatenables efficient batch correction without bias. Anotherproblem is the identification of marker genes. In collaboration
with Alexis Vandenbon at Kyoto University, we have developed
singleCellHaystack, a method to identify differentiallyexpressed genes from multi-dimensional representations of
single cell genomics data.

Mathematical modeling82

The large number of cells obtained in single cell
genomics experiments opens the door to approaches thatstudy the immune system using mathematical modeling andmachine learning. Transcriptional regulatory networks are

Host Defense laboratory, we study the role of the RNase

cells in human Eosinophilic Chronic Rhinosinusitis. With theReg-1 in immune cell development.

Combined Research Fields

Quantitative Immunology

Immune Regulation laboratory, we study the role of CD4 T

Applications to immunology

Figure 1. Single cell transcriptomics defines signatures of NKT cells in the thymus and spleen of WT and SKG mice.

Recent Publications
⿎⿎ Vandenbon A and Diez D. A clustering-independent method for finding
differentially expressed genes in single-cell transcriptome data. Nat
Commun 11(1): 4318 (2020).

⿎⿎ Bahrini I, Song JH, Diez D & Hanayama R. Neuronal exosomes facilitate
synaptic pruning by up-regulating complement factors in microglia.
Scientific reports 5: 7989 (2015).

⿎⿎ Teraguchi S, Saputri DS, Llamas-Covarrubias MA, Davila A, Diez D, etal. Methods for sequence and structural analysis of B and T cell
receptor repertoires. Comput Struct Biotechnol J 18: 2000-2011 (2020).

⿎⿎ Diez D, Agusti A & Wheelock CE. Network analysis in the investigationof chronic respiratory diseases. From basics to application. American
journal of respiratory and critical care medicine 190: 981-988 (2014).

⿎⿎ Nakai W, Yoshida T, Diez D, et al. A novel affinity-based method for theisolation of highly purified extracellular vesicles. Scientific Reports
6(1): 33935 (2016).

83Events & Outreach Activities

Due to the COVID-19 pandemic, travel by researchers has been restricted for the past year, and IFReC is no longer able
to invite researchers as speakers to its academic events. As a result, the IFReC Colloquium, an important event held every
two months allowing researchers of IFReC to gather together, had to be postponed several times to prevent the spread of
COVID-19. Fortunately, the use of online tools has now made it possible to hold various events, although in a different format.

IFReC Colloquia in FY 2020

“ImmunoSeminar” by online
IFReC started a series of online seminars called the “ImmunoSeminar
Series.” As the first speaker of this series, Prof. Jason Cyster (UCSF)
was chosen, and the seminar was held in real time across several
continents. This first seminar of the series was well received and theseries is planned to be continued by inviting world-class immunologists
to speak online at the seminars.

Events & Outreach Activities

Online Academic Events at IFReC

The Memorial Lecture by Prof. Kurosaki

No.

46

47

48

86

49

Date

Sept. 30, 2020

Nov. 25, 2020

Jan. 27, 2021

Mar. 10, 2021

Title

Speakers

Vaccine and Immunotherapy against viruses
and cancer.

Ken J. Ishii (Vaccine Science, Ishii Lab)

All-optical single cell phenotyping and pathwayspecific activation of immune cells.

Nicolas Pavillon (Biophotonics, Smith Lab)

Differential roles of RUBCN isoforms in memory
B and plasma cells differentiation.

Chao-Yuan Tsai (Immune Regulation, KikutaniGroup)

Applications of single cell genomic approaches
to immune cell development and differentiation
processes.

Diego Diez (Quantitative Immunology Research
Unit)

Identification of human CAR cells and anotherspecific transcription factor essential for
maintenance of the HSC cellular niche.

Yoshiki Omatsu, and Kazunari Aoki
(Stem Cell Biology & Developmental Immunology,
Nagasawa lab)

Characterization of the COMMD3/8 complex asa therapeutic target for inflammatory diseases.

Taiichiro Shirai (Immune Response Dynamics,
Suzuki Lab)Metagenome and metaviorme-wide association
studies of autoimmune diseases.

Yukinori Okada (Statistical Immunology, Okada
Lab)

A sublethal mutation in phospholipid flippasethat causes aberrant phosphatidylcholinetranslocation in the plasma membrane.

Katsumori Segawa (Biochemistry & Immunology,
Nagata Lab)In a first for IFReC, a special lecture in celebration of Prof. Tomohiro Kurosaki’s 65th birthday was held on March 18, 2021
in a hybrid format connecting the online audience with the venue. As the demand for events in this type of format is expected
to increase in the future, IFReC intends to continually upgrade the hosting environment for an improved and more productive
online experience.

87

As with IFReC colloquia and seminars, in-person outreach activities such as science cafes have been largely curtailed due
to COVID-19. As a consequence, IFReC began implementing a new approach to holding its outreach activities by making useof the web and online tools. The experience gained from holding these virtual events is expected to be highly useful even after
the COVID-19 restrictions on activities end.

● Enhancement of IFReC’s Official YouTube Channel● Science Agora - Ten days of connecting science and society
Date
Organizer
Speakers

Nov. 13-22, 2020
IFReC & Research Institute for Microbial Diseases (RIMD), Osaka University
Masayuki Miyasaka (IFReC) & Tokiko Watanabe (RIMD)● Research Frontline Lecture for Educators “Let's get to know the latest
research at WPI!”
Date
Nov. 28 & Dec. 5, 2020
Organizer	IFReC (Osaka Univ), IRCN (Univ Tokyo), ITbM (Nagoya Univ),and MANA (NIMS)
Speakers	Toshihide Yamashita (IFReC) & Takamitsu Watabe (IRCN), andothers● Let’s learn about Life Science! “World-class researchers from WPI!”
Date
Organizer
Speakers

Dec. 26, 2020
JSPS and 13 WPI institutes
Mari Murakami (IFReC) and others● T he 9 WPI Science Symposium “Life Science Research in the 21
Century”
thDate
Organizer

88

Events & Outreach Activities

Online Outreach Activities

stFeb. 7 & 8, 2021
ELSI (Tokyo Tech), WPI institutes and JSPS89

Japanese Language Class
These classes are held for overseas researchers/
students to alleviate any stress and inconveniencein their research and in their daily lives that may becaused by the language barrier. Totally, over 300
IFReC members have joined the classes since 2012.
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 eachlevel, Kanji, etc., through provided class materials
and group work. In FY2020, all the classes wereorganized by the use of an online tool.

Message from Ms. Tomomi Tomomune,a Japanese language teacher
I would like to support all of my students by providing
effective lessons to make your life more enjoyable.
During class, there are many activities which ask you toshare your experiences and opinions. By interacting withothers and hearing their stories, you can gain an insight
into unique, intriguing cultures and also widen yourperspective. Let’s expand our horizons through learning
Japanese together!

Information

90

Shimon SAKAGUCHI

Toshio YANAGIDA⃝ Robert Koch Award 2020

⃝ Member of the Japan Academy

Information

Major Awards“The regulatory T-cells (Tregs) discovered by Prof. Sakaguchi are regarded
as peacemakers of the immune system, because they prevent defense cells
patrolling the body from attacking the body’s own tissue. The clinical applicationof Tregs are already being pursued very actively.”

Hisashi ARASE⃝ Hideyo Noguchi Memorial Prize 2020

Tadamitsu KISHIMOTO⃝ The Tang Prize 2020
“Prof. Kishimoto discovered interleukin 6 (IL-6), which plays important roles invarious inflammatory responses, and led to the development of Tocilizumab(commercial name: ActemraR), which may be effective in treating the pneumonia
caused by COVID-19.”

Sujin KANG
⃝ The Young Scientists’ Award (MEXT) 2020
⃝ Osaka University Prize 2020

Yukinori OKADA
Shigekazu NAGATA⃝ A merican Association for Cancer Research (AACR)
Academy Fellow⃝ JSPS Prize 2020
⃝ President award of AMED 2020

“Prof. Nagata identified the Fas membrane protein as a member of the tumor
necrosis factor receptor family, and defined the role of the Fas ligand as an
inducer of apoptotic signaling.”

Masaru ISHIIth⃝ The 38 Osaka Science Prize

92

93

Information

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

As of Feb, 1st

Number of IFReC Staff
350

Researchers (JPN)
Researchers (Overseas)

300

Administrative and Support Staff

250
102

52

150
6

38

100

3

55

4

5

2

50
1

44

48

42

54

54

117

119

128

138

137

2011

2012

2013

2014

2015

90

105
102

50

90

89
86
53
47

43

44

130

124

124

117

2016

2017

2018

2019

43

42

24
16
8

106

92

70

200

117

93

129

142

2020

2021

65

0
2008

2009

2010

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

Finance
Sources

Expenditure

From the University
4.1%

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.
94

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

Main Grant
32.3%

Management
Expenses Grant
14.1%

JPN
3,873,985,055r
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

Grant-in-Aid
for Scientific
Research
8.6%

Contract
Research
31.8%

Donation
9.1%

Personnel
25.2%

Individual
research project
34.9%

JPN
3,873,985,055

Project Activities
21.4%
Equipment
18.1%
95
Travel 0.3%

Original Support Programs for Young Researchers

IFReC has established the Advanced Postdoc system, which offers outstanding young researchers opportunities to work

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

with diverse researchers in IFReC as well as to conduct their own research under their own merit. Under this system, IFReC
has employed excellent postdoctoral fellows from 2018. They were assigned to laboratories in IFReC with an internationallycompetitive salary and research funds (three million JPY per year) to conduct original research.

established two kinds of financial support programs for researchers. 1) “IFReC Kishimoto Foundation Fellowship,” which
has been used to invite international researchers to Osaka. 2) “Program for International Circulation of Young Talented
Researchers” for those who wish to participate in overseas research activities. These programs aim to develop the practicalskills and abilities of young researchers in international collaborative research and to develop their network with researchers
overseas. Since 2009, 127 researchers have received these grants.

Information

Advanced Postdoc Program at IFReC

Support for Paper Submission
Due to the inﬂuence of COVID-19 during 2020, it was difficult for
researchers to ﬁnd opportunities to present their research results atoverseas conferences. This program aims to support the disseminationof research results by the researchers in IFReC.

Website of the Advance Postdoc Program including the members as of March, 2021

Grant for Next Generation Principal Investigators

96

This program aims to foster the next generation of principal investigators at IFReC. In particular, challenging researchthat 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 in FY2018, two in FY2019, and three in FY2020 with the continuous
research funding for three fiscal years. The grant is expected to generate excellent research achievements, raise theinternational recognition of these PIs, and contribute to the acquisition of external research funding.

97

Information

Selected Articles
Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.
Tanaka S, Ise W, Inoue T, et al.

Harrison TE, Mørch AM, Felce JH, Sakoguchi A, et al.

Nature Immunology 21: 950-961 (2020)

Nature 587: 309-312 (2020)

Wataru Ise, Tomohiro Kurosaki and their research groupreported that deficiency of ten-eleven translocation (Tet)
DNA demethylase family members, Tet2 and Tet3, in B
cells led to hyperactivation of B and T cells, autoantibody
production and lupus-like disease. Mechanistically, in theabsence of Tet2/Tet3, down-regulation of CD86, which
normally occurs following chronic exposure of self-reactive
B cells to self-antigen, did not take place. The importanceof the dysregulated CD86 expression in Tet2/Tet3-deficient
B cells was further demonstrated by restraining, albeit
not completely, aberrant T and B cell activation by antiCD86 blocking. Tet2/Tet3-deficient B cells had a decreased
accumulation of histone deacetylase 1 (HDAC1) andHDAC2 at the Cd86 locus. Their findings suggest that
Tet2/Tet3-mediated chromatin modification participates in
repression of CD86 on chronically stimulated self-reactive
B cells, which, at least partly, contributes to preventing
autoimmunity.

The Plasmodium species that cause malaria are obligate
intracellular parasites, and disease symptoms occur as
they replicate within human blood. Despite risking immune
detection, the parasite delivers proteins that bind host
receptors to infected erythrocyte surfaces. In the causative
agent of the most deadly human malaria, Plasmodium
falciparum, RIFINs form the largest erythrocyte surface
protein family (Saito et al. Nature 2017). Some RIFINs canbind inhibitory immune receptors, acting as targets for
unusual antibodies containing a LAIR1 ectodomain, or
as ligands for LILRB1. RIFINs potentially dampen humanimmune responses against malaria.
The research group of University of Oxford and Osaka
University led by Hisahi Arase determined a structure of a
RIFIN bound to LILRB1. They showed that the RIFIN mimics
the natural activating ligand of LILRB1, MHC class I, in its
LILRB1-binding mode. Therefore, LILRB1-binding RIFINs
suppress NK cell function.

A BET family protein degrader provokes senolysis by targeting NHEJ andautophagy in senescent cells.

98

Structural basis for RIFIN-mediated activation of LILRB1 in malaria.

Association of the RPA3-UMAD1 locus with interstitial lung diseases complicatedwith rheumatoid arthritis in Japanese.

Wakita M, Takahashi A, Sano O, et al.

Shirai Y, Honda S, Ikari K, et al.

Nature Communications 11: 1935 (2020)

Ann Rheum Dis 79 (10): 1305-1309 (2020)

Although cellular senescence acts primarily as a tumour
suppression mechanism, the accumulation of senescentcells in vivo eventually exerts deleterious side effects
through inflammatory/tumour-promoting factor secretion.
Thus, the development of new drugs that cause the specificelimination of senescent cells, termed senolysis, isanticipated. Here, by an unbiased high-throughput
screening of chemical compounds and a biofunctional analysis, Eiji Hara group identified BET
family protein degrader (BETd) as a promisingsenolytic drug. BETd provokes senolysis through two
independent but integrated pathways: (i) attenuationof non-homologous end joining (NHEJ), and (ii) upregulation of autophagic gene expression. Notably,
BETd treatment eliminates senescent hepatic stellate
cells in obese mouse livers, accompanied by the
reduction of liver cancer development. Furthermore,

the elimination of chemotherapy-induced senescent cells by
BETd substantially increases the efficacy of chemotherapy
against xenograft tumors in immunocompromised mice.
These results reveal the vulnerability of senescent cells andopen up the possibilities for its control.

It is known that the mortality rate of rheumatoid arthritis
patients increases due to complications from interstitial
pneumonia.
Using genome-wide association analysis (GWAS)
for 5,000 rheumatoid arthritis patients in Japanese, theresearch group led by Yukinori Okada revealed that a generegion RPA3-UMAD1 to be related to interstitial pneumonia
associated with rheumatoid arthritis. The RPA3-UMAD1is strongly associated with the chest CT image pattern of
pulmonary fibrosis. Their research is expected to contributeto elucidating the pathology of interstitial pneumonia
associated with rheumatoid arthritis.

99

Kang S, Tanaka T, Inoue H, et al.

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J Exp Med 218 (1): e20200815 (2021)

Cytokine release syndrome (CRS) is a life-threatening
complication induced by hyperinflammatory responses.
However, no specific immunotherapies are available for its
treatment. Sujin Kang, Tadamitsu Kishimoto and the researchgroup found that interleukin (IL)-6 signaling plays a crucialrole in endothelial cell dysfunction during bacterial andviral CRS. Specifically, they identified that the pathogenesis
of CRS in patients with sepsis, acute respiratory distresssyndrome, and burns involved the IL-6–mediated productionof hyperinflammatory cytokines and plasminogen activator
inhibitor-1 (PAI-1), which indicates that IL-6 signaling
blockade has potential as a therapy for CRS. The groupalso found that the inhibition of IL-6 signaling by Tocilizumab
treatment decreased PAI-1 production and alleviated clinicalmanifestations in severe COVID-19 patients.

Helicobacter pylori (H.pylori) causes gastritis, which has
been attributed to the development of H. pylori-specific T
cells during infection. However, the mechanism underlying
innate immune detection leading to the priming of T cells is
not fully understood, as H. pylori evades TLR detection.
Sho Yamasaki and his research group reported thatH. pylori metabolites modified from host cholesterol
exacerbate gastritis through the interaction with C-type
lectin receptors.
Cholester yl acyl α-glucoside (αCAG) andcholesteryl phosphatidyl α-glucoside (αCPG) wereidentified as noncanonical ligands for Mincle (Clec4e)and DCAR (Clec4b1). During chronic infection, H.
pylori-specific T cell responses and gastritis wereameliorated in Mincle-deficient mice, although bacterial
burdens remained unchanged. Furthermore, a mutant
H. pylori strain lacking αCAG and αCPG exhibited an
impaired ability to cause gastritis. Thus H. pylori-specificMechanism for the establishment of immunological memory.

100

Helicobacter pylori metabolites exacerbate gastritis through C-type lectin
receptors.

Sudo T, Motomura Y, Okuzaki D, et al.

J Exp Med 218 (1): e20200866 (2021)

J Exp Med 218 (5): e20200817 (2021)
achievement provides the underlying mechanism for theestablishment of immunological memory, which will help todevelop new vaccine strategies.

modification of host cholesterol plays a pathophysiologicalrole that exacerbates gastric inflammation by triggering
C-type lectin receptors.

Group 2 innate lymphoid cells support hematopoietic recovery under stressconditions.

Inoue T, Shinnakasu R, Kawai C, et al.

When exposed to antigens, such as viruses or bacteria,“germinal center (GC)” is generated in the secondary
lymphoid organs including spleens and lymph nodes. B cellfate decision occurs during GC reaction, but it is still unclear
how they are selected to differentiate into plasma cells,
memory B cells, or to remain in the GC.
A group of researchers with Takeshi Inoue and Tomohiro
Kurosaki identified and characterized a small GC populationof precursors for memory B cells. They found that the GC
B cells with lower mTORC1 activity and increased survival
signal from surface B cell receptors favor a memory B cellfate. This group previously reported that the transcription
factor Bach2 is required for memory B cell generation(Shinnakasu et al., Nat Immunol, 2016), and in this study,
they clarified that Bach2 has an important role for the
regulation of metabolism during GC responses. This group’s

Information

IL-6 trans-signaling induces plasminogen activator inhibitor-1 in cytokine release
syndrome.

The cell-cycle status of hematopoietic stem andprogenitor cells (HSPCs) becomes activated following
chemotherapy-induced stress, promoting bone marrow(BM) regeneration; however, the underlying molecularmechanism remains elusive. Masaru Ishii and theresearch group showed that BM-resident group 2 innate
lymphoid cells (ILC2s) support the recovery of HSPCs
from 5-fluorouracil (5-FU)–induced stress by secreting
granulocyte-macrophage colony-stimulating factor (GMCSF). Mechanistically, IL-33 released from chemo-sensitive
B cell progenitors activates MyD88-mediated secretionof GM-CSF in ILC2, suggesting the existence of a B cell–
ILC2 axis for maintaining hematopoietic homeostasis. GMCSF knockout mice treated with 5-FU showed severe loss of
myeloid lineage cells, causing lethality, which was rescuedby transferring BM ILC2s from wild-type mice. Further, theadoptive transfer of ILC2s to 5-FU–treated mice accelerates

hematopoietic recovery, while the reduction of ILC2s resultsin the opposite effect. Thus, ILC2s may function by “sensing”
the damaged BM spaces and subsequently support
hematopoietic recovery under stress conditions.

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107

The articles were published between April, 2020 and March, 2021. The data were acquired using Web of ScienceTM Core
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