WPI Immunology Frontier Research Center 2021- 2022

Annual Reportof IFReC
2021 - 2022

Edit : Research Planning & Management Office, IFReC
Published in June, 2022

Osaka University

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

Message from the Director

CON T E N T S
Message from the Director ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・1
Organization ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・3
Laboratories ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・9● Immunology Groups ・
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Events & Outreach Activities ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・81
Information ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・91
Access Map ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・112

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 IFReC joined the WPI Academy in 2017,
we have continued efforts to create history as a research
center through a novel academic-industry partnershipagreement.
More than two years have passed since COVID-19
pandemic began. After several major epidemic peaks, thetotal number of COVID-19 cases in Japan exceeded 6.5
million as of late March 2022. In other words, one out of
every 19 Japanese persons has been infected.
Against this backdrop, the Center for Infectious Diseases
Education and Research (CiDER) was established at Osaka
University in response to the outbreak of COVID-19 andIFReC plays a central role in its research division.

In FY2021, we welcomed Dr. Nobuhiko Kamada, who isactively engaged in research at Michigan University, as a
new principal investigator. Dr. Kamada aims to elucidate
how pathogenic commensal microbiota cause diseases
and how the host immune system fights them.
In this era of global pandemics, we continue our efforts
to conduct basic research in immunology and to seek ways
to contribute to society. Through research and education,
IFReC continues to contribute to the advancement ofscience and grow to lead the world in immunology
research.

Kiyoshi TAKEDA, MD/ PhD
Director
WPI Immunology Frontier Research Center

Although the traditional role of scientists involves
directly discussing their findings across national borders,the COVID-19 pandemic has forced the cancellation or
postponement of many academic events that invite guest
speakers from abroad. Similarly, many outreach activities
such as science cafés have also been put on hold.
In response to these challenges, we launched theonline "Immuno-Seminar Series" to virtually connect thecontinents and welcomed eight world-class researchers
as invited speakers in FY2021. This seminar series was very
well received and will continue in FY2022. We also held
"The 2nd ImmunoSensation2-IFReC Joint Workshop" in an
online format with the University of Bonn. Despite the timedifference between the two countries, many researchers
participated in this event. In 2022, we will jointly hold an
on-site symposium with University College London in
the UK. We also plan to co-host an on-site "International
School on Advanced Immunology" with the University ofBonn on Awaji Island.

1Organization

As of March, 2022

MEXT

Osaka University

Organization

Organization ChartWPI Initiative Program Committee

International Advanced
Research Institute (IARI)

JSPS

Joint Research Chairs with the Pharmaceutical Companies
International
Scientific
Advisory
Board

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

Imaging Group
Informatics Group
Core Instrumentation Facility

Unit for Combined Research Fields

Common Facilities

Animal Resource Center for Infectious Diseases
Network Administration OfficeDirector

Management
Committee

Board of Representatives

Administrative Office

DeputyDirectors

Administrative
Director

General Affairs Section
Administrative
Department• General Affairs Section• Accounting Section• 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
Research Planning and Management Office• 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• Institute for Frontier Life and Medical Sciences, Kyoto University, Japan• RIKEN Center for Integrative Medical Sciences, Japan• University College London, UK• ImmunoSensation 2 , Clusters of Excellence, the Rheinische Friedrich-Wilhelms-University of Bonn, Germany

4• Research promotion and support (Consultation for grants and patents, etc.)• Establishing research environments (Facility and Safety management, Research agreement, etc.)• Fostering young scientists (Winter School, Advanced postdoc program, Orientation, etc.)• Organizing scientific events (Symposia, Colloquia, Seminars, etc.)• Public relations (Publishing, Website, Outreach to citizens, etc.)5Organization

Committees & Advisory Board for IFReC● The World Premier International Research Center Initiative ( WPI)■■ Program Director
Akira UKAWA

As of November, 2021
WPI Program Director and Academy Director■■ Program Committee Members
Michinari HAMAGUCHI(Chairperson)

President, Japan Science and Technology Agency (JST)
Chairperson, The Japanese National Commission for UNESCO

Hiroshi AMANO

Professor, Nagoya University, Tokai National Higher Education and Research System
Nobel laureate in Physics (2014)

Mariko HASEGAWA

President, The Graduate University for Advanced Studies, SOKENDAI● WPI 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 Japan as 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 and outreach activities.

■■ Program Officer for IFReC
Kouji MATSUSHIMA

Professor, Research Institute for Biomedical Sciences, Tokyo University of Science● International Scientific Advisory Board for IFReC
Kazuhiko ISHIMURA

President, National Institute of Advanced Industrial Science and Technology

Maki KAWAI

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

Kiyoshi KUROKAWA

Professor Emeritus, National Graduate Institute for Policy Studies

Hiroshi MATSUMOTO

President, RIKEN

Ryozo NAGAI

6

Lewis LANIER

University of California, San Francisco

Anne O’GARRAThe Francis Crick InstituteJeffrey RAVETCH

Rockefeller University

Art WEISS

University of California, San Francisco/Howard Hughes Medical Institute

Joachim SCHULTZ

DZNE/LIMES Institute, University of Bonn

As of March, 2022

President, Jichi Medical University

Rita COLWELL

Distinguished University Professor, University of Maryland

Richard DASHER

Director, US-Asia Technology Management Center, Stanford University

Victor Joseph DZAU

President, National Academy of Medicine

Klaus von KLITZING

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

Chuan Poh LIM

Chairman, Singapore Food Agency (SFA)

Harriet WALLBERG

Professor, Karolinska Institutet

Jean ZINN-JUSTIN

Scientific adviser, Institute of Research into the Fundamental Laws of the Universe (IRFU/CEA)

7

Laboratories
As of March, 2022

Host Defense

Immunology

macrophage subset that Ceacam1+Msr1+Ly6C-F4/80-Mac1+
monocytes, which we termed SatM (segregated-nucleus-containing
atypical monocytes), share granulocyte characteristics, areregulated 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 RNA-binding
protein RBM7 that is a component of the NEXT (nuclear exosome
targeting) complex. We found that the expression of Rbm7 isincreased in the fibrotic phase. Rbm7-deletion in nonhematopoietic
cells suppresses fibrosis (Fig.2). Dysregulated expression Rbm7
triggers apoptosis via nuclear degradation of noncoding RNA
Neat1. Rbm7 in epithelial cells plays a critical role in thedevelopment of fibrosis by regulating ncRNA decay, thereby
producing of chemokines that recruit SatMs. Inhibition of RBM7
would provide an effective treatment of fibrosis in patients. We
further investigated the fibrotic lungs of mice and humans using
spatial transcriptomics and single-cell RNA-seq. We found closeinteractions of pro-fibrotic macrophages derived from distinct
monocyte subsets, and other immune and non-immune cells
releasing the essential factors maintaining the fibrotic niche.
Now, we are trying to unveil a comprehensive understanding of
the mechanisms involved in the development and maintenanceof fibrosis.

Shizuo Akira, MD/PhD
Professor
Associate Professor

Shizuo Akira
Kazuhiko Maeda
Hiroki Tanaka

Assistant Professor

Kiyoharu Fukushima

Postdoctoral Fellow

2

Research Assistant

6

Visiting Scientist

5

Support Staff

5

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

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

Molecular mechanism of endoribonuclease Regnase-1
in inflammation
Regnase-1 is a member of CCCH-type zinc finger proteins.
Regnase-1 has the endonuclease activity and destabilizes a set ofmRNAs through cleavage of their 3' UTRs such as IL-6 and IL-12
p40 in macrophages and c-Rel, Ox40, and IL-2 in CD4+ T cells.
Recent studies reveal that both static and dynamic control ofmRNA expression by Regnase-1 contributes to robustness ofcellular functions in various types of cells including macrophages,
T cells, intestinal epithelial cells, and etc. And Regnase-1 isthought to be a key contributor to maintenance of homeostasis
in various types of tissues and cells. Based on these findings, we
promote understanding of the precious roles of Regnase-1 in
immune and non-immune cells by using tissue-specific Regnase1-deficient mice and mutant mice.
Regnase-1 is inactivated in response to external stimuli
through posttranslational modifications, however, yet the preciserole of phosphorylation largely remains unknown. We showedthat IL-17 induces the phosphorylation of Regnase-1 in an Act1-

10

TBK1-IKKi-dependent manner, especially in non-hematopoietic
cells. Phosphorylated Regnase-1 is released from the endoplasmic
reticulum into the cytosol, thereby losing its mRNA degradation
function, which leads to expression of IL-17 target genes (Fig.1).
IL-17-induced Regnase-1 phosphorylation is completely blocked
in two Regnase-1-mutant (Regnase-1AA/AA and Regnase-1ΔCTD/ΔCTD)
mice. Thus, Regnase-1 plays a critical role in the development of
IL-17-mediated inflammatory diseases via the Act1-TBK1-IKKi
axis. Blockade of Regnase-1 phosphorylation sites may bepromising for treatment of T helper 17-associated diseases.
We recently found that Regnase-1 also play a critical role incytotoxic activity of natural killer (NK) cells. NK cells are essential
for immunesurveillance against tumor and viral infection by
secretion of several substances associated with cytolysis, such as
Perforin, Granzymes, and Interferons. Regnase-1-deficient in NK
cells (Regnase-1ΔNK) mice were highly resistant to tumor
progression and metastasis by hyprproduction of Perforin andInterferon-γ. Now we try to determine the mechanism how
Regnase-1 gene deficiency results in strong anti-tumor activity.
We are further studying to achieve the goal of a comprehensive
understanding of the innate immune system and to develop an
effective treatment for immune-related inflammatory diseases.

Figure 2.
RBM-7 regulates fibrosis formation through ncRNA decay and cell death. RBM-7 destabilizes ncRNA NEAT1 and induces cell death in lung epithelial
cells, resulting in recruitment of SatM and fibrocytes into lung to promote lung fibrosis.

Recent Publications
1. Kawasaki T. et al. Loss of FCHSD1 leads to amelioration of chronic
obstructive pulmonary disease. Proc Natl Acad Sci U S A 118:e2019167118(2021).

4. Tanaka H, Arima Y, Kamimura D, et al. Phosphorylation-dependent
Regnase-1 release from endoplasmic reticulum is critical in IL-17
response. J Exp Med. 216:1431-1449 (2019).

Heterogeneity of monocyte subsets comprise diverse
macrophages in mouse and human lung fibrosis

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

Fibrosis is a life-threatening disease of unknown aetiology. Its
pathogenesis is poorly understood, and there are few effective
therapies. The development of fibrosis is associated withactivation of monocytes and macrophages. We identified new

3. Fukushima K, Satoh T, Sugihara F, et al. Dysregulated expression of thenuclear exosome targeting complex component Rbm7 in nonhematopoietic cells licenses the development of fibrosis. Immunity
52:542-556 (2020).

5. 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 of mTOR and purine
metabolism. Proc Natl Acad Sci U S A. 115:11036-11041 (2018).

11

B Protein-free liposome

Immunoglycobiology

50% GlcN-PI
CLPTM1L

ER Lumen

GlcN-PI
PI-PLC

Proteoliposome

DAG

0% GlcN-PI

CLPTM1L

PI-PLC[䠏H]GlcN[䠏H]GlcN-IP + DAG
PI
PI-PLC[䠏H]PI[䠏H]inositol-P + DAG

Cytoplasm
PI-PLCCTime courses[䠏H]GlcNPI% [3H]PI hydrolysis

60
40
20
0
0

Taroh Kinoshita
Yoshiko Murakami

3

Biosynthesis pathway of GPI initiates on the cytoplasmic sideof the endoplasmic reticulum (ER), flips into the luminal side at
the third step, and continues on the luminal side beyond. How
the second intermediate glucosaminyl-phosphatidylinositol(GlcN-PI) flips from the cytoplasmic side to the luminal side acrossthe ER membrane has been unclear. We identified CLPTM1L, an
ER membrane protein with eight transmembrane domains, as amajor GlcN-PI scramblase in HEK293 cells. Thus, GPI-anchoredprotein (GPI-AP) levels were clearly reduced but not completely
in CLPTM1L-KO HEK293 cells, suggesting the presence of yet
another scramblase for GlcN-PI. CLPTM1L scrambled not only
GlcN-PI but also PI, GlcNAc-PI, phosphatidylethanolamine andphosphatidylcholine in in vitro scrambling assays using
poroteoliposomes (Figure 1). These results indicate that CLPTM1L
is a new lipid scramblase critical for the GPI biosynthesis pathway(Wang Y et al, Proc. Natl. Acad. Sci. USA, 2022).

Pathophysiological roles of side-chain modification of
GPI-anchors.
The conserved GPI core of mammalian GPI-APs are often
modified by an N-acetylgalactosamine (GalNAc) side chain
attached to the first mannose. We previously reported a Golgiresident GalNAc transferase (PGAP4/TMEM246) that mediates
this modification, however, roles of the side chain have been

12Free, un-protein-linked GPIs as the Emm blood groupantigen.
We previously reported presence of free, un-protein-linked GPIin the various tissues (Wang Y et al, J. Biol. Chem., 2018). In
collaboration with Drs. Peyrard and Azouzi group in Paris, wedemonstrated that free, un-protein-linked GPI, bearing
ethanolamine-phosphate side chain linked to the second
mannose, is expressed on the surface of red blood cells, platelets
and neutrophils. Patients with inherited GPI deficiency caused bynull mutations of PIGG gene did not have this GPI structure andgenerated natural antibody against this type of free GPI. Thus, thefree GPI bearing ethanolamine-phosphate side chain linked tothe second mannose represents the Emm blood group antigen
known as a GPI-related blood group. (Duval R et al, Blood,
137:3660, 2021)

60

2

4

6

8

0

10

2

4

8

6

Protein / phospholipid ratio(mg/mmol)Time (min)Liposome

Figure 1.
CLPTM1L scrambled GlcN-PI and PI in vitro. A. A schematic of CLPTM1L-mediated scrambling of GlcN-PI across the endoplasmic reticulum (ER)membrane during biosynthesis of GPI-anchor. B. Principles of in vitro scrambling assay using liposomes and proteoliposomes. Phosphatidylinositolspecific phospholipase C (PI-PLC) cleaves radiolabeled GlcN-PI and PI in the outer leaflet, releasing radioactive GlcN-inositolcyclicphosphate andinositolcyclicphosphate, respectively from liposomes. C. Time course and CLPTM1L-dose response of hydrolysis of GlcN-PI and PI in liposomes.
(Adopted from Wang Y et al, Proc. Natl. Acad. Sci. USA, 2022)

ALP (IU/L)

Studies on GPI biosynthesis.

[䠏H]GlcNPI

70

40
0

CLPTM1L-proteoliposome

ALP
2500

unclear. To clarify this, we generated PGAP4-knockout (KO) mice.
PGAP4-KO mice showed various phenotypes, including a highblood alkaline phosphatase level, impaired bone formation,
lowered locomotion activity, and impaired memory, despite
normal expression levels and raft-association of various GPI-APs.
Therefore, the GalNAc side chain is required for in vivo functionsof GPI-APs in mammals, especially in bone and brain. Moreover,
PGAP4-KO mice were more vulnerable to prion diseases and diedearlier after intracerebral inoculation of the pathogenic prion
strains than wild-type mice, highlighting the protective roles of
the GalNAc-side chain against prion diseases (Hirata T et al, J. Biol.
Chem., 2022).

10

80

50

Time (min)A

The main topics of research in the Immunoglycobiology laboratory
have been biology and medicine of glycosylphosphatidylinositol(GPI). In FY 2021, we made following progress.

20[䠏H]PI

2000

WT

KO

9.5

***
***

1500
1000

B

Ca
WT

*

KO

*

9.0

8.5

500
0

C

8.0

Shape of ribs and ribcage

Standard
WT: 7/7
KO: 4/7

Body mineral content
0.7

WT

**

Bone area

KO

*

0.6

0.5

14

Bone area (cm2)1

Support Staff

40

90

0.4

KO

*

12

10

D Shape of cervical vertebrae E

Bowed and caved-inStandard
WT: 0/7
WT: 6/7
KO: 3/7
KO: 3/7

WT

8

Deformed ventral
tubercle of atlas
WT: 1/7
KO: 4/7

Tibia length19

Tibia length (mm)Research Assistant

8

6

60

Body mineral content (g)

1

4

80

0

Ca (mg/dL)

Postdoctoral Fellow

2

100

100

80
% hydrolysis

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

CLPTM1L dose responses[䠏H]PI% hydrolysis

100

Professor

Immunology

A

WT

KO

*
18

17

Figure 2.
Abnormal bone formation in PGAP4-knockout mice. A. Blood alkaline phosphatase level (ALP) and blood calcium level (Ca). B. Body mineral content
and bone area. C. Normal (left) and abnormal (right) shapes of ribs and ribcage. The number of male mice showing the phenotypes are indicated.
D. Normal (left) and abnormal (right) shapes of cervical vertebrae. The number of female mice showing the phenotypes are indicated. E. Tibia
length. (Adopted from Hirata T et al, J. Biol. Chem., 2022)

Recent Publications
1. Wang Y, Menon AK, Maki Y, Liu Y-S, Iwasaki Y, Fujita M, Guerrero PA, Varón
Silva D, Seeberger PH, Murakami Y and Kinoshita T. Genome-wide CRISPR
screen reveals CLPTM1L as a lipid scramblase required for efficient
glycosylphosphatidylinositol biosynthesis. Proc Natl Acad Sci USA inpress (2022).
2. Hirata T, Kobayashi A, Furuse T, Yamada I, Tamura M, Tomita H, Tokoro Y,
Ninomiya A, Fujihara Y, Ikawa M, Maeda Y, Murakami Y, Kizuka Y andKinoshita T. Loss of the N-acetylgalactosamine side chain of the GPIanchor impairs bone formation and brain functions and accelerates theprion disease pathology. J Biol Chem. in press (2022).

4. 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).
5. 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).

3. 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).

13

Immunology

Immunopathology

Atsushi Kumanogoh, MD/PhD

Our research team is involved in two approaches, that is, basic
and clinical immunology. As basic aspects of our projects, ourproposed study is the regulation of immune cell motility andmigratory behavior in vivo by soluble and membrane-bound‘immune guidance molecules’ such as semaphorins and their
receptors. Semaphorins were originally identified as axonguidance molecules that function during neuronal development.
However, cumulative evidence indicates that semaphorins also
participate in immune responses, both physiological andpathological, and they are now considered to be potential
diagnostic and/or therapeutic targets for a range of diseases.
Beyond such basic implications, we are trying to apply thefindings from this proposed study into the diagnosis/therapy forhuman immunological disorders, such as autoimmunity, allergy,
immune deficiency, cancer/metastasis, and neurodegenerative
diseases. We recently focus on the crosstalk among neuronal,
immune and metabolic systems since some of the semaphorins’
expression are regulated by a metabolic sensor, mTOR, in which
we investigated the biological and pathological significance ofLamtor1/p18, an amino acid sensor localized at the lysosome.
Functional coupling of the neural, metabolic, and immune
systems is essential for organ development and maintenance,
but the molecular mechanisms controlling this network remain
unclear. Here, we demonstrate that an axonal guidance cue
Semaphorin 6D (Sema6D) combines systemic metabolism andmyelopoiesis by tuning sympathetic innervations. Sema6d-/- mice
fed a high-fat diet (HFD) exhibited attenuated obesity andenhanced myelopoiesis. Combinational analysis of bone marrow

14

Professor

Atsushi Kumanogoh

Assistant Professor

Takahiro Kawasaki

Research Assistant5

Support Staff

5(BM) chimeric and conditional knockout mice revealed that nonhematopoietic and neuron-derived Sema6D is responsible forthese phenotypes through Plexin-A4. Notably, Sema6d deficiency
led to increased sympathetic innervations in the BM. Consistently,
loss of β3-adrenergic receptor signaling resolved metabolic andhematopoietic defects in HFD-fed Sema6d-/- mice. Collectively,
these data demonstrate that Sema6D orchestrates systemic
metabolism and hematopoiesis by tuning sympathetic
innervations. Thus, Sema6D functions as a hub for neural–
metabolic–immune circuits by repelling/pruning sympathetic
innervations. Our findings provide mechanistic and clinicalinsights into the hierarchical regulation of the immune andmetabolic diseases.

Figure.
Sema6D guides sympathetic neurons to regulate systemic metabolism and myelopoiesis.
A schematic view of the neural–metabolic–immune connections regulated by Sema6D. The loss of Sema6D in neurons causes increased
sympathetic innervations in the BM, resulting in enhanced systemic metabolism and myelopoiesis under stress conditions. These metabolic andhematopoietic defects are resolved by blocking β3-adrenergic receptor signaling. Thus, neuron-derived Sema6D fine-tunes sympathetic
innervations in the BM, leading to coupling of metabolic and hematopoietic adaptations under metabolic stress.

Recent Publications
1. 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 rolein leukocyte trafficking by activating myosin II. Nat Commun. 12(1):3333(2021).
2. Tsuda T, Nishide M, Maeda Y, Hayama Y, Koyama S, Nojima S, Takamatsu H,
Okuzaki D, Morita T, Nakatani T, Kato Y, Nakanishi Y, Futami Y, Suga Y, Naito
Y, Konaka H, Satoh S, Naito M, Izumi M, Obata S, Nakatani A, Shikina T,
Takeda K, Hayama M, Inohara H, and Kumanogoh A. Pathological andTherapeutic Implications of Eosinophil-Derived Semaphorin 4D in
Eosinophilic Chronic Rhinosinusitis. J Allergy Clin Immunol. 145(3):843854 (2020).

4. Nishide M, and Kumanogoh A. The role of semaphorins in immune
responses and autoimmune rheumatic diseases. Nat Rev Rheumatol
14:19-31 (2018).
5. Hosen N, Matsunaga Y, Hasegawa K, Matsuno H, Nakamura Y, Makita M,
Watanabe K, Yoshida M, Satoh K, Morimoto S, Fujiki F, Nakajima H, et al.
2017. The activated conformation of integrin β. Nat Med. 23:1436-1443(2017).

3. Kang S, Nakanishi Y, Kioi Y, Okuzaki D, Kimura T, Takamatsu H, Koyama S,
Nojima S, Nishide M, Hayama Y, Kinehara Y, Kato Y, Nakatani T, Shimogori
T, Junichi Takagi J, Toyofuku T. and Kumanogoh A. Semaphorin 6D reverse
signaling controls macrophage lipid metabolism and anti-inflammatorypolarization. Nature Immunol. 19:561-570 (2018).

15

Immunology

Immunochemistry

Hisashi Arase, MD/PhD

Professor

A) Misfolded proteins complexed with MHC class II
molecules are targets for autoimmune diseases.
MHC class II allelic polymorphisms are associated withsusceptibility to many autoimmune diseases. However, it hasremained unclear how MHC class II molecules are involved in
autoimmune disease susceptibility. We found that cellular
misfolded autoantigens are rescued from protein degradation by
MHC class II molecules (Int. Immunol. 2013). Furthermore, we
found that misfolded proteins complexed with MHC class II
molecules are targets for autoantibodies in autoimmune diseases
such as rheumatoid arthritis, antiphospholipid syndrome, andANCA-associated vasculitis (PNAS 2014; Blood. 2015; Br. J.
Dermatol. 2017; Arthritis Rheumatol. 2017; Arthritis Rheumatol.
2021). Furthermore, autoantibody binding to misfolded proteinstransported to the cell surface by MHC class II molecules wasstrongly correlated with susceptibility to autoimmune disease.
Further analyses revealed that self-antigens complexed withMHC II molecules abrogate self tolerance to induce autoimmune
response (Science Advances 2022). In addition, we found that not
only misfolded protein but also DNA is presented on MHC class II
molecules with SLE-risk allele but not with SLE-protective allele(Arthritis Rheumatol. 2022). These findings demonstrated that
misfolded proteins, which normally would not be exposed to theimmune system, are involved in the pathogenicity of autoimmune
diseases as ‘neo-self’ antigens (Figure 1).

B) Studies on host-pathogen interaction
Immune system has evolved with infectious diseases,
indicating that studies on host-pathogen interaction is important

16

Hisashi Arase

Associate Professor

Masako Kohyama

Assistant Professor

Wataru Nakai

Postdoctoral Fellow

2

Research Assistant

5

Visiting Scientist

1

Support Staff

4

to understand immune system. We have found that PILRα, one ofpaired 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 and Sigle-7, one of paired receptors, areinvolved in VZV infection (PNAS 2010; BBRC 2022). LILR is anothertype of paired receptor family. We found that activating LILRA2 isa sensor to detect immunoglobulin abnormalities in microbial
infection (Nature Microbiology 2016). Furthermore, we found that
RIFINs, products of multigene family of Plasmodium falciparum,
are involved in immune evasion through binding to inhibitory
LILRB1 and LILRB2 (Nature 2017; Nature 2020; BBRC 2021). Thesefindings demonstrated that paired receptors play an importantrole in immune regulation as well as viral infection.
SARS-CoV-2 causes severe pneumonia in some infectedindividuals. Therefore, it is important to elucidate the factors thatcause the severe COVID-19. Antibodies against the receptor
binding domain (RBD) of spike protein play an important role inthe defense against SARS-CoV-2 infection. However, we foundthat some antibodies against the N-terminal domain (NTD) of
spike protein cause conformational changes in spike protein andenhance the infectivity. In particular, anti-NTD antibodies thatenhance the infectivity were found more in severe patients,suggesting that the antibodies may be involved in thedevelopment of severe disease (Cell 2021, Figure 2). Thesefindings indicated that the infectivity-enhancing antibodies must
be considered in antibody response against SARS-CoV-2.

Figure 1.
Misfolded proteins transported to the cell surface by MHC class II molecules are targets for autoantibodies.
Cellular misfolded proteins are generally degraded in the cells and are not transported to outside the cells. Therefore, misfolded proteinstransported to the cell surface by MHC class II molecules may be recognized as ‘neo-self’ antigens by immune system, which initiates aberrant
immune response to self-antigens (Int. Immunol. 2013; PNAS 2014; Blood 2015, Br. J. Dermatol. 2017; Arthritis Rheumatol. 2017, Arthritis Rheumatol.
2020; Arthritis Rheumatol. 2022; Science Advances 2022).

Figure 2.
SARS-CoV-2 infectivity enhancing antibodies.
Some antibodies against NTD of spike protein induces conformational change in spike protein to induce the open form of RBD and enhance theinfectivity of SARS-CoV-2 (Cell 2021).

Recent Publications
1. Jin H, Kishida K, Arase N, Matsuoka S, Nakai W, Kohyama M, Suenaga T,
Yamamoto K, Sasazuki T, Arase H. Abrogation of self-tolerance by
misfolded self-antigens complexed with MHC class II molecules. Sci
Advances. 8:eabj9867 (2022) .

4. 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).

2. Liu Y, Soh WT, Tada A, Arakawa A, Matsuoka S, Nakayama EE, Li S, Ono C,
Torii S, Kishida K, Jin H, Nakai W, Arase N, Nakagawa A, Shindo Y, Kohyama
M, Nakagami H, Tomii K, Ohmura K, Ohshima S, Okada M, Matsuura Y,
Standley DM, Shioda T, Arase H. An infectivity-enhancing site on the
SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies.
Cell 184:3452-3466 (2021).

5. Hirayasu K, Saito F, Suenaga T, Shida K, Arase N, Oikawa K, Yamaoka T,
Murota H, Chibana H, Nagai H, Nakamura Y, Katayama I, Colonna M, Arase
H. LILRA2 is an innate immune sensor for microbially cleaved
immunoglobulins. Nat Microbiol. 1:1-7 (2016).

3. Saito F, Hirayasu K, Satoh T, et al. Immune evasion of Plasmodium
falciparum by RIFIN via inhibitory receptors. Nature 552:101-105 (2017).

17

Tadamitsu Kishimoto, MD/PhD

Professor

Endothelial integrity is important for maintaining immune
homeostasis. Previously, we identified that interleukin (IL)-6trans-signaling induced endothelial activation in the context ofcytokine production and activation of the coagulation cascade.
Additionally, treatment with an anti-IL-6R antibody, tocilizumab
inhibited endothelial activation and prevented the developmentof cytokine release syndrome (CRS). In accordance with ourfindings, tocilizumab was recently approved by the United States
Food and Drug Administration for the treatment of SARS-CoV-2
infection-induced pneumonia. Nevertheless, the long-lastingand discriminate suppression of IL-6 signaling in the acute criticalcare setting raises concerns about the increased risk of secondary
infections. To overcome the limitations of IL-6 signaling blockadein acute inflammatory diseases, we generated an anti-IL-6R
antibody (silent anti-IL-6R antibody) with a short half-life to
reduce Fc-mediated side effects. We found that the treatment ofseveral CRS mouse models including thermal injury and sepsis
models with the silent anti-IL-6R antibody significantly improved
the survival rate compared with controls. Additionally, we foundthat silent anti-IL-6R antibody treatment prevented endothelial
damage by inhibiting glycocalyx degradation, increasing
endothelial permeability, and plasminogen activation inhibitor-1
production, compared with the normal half-life anti-IL-6R
antibody. Consequently, treatment with silent anti-IL-6R antibody
showed significant efficacy in several acute inflammatory
diseases by controlling endothelial injury (Fig. 1). However, themechanisms involved in how the silent antibody affects immune

18

Tadamitsu Kishimoto

Associate Professor

Sujin Kang

Postdoctoral Fellow

2

Research Assistant2

Support Staff

3

Figure 1.
Targeting of anti-IL-6R antibody for infectious diseases.

Figure 2.
Role of novel phosphorylation site of STAT1 in vivo.

Immunology

Immune Regulation

A novel insight into endothelial injury at the onset ofcytokine storm

The STAT1 working paradigm has focused on interferon (IFN)
mediated JAK-Tyr701 phosphorylation. However, IFN-JAK signaling
does not explain the full spectrum of STAT1 functions against
inflammatory stimuli. Our recent work identified the novel Thr749(Thr748 in mouse) phosphorylation of STAT1, which triggered
distinct gene-regulatory functions of STAT1 independent of thecanonical Tyr701 phosphorylation. 1) Disruption of Thr748
phosphorylation of STAT1 protects the host against
lipopolysaccharide (LPS)-induced septic shock: We generated
genetically engineered mice expressing a non-phosphomimic
threonine748-to-alanine (T748A) mutant STAT1. We found a
gradual trend of survival against LPS-induced septic shock where
STAT1T748A and heterozygous mice exhibited higher survival
compared with their wild-type littermates, suggesting that pThr
STAT1 promoted the host inflammatory response in septic shock.
Additionally, STAT1T748A mice were resistant to LPS-induced
lethality, albeit to a lesser extent than their STAT1-deficient
littermates. Of note, the expression of STAT1 and other STAT
proteins was unperturbed in STAT1T748A mice. These findings
showed that threonine748 phosphorylation is a major contributor
to STAT1 functions in LPS-induced septic shock. 2) Disruption of

Thr748 phosphorylation of STAT1 promotes colitis: A deficiency
in STAT1 leads to severe DSS-induced colitis, a murine model
which closely resembles human ulcerative colitis. However, colon
mucosal samples from patients with ulcerative colitis exhibitedthe increased expression of JAK-mediated pTyr STAT1. Tofacitinib,
a JAK inhibitor, has been approved for the treatment of patientswith ulcerative colitis. A potential explanation of theseparadoxical findings is that the protective role of STAT1 inulcerative colitis is largely independent of the canonical JAKSTAT1 pathway. In this study, we found that Stat1T748A mice
exhibited severe intestinal inflammation compared with theirwild-type mice. Intriguingly, the Stat1T748A mice phenotype
completely mimicked the detrimental phenotype of their STAT1
knockout mice as shown by their weight loss, colon lengths, andhistopathology analysis. Our data indicate that the threonine748
phosphorylation on STAT1 is a key driver for the STAT1 protectivefunction against intestinal inflammation. Consequently, we
found that pThr STAT1 contributed to STAT1 functions to adifferent extent in vivo in various disease models. The simplest
interpretation of our findings is that cell-dependent threonine
kinase pathways may contribute to the specificity of STAT1
functions, especially those not driven by the canonical JAK
pathway (Fig. 2).

Threonine Phosphorylation of STAT1 Controls the
Host Inflammatory Response

cells and endothelial damage require further study. Additionally,
the efficacy of the silent antibody should be tested in several
infectious diseases.

Arid5a promotes immune evasion by augmenting
tryptophan metabolism and chemokine expression.
The acquisition of mesenchymal traits leads to immune evasionin various cancers, but the underlying molecular mechanisms
remain unclear. In this study, we found that the expression levelsof AT-rich interaction domain-containing protein 5a (Arid5a), an
RNA-binding protein, were substantially increased inmesenchymal tumor subtypes. The deletion of Arid5a in tumor
cell lines enhanced antitumor immunity in immunocompetent
mice, but not in immunodeficient mice, suggesting a role for
Arid5a in immune evasion. Furthermore, an Arid5a-deficienttumor microenvironment was shown to have robust antitumor
immunity, as manifested by suppressed infiltration of granulocytic
myeloid-derived suppressor cells and regulatory T cells. In
addition, infiltrated T cells were more cytotoxic and lessexhausted. Mechanistically, Arid5a stabilized Ido1 and Ccl2
mRNAs and augmented their expression, resulting in enhanced
tryptophan catabolism and an immunosuppressive tumor
microenvironment. Thus, our findings demonstrate the role ofArid5a beyond inflammatory diseases and suggest Arid5a as apromising target for the treatment of immunotolerant malignant
tumors.

Recent Publications
1. Kishimoto T and Kang S. IL-6 Revisited: From Rheumatoid Arthritis to CAR
T Cell Therapy and COVID-19. Annu Rev Immunol. in press (2022).
2. Nyati KK, Hashimoto S, Singh SK, Tekguc M, Metwally H, Liu YC, Okuzaki
D, Gemechu Y, Kang S, Kishimoto T. The novel long noncoding RNA
AU021063, induced by IL-6/Arid5a signaling, exacerbates breast cancer
invasion and metastasis by stabilizing Trib3 and activating the Mek/Erk
pathway. Cancer Lett. 1:520:295-306 (2021).
3. Kang S, Kishimoto T. Interplay between interleukin-6 signaling and thevascular endothelium in cytokine storms. Exp Mol Med. 53(7):1116-1123(2021).

4. Parajuli G, Tekguc M, Wing JB, Hashimoto A, Okuzaki D, Hirata T, Sasaki A,
Itokazu T, Handa H, Sugino H, Nishikawa Y, Metwally H, Kodama Y, Tanaka
S, Sabe H, Yamashita T, Sakaguchi S, Kishimoto T, Hashimoto S.Arid5a
Promotes Immune Evasion by Augmenting Tryptophan Metabolism andChemokine Expression. Cancer Immunol Res. 9(8):862-876 (2021).
5. Masaki T, Okazawa M, Asano R, Inagaki T, Ishibashi T, Yamagishi A, UmekiMizushima S, Nishimura M, Manabe Y, Ishibashi-Ueda H, Shirai M,
Tsuchimochi H, Pearson JT, Kumanogoh A, Sakata Y, Ogo T, Kishimoto T,
Nakaoka Y. Aryl hydrocarbon receptor is essential for the pathogenesis of
pulmonary arterial hypertension. Proc Natl Acad Sci USA.
16;118(11):e2023899118 (2021) .

19

Mucosal Immunology

Kiyoshi Takeda, MD/PhD
Professor

Kiyoshi Takeda

Associate Professor

Hisako Kayama

Assistant Professor

Ryu Okumura

To determine the role of LysoPS in the progression of CD, weanalyzed effect of 18:1 LysoPS on two colitis models, such as
2,4,6-trinitrobenzenesulfonic acid solution (TNBS)-induced colitis
and adoptive T cell transfer-induced colitis. In both colitis models,
intraperitoneally injection of LysoPS aggravated large intestinal
pathology with increased number of IFN-γ-producing CD4+ T
cells. In Th1-polarized cells, LysoPS augmented glycolytic activity
through P2Y10 receptor, which linked to elevated IFN-γ andintracellular ROS production and promoted cell proliferation(Figure 1). To assess the impact of P2ry10 deficiency in CD4+ T
cells on LysoPS-induced intestinal pathology, Rag2-/- mice weretransferred wild-type or P2ry10-/yP2ry10b-/y naive T cells and somegroups were administered LysoPS intraperitoneally. LysoPS
drastically enhanced body weight loss and worsened large
intestinal pathology in Rag2-/- mice that received wild-type naive
T cells but not in mice that received P2ry10-/yP2ry10b-/y naive T
cells. In this context, LysoPS increased the number of IFN-γ+ CD4+
T cells in the large intestinal lamina propria of Rag2-/- mice thatreceived wild-type cells, whereas it did not affect the number ofthese cells in mice that received P2ry10-/yP2ry10b-/y CD4+ T cells.
Thus, P2Y10 receptor activation by LysoPS elicits immunopathologicalTh1 responses, which leads to the aggravation of intestinal
inflammation.
Regulation of lipid metabolites, including leukotrienes andprostaglandins, is an emerging therapeutic approach for
inflammatory disorders. Thus, the bioactive lipid LysoPS could bea putative diagnostic biomarker and a therapeutic target for CD.

Escherichia coli with ECSF_3660

Mari Murakami

Lumen

Taiki Sakaguchi
Postdoctoral Fellow

1

Research Assistant

12

Support Staff

2

Immunology

LysoPS exaggerates colitis by driving immunopathologicalTh1 responses through P2Y10 receptor-dependent
metabolic reprograming

Phosphatidylserine
Epithelial
cell

PLA

LysoPS
Lamin propria

P2Y10
Th1
We previously demonstrated that microbial metabolites:
adenosine triphosphate (ATP) and lactate/pyruvate exert theirimmunomodulatory activities through P2X/P2Y receptor and
GPR31, respectively. Activation of the lactate/pyruvate-GPR31
signaling pathway facilitates uptake of lumina bacteria by CX3CR1
phagocytes and thereby protects the host against pathogenic
bacteria. Although induction of inflammatory responses byextracellular ATP is essential for host defense, aberrant ATP
signaling results in disruption of intestinal immune system.
Therefore, luminal ATP is tightly regulated by ATP-hydrolyzing
ectoenzymes in epithelial cells, such as E-NPP3, E-NTPD7, and
E-NTPD8, suggesting that microbial metabolites in intestinal
lumen are strictly tuned to maintain the gut homeostasis.
Crohn’s disease (CD), the main inflammatory bowel disease
clinical phenotype, is a chronic gastrointestinal tract disorder.
IFN-γ-producing CD4+ T (Th1) cells is associated with CD severity.
Along with increased number of immunopathological Th1 cells,
alterations of intestinal metabolites caused by microbiota
community alterations (dysbiosis) are a feature of CD. However,the physiological or pathological functions of dysbiotic
microbiota-derived metabolites in the gut of patients with CDremain poorly understood.

Dysbiotic microbiota promote lysophosphatidylserine
generation in patients with CD
Some lysophospholipid species were identified to elevate in
the plasma of patients with CD. To define whether plasma lipid
profiles reflect intestinal lipid profiles, we performed a fecal
lipidomic analysis. Fifteen lipid species were upregulated in fecal

20

samples from patients with CD relative to those from healthy
controls (HCs). Among these lipid species, 18:0 and 18:1
lysophosphatidylserine (LysoPS) were commonly augmented inboth the fecal and plasma samples from patients with CD.
Microbial phospholipase A (PLA) generates lysoglycerophospholipid
species including LysoPS through the hydrolysis of host cell
membrane phospholipids. Therefore, to define whether dysbiosis
in patients with CD lead to the generation of more intestinal
LysoPS by microbial PLA, we performed whole-genome shotgun
sequencing of fecal DNA extracts. Lower fecal microbiota species
diversity was observed in CD samples compared with HC samples.
Additionally, a β-diversityanalysis demonstrated that the
microbial community profiles of CD samples substantiallyseparated from those of HC samples. Furthermore, ECSF_3660
gene predicted to encode PLA (mainly derived from Escherichiacoli) was increased in the fecal samples of patients with CDcompared with HCs. Gnotobiotic mice colonized with fecal
microbiota from patients with CD showed increased fecal level of
18:0/18:1 LysoPS compared to those in mice transplanted healthy
microbiota. Similarly, germ-free mice inoculated with E. coli with
ECSF_3660 isolated from feces of a patient with CD exhibited
higher fecal level of 18:0/18:1 LysoPS than those in untreatedgerm-free mice. These findings indicate that colonization by thedysbiotic microbiota found in patients with CD links to thepromotion of LysoPS generation in the intestine.

Th1

Promotion of Glycolysis
ʀIFN-J production (↑)ʀProliferation (↑)ʀOxidative stress (↑)
Aggravation of colitis
Figure.
LysoPS aggravates Th1-dependent colitis via P2Y10 receptor.
Elevation of intestinal lysophosphatidylserine (LysoPS) concentration due to microbial phospholipase A (PLA)-mediated hydrolysis ofphosphatidylserine in the host cell membrane causes progression of intestinal inflammation by eliciting immunopathological Th1 responses
through promotion of glycolysis via P2Y10 receptor.

Recent Publications
1. Otake Y, Kayama H, Kishikawa T, Shinzaki S, Tashiro T, Amano T, Tani M,
Yoshihara T, Li B, Tani H, Liu L, Hayashi A, Okuzaki D, Motooka D, Nakamura
S, Okada Y, Iijima H, Takeda K, Takehara T. Lysophosphatidylserines
derived from microbiota in Crohn’s disease elicit pathological Th1
response. J. Exp. Med. (2022) in press.

4. 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).

2. Tani H, Li B, Kusu T, Okumura R, Nishimura J, Okuzaki D, Motooka D,
Arakawa S, Yoshihara T, Ogino T, Tsai SH, Furuta Y, Muneta M, Nakamura S,
Fukusaki E, Yamamoto K, Yagita H, Kayama H, Takeda K. The ATPhydrolyzing ectoenzyme E-NTPD8 attenuates colitis through modulationof P2X4 receptor-dependent metabolism in myeloid cells. Proc. Natl.
Acad. Sci. USA. 118:e2100594118 (2021).

5. 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. Heme ameliorates dextran
sodium sulfate-induced colitis through providing intestinal macrophages
with non-inflammatory profiles. Proc. Natl. Acad. Sci. USA. 115:8418-8423(2018).

3. Kayama H, Okumura R, Takeda K. Interaction between the microbiota,
epithelia, and immune cells in the intestine. Ann. Rev. Immunol. 38:23-48(2020).

21

Immunology

Experimental Immunology

CTLA-4 facilitates Treg-APC conjugation and immune synapse
formation. The immune synapses thus formed provide a stable
platform whereby Tregs are able to deplete CD80/CD86
molecules on APCs by extracting them via CTLA-4-dependent
trogocytosis. The depletion occurs even with Tregs solely
expressing a mutant CTLA-4 form lacking the cytoplasmic portion
required for its endocytosis. Furthermore, CD80 downregulationor blockade by Treg-expressed membrane CTLA-4 or soluble
CTLA-4 Ig, respectively, disrupts cis-CD80/PD-L1 heterodimers
and increases free PD-L1 on dendritic cells (DCs), expanding aphenotypically distinct population of CD80lo free-PD-L1hi DCs.

Taken together, Tregs are able to inhibit the T-cell stimulatory
activity of APCs by reducing their CD80/CD86 expression via
CTLA-4-dependent trogocytosis. This CD80/CD86 reduction on
APCs is able to exert dual suppressive effects on T-cell immune
responses by limiting CD80/CD86 costimulation to naïve T cells
and by increasing free-PD-L1 available for the inhibition of PD-1
expressing effector T cells. Blockade of CTLA-4 and PD-1/PD-L1 in
combination may therefore synergistically hinder Treg-mediatedimmune suppression, thereby effectively enhancing anti-tumor
immune responses, with occasional development of
autoimmunity (Tekguc et al., PNAS, 2021).

Shimon Sakaguchi, MD/PhD
Thymic Treg generation
DN-DP
thymocytes
Professor

22

Thymic
Tregs

Shimon Sakaguchi

Associate Professor

Kenji Ichiyama

Assistant Professor

Yusuke Takeshima

Postdoctoral Fellow

4

Research Assistant

3

Visiting Scientist

14

Support Staff

6

Foxp3

TCR-independent epigenetic priming
Pioneering Factor

H3K4me1This laboratory studies: (i) the cellular and molecular basis of
immunologic self-tolerance, in particular the roles of regulatory T
cells (Treg); (ii) the strategy for eliciting effective immune
responses to autologous tumor cells, or inducing immunologic
tolerance to organ transplants, by manipulating the mechanismof immunologic self-tolerance; and (iii) the cause andpathogenetic mechanism of systemic autoimmune diseases,
such as rheumatoid arthritis, by utilizing an animal model
established in our laboratory.
Treg cells, which specifically express the transcription factor
Foxp3, are actively engaged in the maintenance of immunologicalself-tolerance and homeostasis. Treg development and function
is coordinated by the establishment of the Treg-specificepigenetic landscape and the transcription factor network
involving FoxP3. While FoxP3 functionally acts as a strong
repressor of Il2, Ifng, and other genes upon Treg activation, theTreg epigenome encompassing Treg-specific CpG hypomethylation,
chromatin accessibility and histone modifications, reinforces
activation of enhancers, especially super-enhancers, associatedwith Treg signature genes such as Foxp3, Cd25 and Ctla4, andstabilizes their expression. Notably, the establishment of such
Treg-specific epigenome is FoxP3-independent and a large
portion of Treg-specific super-enhancers are concurrently
activated at the precursor stage, prior to FoxP3 expression(Kitagawa et al., 2017). These findings suggest that genetic andepigenetic alterations at Treg function-associated genes may becausative of autoimmune and other inflammatory diseases byaffecting Treg development and function (reviewed in Sakaguchi2020).

CD4SP
thymocytes

In 2021, we addressed how Treg signature gene loci, inparticular the Foxp3 locus, become activated along Treg celldifferentiation in the thymus. At the Foxp3 gene locus, themammalian conserved non-coding sequences (CNSs) have beenhighlighted as the key functional enhancer elements for
induction and stabilization of Foxp3 expression. To determine
how they developmentally contribute to Treg lineage
specification, we showed that among Foxp3 CNSs, the promoterupstream CNS0 and the intergenic CNS3, which bound distinct
transcription factors, were activated at early stages of thymocyte
differentiation prior to Foxp3 promoter activation, with sequential
genomic looping bridging these regions and the promoter. Whiledeletion of either CNS0 or CNS3 partially compromised thymic
Treg cell generation, deletion of both completely abrogated thegeneration and impaired the stability of Foxp3 expression inresidual natural Treg cells. As a result, CNS0/3-double-deleted
mice succumbed to lethal systemic autoimmunity/inflammation.
Thus, coordinated activation of Foxp3 CNS0 and CNS3 initiates
and stabilizes Foxp3 gene expression, thereby cruciallycontrolling Treg cell development, maintenance, andconsequently immunological self-tolerance (Kawakami, Kitagawa
et al., Immunity, 2021).
Foxp3-expressing CD4+CD25+ Tregs constitutively and highly
express the immune checkpoint receptor CTLA-4, whose Tregspecific deficiency causes severe systemic autoimmunity (Winget al., Science 2008). As a key mechanism of Treg-mediatedsuppression, Treg-expressed CTLA-4 downregulates theexpression of CD80/CD86 costimulatory molecules on antigenpresenting cells (APCs). We have shown that Treg-expressed

CNS0

IL-2

H3K4me1

Foxp3

TCR- & IL-2-dependent Foxp3 induction

DNA

Stat5

CNS3

CNS0

Primed Enhancer with H3K4me1(Closed Chromatin)

TCR

DNA

CNS3

Foxp3

Active Enhancer
with H3K27ac(Open Chromatin)

Transcription
Factors

Deletion of ・Loss of thymic Tregs
CNS0+CNS3 ・Instability of peripheral Tregs
・Systemic autoimmunity

Figure.
At the Foxp3 gene locus, the promoter-upstream CNS0 and then the intergenic CNS3 are activated at early stages of thymocyte differentiation
prior to Foxp3 promoter activation, with sequential genomic looping among these regions. While deletion of either CNS0 or CNS3 partially impairs
thymic Treg cell generation, deletion of both completely abrogates the Treg generation, evoking systemic autoimmunity/inflammation.

Recent Publications
1. Kidani Y, Nogami W, Yasumizu Y, Kawashima A, Tanaka A, et al. CCR8targeted specific depletion of clonally expanded Treg cells in tumor
tissues evokes potent tumor immunity with long-lasting memory. Proc
Natl Acad Sci U S A. 119(7):e2114282119 (2022).

4. Ohkura N, Yasumizu Y, Kitagawa Y, Tanaka A, Nakamura Y, Motooka D,
Nakamura S, Okada Y, Sakaguchi S. Regulatory T Cell-Specific Epigenomic
Region Variants Are a Key Determinant of Susceptibility to Common
Autoimmune Diseases. Immunity. 52(6):1119-1132.e4 (2020).

2. Tekguc M, James Badger Wing JB, Osaki M, Long J, Sakaguchi S. Tregexpressed CTLA-4 depletes CD80/CD86 by trogocytosis, releasing free PD-L1
on antigen-presenting cells. Proc Natl Acad Sci U S A. 118(30):e2023739118(2021).

5. Sakaguchi S, Mikami N, Wing JB, Tanaka A, Ichiyama K, and Ohkura N.
Regulatory T Cells and Human Disease. Ann. Rev. Immunol. 38:541-566(2020).

3. 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 selftolerance. Immunity. 54(5):947-961.e8 (2021).

23

Immunology

Cell Signaling

Takashi Saito, PhD

A

B
LAG3

TCR

Merge

Ab -

Professor

Takashi Saito

Ab +Figure.
Negative regulation of T cell activation through LAG3 colocalized with TCR microcluster.
A. LAG3 generates clusters colocalized with TCR-microcluster (MC) upon T cell activation. anti-LAG3 Ab inhibited the colocalization between TCRMC and LAG3 clusters while enhanced IL-2 production. B. Schematic illustration indicating that colocalization of LAG3 clusters with TCR-MC is
critical for LAG3 to induce suppressive function of T cell activation which is inhibited by anti-LAG3 Ab.

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 such as
autoimmunity and allergic inflammation. For this purpose, we
analyzed the regulation of T cell activation/function from asignaling perspective.

1. Regulation of T cell activation through inhibitory
checkpoint receptors
Our finding that TCR-microclusters (MC) initiate T cell activation
led us to analyze the dynamics of signaling molecules at theimmune synapse. Similar to our previous studies of CTLA4 andPD-1, we have analyzed the dynamic regulation of otherinhibitory co-stimulation receptor, LAG3. This inhibitory receptor
was also colocalized with the TCR-MC upon TCR stimulation to
mediate inhibition of T cell activation. Since the association is
critical for the inhibitory function, anti-LAG3 Ab induced
separation of LAG3 cluster from TCR-MC and consequently
enhancement of T cell activation. Our analyses provide a dynamic
view of signal regulation to define inhibitory mechanism (Figure).

2. Inhibitory mechanism of T cell activation throughphosphatases
We have analyzed negative regulation of T cell activation,
particularly by autoimmune-related PTPN22. Its deficiency
resulted in enhanced activation and an increase in effector/
memory T cells. 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 activation. A
PTPN22 mutant causing susceptibility to autoimmune diseases
was defective in recruitment to the TCR-MC. These studies help
define the autoimmune susceptibility caused by the mutation.

3. Regulation of T cell function by innate signaling
We have also analyzed the modulation of T cell function by
innate-like signals. During such analyses, we found that Receptorinteracting serine/threonine-protein kinase 1 (RIPK1) plays acritical role in T cell function and differentiation, particularly on
metabolic regulation and senescence. We are analyzing T cellspecific RIPK1-deficient mice to analyze its function of T cells on
metabolism and aging.

Recent Publications
1. Takemori T, Sugimoto-Ishige A, et al. Establishment of a monoclonal
antibody against human NTCP that blocks HBV infection. J Virol.
96:e0168621 (2022).
2. Kumagai A, Nara T, Uematsu M, Kakinuma Y, Saito T, and Masuda K.
Development and characterization of a unique anti-IgE monoclonal
antibody cross-reactive between human and canine IgE. Immun Inflamm.
Dis. 9:1740-1748 (2021).

4. Imanishi T and Saito T. T Cell co-stimulation and functional modulation
by innate signals. Trends lmmunol. 41:200-212 (2020).
5. 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).

3. Imanishi T, Unno M, Kobayashi W, Yoneda N, Akita S and Saito T. mTORC1
signaling controls TLR2-Mediated T-cell activation by inducing TIRAP
expression. Cell Reports. 32:107911-107911 (2020).

24

25

Immunology

Lymphocyte Differentiation
Glycan engineering
SARS-CoV-2
Spike RBD

Wild type
SARS-CoV-2
Spike RBD
Head region

Tomohiro Kurosaki, MD/PhD

Professor
Associate Professor

N-GlycanCore region

Immunization

Tomohiro Kurosaki
Takeshi Inoue
Ryo Shinnakasu

Postdoctoral Fellow

2

Research Assistant2

Support Staff

4

SARS-CoV-2
head region
reactive Ab
SARS-CoV-2
core region
reactive Ab

The coronavirus disease 2019 (COVID-19) pandemic, caused by
the β-coronavirus severe acute respiratory syndrome coronavirus
2 (SARS-CoV-2), is a global health crisis. In this year, in order tomake broadly protective vaccines against SARS-related
coronaviruses, we designed new types of vaccines, and tested
whether they work in the mouse model system.
SARS-related coronaviruses may cause future outbreaks,
therefore broadly protective vaccines are urgently needed. Whenlooking carefully at the structure of the receptor binding region(RBD) of the spike protein of the SARS-CoV-2 virus, it is composedof the head and the core sub-region, the former and the latter ofwhich can directly block the entry of SARS-CoV-2, and contributeto the overall structural stability of the RBD region, respectively.

Given that the core sub-domain is structurally well conserved
among SARS-related viruses, we intended to make new type of
vaccines to elicit neutralizing antibodies against the core subdomain. For this purpose, we introduced N-linked glycans onto
the SARS-CoV-2 RBD surfaces and used them as immunogens in amouse model. These types of vaccines indeed elicited significant
neutralizing activity for not only SARS-CoV-2 but SARS-related
viruses such as bat WIV1-CoV.

SARS-CoV-2
Spike RBD

SARS-CoV
Spike RBD

WIV1-CoV
Spike RBDʹ ʹ

Head region
Core region(Conserved
region)Figure.
How to make protective vaccines against not only SARS-CoV-2, but SARS-CoV and WIV1-CoV viruses.
In order to induce the antibodies predominantly against the regions that are structurally conserved in RBD among SARS-related viruses, glycan
engineering was performed to mask the dominant epitope on the RBD head region which is a structurally non-conserved region. When mice are
immunized with this modified RBD vaccine, as expected, antibodies that recognize the structurally conserved core-RBD region of not only SARSCoV-2 but also other related SARS-related viruses such as SARS-CoV, WIV1-CoV are predominantly induced and also showed a high protective
effect against these SARS-related viruses.

Recent Publications
1. 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 tobecome quiescent memory B cells depends on metabolic reprograming
and provision of a survival signal. J Exp Med. 218(1):e20200866 (2021).
2. Shinnakasu R, Sakakibara S, Yamamoto H, Wang PH, Moriyama S, Sax N,
Ono C, Yamanaka A, Adachi Y, Onodera T, Sato T, Shinkai M, Suzuki R,
Matsuura Y, Hashii N, Takahashi Y, Inoue T, Yamashita K, Kurosaki T. Glycan
engineering of the SARS-CoV-2 receptor-binding domain elicits crossneutralizing antibodies for SARS-related viruses. J Exp Med.
218(12):e20211003 (2021).

26

3. Tanaka S, Ise W, Baba Y, Kurosaki T. Silencing and activating anergic B
cells. Immunol Rev. doi: 10.1111/imr.13053 (2021).
4. Yeh CH, Finney J, Okada T, Kurosaki T, Kelsoe G. Primary germinal centerresident T follicular helper cells are a physiologically distinct subset of
CXCR5hiPD-1hi T follicular helper cells. Immunity 55(2):272-289 (2022).
5. Tsao HW, Kaminski J, Kurachi M, Barnitz RA, DiIorio MA, LaFleur MW, Ise W,
Kurosaki T, Wherry EJ, Haining WN, Yosef N. Batf-mediated epigenetic
control of effector CD8+ T cell differentiation. Sci Immunol. 7(68):eabi4919(2022).

27

Immunology

Malaria Immunology

Cevayir Coban, MD

Professor

Malaria infection often leads to severe complications such ascerebral malaria. Even after the recovery from severe illness,
individuals are repeatedly re-infected. The immune pathology
causing severe complications and the reasons of the lack ofsterile immunity against malaria have been an intense research
topic in our laboratory.
To investigate the molecular pathogenesis underlying cerebral
malaria, we investigated the spatial distribution of the parasites
in the whole-brain microvessels by utilizing the new tissueclearing method CUBIC (Clear, Unobstructed, Brain/Body Imaging
Cocktails and Computational analysis) with light-sheet
fluorescent microscopy (LSFM). We demonstrated that
Plasmodium berghei ANKA (PbANKA) parasites significantly
accumulated in the olfactory bulb (OB) of mice, compared withthe other parts of the brain, including the cerebral cortex,
cerebellum and brainstem (Matsuo-Dapaah et al., Int Immunology,2021). We expect that CUBIC clearing of whole ECM brain might
greatly advance our understanding of human cerebral malaria(Figure 1).

Cevayir Coban

The germinal center (GC) in secondary lymphoid organs is avery important structure where somatic hypermutation andclonal selection are coupled for antibody affinity maturation
against infections including malaria. However, how GCs areformed and regulated is incompletely understood. We recently
identified an unexpected role of Tank-binding kinase-1 (TBK1) asa crucial B cell-intrinsic factor for GC formation. In the absence of
TBK1 in B cells, B cells failed to form GC despite normal Tfh celldifferentiation. The TBK1 phosphorylation elevates in B cells
during GC differentiation and regulates the balance of IRF4/BCL6
expression by limiting CD40 and BCR activation throughnoncanonical NF-κB and AKTT308 signaling. In the absence of
TBK1, CD40 and BCR signaling synergistically enhanced IRF4
expression in Pre-GC, leading to BCL6 suppression, and therefore
failed to form GCs. As a result, memory B cells generated from
TBK1-deficient B cells fail to confer sterile immunity upon
reinfection, suggesting that TBK1 determines B cell fate topromote long-lasting humoral immunity (Lee et al., J Exp. Medicine,2022).

Figure 1.
Using a new three-dimensional CUBIC tissue-clearing method to examine the brain during experimental cerebral malaria (Matsuo-Dapaah et al.,Int. Immunology, 2021). The 3-dimensional (3D) images of single microvessels deep in the whole brain after CUBIC cleared in Naïve and PbANKA(GFP tagged)-infected mice brains. The GFP-tagged parasites are preferentially accumulated in the capillaries and tissue of the olfactory bulb.

Figure 2.
B cell-intrinsic TBK1 is essential for germinal center formation during infection and vaccination in mice (Lee et al., J Exp. Medicine, 2022).
A schematic diagram illustrating the mechanism of B cell fate decision controlled by TBK1 through fine tuning of CD40/BCR signaling to negatively
regulate IRF4 and c Myc in Pre GC. As a result, in the absence of TBK1 in B cells GC is not formed and high quality antibodies which protects against
re-infection could not be produced.

Recent Publications
1. Lee MSJ, Inoue T, Ise W, Matsuo-Dapaah J, Wing JB, Temizoz B, Kobiyama
K, Hayashi T, Patil A, Sakaguchi S, Simon AK, Bezbradica JS, Nagatoishi S,
Tsumoto K, Inoue JI, Akira S, Kurosaki T, Ishii KJ, Coban C. B cell intrinsic
TBK1 is essential for germinal center formation during infection andvaccination in mice. J Exp Med. 219(2):e20211336 (2022).
2. Matsuo-Dapaah J, Lee MSJ, Ishii KJ, Tainaka K, Coban C. Using a new
three-dimensional CUBIC tissue-clearing method to examine the brain
during experimental cerebral malaria. Int Immunol. dxab060 (2021).

4. Lee MSJ, Natsume-Kitatani Y, Temizoz B, Fujita Y, Konishi A, Matsuda K,
Igari Y, Tsukui T, Kobiyama K, Kuroda E, Onishi M, Marichal T, Ise W, Inoue
T, Kurosaki T, Mizuguchi K, Akira S, Ishii KJ, Coban C. B cell-intrinsic MyD88
signaling controls IFNγ-mediated early IgG2c class switching in response
to a particulate adjuvant. Euro J Immunol. doi:10.1002/eji.201848084(2019).
5. Coban C, Lee MSJ, Ishii KJ. Tissue-specific immunopathology duringmalaria infection. Nat Rev Immunol. doi:10.1038/nri.2017.138 (2018).

3. Coban C. The host targeting effect of chloroquine in malaria. Curr Opin
Immunol. 66:98-107 (2020).

28

29

Vaccine Science

Immunology

Our findings demonstrate a novel adjuvant discovery with thecomplementary strand conjugated to a lipid, which enableddraining LN targeting and increased ODN2006 accumulation indraining LNs, thereby enhancing the adjuvant effect. Our findings
imply that S-540956 is a promising adjuvant for cancer peptide
vaccines and has a high potential for applications in various
vaccines, including recombinant protein vaccines.

COVID-19 owe part of their success to methylation that masks
immunostimulatory properties of the mRNA, but theimmunological mechanisms of adjuvanticity are unclear. Two
new studies reveal distinct mechanisms for innate sensing of thishidden adjuvant. With two recent papers, we proposed a newmechanism by which LNP and RNA exert the adjuvanticity in vivo
(Figure).

3. Mechanisms of immunogenicity of, and reactogenicity
to, LNP-mRNA vaccines
Messenger (m) RNA vaccines such as those used to prevent

Ken J. Ishi, MD/PhD

Professor

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 to adjuvants.
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.
Below are some of our recent works conducted in FY2021

1. Primary Cilia in the Skin: Functions in Immunity andTherapeutic Potential
The skin is the biggest organ and provides a physical andimmunological barrier against pathogen infection. The distributionof primary cilia in the skin of mice has been reported, but whichcells in human skin have them has not, and we still know very
little about how they change in response to immune reactions or
disease. This review introduces several studies that describe
mechanisms of cilia regulation by immune reaction and thephysiological relevance of cilia regulating proliferation anddifferentiation of stroma cells, including skin-resident Langerhans
cells. Based on such background and potential of research on
primary cilia, we published the review article as shown in thesubheading and research article entitled; Increase in primary cilia
in the epidermis of patients with atopic dermatitis and psoriasis,
where we elucidate the incidence of primary cilia in skininflammation and the potential mechanism underlying thedysregulation of keratinocytes. Significant increases in ciliatedcells were observed in the patients with atopic dermatitis and30

Ken J. Ishii

Associate Professor

Kouji Kobiyama

Assistant Professor

Burcu Temizoz

Postdoctoral Fellow

1

Research Assistant

3

Visiting Scientist

2

Support Staff

2

psoriasis skin samples compared with normal skin samples. An
increase of ciliated cells in the epidermis may impair keratinocyte
differentiation under stress conditions caused by inflammation inboth AD and psoriasis patients.

2. A CpG oligonucleotide annealed to a complementarystrand with an amphiphilic chain unit, acts as a potent
cancer vaccine adjuvant by targeting draining lymph
nodes
Robust induction of cancer-antigen-specific CD8+ T cells isessential for the success of cancer peptide vaccines, which arecomposed of a peptide derived from a cancer-specific antigen
and an immune-potentiating adjuvant, such as a Toll-likereceptor (TLR) agonist. Efficient delivery of a vaccine antigen andan adjuvant to antigen-presenting cells in the draining lymph
nodes (LNs) holds key to maximize vaccine efficacy. We developed
S-540956, a novel TLR9-agonistic adjuvant consisting of B-type
CpG ODN2006 (also known as CpG7909), annealed to its
complementary sequence oligodeoxynucleotide (ODN) conjugatedto a lipid; it could target both a cancer peptide antigen and a
CpG-adjuvant in the draining LNs. S-540956 accumulation in thedraining LNs and activation of plasmacytoid dendritic cells (pDCs)
were significantly higher than that of ODN2006. Mechanistic
analysis revealed that S-540956 enhanced the induction of MHC
class I peptide-specific CD8+ T cell responses via TLR9 in a CD4+
T cell-independent manner. In mice, the therapeutic effect of
S-540956-adjuvanted with a human papillomavirus (HPV)-E7
peptide vaccine against HPV-E7-expressing TC-1 tumors was
significantly better than that of an ODN2006-adjuvanted vaccine.

Figure.
In the lymph nodes (LNs), modified RNA sensed by MDA-5 results in the production of type I interferons (IFNs). Type I interferons induce antigenspecific CD8+ T cell responses. LNPs can activate innate immune responses in lymph nodes and elicit production of IL-6 that is essential for the
induction of TFH cells and GC B cells. These pathways are important for the immunogenicity of LNP-mRNA vaccines. When the mRNA is unmodified
and a specific ionizable lipid (such as SM-102) is contained in the LNP, the mRNA is recognized by TLR7 and/or TLR8 and the ionizable lipid isrecognized by the NLRP3 inflammasome in monocytes. Inflammasome-induced members of the IL-1 family trigger further inflammatory cytokine
production. IL-1Ra is also produced as a negative feedback loop to inhibit IL-1 signaling and reduce inflammation. Unmodified mRNA is also
detected by RIG-I, which results in the production of type I interferons and thus interferes with antigen translation. After booster vaccination,
modified RNA–induced type I interferons also activate natural killer (NK) cells to produce IFN-γ. These signaling pathways might affect thereactogenicity of LNP-mRNA vaccines. APC, antigen-presenting cell; DC, dendritic cell; pDC, plasmacytoid dendritic cell; cDC, conventional
dendritic cell; CARD, caspase-recruitment domain; PRR, proline-rich region; TM, transmembrane domain; TIR, Toll–IL-1R domain; DD, death
domain; TH1 cells, type 1 helper T cells; NACHT, nucleotide-binding domain; PYD, pyrin domain; LRR, leucine-rich repeat; ASC, apoptosis-associatedspeck-like protein containing a caspase-recruitment domain.

Recent Publications
1. Hioki K, Hayashi T, et al. Machine learning-assisted screening of Herbal
MedicineExtracts as vaccine adjuvants Frontiers in Immunology 2022 inpress.

4. Lee MSJ, Inoue T, Ise W, et al. B cell-intrinsic TBK1 is essential for germinal
center formation during infection and vaccination in mice. J Exp Med.
219(2):e20211336 (2022).

2. Kobiyama K, Ishii KJ. Making innate sense of mRNA vaccine adjuvanticity.
Nat Immunol. 23(4):474-476 (2022).

5. Nakagawa T, Tanino T, Onishi M, et al. S-540956, a CpG Oligonucleotide
annealed to a complementary strand with an amphiphilic chain unit, acts
as a potent cancer vaccine adjuvant by targeting draining lymph nodes.
Front Immunol. 12:803090 (2021).

3. Temizoz B, Hioki K, Kobari S, Jounai N, Kusakabe T, Lee MSJ, Coban C,
Kuroda E, Ishii KJ. Anti-tumor immunity by transcriptional synergy
between TLR9 and STING activation. Int Immunol. 2022 in press.

31Immunology

Immunoparasitology

Masahiro Yamamoto, PhD

Professor

T cells play a critical role in adaptive immunity for pathogen
elimination and tumor surveillance (Gaud et al., 2018). T cells
express a TCR-CD3 complex containing TCRαβ and CD3ζγεδεζ
subunits on their surfaces (Fu et al., 2014). Under physiologicalconditions, T-cell activation is initiated by engagement of TCRs
on T cells and peptides in complex with major histocompatibility
complex (pMHC) molecules on antigen-presenting cells (APCs)(Samelson and Klausner, 1988). After binding of the TCR to thepMHC, tyrosine residues of immunoreceptor tyrosine-based
activation motifs in the cytosolic region of the CD3ζ chain arephosphorylated by lymphocyte protein tyrosine kinase (Lck),recruiting zeta-chain-associated protein kinase of 70 kDa (ZAP70)(Courtney et al., 2018). Subsequently, ZAP70 phosphorylates thetransmembrane adaptor molecule linker for the activation of T
cells (LAT), which serves as a signaling hub and recruits othersignaling molecules to the plasma membrane, including SH2domain-containing leukocyte protein of 76 kDa (SLP76) andphospholipase C γ1 (PLCγ1) (Dustin and Choudhuri, 2016). The
LAT/SLP76 complex together with interleukin-2-inducible T-celltyrosine kinase (ITK) activates PLCγ1, catalyzing hydrolysis ofphosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol
1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (Rhee, 2001).
IP3 and DAG are important for incrementing cytosolic Ca2+ levels
to activate NFAT and phosphorylate MAP kinases and protein
kinase C θ (PKCθ), which activates NF-κB, resulting in productionof various cytokines, T-cell proliferation, and T-cell effector
function (Rao and Hogan, 2009; Samelson, 2011).
Although these core TCR signaling molecules are shared
between CD4+ and CD8+ T cells, it is widely assumed that CD4 and32

Masahiro Yamamoto

Associate Professor

Miwa Sasai

Postdoctoral Fellow

1

Support Staff

2

CD8 are not accessory molecules but co-receptors that potentiate
activation signals (Eichmann et al., 1989; Janeway, 1992; Parnes,
1989). Both CD4 and CD8α co-receptors augment CD3-TCR
signaling, since the cytoplasmic regions of both CD4 and CD8α
interact with Lck (Rudd et al., 1988; Veillette et al., 1988). Since Lck
preferentially binds CD4 rather than CD8α (Veillette et al., 1989),
TCR signaling pathways in CD4+ and CD8+ T cells are quantitatively
different. However, given the shared signaling molecules, it is
widely accepted that TCR signaling pathways in CD4+ and CD8+ T
cells are qualitatively identical (Artyomov et al., 2010; Fu et al.,2014; Gaud et al., 2018). The cytoplasmic tail of CD8α contains a
CXCP motif important for Lck binding (Shaw et al., 1990; Turner etal., 1990). CD8α-deficient mice restored with a mutant CD8α in
which the Lck-binding motif is replaced with alanine display
milder phenotypes in terms of CD8+ T-cell activation and T-celldevelopment in the thymus than do CD8-deficient mice restoredwith tailless CD8α (Chan et al., 1993; Fung-Leung et al., 1993),
suggesting that there are other signaling molecules that bind tothe cytoplasmic regions of CD8α (Zamoyska, 1994). However,such signaling molecules that specifically bind CD8α anddifferentiate the CD8+ T-cell receptor signaling pathway are
unknown.
Phospholipase C (PLC) regulates cellular concentrations of PIP2
as substrate or IP3 and DAG as products to control complex
cellular homeostasis. IP3 and DAG act as second messengers(Kadamur and Ross, 2013). IP3 binds to the IP3 receptor on theendoplasmic reticulum and increases cytosolic Ca2+ levels. Ca2+and DAG activate protein kinase C (Rhee, 2001). The mammalian
PLC family consists of thirteen members classified into six

subtypes (β, γ, δ, ε, ζ, and η) based on amino acid sequence anddomain structure (Kadamur and Ross, 2013). All PLCs possess ahighly conserved catalytic core containing an N-terminal
pleckstrin homology (PH) domain, an EF-hands motif, a split X+Y
catalytic domain, and a C2 domain (Kadamur and Ross, 2013). In
TCR signaling, PLCγ1 plays a critical role in activation of both
CD4+ and CD8+ T cells under physiological conditions (Samelson,
2002). In contrast, bacterial superantigens activate TCR signaling
pathways independently of PLCγ1 (Bueno et al., 2006). Moreover,such superantigen-induced activation does not require Lck,
ZAP70, or CD4. Furthermore, PLCβ inhibition and PLCβ1 silencing
each partially block superantigen-induced T-cell activation(Bueno et al., 2006), suggesting that PLCβ1 is involved in
activation under pathological conditions. PLCβ4 is the last

member of the PLCβ family and is highly expressed in the retina
and cerebellar Purkinje cells (Peng et al., 1997; Watanabe et al.,1998). PLCβ4 deficiency leads to defects in the visual process andataxia, suggesting its neurological significance (Jiang et al., 1996;
Kano et al., 1998; Kim et al., 1997). Despite these establishedneurological roles, the immune functions of PLCβ4, especially in
T-cell signaling pathways, remain unexplored (Kawakami and
Xiao, 2013).
In this study, we examine the roles of PLCβ4 in TCR signaling
and immune response and find that PLCβ4 is selectively required
for activation of TCR signaling in CD8+ T cells and physiologicallyimportant for anti-parasitic and anti-tumor adaptive immune
responses.

Figure.
Uncovering a novel role of PLCβ4 in selectively mediating TCR signaling in CD8+ but not CD4+ T cells.

Recent Publications
1. Sasai M, Ma JS, Okamoto M, Nishino K, Nagaoka H, Takashima E, Pradipta
A, Lee Y, Kosako H, Suh PG, Yamamoto M. Uncovering a novel role of
PLCβ4 in selectively mediating TCR signaling in CD8+ but not CD4+ T
cells. J Exp Med. 218:e20201763 (2021).

4. Sakaguchi N, Sasai M, Bando H, Lee Y, Pradipta A, Ma JS, Yamamoto M.
Role of Gate-16 and Gabarap in Prevention of Caspase-11-Dependent
Excess Inflammation and Lethal Endotoxic Shock. Front Immunol.
11:561948 (2020).

2. Pradipta A, Bando H, Ma JS, Tanaka S, Sasai M, Yamamoto M. Plasmodium
UIS3 avoids host cell-autonomous exclusion that requires GABARAPs but
not LC3 and autophagy. Parasitol Int. 83:102335 (2021).

5. Bando H, Pradipta A, Iwanaga S, Okamoto T, Okuzaki D, Tanaka S, VegaRodríguez 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).

3. Pradipta A, Sasai M, Motani K, Ma JS, Lee Y, Kosako H, Yamamoto M. Cellautonomous Toxoplasma killing program requires Irgm2 but not its
microbe vacuolar localization. Life Sci Alliance. 4:e202000960 (2021).

33

Wild-type
ATP11A (Q84)

Immunology

Biochemistry & Immunology

cleft on the surface exposed to the outer leaflet of the plasma
membrane. Nine charged residues are placed from top to bottom
as a staircase inside the molecule. Two were essential forstabilizing the complex, while six were for scrambling
phospholipids in inward and outward directions, providing apathway for the translocation of phospholipids. A tryptophan

residue (W45) was present between the head group of PtdCho
and the extracellular end of the staircase. Its mutation to alanine
made the Xkr8-Basigin complex constitutively active, indicatingthat the W45 may play a gatekeeper in regulating its scramblase
activity. The structure of Xkr8-Basigin would provide insights into
the molecular mechanisms underlying phospholipid scrambling.

MutantATP11A (Q84E)

Shigekazu Nagata, PhD

(1)

(3)

Professor

Shigekazu Nagata

Assistant Professor

Takaharu Sakuragi(2)

Postdoctoral Fellow

1

Research Assistant

3

Visiting Scientist

2

Support Staff

1

Figure 1.
A sublethal point mutation of human ATP11A that changes the substrate specificity. The ATP11A flippase translocates phosphatidylserine (PtdSer),but not phosphatidylcholine (PtdCho), from the outer to the inner leaflet of plasma membranes, maintaining the asymmetric distribution of
PtdSer. A point Q84E mutation of ATP11A found in a patient with developmental delays and neurological deterioration allows PtdCho binding at
the substrate entry site for flipping (1). The PtdCho flipped into the inner leaflet of the plasma membranes up-regulates the expression of thesphingomyelin (SM) synthase (SGMS1) gene (2). The SGMS1 then converts PtdCho to SM at the Golgi apparatus (3). SM then moves to the outerleaflet of the plasma membranes. The reduced PtdCho and increased SM concentrations at the outer leaflet of plasma membranes have adeleterious effect on cells, including cell growth, cholesterol homeostasis, and sensitivity to sphingomyelinase.

Phospholipids are asymmetrically distributed between innerand outer leaflets of plasma membranes with exclusive
phosphatidylserine (PtdSer) localization in the inner leaflet. Thisasymmetrical distribution of phospholipids is maintained by ATPdependent phospholipid flippases, which translocate PtdSer
from outer to inner leaflets. When cells undergo apoptosis, theasymmetrical distribution of phospholipids is disrupted by ascramblase(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 for
apoptotic cells to macrophages. The PtdSer-exposure is also
observed in activated platelets, lymphocytes and mast cells,
capacitated sperm, aged erythrocytes, exosomes, and enveloped
viruses.
We previously identified two P4-type ATPases (ATP11A and11C) and their subunit CDC50A as flippases at plasma membranes(Segawa et al. Science 2014). ATP11A and 11C flip PtdSer, but notphosphatidylcholine (PtdCho), maintaining the asymmetric
distribution of PtdSer. A group in Tohoku University Medical
School detected a de novo heterozygous point mutation of
ATP11A in a patient with developmental delays and neurologicaldeterioration. Mice carrying the corresponding mutation diedperinatally of neurological disorders. This mutation caused anamino acid substitution (Q84E) in the first transmembranesegment of ATP11A, and the mutant ATP11A flipped PtdCho(Figure 1). Aberrant PtdCho flipping markedly decreased theconcentration of PtdCho in the outer leaflet of plasma
membranes, whereas sphingomyelin (SM) concentrations in theouter leaflet increased. This change in the distribution of34

phospholipids altered cell characteristics, including cell growth,cholesterol homeostasis, and sensitivity to sphingomyelinase.
These results provided insights into the physiological importanceof the substrate specificity of plasma membrane flippases for theproper distribution of PtdCho and SM (Segawa et al. J. Clin. Invest.
2021).
Two families of membrane proteins (TMEM16 and XKR) support
the scrambling of phospholipids at plasma membranes. Amongten members of the TMEM16 family, TMEM16F ubiquitously
expressed and functions as Ca2+-dependent scramblases. The
TMEM16F gene is mutated in human Scott syndrome patients, acongenital bleeding disorder, indicating that TMEM16F is
responsible for exposing PtdSer to activate blood clotting factors(Suzuki et al. Nature 2010). The XKR family comprises nine
members. XKR8 is ubiquitously expressed and forms a binary
complex with Basigin or Neuroplastin, an Ig-super family protein(Suzuki et al. Science 2013; PNAS 2016). XKR8 carries a caspaserecognition site at the C-terminal tail region, and its cleavage
during apoptosis is necessary to function as a scramblase. The
PtdSer, thus on the dead cell’s surface, is recognized bymacrophages for engulfment. A loss-of-function mutation of
Xkr8 causes inefficient engulfment of apoptotic cells leading to
the activation of autoimmunity and male infertility (Kohno et al.
PNAS 2019; Yamashita et al. MCB 2020).
Combining cryo-EM and X-ray crystallography, we determined
the tertiary structure of the human XKR8-Basigin complex at aresolution of 3.8 Å (Sakuragi et al. Nat. Struct. Mol. Biol. 2021). Its
membrane-spanning region adopts a cuboid-like structure(Figure 2). A single PtdCho molecule was present in a hydrophobic

D

(C)

(A)

D
D

Hydrophilic
pore

XKR8

PtdChoW45

D

D1

Basigin

D

D
PtdCho

D3

D2
D7

(B)
39

44

156

D11
161
224

2

1

96

N

69

83

3b

322

268

5

141
139

91

3a
7

306

227

4b
6

7

195

247

9

10

8

4a

348
187

112

258

117

283

11

340

358

C

Figure 2.
The tertiary structure of the human XKR8-Basigin complex that scrambles phospholipids. (A) The tertiary structure of the human XKR8-Basigin
complex is represented in a ribbon diagram. There is a cleft surrounded by hydrophobic residues in the upper middle region of the molecule, andphosphatidylcholine (PtdCho) is inserted in the cleft. (B) α-helices of hXkr8 are numbered and schematically shown. (C) Close-up side (lower panel)and top views of the hydrophilic pore (boxed area) carrying 9 charged amino acids (Glu, Asp, and Arg) in the α1, α 4, and α 5 helices. The side chainsof charged amino acids are represented in a colored ball and stick model. PtdCho is present in the cleft on the surface. W45 (green) at the top of α2seems to serve as a gatekeeper for scrambling phospholipids through the pore.

Recent Publications
1. Ochiai Y, Suzuki C, Segawa K, Uchiyama Y, and Nagata S. Inefficientdevelopment of syncytiotrophoblasts in the Atp11a-deficient mouseplacenta. Proc Natl Acad Sci. USA. 119 in press.

4. Sakuragi T, Kanai R, et al. The tertiary structure of the human Xkr8-Basigin
complex that scrambles phospholipids at plasma membranes. Nat Struct
Mol Biol. 28:825-834 (2021).

2. Ryoden Y, Segawa K, and Nagata S. Requirement of Xk and Vps13a for theP2X7-mediated phospholipid scrambling and cell lysis in mouse T cells.
Proc Nat Acad Sci. USA. 119: e2119286119 (2022).

5. Caronni N, Piperno GM, et al. TIM4 expression by dendritic cells mediates
uptake of tumor-associated antigens and anti-tumor responses. Nat
Commun. 12:2237 (2021).

3. Segawa K, Kikuchi A, et al. A sublethal ATP11A mutation associated withneurological deterioration causes aberrant phosphatidylcholine flippingin plasma membranes. J Clin Invest. 131:e148005 (2021).

35

Immunology

Molecular Neuroscience
Toshihide Yamashita, MD/PhD
Professor

Toshihide Yamashita

Associate Professor

Takahide Itokazu

Assistant Professor

Hiroshi Nakagawa
Oluwaseun Fatoba
Hiroshi Tsujioka
Yosuke Ohtake
Toru Nakanishi
Shinichiro Hiraga
Nao Shibuya
Abdellatif Abbaoui

Disorders of the central nervous system, such as cerebrovasculardiseases, cerebrospinal trauma, and encephalomyelitis, oftencause spatiotemporal changes in the nervous system and invarious biological systems, such as the immune system andvascular system. We have analyzed disorders of the neuralnetworks in the central nervous system and the subsequent
restoration process from the perspective of the functional
network of biological systems (Fig. 1). Further, we have analyzed
the mechanism by which the spatiotemporal dynamics in thosebiological systems control a series of processes (Fig. 2).
Particularly, the ultimate goal of this study is to elucidate themanner in which the control mechanism is affected by the
associations among the nervous system, immune system, andvascular system. Additionally, we aim to elucidate the processes
involved in the functioning of living organisms with neural

Postdoctoral Fellow

1

Research Assistant

2

Visiting Scientist

6

Support Staff

2

Figure 1.
The mechanism of spontaneous functional recovery.

network disorders within the central nervous system by observing
such disorders and their functional recovery process with respect
to the dynamics of the entire biological system and by conducting
a comprehensive analysis of the association between eachsystem.
We observe the central nervous system as a single organ withina biological system. Further, studies from the perspective of how
the entire biological system is involved in disorders and recoveryof neural networks are scarce. By observing disorders in neuralnetworks and the biological reactions during the subsequent
recovery process as a “scrap-and-build” strategy, we aim toelucidate the mechanisms behind a series of reactions as well as
their significance 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

36

1. Iwamoto S, Itokazu T, Sasaki A, et al. RGMa signal in macrophages induces
neutrophil-related astrocytopathy in NMO. An Neurol. 91:532-547 (2022).

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

2. 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 mediatedby apelin-APJ signaling. Nat Aging. 1:284-294 (2021).

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

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

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

37

Immunology

Molecular Immunology

Sho Yamasaki, PhD

Professor
Assistant Professor

Convalescent COVID-19 patientsfrom mild symptoms

Healthy donors

Sho Yamasaki
Masamichi Nagae
Eri Ishikawa

Postdoctoral Fellow

2

Research Assistant

8

Visiting Scientist

5

Support Staff

5

Clonal expansion

PublicTfh clonotypes
TCRα

Symbiotic
bacteria

Identification of conserved SARS-CoV-2 spike epitopes
that expand public cTfh clonotypes in mild COVID-19
patients
Adaptive immunity is a fundamental component in controlling
COVID-19. In this process, follicular helper T (Tfh) cells are a subsetof CD4+ T cells that mediate the production of protective
antibodies; however, the SARS-CoV-2 epitopes activating Tfh
cells are not well characterized. Here, we identified andcrystallized TCRs of public circulating Tfh (cTfh) clonotypes thatare expanded in patients who have recovered from mild
symptoms. These public clonotypes recognized the SARS-CoV-2
spike (S) epitopes conserved across emerging variants. The
epitope of the most prevalent cTfh clonotype, S864-882, waspresented by multiple HLAs and activated T cells in most ofhealthy donors, suggesting that this S region is a universal T cell
epitope useful for booster antigen. SARS-CoV-2-specific publiccTfh clonotypes also cross-reacted with specific commensal
bacteria. In this study, we identified conserved SARS-CoV-2 S
epitopes that activate public cTfh clonotypes associated withmild symptoms.

cross-react

TCRβ

SARS-CoV-2
S epitope

Symbiotic
bacteria

Symbiotic bacteria-dependent expansion of MR1reactive T cells causes autoimmune pancreatitis
MHC class I-related protein 1 (MR1) is a metabolite-presenting
molecule that restricts MR1-reactive T cells including mucosalassociated invariant T (MAIT) cells. In contrast to MAIT cells, thefunction of other MR1-restricted T cell subsets is largely unknown.
Here, we report that mice in which a T cell-specific transcription
factor, B-cell lymphoma/leukemia 11B (Bcl11b), was ablated inimmature thymocytes (Bcl11bΔiThy mice) developed chronic
inflammation. Bcl11bΔiThy mice lacked conventional T cells andMAIT cells, whereas CD4+IL-18R+ αβ T cells expressing a skewed
Traj33 (Jα33)+ T cell receptors (TCR) accumulate in the periphery,
which are necessary and sufficient for the pathogenesis. The
disorders observed in Bcl11bΔiThy mice were ameliorated by MR1deficiency, transfer of conventional T cells, or germ-free
conditions. We further determined the crystal structure andidentified the antigen of the TCR expressed by pathogenic T cells.
Here, we show that MR1-reactive T cells become pathogenic in a
symbiotic bacteria-dependent manner.

Prevention of COVID-19 aggravation

Recent Publications
1. Lu X, Hosono Y, Nagae M, Ishizuka S, et al. Identification of conserved
SARS-CoV-2 spike epitopes that expand public cTfh clonotypes in mild
COVID-19 patients. J Exp Med. 218:e20211327 (2021).
2. Nagata M, Toyonaga K, Ishikawa E, et al. Helicobacter pylori metabolites
exacerbate gastritis through C-type lectin receptors. J Exp Med.
218:e20200815 (2021).

4. Nagata M, Izumi Y, Ishikawa E, et al. Intracellular metabolite
β-glucosylceramide is an endogenous Mincle ligand possessing
immunostimulatory activity. Proc Natl Acad Sci U S A.114: E3285-E3294(2017).
5. Toyonaga K, Torigoe S, Motomura Y, et al. C-Type Lectin Receptor DCAR
Recognizes Mycobacterial Phosphatidyl-Inositol Mannosides to Promotea Th1 Response during Infection. Immunity 45:1245-57 (2016).

3. Imai T, Matsumura T, Mayer-Lambertz S, et al. Lipoteichoic acid anchor
triggers Mincle to drive protective immunity against invasive group A
Streptococcus infection. Proc Natl Acad Sci U S A. 115: E10662-E10671(2018).

38

39

Immunology

Stem Cell Biology and Developmental Immunology

associated with a poor prognosis. These results strengthen theclaim that CAR cells are the bone marrow specific fibroblastic
reticular cells, which express specific and critical transcription

factors, including Foxc1, Ebf 1/3, and Runx1/2, providing HSC
niches and bone.

Adipocyte

CAR cell
Perichondrial
progenitor

Takashi Nagasawa, MD/PhD

Fibrotic cell

Runx1/2

HSC
Runx2
Osteoblast

Professor

Takashi Nagasawa

Associate Professor

Yoshiki Omatsu

Support Staff

1

Osteoblast

Runx2
Osxhi

Runx2
Osxhi

Figure 1.
The functions of Runx1/2 in CAR cell maintenance.
CAR cells require Runx1 or Runx2 to prevent their fibrotic conversion and maintain HSCs and hematopoiesis in adult bone marrow.

2VWHREODVWV
3HULFKRQGULXP
FHOOV
Runx2
Special microenvironments known as niches are essential forthe maintenance of hematopoietic stem cells (HSCs), which giverise to blood cells, and lympho-hematopoiesis within bonemarrow cavities. We isolated a chemokine, CXCL12 (SDF-1/PBSF)
as a molecule that stimulates the growth of B cell precursors(Nagasawa et al. PNAS 1994) and found that CXCL12 and its
receptor CXCR4 are essential for colonization of bone marrow by
HSCs during embryogenesis (Nagasawa et al. Nature 1996; Ara etal. Immunity 2003), maintenance of a pool of HSCs (Sugiyama etal. Immunity 2006), and development of immune cells, including
B cells, plasmacytoid dendritic cells (pDCs) and NK cells in bonemarrow as well as vascular formation and cardiogenesis(Tachibana et al. Nature 1998). Based on these findings, we
identified a population of fibroblastic 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) and found that CAR cells are themajor producer of CXCL12 and SCF (Omatsu at al. Immunity2010), and the major cellular components of niches for HSCs andimmune cells (Omatsu at al. Immunity 2010; Shimoto et al. Blood2017).
In addition, we determined the nature of CAR cells, showingthat CAR cells are mesenchymal stem cells, which give rise toadipocytes and osteoblasts (Seike et al. Genes Dev. 2018) and thattranscription factors, Foxc1 and Ebf3 are preferentially expressedin CAR cells and play a critical role in the formation andmaintenance 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.

402018). These studies clarified the nature and functions of CAR
cells in murine bone marrow.
In addition to mouse, we revealed the human counterpart ofCAR cells were the major component of nonhematopoietic cells
in human bone marrow and enabled the evaluation of theiralterations in various hematological disorders by flow cytometricand histological analyses (Aoki et al., Br J Haematol 2021).
The results that Runx2, which is essential for generation of
osteoblasts, is highly expressed in CAR cells prompted us to
examine the role of Runx transcription factors in the development
and/or maintenance of CAR cells. We showed that Runx1, which
is known to be essential for the establishment of definitive
hematopoiesis in hemogenic endothelial cells, is predominantly
expressed in CAR cells. CAR cells and HSC niches are normallyformed and maintained in mice lacking Runx1 or Runx2 in CAR
cells. However, mice lacking both Runx1 and Runx2 in CAR cells(tamoxifen-treated Ebf3-CreERT2;Runx1f/fRunx2f/f mice) displayed
an increase in fibrosis and bone formation with markedly reduced
HSCs and hematopoietic progenitor cells in bone marrow.
Consistent with this, CAR cells from the mutants displayed
markedly increased expression of fibrotic genes, including
Col1a1, Col3a1, and Col6a3. In vitro, Runx1 is induced by Foxc1
and enforced expression of Runx1 decreases fibrotic gene
expression in cultured CAR cells. Thus, CAR cells require Runx1 orRunx2 to prevent their fibrotic conversion and maintain HSCs andhematopoiesis in adults. Clinically, expression of Runx1 andRunx2 was reduced in CAR cells in a mouse model of marrow
fibrosis, which is a significant complication of myeloproliferative
neoplasns (MPN) that affects up to 20% of patients and isEbf3/12VWHREODVWVCXCL12
SCF

Erythroid, Myeloid
progenitors

Runx2/1
Foxc1

CAR cells
Foxc1hiEbf3/1hi
Runx2/1hi
CXCL12hiSCFhi

Lymphoid progenitors

HSCs

Bone marrow$GLSRF\WHV

Figure 2.
The development and functions of CAR cells.
CAR cells are the major cellular component of non-hematopoietic cells in bone marrow characterized by several salient features in both mouse andhuman. The transcription factors, Foxc1, Ebf1/Ebf3, and Runx1/2 and cytokines, CXCL12 and SCF are preferentially expressed in CAR cells and criticalfor formation and maintenance of niches for HSCs and immune cells, within the bone marrow.

Recent Publications
1. Omatsu Y, Aiba S, Maeta T, Higaki K, Aoki K, Watanabe H, et al. Runx1 andRunx2 inhibit fibrotic conversion of cellular niches for hematopoietic
stem cells. Nat Commun. in press (2022).
2. Omatsu Y, Higaki K, Nagasawa T. Cellular Niches for Hematopoietic StemCells and Lympho-Hematopoiesis in Bone Marrow During Homeostasis
and Blood Cancers. Curr Top Microbiol Immunol. 434:33-54 (2021).
3. Nakagawa T, Jörg DJ, Watanabe H, Mizuno S, Han S, Ikeda T, Omatsu Y,
Nishimura K, Fujita M, Takahashi S, Kondoh G, Simons BD, Yoshida S,
Nagasawa T. A multistate stem cell dynamics maintains homeostasis inmouse spermatogenesis. Cell Rep. 37(3):109875 (2021).

4. Omatsu Y, Nagasawa T. Identification of microenvironmental niches forhematopoietic stem cells and lymphoid progenitors-bone marrow
fibroblastic reticular cells with salient features. Int Immunol. 33(12):821826 (2021).
5. Aoki K, Kurashige M, Ichii M, Higaki K, Sugiyama T, Kaito T, Ando W,
Sugano N, Sakai T, Shibayama H; HANDAI Clinical Blood Club, TakaoriKondo A, Morii E, Kanakura Y, Nagasawa T. Identification of CXCL12abundant reticular cells in human adult bone marrow. Br J Haematol.
193(3):659-668 (2021).

41

Immunology

species overgrowth and colorectal tumourigenesis, and butyrateinduced senescence may be one of the mechanisms.

Aging Biology

Healthy individuals (HI)n=384

CRC patients
n=380

Gut microbiome analysis

Eiji Hara, PhD

P. micra(%)
15.0

P. intermedia
(%)

(%)

P. asaccharolytica
(%)

10.0

20.0

5.0

10.0

P. stomatis

S. moorei

G. morbillorum
(%)

(%)

6.0
30.0
10.0

6.0

Assistant Professor

Eiji Hara
Shimpei Kawamoto

0.0

0.0

0.0

0.0

P. anaerobius(%)

P. gingivalis(%)

A. tannerae(%)

D. pneumosintes(%)

2

Support Staff

4

F. nucleatum subsp.
(%)

2.0
1.5

5.0

0.0

7.5
3.0

4.0
5.0

2.5

Research Assistant

6.0
7.5

Masahiro Wakita
1

0.0

0.0
F. nucleatum subsp.
(%)
4.0

7.5

Tomonori Matsumoto

Postdoctoral Fellow

2.0

2.0

10.0

Professor

4.0

4.0

20.0
5.0

5.0
1.0

2.5

2.0

0.0

0.0

2.0

0.5

1.0

0.0

0.0

2.5

0.0

HI
CRC patients

Figure 1.
Twelve bacterial taxa that were abundant in CRC patients and rarely present in healthy individuals were identified.

Cellular senescence is a state of irreversible cell-cycle arrest
induced by a variety of potentially oncogenic stimuli and istherefore thought to serve as an important tumour suppression
mechanism. On the other hand, however, senescent cells also
cause senescence-associated secretory phenotypes (SASP),
which secrete a variety of pro-inflammatory factors. Therefore,
the accumulation of senescent cells, which is often seen withageing and obesity, ultimately leads to harmful side effects, andthere is currently a worldwide effort to develop drugs that
selectively kill senescent cells. However, since senescent cells also
play beneficial roles, for example, in promoting wound healing,
maintaining the blood-tissue barrier, and activating the immune
system, blindly removing senescent cells may be harmful.
Therefore, identifying and preventing the causes of cellularsenescence in vivo, rather than killing senescent cells, may be asafer and more efficient way to address the harmful side effects ofsenescent cells. However, to date, the triggers of cellularsenescence, particularly with ageing, are poorly understood. This
year we discovered that both gut bacteria and respiratory viruses
induce cellular ageing in vivo through the following two studies.
(1) Reports of "post-acute COVID-19 syndrome," in which the
inflammatory response persists even after SARS-CoV-2 hasdisappeared, are increasing but the underlying mechanisms of
post-acute COVID-19 syndrome remain unknown. Here we showthat SARS-CoV-2 infected cells trigger senescence-like cell-cycle
arrest in neighboring uninfected cells in a paracrine manner via
virus-induced cytokine production. In cultured human cells or
bronchial organoids, these SASR-CoV-2 infection-induced
senescent cells express high levels of a series of inflammatory

42

factors known as senescence-associated secretory phenotypes(SASPs), in a sustained manner, even after SARS-CoV-2 is nolonger detectable. We also show that the expression of thesenescence marker CDKN2A and various SASP factor genes isincreased in the pulmonary cells of patients with severe postacute COVID-19 syndrome. Furthermore, we find that mice
exposed to a mouse-adapted strain of SARS-CoV-2 exhibit
prolonged signs of cellular senescence and SASP in the lung at 14days post-infection when the virus was undetectable, which
could be substantially reduced by the administration of senolytic
drugs. The sustained infection-induced paracrine senescencedescribed here may be involved in the long-term inflammation
caused by SARS-CoV-2 infection.
(2) Emerging evidence is revealing that alterations in gut
microbiota are associated with colorectal cancer (CRC). However,very little is currently known about whether and how gut
microbiota alterations are causally associated with CRC
development. Here we show that 12 faecal bacterial taxa areenriched in CRC patients in two independent cohort studies.
Among them, 2 Porphyromonas species are capable of inducing
cellular senescence, an oncogenic stress response, through thesecretion of the bacterial metabolite, butyrate. Notably, theinvasion of these bacteria is observed in the CRC tissues,coinciding with the elevation of butyrate levels and signs ofsenescence-associated inflammatory phenotypes. Moreover,although the administration of these bacteria into ApcΔ14/+ miceaccelerate the onset of colorectal tumours, this is not the casewhen bacterial butyrate-synthesis genes are disrupted. These
results suggest a causal relationship between Porphyromonas

Figure 2.
Induction of cellular senescence and persistence of inflammatory response by SARS-CoV-2

Recent Publications
1. Tsuji S, Minami S, Hashimoto R, et al. SARS-CoV-2 infection triggers
paracrine senescence and leads to a sustained senescence-associatedinflammatory response. Nat. Aging, 2:115-124 (2022).

4. Takahashi A, Loo TM, Okada R, Kamachi F, Watanabe Y, Wakita M,
Watanabe S, Kawamoto S, Miyata K, Barber G N, Ohtani N, Hara E.
Downregulation of cytoplasmic DNases is implicated in cytoplasmic DNA
accumulation and SASP in senescent cells. Nat. Commun. 9:1249 (2018).

2. Okumura S, Konishi Y, Narukawa M, et al. Gut bacteria identified incolorectal cancer patients promote tumourigenesis via butyrate
secretion. Nat Commun. 12:5674 (2021).
3. 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 and autophagy
in senescent cells. Nat. Commun. 11:1935 (2020).

43

Immunology

Oncogene Research

Masato Okada, PhD

Professor

Masato Okada

Associate Professor

Shigeyuki Nada

Assistant Professor

Kentaro Kajiwara

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

(S.A.) Tetsuya Kimura(S.A.) Makoto Matsuda
Research AssistantRole of Src tyrosine kinase in tumor progression
We have investigated the role of Src tyrosine kinase in tumor
progression. Src is the first-identified oncogenic tyrosine kinase,but no significant mutation of the SRC gene occurs in any type ofhuman cancer. Nonetheless, the function of Src is frequently
upregulated in various malignant cancers, and it is appreciatedthat upregulated Src plays a crucial role in tumor progression,
particularly in the acquisition of invasive and metastatic features.
To elucidate the molecular mechanisms underlying upregulationof Src, we investigated the regulatory mechanism of SRC gene
expression and searched for Src-activating factors. We found that
TGF-β treatment directly induces SRC gene expression via the
Smad pathway in coupled with additional transcription factors,
and determined the promoter and enhancer regions locatedupstream of the SRC gene. The upregulation of Src contributes tothe progression of TGF-β–induced epithelial-mesenchymaltransition. 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 morphologicalchanges and cell growth, and attenuates membrane presentationof MET, resulting in inhibition of invasive activity induced by HGF.
These findings suggest that 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 this study toidentify a potential therapeutic target in some types of cancer.

44

1

Role of p18 in the regulation of mTORC1 nutrient
signaling
We previously identified a new Src substrate termed p18/
Lamtor1, which exclusively localizes to lipid rafts of lysosomes.
Subsequent analysis revealed that p18 functions by forming ahetero-heptamer complex (Ragulator), consisting of p18, p14,
MP1, HBXIP, p10, RagA/C, and it is required for activation ofmTORC1 on lysosomes. Conditional KO of p18 in the epidermis
showed that p18-mTORC1 is crucial not only for anabolism of biomaterials, but also for catabolism via autophagy, indicating that
p18 is tightly associated with the regulation of mTORC1 nutrient
signaling in vivo. Recent studies in the 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 theregulation of metabolic homeostasis in various tissues and cells.
To further analyze the regulation of the p18 complex at themolecular level, we previously determined the crystal structure of
Ragulator. This revealed that p18 wraps around the othercomponents of Ragulator and provided significant insights into
the role of p18-mediated regulation of mTORC1 on lysosomes.
Recent analysis using p18 KO cells that lack regulatorycomponents of Rag GTPase, such as GATOR1 and FLCN, showedthat p18-Ragulator complex provides a regulatory platform thatis indispensable for amino acid-dependent regulation of mTORC1(Fig. 2). These findings identified the interacting molecularsurface as a potential therapeutic target in lifestyle diseases, suchas diabetes mellitus and cancer, both of which are linked todysfunction of the mTORC1 pathway.

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

Recent Publications
1. Kimura T, Hayama Y, Okuzaki D, Nada S, Okada M. The Ragulator complex
serves as a substrate-specific mTORC1 scaffold in regulating the nuclear
translocation of transcription factor EB. J Biol Chem. 298(3):101744(2022).
2. Kawase N, Sugihara A, Kajiwara K, Hiroshima M, Akamatsu K, Nada S,
Matsumoto K, Ueda M, Okada M. SRC kinase activator CDCP1 promotes
hepatocyte growth factor-induced cell migration/invasion of a subset ofbreast cancer cells. J Biol Chem. 298(3):101630 (2022).

4. Nakatani T, Tsujimoto K, Park J, Jo T, Kimura T, Hayama Y, Konaka H,
Takayoshi Morita T, Kato Y, Nishide M, Koyama S, Nada S, Okada M,
Takamatsu H, Kumanogoh A. The lysosomal Ragulator complex plays anessential role in leukocyte trafficking by activating myosin II. Nature
Commun. 12(1):3333 (2021).
5. Kajiwara K, Yamano S, Aoki K, Okuzaki D, Matsumoto K, Okada M. CDCP1
promotes compensatory renal growth by integrating Src and Met
signaling. Life Sci Alliance. 4(4):e202000832 (2021).

3. Liu Y, Soh WT, Kishikawa JI, et al. An infectivity-enhancing site on the
SARS-CoV-2 spike protein targeted by antibodies. Cell. 184(13):34523466.e18 (2021).

45

Immunology

Signal Transduction
A

Nobuyuki Takakura, MD/PhD

Professor

Nobuyuki Takakura

Assistant Professor

Fumitaka Muramatsu
Weizhen Jia
Bal Zeynep

B

Keigo Akuta

Angiogenesis, a process of new blood vessels from preexistingvasculature, is involved in the progression of many diseases, suchas cancer, retinopathy, inflammatory diseases, and so on and is
one of drug target to control these diseases. Many types of cells
such as pericytes, fibroblasts, leukocytes, platelets, and organ
specific cells including cancer cells interact with endothelial cells
to generate tissue and disease specific blood vessels. Since
angiogenesis is a complex process, it is difficult to recapitulate it
in vitro. Therefore, in vivo models are required for the study ofvascular change and drug development.
Several in vivo models analyzing angiogenesis have beendeveloped so far, i.e., tumor model, ischemia hind limb model,
corneal transplantation model and so on in mice; however, it isdifficult to translate experimental results from mouse to humanblood vessels. In order to overcome this issue, we tried toestablish a new model visualizing human blood vessels by using(cancer) patient derived xenograft (PDX) in immunodeficient

Postdoctoral Fellow

2

Research Assistant

6

Support Staff

3

mice. The reason why we used tumor tissue is because tumor
blood vessels have highly angiogenic potential. We chose thecranial window model to monitor human blood vessels because
this uses skull bone for fixing the cover glass to enable a stable
rigid fixation allowing long-term observation for up to a year.
Using this method, we were able to observe human bloodvessels in tumor tissues over the long term, for at least 49 days. As
depicted in Figure, human blood vessels continuously grew andconnected with mouse blood vessels. Human specific blocking
antibody against vascular endothelial growth factor receptor 2(VEGFR2) inhibited angiogenesis in a human endothelial cell
specific manner. Therefore, this model provides images of the
kinetics of changes to the vasculature within the tissue of thesame individual mouse, suggesting that we can analyze themolecular mechanism of human angiogenesis precisely andsearch the methods to accelerate or suppress angiogenesis byusing this model.

Figure.
Visualization of human blood vessels formed in PDX mouse model.
(A) Tumors freshly isolated from patient were cut into 1-2 mm fragments and were transplanted into a cranial window in NOD-Scid mice. In several
time points, mice were intravenously injected with anti-human CD31 monoclonal antibody to visualize the human blood vessels (green) specificallyin vivo. Blood vessels were observed by confocal microscopy under isoflurane anesthesia. (B) Mice as described above were intravenously injectedwith human specific anti-CD31 antibody (green) and mouse specific anti-CD31 antibody (red) and connection between human and mouse bloodvessels was observed in PDX mouse model.

Recent Publications
1. 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).

4. 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 and maintains vascular
integrity. Dev Cell 48:151-166 (2019).

2. Naito H, Wakabayashi T, Ishida M, Gil CH, Iba T, Rahmawati FN, Shimizu S,
Yoder MC, Takakura N. Isolation of tissue-resident vascular endothelial
stem cells from mouse liver. Nat Protoc. 15:1066-1081 (2020).

5. 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).

3. 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. Regnase1-mediated post-transcriptional regulation is essential for hematopoietic
stem and progenitor cell homeostasis. Nat Commun. 10:1072 (2019).

46

47

Cutaneous Immunology

GD3 May Suppress the Functional Activities of Benign
Skin T Cells in Cutaneous T-Cell Lymphoma

Manabu Fujimoto, MD/PhD

In cutaneous T-cell lymphoma (CTCL), which arises from skintropic memory T cells, malignant T cells and benign T cells are
confined in the same skin lesions. Disialoganglioside with threeglycosyl groups (GD3) is increasingly expressed on the surface ofsolid malignant tumor cells and takes part in tumor progression
and suppression of tumor immunity. However, the role of GD3 in
CTCL is not well-understood. Therefore, we analyzed the role of
GD3 in cutaneous lymphoma. We found that GD3 from themalignant T cells was involved in suppressing the Th17 activity ofthe benign T cells independent of the regulation of resident
memory T cell differentiation in CTCL.

Distribution of hypomelanotic macules in tuberous
sclerosis complex
Professor

Manabu Fujimoto

Associate Professor

Yumi Matsuoka-Nakamura

Research Assistant

4

Visiting Scientist

1

Support Staff

1

We treat a hereditary disorder called TSC with tumors,neurological symptoms and vitiligo due to the activation of

Immunologyhydrocarbons.

mTORC1. However, the pathophysiology of the neurologicalsymptoms and vitiligo in TSC are still unknown. In order toinvestigate the pathogenesis of vitiligo in TSC, which shows aunique shape and distribution, we reported that the activation ofmTORC1 during development might cause the characteristic
distribution of vitiligo via abnormal melanocyte migration.

Collagen homeostasis paradoxically maintains invitiligo lesion (On-going)
In a daily clinic experience, the lesional skin of vitiligo would be
more resilient than uninvolved lesion even in sun-exposed areas.
Therefore, we hypothesized that a collagen homeostasis mightparadoxically maintain in vitiligo lesion irrespective of substantial
excessive oxidative stress condition without melanin protection.
Single-cell RNA sequencing analysis showed that the expressionsof collagen-related and anti-oxidative enzyme genes were upregulated in the fibroblasts of vitiligo lesional skin compared tothose of uninvolved skin. Our results suggest that the dermal
fibroblasts in vitiligo may play a protective role against the
locally-exposed oxidative stress.

Subtypes ofantiInflammatory myopathies
Ro52/IgG/HLA-DR
specific autoantibodies
IgG

ILD

Cell surface-expressed Ro52/IgG/HLA-DR complex istargeted by autoantibodies in patients with inflammatory
myopathies
Cellular misfolded proteins are transported to the cell surface
by HLA class II molecules and recognized by autoantibodies. We
found that the specific autoantibodies against the Ro52/IgG/
HLA-DR complex were positive in 90% and 93% of patients withdermatomyositis who were positive for anti-MDA5 and anti-ARS
antibodies, respectively. Furthermore, changes in the serum
antibody titers correlated with laboratory values reflecting
symptoms. These results suggest that antibodies to Ro52/IgG/
HLA-DR may be involved in the pathogenesis of a subgroup of
inflammatory myopathies.

Clinical and laboratory parameters predicting cancer
in dermatomyositis patients with anti-TIF1γ antibodies
Dermatomyositis is an autoimmune disorder strongly
associated with cancer development. We reported that a closepathophysiological relationship among myositis, cancer and skininvolvements in dermatomyositis patients with anti-TIF1γ
antibodies. Some inflammatory cytokines, particularly TNF and
TNF receptor families may support cancer prediction indermatomyositis patients with anti-TIF1γ antibodies.

Regulatory B cells in immune-mediated cutaneous
disorders (On-going)
Regulatory B cells (Bregs) are regarded as a group of B cells
which converge accelerated immune reactions by secreting IL10. Dysfunction of Bregs has been reported in autoimmune

48

disorders including systemic lupus erythematosus both inpatients and murine models. However, since B cells are rarely
found in the skin lesions, the role of B cells in cutaneous disorders
has not been established. We found that Breg population isskewed in immune-mediated cutaneous disorders such as drug
eruption and alopecia areata and B cells are presumed to exert
causative role in the augmented T1 response in these disorders.

HLA-DR

Myopathy
Ro52
Ro52/IgG/HLA-DR
complex

Other
symptoms?

Keratinocyte IL-36 receptor/MyD88 signaling mediates
Malassezia-induced skin inflammation
Among skin commensal fungi, Malassezia species exist on
nearly all human skin surfaces. The pathophysiology ofMalassezia-associated skin diseases remains poorly understood
due in part to the lack of appropriate animal models. We newly
developed a Malassezia skin infection model and found that
Malassezia-induced IL-17- dependent skin inflammation andcontrol of fungal infection are mediated via keratinocyte IL-36
receptor/MyD88 signaling.

Loricrin and NRF2 coordinate epidermal cornification
The skin epidermis undergoes a specialized mode of the celldeath program cornification, producing the stratum corneum(SC) compared to the “protein alloy.” The keratin-associatedprotein loricrin (LOR) promotes the maturation of the SC bygenerating extensive disulfide cross-linkages and appears
indispensable for cornification. Following previous researchsuggesting LOR’s antioxidative property, we have found that LOR
confers protection against carcinogenic electrophiles produced
by epidermal resident Langerhans cells capturing polyaromatic

Figure.
Ro52, an intracellular Fc receptor, formed Ro52/IgG/HLA-DR complex and was transported to the cell surface. Sequential changes in serum antiRo52/IgG/HLA-DR specific autoantibody titers were correlated with changes in the disease status in patients with dermatomyositis.

Recent Publications
1. Arase N, Tsuji H, Takamatsu H, Jin H, Konaka H, Hamaguchi Y, Tonomura K,
Kotobuki Y, Ueda-Hayakawa I, Matsuoka S, Hirano T, Yorifuji H, Murota H,
Ohmura K, Nakashima R, Sato T, Kumanogoh A, Katayama I, Arase H,
Fujimoto M. Cell surface-expressed Ro52/IgG/HLA-DR complex istargeted by autoantibodies in patients with inflammatory myopathies. J
Autoimmun. 126:102774 (2022).
2. Miyachi H, Wakabayashi S, Sugihira T, Aoyama R, Saijo S, KoguchiYoshioka H, Fujimoto M, Núñez G, Matsue H, Nakamura Y. Keratinocyte
IL-36 Receptor/MyD88 Signaling Mediates Malassezia-Induced IL-17Dependent Skin Inflammation. J Infect Dis. 223(10):1753-1765 (2021).

4. Takahashi A, Hattori S, Sakai E, Yang L, Katayama I, Fujimoto M, WatayaKaneda M. Distribution of hypomelanotic macules in tuberous sclerosis
complex: A retrospective cohort study. J Am Acad Dermatol. S01909622(21)02282-9 (2021).
5. Okiyama N, Ichimura Y, Shobo M, Tanaka R, Kubota N, Saito A, Ishitsuka Y,
Watanabe R, Fujisawa Y, Nakamura Y, Murakami A, Kayama H, Takeda K,
Fujimoto M. Immune response to dermatomyositis-specific autoantigen,transcriptional intermediary factor 1γ can result in experimental myositis.
Ann Rheum Dis. 80(9):1201-1208 (2021).

3. 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).

49

Immunology

Innate Immune Systems
osteoclasts by GM-CSF and IL-13 production. These results
suggest that ILC2s regulate osteoclast activation and contributeto bone homeostasis in both steady-state and IL-33-induced
inflammation.

Kazuyo Moro, PhD

Professor

We have investigated the remarkable properties of innate
immunity through studying group 2 innate lymphoid cells (ILC2),
an innate lymphocyte lineage that we identified in 2010. ILC2
contribute to immune responses by secreting effector cytokines
such as IL-5 and IL-13 and regulate the functions of both immune
and non-immune cells. ILC2 play a pathogenic role in allergic
diseases in barrier tissues including lungs, intestines, and skin.
Aiming at advancing therapeutic strategies, we dissect how ILC2
form communication networks with other cells and how these
networks malfunction in disease.

1. Posttranscriptional regulation of ILC2 homeostatic
function via TTP
ILC2s are unique in their ability to produce low levels of type 2
cytokines at steady-state. However, it was unknown how thisconstitutive cytokine production is regulated. In this study, wedemonstrate post-transcriptional regulation through tristetraprolin(TTP) is crucial for the regulation of constitutive type 2 cytokine
production by ILC2s at an appropriate level for homeostasis. TTP
binds to AU-rich elements (AREs) in the 3' UTRs of the mRNAs andpromotes their degradation, often acting as a component of anegative feedback loop for cytokine production by destabilizing
mRNA. However, we found that ILC2s highly express TTP in thesteady state, while losing this expression following IL-33
stimulation, suggesting that continuous mRNA degradation by
TTP prevents cytokine production in the steady state, while lossof TTP by IL-33 stimulation allows ILC2s to produce large amountsof type 2 cytokines. As a result, retroviral overexpression of TTP in
ILC2s markedly suppresses IL-5 and IL-13 production under IL-33

50

Kazuyo Moro

Postdoctoral Fellow

1

Research Assistant

2

Visiting Scientist

3

Support Staff

2

stimulation through their mRNA degradation. Luciferase assay
further demonstrates that TTP directly regulates Il5 expression
through AREs in the 3' UTR of Il5 mRNA, suggesting that IL-5 is a
novel target of TTP. Moreover, Il13 expression was found to beindirectly regulated by TTP, however, we did not focus on
elucidating the associated detailed mechanism in this study.
Finally, we confirm that ILC2s from TTP-deficient mice
overproduce IL-5 and IL-13 even in steady state, leading to thedysregulation of eosinophil homeostasis. Collectively, TTP
maintains the homeostatic function of ILC2s by suppressing IL-5
and IL-13 production via mRNA degradation.

Figure.
IL-33 induces a large amount of IL-5 from activated-ILC2s by transcriptional regulation. On the other hand, in steady-state ILC2s, excessive cytokine
production is suppressed by TTP-mediated posttranscriptional regulation.

2. ILC2 in bone marrow regulate osteoclastogenesis in
a reciprocal manner via RANKLILC2s are tissue-resident cells that play different roles indifferent organs by sensing environmental factors in theirsurroundings. Initially, it was thought that ILC2s in bone marrow(BM) are progenitors for systemic ILC2s, which migrate to otherorgans and acquire effector functions. However, accumulating
evidence that ILC2s differentiate in peripheral tissues suggeststhat BM ILC2s may play a specific role in the BM as a uniqueeffector per se. We demonstrated that BM ILC2s highly express
RANKL, a robust cytokine for osteoclast differentiation andactivation, and that RANKL expression on ILC2s is up-regulated
by IL-2 and IL-7. BM ILC2s co-cultured with BMMs in the presenceof IL-7 induce the differentiation of TRAP-positive osteoclasts in a
RANKL-dependent manner. In contrast, BM ILC2s stimulated with
IL-33 down-regulate RANKL expression and convert BMM
differentiation into M2 macrophage-like cells rather than

Recent Publications
1. Momiuchi Y, Motomura Y, Suga E, Mizuno H, Kikuta J, Morimoto A,
Mochizuki M, Otaki N, Ishii M & Moro K. Group 2 innate lymphoid cells inbone marrow regulate osteoclastogenesis in a reciprocal manner via
RANKL, GM-CSF and IL-13. Int Immunol. 33:573-585 (2021).
2. Matsuyama T, Machida K, Motomura Y, Takagi K, Doutake Y, TanoueHamu A, Kondo K, Mizuno K, Moro K & Inoue H. Long-acting muscarinic
antagonist regulates group 2 innate lymphoid cell-dependent airway
eosinophilic inflammation. Allergy. 76:2785-2796 (2021).

4. Kiniwa T & Moro K. Localization and site-specific cell-cell interactions ofgroup 2 innate lymphoid cells. Int Immunol. 33:251-259 (2021).
5. Hikichi Y, Motomura Y, Takeuchi O & Moro K. Posttranscriptional
regulation of ILC2 homeostatic function via tristetraprolin. J Exp Med.
218 (2021).

3. Kobayashi T, Motomura Y & Moro K. The discovery of group 2 innate
lymphoid cells has changed the concept of type 2 immune diseases. Int
Immunol. 33:705-709 (2021).

51

Immunology

Human Single Cell Immunology

James Badger Wing, PhD

Associate Professor

The Human Single Cell Immunology Lab focuses on single cellbiology and its applications to human immunology. Recent
advances in single cell technologies, such as mass cytometry(also known as CyTOF) have revealed high levels of heterogeneity
among immune cells. This makes it uniquely well placed to obtaina wide-ranging picture of the human immune system. Primarily
we use mass cytometry to explore two areas, autoimmunity andinfectious diseases.
The devastating outbreak of SARS-CoV2 and the associated
COVID-19 disease has had a severe impact on both the global
community and Japan. While recent advances in vaccination
have given some hope of respite the proven ability of the virus tomutate and potentially generate vaccine resistant strains
indicates that there can be no relaxation of our urgent efforts tobetter understand the effects of this disease (Grubaugh et al, Cell,
2021). A critical part of this effort is to understand the changes tothe immune system of infected patients since both viral clearance
and most symptoms of the disease are mediated by the immune
system (Mathew et al. Science, 2020).

James Badger Wing

Assistant Professor

Jonas N. Søndergaard

Postdoctoral Fellow

2

Research Assistant1

Support Staff

1

In order to advance our ability to understand these complex
systems, we have designed and optimized mass cytometry
protocols for the detailed assessment of immune cell phenotypes
in humans. Using our expertise in mass cytometry to respond tothe challenge of the COVID-19 pandemic we have assessedchanges to the immunophenotype of COVID-19 patients incollaboration with the Okada, Kumanogoh, Sakaguchi andOkuzaki groups. Importantly we are simultaneously able to assessmany other types of cells such as cytotoxic CD8 T-cells andantibody producing B-cells all of which may be critical to theoutcome of COVID-19. Figure 1. As part of this we found that
Tregs appear to be central hubs of related to the severity sex andage of patients. In particular reference to the role of T-follicularregulatory cells (Tfr) we found that there is a sex biased imbalanceof Tfr and antibody producing plasma cells in patients with
Severe COVID-19. Figure 2. Since Tfr are known to regulate thefrequency of plasma cells this may give some indication of thereason for the sex imbalanced production of autoantibodies thathas been seen in COVID-19 patients.

Figure 1.
Multi-level assessment of immunophenotype of COVID-19 patient peripheral blood mononuclear cells.

Figure 2.
Sex correlations with cellular phenotypes in COVID-19.
Top 5 cellular correlations with male and female sex in severe COVID-19 patients. Edge width is proportional to correlation strength. Edges connecting toother cellular subsets are positive correlations between indicated subsets. Layout by ForceAtlas2 using edge weights as input. Violin plots of sex specificdifferences of indicated cellular populations in severe COVID-19 patients. Data from JN. Søndergaard et al. Preprint on MedRXiv 2022.

Recent Publications
1. Søndergaard JN, et al. Regulatory T-cells are central hubs for age-, sexand severity-associated cellular networks during COVID-19. Preprint on
MedRXiv (2022). Currently under review.

4. Wing JB, et al. Scalable, multimodal profiling of chromatin accessibility,
gene expression and protein levels in single cells. Nature Biotechnology
39(10):1246-1258 (2021).

2. Wing JB & Sakaguchi S. Using mass cytometry to address Tfh and Tfr
heterogeneity. Methods in Molecular Biology. “T follicular helper cells”.
47-57 (2022).

5. Tulyeu J, Søndergaard JN, Sakaguchi S and Wing JB. Isolation andCharacterization of Both Human and Mouse Tfh/Tfr Cells. Current
Protocols 1(11):e283 (2021).

3. Tekguc M, Wing JB, Osaki M, Long J, and Sakaguchi S. Treg-expressed
CTLA-4 depletes CD80/CD86 by trogocytosis, releasing free PD-L1 on
antigen-presenting cells. PNAS. 118 (30) e2023739118 (2021).

52

53

Immunology

Human Immunology (Single Cell Genomics)

Daisuke Okuzaki, PhD

Associate Professor

The Laboratory of Human Immunology was established in
November 2019 for the purpose of accelerating the applicationof single cell sequencing technology to improve the connection
between fundamental science research and clinical applications.
One of the major research projects we are currently focused on isa genomics project in collaboration with the Trauma and Acute
Critical Care Center in the Osaka University Graduate School of
Medicine. Many of the patients sent to the Trauma and Acute
Critical Care Center have acute inflammation and suffer fromsystemic inflammatory response syndrome (SIRS). The severity of
SIRS usually has to be clinically classified within several hours
upon patient arrival. Classification is based on clinical criteriasuch 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, progressin the research of its pathological classification and associatedmolecular mechanism has not advanced in Japan as it has inEurope and America.
We hereby kick started a collaboration research project
involving the measurement and collection of biomolecular datasuch as DNA, RNA and protein from patients biomaterials, while
integrated with cutting edge single cell sequencing technology.
In the past year prospective observational study, we collected
ELISA, RNA sequencing, Olink and single cell sequencing datafrom patients three days and seven days post arrival. The resulting
multiomics data, combined with clinical pathology andbioinformatic multi-dimensional analysis, were utilized to classify
SIRS patients based on molecular properties into pathological54

On the other hand, the importance of evaluating vaccination
efficacy is gradually increasing given the current nationwide
vaccination efforts. In order to elucidate the pathologicalmechanism of the novel coronavirus as well as develop potential
treatments, single cell RNA sequencing was performed for
peripheral blood samples that were taken from healthy
individuals prior and post vaccination. In connection with the
Joint Research Coronavirus Task Force and serving as a memberof 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 of Medicine, and analyzing
the peripheral blood samples taken from the patients sent toOsaka University Hospital during all four waves of COVID-19 inJapan. Currently, we are analyzing multiple aspects of themolecular properties of immune cells from dozens of patients.
We have co-authored two papers in which we applied single
cell RNA sequencing. They include a study of PTH-induced boneformation conducted by Morimoto et al., published in Nat
Commun. 2021 and a successful observation of hematopoietic
recovery supported by innate lymphoid cells under stressconditions performed by Sudo T, et al., published on J Exp Med.
2021.

Daisuke Okuzaki

Postdoctoral Fellow

3

Visiting Scientist

2

Support Staff

1classifications such as sepsis, ARDS, PCAS, trauma, heat stroke orburn. We are currently working on improving the classifications,which can be used as a guideline for proper treatment selection
upon arrival.
Our proposal to search for appropriate 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 andAcute Critical Care Center at Osaka University and the Osaka
Prefectural Nakakawachi Emergency and Critical Care Center.
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. Currently there are no conventionallyestablished biomarkers or drug treatments for COVID 19. In order
to search for the appropriate biomarkers to reflect thepathological classification for COVID-19, cohorts were divided
into the three groups of research, development and verification.
Applying the same strategy as the aforementioned SIRS studies,
we collected experimental data using RNA sequencing, mass
spectrometry, Olink proteomics, and single cell RNA sequencing.
Through the integration with panomics data, a set of “pathologicalmolecular scores” and “pathological correlated clusters” were
defined. The novel “pathological classification” is determined by
the defined clusters and scores, and enables selective therapeuticintervention depending on the COVID-19 symptoms.

Figure.
Overview of the research process.

Recent Publications
1. Uenaka M, Yamashita E, Kikuta J, Morimoto A, Ao T, Mizuno H, Furuya M,
et al. Osteoblast-derived vesicles induce a switch from bone-formation
to bone-resorption in vivo. Nat Commun. 13(1):1066 (2022).

4. Al Kadi M, Ishii E, Truong DT, Motooka D, Matsuda S, Iida T, Kodama T,
Okuzaki D. Direct RNA Sequencing Unfolds the Complex Transcriptomeof Vibrio parahaemolyticus. mSystems. 6(6):e0099621 (2021).

2. Kidani Y, Nogami W, Yasumizu Y, Kawashima A, Tanaka A, Sonoda Y, Tona
Y, et al. CCR8-targeted specific depletion of clonally expanded Treg cells
in tumor tissues evokes potent tumor immunity with long-lasting
memory. Proc Natl Acad Sci USA. 119(7):e2114282119 (2022).

5. Morimoto A, Kikuta J, Nishikawa K, Sudo T, Uenaka M, Furuya M,
Hasegawa T, et al. SLPI is a critical mediator that controls PTH-inducedbone formation. Nat Commun. 12(1):2136 (2021).

3. Ebihara T, Matsumoto H, Matsubara T, Togami Y, Nakao S, Matsuura H,
Kojima T, et al. Cytokine Elevation in Severe COVID-19 From Longitudinal
Proteomics Analysis: Comparison With Sepsis. Front Immunol. 12:798338(2022).

55

Immunology

Immune Homeostasis

Yasutaka Okabe, PhD

Associate Professor

Yasutaka Okabe

Postdoctoral Fellow

1

Research Assistant2

Support Staff

1

7LVVXHPLFURHQYLURQPHQW

7LVVXHDGDSWDWLRQ

Tissue Macrophage Biology
Tissue-resident macrophages are present in virtually every
mammalian tissue and they are essential components for the
maintenance of tissue homeostasis. They perform tissue-specificfunctions that are critical for normal tissue physiology. The
abnormalities of tissue-resident macrophage functions are often
linked to various pathologies including osteopetrosis, type 2
diabetes, immune deficiency, and neurodevelopmental diseases.
The focus of the laboratory is to understand molecularmechanisms that generate a diversity of tissue-resident
macrophage phenotypes as well as the roles of macrophages intissue homeostasis. The heterogeneity of tissue-resident
macrophage phenotypes is considered to be functional
specialization as a consequence of their adaptation to local tissue
environments (Figure). Tissue-resident macrophages, in response
to tissue environmental cues, activate the corresponding
functional polarization programs which accompany the inductionof specific gene-expression programs. We have characterizedtissue-specific transcriptional programs of resident macrophages
and identified hundreds of genes that are selectively expressed intissue macrophages (Okabe and Medzhitov, Cell, 2014). Amongthese genes, we found transcription factor GATA6 is uniquely
expressed in macrophages that reside in the peritoneal cavity.
We reported that GATA6 acts as a master transcriptional regulator
for functional specialization of peritoneal macrophages, as thedeletion of GATA6 gene in peritoneal macrophages resulted in
the defect of peritoneal specific gene expression program.

Additionally, we and other groups found GATA6-mediating
program control macrophage positioning within the peritoneal
cavity, local proliferation, and peritoneal-specific immune
responses. We identified retinoic acid, a lipophilic molecule
derived from vitamin A, plays an essential role in the induction of
GATA6 gene in peritoneal macrophages. Together, these results
provide insight into the mechanism of generation of tissue
specialization of resident macrophages.

QFWLRQDOVSHFLDOL]DWLRQ)XQFWLRQDOVSHFLDOL]DWLRQ
Figure.
Local tissue environments induce functional specialization of tissue-resident macrophages.

Regulation of Body Cavity Immunity
Body cavities (peritoneal, pleural, and pericardial cavities) are
fluid-filled spaces lined with a layer of mesothelial cells, andaccommodate visceral organs and other structures in the animal
body. The body cavity represents a unique place for immune cell
distribution; some of the immune cells such as B-1 lymphocytes
are predominantly present in the body cavity but they are rarely
found in the other lymphoid organs. These lymphocytes
constantly migrate from the body cavity into peripheral tissues
and contribute to whole-body immune regulation such as natural
antibodies and gut IgA production. Therefore, it is increasingly
appreciated that the body cavity plays unreplaceable functions in
the immune system. However, the mechanisms regulating thedevelopment, localization, and activation of immune cells in the
body cavity remain largely unknown. We study the regulating
mechanisms of the body cavity immune system and its roles in
the infection and injury.

Recent Publications
1. Okabe Y. Immune Niche Within the Peritoneal Cavity. Current Topics ofMicrobiology and Immunology. 434:123-134 (2021).

4. Okabe Y & Medzhitov R. Tissue-specific signals control reversible programof localization and functional polarization of macrophages. Cell 157:832844 (2014).

2. Okabe Y. Molecular Control of the Identity of Tissue-Resident
Macrophages. Int Immunol. 30:485-491 (2018).
3. Okabe Y & Medzhitov R. Tissue Biology Perspective on Macrophages. Nat
Immunol. 17:9-17 (2016).

56

57

Immunology

Cellular Immunotherapy
cells to tumor sites and immune-suppressive tumor
microenvironment hamper development of effective CAR-T celltherapy, the most critical problem is lack of appropriate cancerspecific target antigens. We start trying to find cell surface antigen

structures for various types of cancers in collaboration withseveral departments treating cancers in Osaka University
Hospital.

Naoki Hosen, MD/PhD

Professor

Naoki Hosen

Associate Professor

Hisashi Kato

Assistant Professor

Kana Hasegawa
Kentaro Fukushima$%

Jiro Fujita&RQWURO

Michiko Ichii
Yasutaka Ueda
TomoakiUeda
Support Staff

We are focusing on cellular immunotherapy, especiallychimeric antigen receptor (CAR)-T cell therapy for cancers. CAR-T
cells specifically recognize cancer cells using the cancer-specificmAb-derived antigen-recognition domain, and are activated.
Activated CAR-T cells kill tumor cells and also proliferate
extensively. CD19 CAR-T cells showed surprisingly higheffectiveness against acute lymphocytic leukemia and malignant
lymphoma. We discovered that the active conformer of an
integrin could serve as a specific therapeutic target for multiple
myeloma (MM), which is an incurable hematological cancer
characterized by the accumulation of neoplastic plasma cells inthe bone marrow (BM). Clinical trial of MMG49 CAR T-cell for MM
is now on-going.

Identification of a novel target antigen for multiple
myelomaCancer-specific cell surface antigens are ideal therapeutictargets for monoclonal antibody (mAb)-based therapy. However,most transcripts or proteins highly specific for cancer cells havealready been identified after extensive efforts using transcriptome
or proteome analyses. We recently found that MM can be
specifically targeted by a new monoclonal antibody thatrecognizes a ubiquitously expressed protein CD98 heavy chain(hc)/SLC3A2. We screened more than 10,000 mAb clones raised
against MM cells, and identified R8H283 as a mAb that bound to
MM cells but not to normal hematopoietic or non-hematopoietic
cells. R8H283 specifically recognized CD98hc. R8H283 did notreact with CD98hc monomer, but bound to CD98hc forming
heterodimers with the light chains, which are amino-acid58

3transporters. MM cells abundantly expressed CD98 heterodimers
to intake amino acids for constitutive production of
immunoglobulin. Although CD98 heterodimers were also
expressed in normal leukocytes, R8H283 did not react with them.
Normal leukocytes expressed CD98hc glycoforms different fromthose expressed in MM cells, which may be a cause for lack of
R8H283 reactivity in normal leukocytes. R8H283 exerted
significant anti-MM effects without damaging normal
hematopoietic cells. These findings not only suggest that R8H283
is a new source for mAb-based therapies such as CAR-T celltherapy against MM, but also that a cancer-specific conformational
epitope in a ubiquitous protein, which cannot be identified bytranscriptome or proteome analyses, can be found by extensive
screening with primary human tumor samples.

00

1RUPDOOHXNRF\WHV
5+

5+
&'KF&'KF5+

Figure.
R8H283, a novel antibody against CD98hc, a widely expressed protein, can specifically targets myeloma cells.
(A) The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of
R8H283 reactivity to normal leukocytes. Indicated glycan structure is just a conceptual scheme. Glycans actually attached to CD98hc hashuge varieties. (B) R8H283 exerts anti-MM effectsn in a mouse xenograft model.

Development of CAR-T cell therapy targeting antigen
structures formed as results of post-translational
events in various cancers
The above findings suggest that cancer immunotherapeutictargets may yet be identified in many cell-surface proteins that
undergo post-translational changes, even if the expression of theproteins themselves is not cancer-specific. Thus, we applied thesame strategy to various types of cancers. For hematologicalcancers such as acute myeloid leukemia (AML), the only hurdle
for developing CAR-T cell therapy is lack of an appropriate cancerspecific cell surface antigen. We have already established huge
numbers of mAbs reacting with AML cells, and are now selecting
ones recognizing antigen structures highly specific for AML cells.
In the case of solid tumors, while inefficient trafficking of CAR-T

Recent Publications
1. Hasegawa K, Ikeda S, Yaga M, et al. Selective targeting of multiple
myeloma cells with a monoclonal antibody recognizing the ubiquitous
protein CD98 heavy chain. Sci Transl Med. 14:eaax7706 (2022).

3. Shingai Y, Yokota T, Okuzaki D, et al. Autonomous TGFbeta signaling
induces phenotypic variation in human acute myeloid leukemia. StemCells 39:723-736 (2021).

2. Ozawa T, Fujii K, Sudo T, et al. Special AT-Rich Sequence-Binding Protein 1
Supports Survival and Maturation of Naive B Cells Stimulated by B Cell
Receptors. J Immunol 208:1937-1946 (2022).

59Immunology

Microbiology and Immunology

Nobuhiko Kamada, PhD

Professor

Our team has been studying the role of the gut microbiota inthe pathogenesis of the gastrointestinal disease, such as
inflammatory bowel disease (IBD). We developed a humanized
gnotobiotic mouse model that enables one to examine thefunctional impact of gut dysbiosis associated with IBD. Using thismodel, we identify potential pathobionts enriched in IBD patientsand are responsible for disease induction and progression. We
have identified potential pathobionts that elicit inflammasome
activation in host immune cells (e.g., macrophages) and theintestinal epithelium. Some of the identified pathobionts appear
to have adherent–invasive E. coli (AIEC)–like phenotypes. In
addition to intestinal inflammation, we observed that persistent
gut colonization by AIEC bacteria leads to the development ofintestinal fibrosis. Moreover, we have identified unique metabolic
pathways used by pathobionts to maximize their fitness in the
inflamed gut. We discovered that AIEC bacteria reprogram their
metabolism in the inflamed gut and upregulate genes related to
L-serine catabolism. AIEC, but not commensal E. coli strains, use
L-serine catabolism; a unique metabolic function that confers afitness advantage on AIEC over commensal competitors.
Also, we focus on the microbial and immunological cross-talkbetween mucosal tissues in the pathogenesis of gastrointestinal
diseases. In this regard, we study the oral–gut axis in IBD
pathogenesis. Recent studies have demonstrated that theenrichment of oral bacteria in the intestinal mucosa of IBD
patients may contribute to proinflammatory processes in the gut.
We aim to identify the mechanisms by which oral disease
exacerbates intestinal inflammation. We have discovered that

60

Nobuhiko Kamada

periodontitis results in the expansion of pathobionts in the oralcavity. Amassed oral pathobionts are ingested and colonize thecolonic mucosa. Ectopic colonization of oral pathobionts elicits
the activation of the inflammasome in lamina propria
macrophages, which may compound their colitogenic capacities.
In parallel, oral bacteria–reactive Th17-skewed T cells arise denovo in the oral cavity during periodontitis. Oral bacteria–reactive
T cells are imprinted with gut tropism and migrate to the inflamedgut. Once in the gut, the oral bacteria–reactive T cells areactivated by ectopically colonized oral pathobionts. Thus,
together, oral pathobionts and pathogenic T cells, which are
generated as a result of oral inflammation, exacerbate gut
inflammation.
Note: The PI is cross-appointed at the IFReC and the Universityof Michigan (USA) from September 2021. These research projects
were conducted at the University of Michigan. We are currently
establishing new projects on host-microbe interactions ingastrointestinal disease at the IFReC.

Figure.
(Left) Ectopic gut colonization by oral pathobionts elicits inflammasome activation in the colonic mucosa. (Right) Gut resident pathobionts, suchas adherent-invasive Escherichia coli, reprogram their metabolism in the inflamed gut. L-serine catabolism up-regulated during inflammation
confer pathobionts a competitive fitness advantage over competing commensals. Gut pathobionts promote intestinal inflammation via theactivation of the inflammasome.

Recent Publications
1. Imai J, Ichikawa H, Kitamoto S, Golob JL, Kaneko M, Nagata J, Takahashi
M, Gillilland MG, Tanaka R, Nagao-Kitamoto H, Hayashi A, Sugihara K,
Bishu S, Tsuda S, Ito H, Kojima S, Karakida K, Matsushima M, Suzuki T,
Hozumi K, Watanabe N, Giannobile WV, Shirai T, Suzuki H, Kamada N. A
potential pathogenic association between periodontal disease andCrohn's disease. JCI Insight. 6(23):e148543 (2021).

4. Nagao-Kitamoto H, Leslie JL, Kitamoto S, Jin C, Thomsson KA, Gillilland
MG 3rd, Kuffa P, Goto Y, Jenq RR, Ishii C, Hirayama A, Seekatz AM, Martens
EC, Eaton KA, Kao JY, Fukuda S, Higgins PDR, Karlsson NG, Young VB,
Kamada N. Interleukin-22-mediated host glycosylation prevents
Clostridioides difficile infection by modulating the metabolic activity of
the gut microbiota. Nat Med. 26(4):608-617 (2020).

2. Hayashi A, Nagao-Kitamoto H, Kitamoto S, Kim CH, Kamada N. The
butyrate-producing bacterium Clostridium butyricum suppresses
Clostridioides difficile infection via neutrophil- and antimicrobial
cytokine-dependent but GPR43/109a-independent mechanisms. J
Immunol. 206(7):1576-1585 (2021).

5. Kitamoto S, Alteri CJ, Rodrigues M, Nagao-Kitamoto H, Sugihara K,
Himpsl SD, Bazzi M, Miyoshi M, Nishioka T, Hayashi A, Morhardt TL, Kuffa
P, Grasberger H, El-Zaatari M, Bishu S, Ishii C, Hirayama A, Eaton KA,
Dogan B, Simpson KW, Inohara N, Mobley HLT, Kao JY, Fukuda S, Barnich
N, Kamada N. Dietary L-serine confers a competitive fitness advantage toEnterobacteriaceae in the inflamed gut. Nat Microbiol. 5(1):116-125(2020).

3. Kitamoto S, Nagao-Kitamoto H, Jiao Y, Gillilland III MG, Hayashi A, Imai J,
Sugihara K, Miyoshi M, Brazil JC, Kuffa P, Hill BD, Rizvi SM, Wen F, Bishu S,
Inohara N, Eaton KA, Nusrat A, Lei YL, Giannobile WV, and Kamada N. Theintermucosal connection between the mouth and gut in commensal
pathobiont-driven colitis. Cell. 182(2):447-462 (2020).

61

Imaging

Single Molecule Imaging

Toshio Yanagida, PhD
Ben Seymour, MD/PhD

One of the major puzzles in medicine is why people are sosusceptible to developing chronic pain after injury or
inflammation. Indeed around 20-30% of the population develop
chronic pain at some point in their life, making it the leading
cause of disability and one of the greatest unmet needs acrossthe entire medical spectrum. Our lab focuses on understanding
the core mechanisms of pain in the brain, and testing the broad
hypothesis that injury and inflammation set-off hyper-protective
behaviours that evolved to protect us during recuperation, butthat are extremely prone to cause continuation of pain after the
insult has resolved.
We have received a generous award from the UK Medical
Research Council / Versus Arthritis, to test this idea in more detail.
Recently, we have been exploring brain-mediated hyperprotective behaviours in patients with rheumatoid arthritis, andfound both behavioural and brain evidence that characterizes
precisely how this is implemented, and how it is likely to generatea suite of symptoms that includes pain, fatigue, and moodchanges. To probe this idea more robustly – especially to test
whether these changes plays a causative role in chronification ofpain after injury or inflammation, we have developed a new
digital experimental platform for studying behaviour in patients.
This platform is an open digital laboratory that can be used byresearchers and applied to multiple cross-sectional andlongitudinal clinical cohorts. This exists on a free software
platform that also includes sophisticated computational analysis
tools, as well as a data sharing database that allows normativeand comparative data analysis.

62

Professor

Toshio Yanagida
Ben Seymour

Associate Professor

Mitsuhiro Iwaki

Support Staff

2

An inherent prediction of our results is that brain mechanismsthat cause pain and fatigue can be targeted by technology-based
approaches. We have received a generous award from the UK
Engineering and Physical Sciences Research Council to establish
an infrastructure for developing this, exploiting UK-Japan
collaboration. In particular, we are focusing on non-invasive
technologies that include brain stimulation, virtual reality, digital
therapy and neurofeedback tools, which are integrated andapplied in a mechanism-targeted strategy. Our ultimate goal is todesign and deliver low-cost, accessible technologies that canhave a transformative impact on chronic pain across different
societies and populations.

of the NS and adsorbed to the glass bottom dish, then monitored
the single molecule traction force of human skin fibroblast via
integrin receptors (Figure 2). We could successfully observe theextension of the fluorescently labeled NS underneath focal
adhesions using total internal reflection microscopy. The NS
length was dynamically changed at second time scale and theforce varied at the range of 0.1 to 25 pN. This is the first report toobserve dynamical change of the traction force at the single

molecule level. The observation reports us fruitful information
regarding the molecular mechanism of cellular mechanosensing.
Recently, it is known that T cell receptor (TCR) senses specificmagnitude and direction of force via membrane receptor when T
cell interact with antigen presenting cell (APC). Our new
methodology reported here should be powerful to clarify themechanical-induced immune response.

Figure.
Single molecule traction force microscopy measured by DNA-based force sensor, Nanospring to clarify the mechanicalinduced immune response.

In our other work, we are also studying fundamental pathways
for adaptive learning in the brain. Diseases such as inflammatory
arthritis causes a complex fluctuating pattern of pain. We havebeen studying how the brain can learn this temporal pattern, andhave found that the brain generates an internal model of thepain-causing state using Bayesian statistical learning. This isformed in the front-parietal cortex (Mancini et al, 2021).
We are also developing single molecule imaging techniques
for mechanobiology of immune cell. To visualize the mechanicalforce in cells, we previously developed DNA-based force sensor,
Nanospring (NS). This is a protein-sized spring molecule and canbe fully stretched by pN force. The force-extension relation wasquantified using acoustic force spectroscopy and we confirmed
the stiffness and the robustness against solution condition. We
attached RGDfK peptide (ligand of integrin receptor) to the end

Recent Publications
1. Marcucci L, Fukunaga H, Yanagida T, Iwaki M. The synergic role ofactomyosin architecture and biased detachment in muscle energetics:
insights in cross bridge mechanism beyond the lever-arm swing. Int J
Mol Sci. 22:7037 (2021).

3. Mancini F, Zhang S, Seymour B. Learning the statistics of pain:
computational and neural mechanisms. bioRxiv 2021.

2. Tanaka SC, Yamashita A, Yahata N, Itahashi T, Lisi G, Yamada T, et al. A
multi-site, multi-disorder resting-state magnetic resonance image
database. Scientific data 8 (1):1-15 (2021).

63

Immunology and Cell Biology
Masaru Ishii, MD/PhD

Professor

Masaru Ishii

Associate Professor

Junichi Kikuta

Assistant Professor

Yutaka Uchida
Akito Morimoto
Kentaro Fujii (SA)

1. Intravital bone imaging revealing osteoclast andosteoblast dynamics in vivo
We developed a novel imaging system to visualize insidebones using intravital multiphoton microscopy. Using thismethodology, we succeeded in visualizing the in vivo behaviors
of osteoclast and osteoblast as well as the activity of boneresorbing by osteoclasts. In addition to development ofmethodology, we are also updating various materials for bone
imaging such as mouse models to visualize osteoclast andosteoblast and probes to visualize the bone resorption activity ofosteoclasts. Our major contributions to the field of bone biology
research are as follows:· Identifying S1P (Sphingosine-1-phosphate) as a key factor incontrolling the migratory behavior of osteoclast precursors(Nature 2009, J. Exp. Med. 2010).
· Showing the substantial contribution of S1P-mediatedmigration 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 non-resorbing mature
osteoclasts (J. Clin. Invest. 2013).
· Showing the importance of epigenetic processes of osteoclast
in the regulation of cellular metabolism and differentiation(Nat. Med. 2015).
· Developing a new fluorescence probe for detecting bone
surface pH to visualize bone resorption by mature osteoclasts

64

Postdoctoral Fellow

3

Research Assistant

5

Visiting Scientist

1

Support Staff

2(Nat. Chem. Biol. 2016).
· Applying intravital bone imaging to drug efficacy evaluationand 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 for bonehomeostasis (Nat. Commun. 2018).
· Identifying SLPI (secretory leukocyte protease inhibitor) as a
key mediator for PTH-induced bone formation (Nat. Commun.
2021).
Bone metabolism is regulated by the cooperative activity
between bone-forming osteoblasts and bone-resorbing
osteoclasts. However, the mechanisms mediating the switch
between the osteoblastic and osteoclastic phases have not been
fully elucidated. We identified a specific subset of mature
osteoblast-derived extracellular vesicles that inhibit boneformation and enhance osteoclastogenesis. Intravital imaging
reveals that mature osteoblasts secrete and capture extracellular
vesicles, referred to as small osteoblast vesicles (SOVs). Co-culture
experiments demonstrate that SOVs suppress osteoblast
differentiation and enhance the expression of receptor activatorof NF-κB ligand, thereby inducing osteoclast differentiation. We
also elucidate that the SOV-enriched microRNA miR-143 inhibits
Runt-related transcription factor 2, a master regulator ofosteoblastogenesis, by targeting the mRNA expression of its
dimerization partner, core-binding factor β. In summary, we
identified SOVs as a mode of cell-to-cell communication,
controlling the dynamic transition from bone-forming to boneresorbing phases in vivo (Nat. Commun. 2022).

Intravital imaging provides information of dynamic parameters,
is useful for identifying a novel subset of macrophages. Inside live
bones, osteoclasts and osteoblasts are constantly generated.
Under well-controlled bone destruction and formation processes,
bone constantly undergoes destruction and formation tomaintain homeostasis. In our previous study, we developed aunique technology to collect and analyze cells from arthritic
joints, successfully identifying a new type of bone-destroying
osteoclast that contributes to RA (rheumatoid arthritis), called
Arthritis-associated osteoclastogenic Macrophages, or “AtoMs”(Nat. Immunol. 2019). We found that the arthritis-inducing
abnormal osteoclasts have distinct properties and origins fromnormal osteoclasts involved in bone metabolism. By targetingthe differentiation and functions of arthritis-inducing abnormal
osteoclasts, we will be able to establish innovative therapeuticstrategies and next-generation of anti-RA drugs that do not affectnormal osteoclasts and normal bone homeostasis.

3. Application of multiphoton microscopy for humancancer diagnosis
Intravital imaging with multiphoton microscopy is an
undoubtedly powerful tool for dissecting live cellular dynamics in

Imaging

2. Identification of disease associated macrophages

intact tissues and organs and thus useful for studying immune
system dynamics in vivo. However, the application is currently
limited in animal models and may not be for analyzing humansamples. By collaborating with companies (supported by AMED)
we are developing a new microscopy system for applying humantissues and organs in vivo. Currently, we have succeeded invisualizing non-labelled human normal and cancer tissues, which
can be used for differential diagnosis (Sci. Rep., 2017). In thefollow-up study (Cancer Res.2021), we applied near-infrared
excitation and nonlinear optics to visualize unstained humanepithelial tissues of the cervix uteri by constructing images withthird-harmonic generation (THG) and second-harmonic
generation (SHG). THG images enabled evaluation of nuclear
morphology in a quantitative manner with six parameters afterimage analysis using deep learning. It was also possible toquantitatively assess intraepithelial fibrotic changes based on
SHG images and another deep learning analysis. Our method
enables real-time noninvasive diagnosis of cervical lesions, thus
constituting a potential tool to dramatically change early
detection. This study proposes a novel method for diagnosing
cancer, which enables visualization of histologic features of living
tissues without the need for any biopsy or staining dye.

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 cells by visualizing in vivo behaviors using advanced imaging
techniques.

Recent Publications
1. Uenaka M, et al. Osteoblast-derived vesicles induce a switch from boneformation to bone-resorption in vivo. Nat Commun. 13(1):1066 (2022).
2. Morimoto A, et al. SLPI is a critical mediator that controls PTH-inducedbone formation. Na. Commun. 12:2136 (2021).
3. Sudo T, et al. Group 2 innate lymphoid cells support hematopoietic
recovery under stress conditions. J Exp Med. 218:e20200817 (2021).

4. Matsui T, et al. Nonlinear optics with near-infrared excitation enable realtime quantitative diagnosis of human cervical cancers. Cancer Res.
80:3745-3754 (2020).
5. Hasegawa T, et al. Identification of a novel arthritis-associated osteoclast
precursor macrophage regulated by FoxM1. Nat Immunol. 20:1631-43(2019).

65

Imaging

Nuclear Medicine
Phase I Clinical Trial of Na211At
Jun Hatazawa, MD/PhD

Na211At

Phase I Clinical Trial of Na211At for intractable Thyroid Cancers
Since the first use of 131I for thyroid diseases (1951) and 60Co forexternal irradiation source (1953) in Japan, medical radioisotopes
were mainly imported from abroad. Due to senile nuclear reactors
in Canada, Europe, and South Africa, reduced workers during
COVID-19 pandemic, and limited transport capacity by approved
airlines, a supply of medical RI became limited and unstable. Afterthe policy change by Japanese government from import todomestic production (July, 2021), a rebuild of supply chain of
medical RI is promoted under the Japan Atomic Energy
Commission and regulatory authorities of the government. It
consisted of target production for 99Mo and 225Ac, neutron/
proton/deuteron/electron irradiation, purification and quality
control of target RI, labeling and distribution ofradiopharmaceuticals, and waste disposal management. The
action plan will be finalized by the end of May 2022.
In this situation, Phase I clinical trial of Astatine-211 (211At) NaAt
started in December 2021 at Osaka University Hospital for
intractable thyroid cancers under the support of AMED (Principal
Investigator: Dr. Tadashi Watabe). This is the first clinical trial in
the world. The first patient study was completed without any

Professor

Jun Hatazawa

Associate Professor

Hiroki Kato

Assistant Professor

Tadashi Watabe

Postdoctoral Fellow

2

Research Assistant2

Support Staff

1

adverse event. Another project to use of 211At is to treat brain
tumors by means of 211At labelled gold nanoparticle (Kato H, et al.,2021). 211At is an alpha particle emitting radioisotope in the
halogen family with 20 times higher biological effect than
gamma-ray. Chemical and biological nature of Astatine-211 issimilar to Iodine-131, a beta-emitting radioisotope used for thetreatment of well-differentiated thyroid cancers. Astatine-211
production and purification procedures were developed in
Research Center for Nuclear Physics, Osaka University (Ikeda H, etal.). Astatine-211 labeled cancer cell specific compounds are now
under development in Osaka University.
・Watabe T, Kaneda-Nakashima K, Liu Y, et al. Enhancement of211
At Uptake via the Sodium Iodide Symporter by the Additionof Ascorbic Acid in Targeted α-Therapy of Thyroid Cancer. J
Nucl Med. 2019 Sep;60(9):1301-1307
・Hiroki Kato, Xuhao Huang, Yuichiro Kadonaga, et al.
Intratumoral administration of astatine-211-labeled gold
nanoparticle for alpha therapy, J Nanobiotechnology. 2021 Jul
28;19(1):223.
・Ikeda H, Hayashi Y, Takahashi N, et al. Appl Radiat Isot. 2018
Sep;139:251-255.

Figure.
Na211At solution is intravenously administered to a patient with metastatic thyroid cancer. Na211At is specifically transported into metastatic thyroid
cancer cells similar to Na131I. Since 211At has short pass length of alpha-particle within several cells and higher energy, it has higher cell killing effectassociated with less normal cell damage.

Recent Publications
1. Hiroki Kato, Xuhao Huang, Yuichiro Kadonaga, Daisuke Katayama,Kazuhiro Ooe, Atsushi Shimoyama, Kazuya Kabayama, Atsushi
Toyoshima, Atsushi Shinohara, Jun Hatazawa, Koichi Fukase. Intratumoral
administration of astatine-211-labeled gold nanoparticle for alphatherapy, J Nanobiotechnology. 19(1):223 (2021).

3. Hatazawa J. The Clinical Value of Breast Specific Gamma Imaging andPositron Imaging: An Update. Seminars in Nuclear Medicine. https://doi.
org/10.1053/j.semnuclmed.2022.02.005 (Review article)

2. Shirakami Y, Watabe T, Obata H, Kaneda K, Ooe K, Liu Y, Teramoto T,
Toyoshima A, Shinohara A, Shimosegawa E, Hatazawa J, Fukase K.
Synthesis of [211At]4-astato-L-phenylalanine by dihydroxyboryl-astatine
substitution reaction in aqueous solution. Sci Rep. 11(1):12982 (2021).

66

67

Imaging

Chemical Imaging Techniquestime. Time-lapse imaging of the transfected cells using confocal
microscopy revealed that the green fluorescence intensity of thecell nuclei decreased steadily over time (Fig. 2C). The rapid
protein labeling kinetics, high fluorescent intensities and modular
nature of this new probe system means it should find widespreaduse for real-time fluorescence visualization of the expression/
degradation of a wide range of short-lived PYP-tag proteins, thus
enabling it to be used as a diagnostic tool to inform numerous
biological studies and drug discovery programs.

Kazuya Kikuchi, PhD

Protein degradation plays an essential role in maintaining
cellular homeostasis, with protein misfolding and uncontrolled
protein aggregation responsible for a range of neurodegenerative
diseases, including Huntington's disease, Alzheimer's disease andamyotrophic lateral sclerosis (ALS). Damaged or misfolded
proteins in cells are normally removed by the ubiquitinproteasome and/or lysosomal proteolytic pathways, whichhydrolyse them into small peptides and amino acids. The ability
to use a probe to fluorescently detect the production/degradationof short-lived proteins (SLPs) in real time would be particularly
useful, because many of the key roles they play in cell regulatory
processes are not fully understood.
We here developed a new trifunctional ‘contact quenching’“OFF–ON–OFF” fluorescence probe (F3-DNB) for the rapid
labeling of PYP-tag proteins (Fig. 1). This probe contains a PYPtag binding 7- hydroxycoumarin ligand connected to afluorescein fluorophore through a PEG linker. The central
coumarin ligand unit is attached to a pendant dinitrobenzene(DNB) quenching unit through a labile thiophenol ester bondthat can undergo a trans-thioesterification reaction with the thiolgroup of the Cys69 residue of a PYP-tag protein, which cleaves
the quenching unit to produce a fluorescent PYP-tag-boundprobe. A DNB quencher moiety was included in the probe design
to avoid steric interactions between the fluorophore and thecoumarin ligand that were known to result in slow PYP-tag
protein labeling kinetics. A structurally related F3-DNB2 probe
was also prepared containing a more activated thiophenol
leaving group that we reasoned would label PYP-tag proteins
more effectively. Finally, a third F5-DNB2 probe containing a

68

Professor

Kazuya Kikuchi

Associate Professor

Yuichiro Hori

Assistant Professor

Masafumi Minoshima

Postdoctoral Fellow

2

Research Assistant

1

Visiting Scientist

1

Support Staff

1

longer PEG5 linker was synthesized, because previous studies
had shown that close interaction between the probe's
fluorophore and ligand units could produce steric congestionthat decreased the rate of labeling of PYP-tag.
In vitro treatment of PYP-tag with F5-DNB2, the most reactive
probe, resulted in rapid formation of fluorescent PYP-tag–probe
conjugates that gave maximal fluorescence intensities after 60
min. These fluorescent conjugates were then treated with trypsin
to induce proteolytic digestion, with the fluorescence intensities
of both PYP-tag–probe conjugates decreasing gradually overtime, reaching stable fluorescence minima after 130 min (Fig. 2A).
SDS-PAGE analyses of the protease cleavage products revealed
no gel bands present for intact probe-labeled PYP proteins, thus
confirming that both had undergone proteolytic digestion (Fig
2B). These results demonstrate that proteolysis of the fluorescent
PYP-tag–probe conjugates produces the desired ‘ON–OFF’ switch
in fluorescence in probe cleavage products.
Next, real-time imaging of the proteolytic degradation of SLPs
in cellular systems using an OFF–ON–OFF fluorescence switch
was performed. Mouse ornithine decarboxylase (MODC) waschosen as a potential SLP to explore degradation studies because
its C-terminal domain is rich in Pro, Glu, Ser and Thr (PEST
sequence) residues that are known to induce rapid proteasomal
degradation. Efficient proteasomal degradation of the expressedfusion construct between PYP-tag and MODC422-461 was confirmedwith western blot analyses of cellular extracts from the transfected
HEK293T cells. F5-DNB2 probe was then used to visualize theexpression (fluorescence ‘ON’) and proteolytic degradation(fluorescence ‘OFF’) of a PYP-tag-MODC422-461 in cell nuclei in real

Figure 1.
Structures and mechanism of action of the modular PYP-tag ligands (F3-DNB, F3-DNB2 and F5-DNB2) used as “OFF–ON–OFF” probes for the
fluorescent detection of protein degradation.

Figure 2.
(A) Protein degradation detected from the “OFF–ON–OFF” fluorescence response of F5-DNB2. PYPNQN incubated with F5-DNB2 for 120 min and thenreacted with trypsin in 20 mM HEPES buffer (pH 7.4), 150 mM NaCl, and 1.0% DMSO at 37 °C. Fluorescence intensities recorded using excitation andemission wavelengths of 500 and 521 nm. (B) SDS-PAGE analyses of protein degradation products. CBB = Coomassie Brilliant Blue; FL = Fluorescence.
(C) Time-lapse fluorescence imaging studies of the degradation of a short-lived PYP-tag protein in cells. HEK293T cells expressing HA-PYPNQN-NLSMODC422-461 in presence of dicetylated F5-DNB2 (1.0 μM). Excitation at 473 nm. Scale bar = 20 μm. PC = Phase Contrast; FL = Fluorescence.

Recent Publications
1. Reja SI, Hori Y, Kamikawa T, Yamasaki K, Nishiura M, Bull SD, & Kikuchi K.
An "OFF-ON-OFF" fluorescence protein-labeling probe for real-timevisualization of the degradation of short-lived proteins in cellular
systems. Chem Sci., 13:1419-1427 (2022).

4. Kowada T, Arai K, Yoshimura A, Matsui T, Kikuchi K, & Mizukami S. Opticalmanipulation of subcellular protein translocation using aphotoactivatable covalent labeling system. Angew Chem Int. Ed.
60:11378-11383 (2021).

2. Tsukazaki H, Kikuta J, Ao T, Morimoto A, Fukuda C, Tsuda E, Minoshima M,
Kikuchi K, Kaito T, & Ishii M. Anti-Siglec-15 antibody suppresses bone
resorption by inhibiting osteoclast multinucleation without attenuatingbone formation. Bone 152:116095 (2021).

5. Hori Y, Nishiura M, Tao T, Baba R, Bull SD, & Kikuchi K. Fluorogenic probes
for detecting deacylase and demethylase activity towards posttranslationally-modified lysine residues. Chem Sci. 12:2498-2503 (2021).

3. Konishi Y, Okunishi A, Sugihara F, Nakamura T, Akazawa K, Minoshima M,& Kikuchi K. Development of off-on switching 19F MRI probes forcathepsin K activity detection. Bull Chem Soc. Jpn. 94:1690-1694 (2021).

69

Kazuhiro Suzuki, MD/PhD

Professor

Our laboratory has been studying the interactions between
the nervous and immune systems with a special focus on theroles of adrenergic nerves, which constitute the efferent arc of
the sympathetic nervous system, in the control of adaptive
immune responses. Our study revealed a mechanism by which
adrenergic nerves control lymphocyte trafficking through lymph
nodes. Inputs from adrenergic nerves to the β2-adrenergic
receptor expressed on lymphocytes enhance the responsivenessof a specific set of chemokine receptors and inhibit lymphocyte
exit from lymph nodes (Nakai et al., J. Exp. Med. 2014). Thismechanism was found to generate diurnal variations inlymphocyte numbers in lymph nodes and consequently themagnitude of adaptive immune responses in phase with thecircadian oscillation of adrenergic nerve activity (Suzuki et al, J.
Exp. Med. 2016). In search of factors that mediate the crosstalk ofsignaling between the two different types of G protein-coupled
receptors (GPCRs), the β2-adrenergic receptor and chemokine
receptors, we identified a protein complex consisting of copper
metabolism MURR1 domain-containing (COMMD) 3 andCOMMD8 (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 the interaction of
COMMD8 with COMMD3. We found that these proteins
constitutively formed a complex in the cytosol, but weretranslocated to the plasma membrane after stimulation of CXCR4.
The COMMD3/8 complex interacted with not only CXCR4 but also

70

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-specific antibodies (Fig.

Imaging

Immune Response Dynamics

chemotaxis (Fig. B). It has been suggested that the specificity of
GRK recruitment to GPCRs is determined by the relative
expression levels of individual GRKs, which vary among cell type,and distinct receptor conformations induced by ligand binding.
Our study identifies a GRK-recruiting adaptor, the COMMD3/8
complex, as an additional determinant of GRK specificity for
GPCRs (Nakai et al., J. Exp. Med. 2019).

D), which was accompanied with reduced formation of germinal
centers (Fig. E). Therefore, the COMMD3/8 complex plays
essential roles in the control of B cell migration and induction ofhumoral immune responses (Nakai et al., J. Exp. Med. 2019). To
test the involvement of the COMMD3/8 complex in autoimmune
diseases, we established 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 progression with
suppressed production of autoantibodies. These findings suggestthat the COMMD3/8 complex is a potential therapeutic target for
autoimmune diseases.

Kazuhiro Suzuki

Assistant Professor

Akiko Nakai

Research Assistant2

Support Staff2other G protein-coupled chemoattractant receptors, including
CXCR5, CCR7, and the oxysterol receptor EBI2, after activation of
the receptors. Interestingly, COMMD3 and COMMD8 weredegraded by the proteasome in the absence of the other, anddeficiency of either protein produced the same functional
consequences, indicating that both COMMD3 and COMMD8 arerequired for the stability and functions of their complex.
Deficiency of COMMD3 or COMMD8 in B cells reduced theirchemotactic responses mediated by the receptors to which the
COMMD3/8 complex was recruited. These findings reveal that
the COMMD3/8 complex is a positive regulator of chemoattractant
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 are phosphorylated
by GPCR kinases (GRKs) and subsequently recruit β-arrestins thatserve 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 outcomes of
β-arrestin engagement. Thus, specific targeting of GRKs toactivated GPCRs is crucial for signal transduction. Our analysis
on the mechanism of action of the COMMD3/8 complex
demonstrated that this protein complex functions as an adaptor
that selectively recruits GRK6 to chemoattractant receptors,
which promotes MAPK activation and consequently lymphocyte

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) Role of the COMMD3/8 complex in GRK6 recruitment to GPCRs.
(C) Defective intrafollicular migration of COMMD8-deficient (Commd8Δ) B cells at an early time point (3 h) after immunization.
(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
1. Nakai A, Leach S and Suzuki K. Control of immune cell trafficking throughinter-organ communication. Int Immunol. 33:327-335 (2021).
2. Nakai A, Fujimoto J, Miyata H, Stumm R, Narazaki M, Schulz S, Baba Y,
Kumanogoh A and Suzuki K. The COMMD3/8 complex is a determinant of
GRK6 specificity for chemoattractant receptors. J Exp Med. 216:16301647 (2019).

4. Suzuki K, Hayano Y, Nakai A, Furuta F and Noda M. Adrenergic control of
the adaptive immune response by diurnal lymphocyte recirculation
through lymph nodes. J Exp Med. 213:2567-2574 (2016).
5. Nakai A, Hayano Y, Furuta F, Noda M and Suzuki K. Control of lymphocyte
egress from lymph nodes through β2-adrenergic receptors. J Exp Med.
211:2583-2598 (2014).

3. Suzuki K and Nakai A. Immune modulation by neuronal electric shockwaves. J Allergy Clin Immunol. 141:2022-2023 (2018).

71

Imaging

Biophotonics

Nicholas Isaac Smith, PhD

Associate Professor
Assistant Professor

Nicholas Isaac Smith
Nicolas Pavillon
Alison Hobro

The Biophotonics laboratory develops tools for label-free
analysis of single cells. Single-cell analysis is a popular target for alarge number of researchers, usually pursued by labeling surface
markers, by introducing fluorescent dyes into the cell, or byinvasive, yet comprehensive, techniques such as single cell RNA
sequencing. In contrast, our tools are based on label-free opticalmethods, which aim to produce some of the same discriminatory
capability as the more invasive methods. Additionally, label-freemethods are based on endogenous contrasts of the cell, and canalso find novel features that can be used to discriminate between
cell phenotypes or cell states.
In the last year we completed work on several projects that
advance the sensitivity and interpretability of label-free single
cell analysis. We showed that label-free evaluation of immune
cells can give us insight at the single-cell level, into a cell
phenotype or state. When performing this type of experiment
there are some aspects that arise which are not necessarily
intuitive. For example, different cell types that are known to befunctionally different can turn out to be challenging todiscriminate with our methods, while the detection of a change
in state (such as activation) can turn out to be easier to detect.
The sensitivity of our detection depends on how much difference
exists at the molecular level between two targets. The use of
optical methods based on spectroscopy means that we do nothave single molecule sensitivity, but rather see the ensemble ofmolecules in the cell that are excited by the laser beam.

72

Postdoctoral Fellow

1

Support Staff

1

We made further improvements towards higher throughput
measurement. This was achieved through additional automationof our systems, using machine vision, and automatic targeting ofindividual cells for Raman analysis. Although fundamentally notcomparable to true high-throughput techniques such as FACS,
we nevertheless managed to increase the speed of measurement
to acquire several thousand cells per hour, without need of
constant human intervention. This is beneficial not only forconvenience but can also help to improve repeatability.
We completed several works with collaborators including
advancements in microscopy techniques with the Fujita group in
Applied Physics, in both label-free and probe-based modalities.
Dr Lelliot also completed a study with collaborators in the
Kumanogoh group, elucidating the role of adhesion in neutrophil
extracellular traps (NETs). Neutrophils are known to produce
extracellular debris that resembles a physical net, and this process
has been implicated in a wide variety of disease conditions,
including COVID-19. It is still relatively early in terms of existingknowledge into how NETs form, whether there are different types
of NETs, and the pathways involved with them. Here, the work
clarified that for NET stimulation specifically related to antineutrophil cytoplasmic antibody associated vasculitis, neutrophil
adhesion is required. This has implications for the disease
pathology and suggests that clinical studies targeting neutrophil
adhesion factors may be a promising direction for treatment.

Figure.
Classification of cell phenotypes, cell states, and immune responses. Panel (a) shows a schematic of our higher throughput optical spectroscopymode giving one signature spectra per cell. This data leads to the ability to classify each cell (b). The sensitivity and specificity can be very high,considering that the whole measurement is label-free. Here, we show the accuracy of a model that determines whether a cell had been exposed
to LPS or LPS combined with cycloheximide which inhibits the normal protein synthesis response to LPS (d). Even when looking at data from an
independent day’s experiments, the accuracy remains high. Depending on the method chosen to create the classification model, it is possible toextract information that tells us which molecular bands are most indicative of the inhibition process (e, see Pavillon et al 2021 for details). Panel fshows data from the NET project, here using labels, where the degree of NET formation was quantified by image analysis, so that accuratequantitative characterization of the NET response to different stimuli could be achieved (g).

Recent Publications
1. Lelliott PM, Nishide M, Pavillon N, Okita Y, Shibahara T, Mizuno Y,
Yoshimura H, Obata S, Kumanogoh A, and Smith NI. Cellular Adhesion Is
a Controlling Factor in Neutrophil Extracellular Trap Formation Inducedby Anti-Neutrophil Cytoplasmic Antibodies. ImmunoHorizons 6 (2):170183 (2022).
2. Pavillon N, and Smith NI. Deriving accurate molecular indicators ofprotein synthesis through Raman-based sparse classification. Analyst
146:3633-3641 (2021).

4. Pavillon N and Smith NI. Immune cell type, cell activation, and single cell
heterogeneity revealed by label-free optical methods. Sci Rep. 9:17054(2019).
5. Pavillon N, Hobro AJ, Akira S, and Smith NI. Noninvasive detection ofmacrophage activation with single-cell resolution through machinelearning. Proc Natl Acad Sci USA. 115(12):E2676-E2685 (2018).

3. 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).

73

Bioinformatics

antibody-antigen interactions using combination of structuralmodeling, molecular docking and Deep Learning (Davila, A. and
Xu, Z. et al. Bioinform. Adv 2022). More recently, we have shownthat such predictions can be significantly improved by use of theAlphaFold software (Xu, Z. et al in prep) (Figure 3).

Systems Immunology

Daron M. Standley, PhD
Green light
590 nmUV
365 nmProfessor

Daron Standley

Associate Professor

Kazutaka Kato
Li Songling
Soyoung Park

A. Reversible optical control of T cell activation
Light is an attractive trigger for controlling the reversible
assembly and disassembly of biomolecules in a spatiotemporalmanner. Our interest is to decipher how the dynamics of T cell
receptor (TCR) clustering affects downstream T cell signaling andgene expression. For this purpose, we have devised an opticallycontrollable T cell activation system using TCR-binding aptamers
or anti-TCR-antibodies and a light-responsive arylazopyrazole(AAP) module (Park, S. et al. 2021). This technology will provide a
structural understanding of T cell activation, which can be applied
to single-chain antibody (scFV) receptors in CAR T cells. In thisway, we ultimately aim to open the door for a new area of
Optoimmunology research.

B. COVID-19 Pandemic
The 2021 year was dominated by efforts to characterize SARSCoV2 as well as host responses to the virus. In the beginning, we
had very little information on B or T cell receptors that targeted
SARS-CoV2, so we focused our attention on the main antigen: thespike protein. We first examined the evolution of the spike
protein in order to identify residues that appeared to be underselection pressure during evolution (Saputri, D. et al Front
Microbiol, 2020). We subsequently observed that a number ofsuch “evolutionarily important” residues were among the first to
mutate in emerging viruses of concern and proposed thatsurveying viruses in non-human hosts my provide an opportunity
to predict their evolution in humans (Katoh, K. & Standley, D. M.
Commun Biol, 2021). As the sequences of antibodies that target
the spike protein began to trickle in to the public domain, we

74

Postdoctoral Fellow

2

Research Assistant3

started to work closely with other groups at IFReC to identify theirfunctions. In particular, Prof. Arase noticed a subset of antibodies
that targeted the spike N-terminal domain (NTD) that enhanced,
rather than neutralized SARS-CoV2 infection (Liu, Y. et al Cell
2021). We hypothesized that the function of such enhancing
antibodies was mediated by spike-spike crosslinking, leading todecoupling between the NTD and receptor-binding domain(RBD). We have carried out extensive simulations of antibodycrosslinked spike proteins in the Fugaku supercomputer in orderto validate our hypothesis. Encouragingly, spike bound NTDs
exhibited significantly larger distances from neighboring RBS
than did unbound NTDs, in support of the proposed mechanism(Figure 2). The project is still ongoing, but, if further experimental
support for this mechanism is found, it would represent a novel
form of antibody-dependent enhancement (Li, S. and Van Eerden,
F.J. et al in prep). Finally, in collaboration with Assist. Prof. Atsushi
Hoshino at the Kyoto Prefectural University of Medicine, Assoc.
Prof. Toru Okamoto at Osaka Univ. RIMD and Prof. Junichi Takagi
at Osaka Univ. IPR, we helped to characterize mutations in the
Omicron variant in terms of immune escape and ACE2 binding(Ikemura, N.T. et al Sci. Transl. Med 2022).

C. Antibody structural modeling
We have continued to work on antibody structural modeling at
various levels. In 2021, we participated in an international
collaborative study of CAR T cells that strongly suggested theimportance of CAR dimerization in the treatment of B cell acutelymphoblastic leukemia (Singh, N. et al Nat Med, 2021). We havealso been developing new tool, AbAdapt, for elucidating

Figure 1.
Conceptual scheme for reversible optical switch for T cell activation using TCR-binding aptamers.

Figure 2.
RBD-NTD distance in the three different spike subunits of the right spike. Spike A(red) is bound by the antibody and has clearly a larger separation between NTD
and RBD separation and then the two other spikes.

Figure 3.
Antibody-specific epitope prediction. The three bars indicate the performance(Median ROC AUC) for predicting epitope residues on a holdout set of 100
antigens. “Initial” means no antibody information; “Docking” means informationfrom docking antibodies built with Repertoire Builder was used; “AF + Docking”
means information from docking antibodies built with AlphaFold was used.

Recent Publications
1. Ikemura NT, Inaba T, Arimori T, Motooka D, Katoh K, et al. An engineered
ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers
protection against infection in vivo. Sci Transl Med. in press (2022).

4. Singh N, Frey NV, Engels B, et al. Antigen-independent activation
enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells. Nat Med.
27:842-850 (2021).

2. Davila A, Xu Z, Li S, Rozewicki J, et al. AbAdapt: an adaptive approach topredicting antibody–antigen complex structures from sequence.
Bioinformatics Advances 2 (2022).

5. Liu Y, Soh WT, Kishikawa JI, Hirose M, et al. An infectivity-enhancing siteon the SARS-CoV-2 spike protein targeted by antibodies. Cell 184:34523466 e3418 (2021).

3. Chong YK, Tartey S, Yoshikawa Y, Imami K, Li S, et al. Cyclin J-CDK
complexes limit innate immune responses by reducing proinflammatorychanges in macrophage metabolism. Sci Signal. 15, eabm5011 (2022).

75

Bioinformatics

the biobank resources, but found no significant genotypephenotype correlations (Sakaue S, et al. Cell Genomics 2022).

Statistical Immunology

Yukinori Okada, MD/PhD

Goal of our laboratory
Genetic backgrounds of individuals have substantial impacts
on risk of a wide range of immune-related diseases. Statisticalimmunology is a research field that evaluates causality of human
genetic variations on immune-related diseases, using statisticaland bioinformatics approaches. The goal of our laboratory is todevelop such methods and apply to the latest large-scale disease
genome and multi-layer omics data.

Metagenome-wide association study of gut microbiome
and virome revealed disease-specific features
Microbiomes play substantial roles in biology of a variety ofhuman immune-related diseases. We conducted metagenomewide association studies (MWAS) of gut microbiome of systemic
lupus erythematosus (SLE) utilizing whole-genome shotgun
sequencing. We identified increases of Streptococcus intermedius
and Streptococcus anginosus in SLE. Microbiome-metabolome
association analysis identified positive dosage correlation ofacylcarnitine with Streptococcus intermedius (Tomofuji Y et al.
Ann Rheum Dis 2021). Further, we constructed an in silico pipeline
to quantify gut virome from the existing shotgun sequencing
data. We conducted virome-wide association study (VWAS)
based on the shotgun sequencing of 476 Japanese which
included patients with rheumatoid arthritis (RA), SLE, multiple
sclerosis and healthy control subjects. Our VWAS revealed that
crAss-like phages, one of the main components of a healthy gut
virome, significantly decreased in RA and SLE (Figure 1, Tomofuji
Y et al. Ann Rheum Dis 2022).

76

Professor

Yukinori Okada

Associate Professor

Qingbo S Wang

Assistant Professor

Kenichi Yamamoto

Research Assistant5

Support Staff

2

Figure 1.
An in silico pipeline to quantify gut virome from metagenome shotgun sequencing

A cross-population atlas of human genotype-phenotype
associations.
Construction of human genotype-phenotype catalog isessentially important to elucidate genetic backgrounds andunderlying biology of diseases. By conducting cross-populationand cross-biobank genome-wide association study (GWAS) of>200 human phenotypes for >600,000 individuals of Asian andEuropean ancestries, we identified ~5,000 new associated loci. By
applying deconvolution analysis of the GWAS summary statistics,
we extracted the variants and genes sharing the related disease
etiology, such as RA and SLE (Sakaue S et al. Nat Genet 2021). As
for our current challenges in the COVID-19 pandemic, weorganized the all Japan consortium and massively collected
omics resources from the patients with COVID-19. We conducted
multi-omics analysis including GWAS, part of which contributed
to international collaborations (COVID-19 Host Genetics Initiative.
Nature 2021).

Deep sequencing of killer cell immunoglobulin-likereceptor genes
The killer cell immunoglobulin-like receptor (KIR) gene region
are one of the most complex genomic region in human genome.
We conducted deep sequencing of KIR genes in >1,100
individuals of the Japanese population. We implemented a novelin silico pipeline to genotype KIR gene variants from thesequencing reads (Figure 2). By combining with the wholegenome sequencing data, we constructed an imputationreference panel of the KIR gene region. We conducted aphenome-wide association study of the KIR gene variants using

Figure 2.
KIRAP (Killer Immunoglobulin-like Receptor variant Analytical Platform)

Recent Publications
1. Sakaue S, et al. Decoding the diversity of killer immunoglobulin-likereceptors by deep sequencing and a high-resolution imputation method.
Decoding the diversity of killer immunoglobulin-like receptors by deep
sequencing and a high-resolution imputation method. Cell Genomics
2:100101 (2022).

4. Tomofuji Y, et al. Metagenome-wide association study revealed diseasespecific landscape of the gut microbiome of systemic lupus
erythematosus in Japanese. Ann Rheum Dis 80:1575-1583 (2021).
5. Mari E K Niemi, et al. COVID-19 Host Genetics Initiative. Mapping thehuman genetic architecture of COVID-19. Nature 600:472-477 (2021).

2. Tomofuji Y, et al. Whole gut virome analysis of 476 Japanese revealed alink between phage and autoimmune disease. Ann Rheum Dis 81:278288 (2022).
3. Sakaue S, et al. A cross-population atlas of genetic associations for 220
human phenotypes. Nat Genet 53:1415-1424 (2021).

77UnitsQuantitative Immunology
Chronic Rhinosinusitis. With the Host Defense laboratory, we
study the role of the RNase Reg-1 in immune cell development.

Our group applies 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 repertoire and spatialtranscriptomics) with publicly available information into networkmodels of immune regulation. We apply this framework to study
gene regulatory networks controlling immune cell development
and function.

Development of computational methods
An 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 methodthat leverages a fuzzy logic framework that enables efficientbatch correction without bias. Another problem is theidentification of marker genes. In collaboration with Alexis
Vandenbon at Kyoto University, we have developed
singleCellHaystack, a method to identify differentially expressed
genes from multi-dimensional representations of single cell
genomics data.

Mathematical modeling
The large number of cells obtained in single cell genomics
experiments opens the door to approaches that study the
immune system using mathematical modeling and machinelearning. Transcriptional regulatory networks are critical
determinants of cell identity and function. We use linear

78

Associate Professor

Diego Diez

Postdoctoral Fellow

1

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 transcriptional networks change during immune celldifferentiation and disease.

Applications to immunology
Using single cell transcriptomics, protein expression, immune
repertoire and chromatin accessibility we study the differentiationof NKT cells in the thymus and spleen of SKG and WT mice. SKG
mice have a mutation in ZAP70 molecule that causes weakened
TCR signaling, resulting in a bias towards development of NKT1
compared to NKT2 in the wild type. This approach enables us tounderstand how changes in regulatory networks during
development effect NKT type biases. In a clinical setting, we apply
single cell genomics to get insight into IgA nephritis onset andtherapies. We analyze the transcriptome and protein expressionof immune cells in PBMCs and tonsils from IgAN patients before
and after treatment with e.g., tonsillectomy and/or steroid
immunosuppression. We combine these datasets with spatialtranscriptomics from tonsils to identify factors that lead tosuccessful therapy.
We collaborate with other groups at IFReC to study diverse
aspects of immune responses in a basic and clinical setting. With
the Experimental Immunology laboratory and the Systems
Immunology laboratories, we study how TCR signaling impacts T
cell repertoire in SKG mice. With the Immune Regulationlaboratory, we study the role of CD4 T cells in human Eosinophilic

Figure.
Single cell genomics identifies signatures of NKT cells in the thymus and spleen of WT and SKG mice.

Recent Publications
1. Loza M, Teraguchi S, Standley DM & Diez D. Unbiased integration of
single cell transcriptome replicates. NAR Genom Bioinform. 4, lqac022,
doi:10.1093/nargab/lqac022 (2022).

4. Teraguchi S, Saputri DS, et al. Methods for sequence and structural
analysis of B and T cell receptor repertoires. Comput Struct Biotechnol J.
18:2000-2011 (2020).

2. Diez D, Morte B & Bernal J. Single-Cell Transcriptome Profiling of Thyroid
Hormone Effectors in the Human Fetal Neocortex: Expression of
SLCO1C1, DIO2, and THRB in Specific Cell Types. Thyroid 31:1577-1588(2021).

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

3. 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).

79Events & Outreach Activities

Events & Outreach Activities

The 2nd ImmunoSensation2-IFReC Joint Workshop
The 2nd ImmunoSensation2- IFReC Joint Workshop was jointly organized by IFReC and ImmunoSensation2 at the University ofBonn. The two institutes had signed an academic exchange agreement in 2018, and organized this online workshop simultaneously
both in Japan and Germany. The workshop provided an opportunity to showcase the latest immunology researches in bothcountries.

● Date: December 16-17, 2021

Speaker82

Title

Kiyoshi Takeda

Opening Remarks

Joachim Schultze

Are we ready for swarm immunology?

Masaru Ishii

Identification of pathogenic tissue‐resident macrophages visualized by intravital
multiphoton imaging technology.

Christoph Wilhelm

Fasting and ketogenesis as regulators of protective immunity in severe respiratory viral
infections.

Elvira Mass

Developmental programming of tissue‐resident macrophages.

Kazuyo MoroGroup 2 innate lymphoid cells drive spontaneous pulmonary fibrosis.

Shizuo Akira

Regnase-1, an endoribonuclease involved in inflammation, immunity and metabolism

Eicke Latz

Cytosolic exposure of mitochondrial matrix contents activates the NLRP10 and AIM2
inflammasomes.

Sho Yamasaki

Innate immune sensing of metabolites causes neuroinflammatory diseases.

Eva Bartok

Characterizing the Innate Immune Response to mRNA Vaccination.

Hiroki Kato

RNA methyltransferases are critical for Influenza A virus and SARS‐CoV‐2 replication.

Irmgard Foerster

Environmental control of immune regulation and metabolism by the AhR/AhRR pathway.

Sujin Kang

IL‐6‐targeting therapy for cytokine storms by COVID‐19, CAR‐T cell therapy and otherdiseases.

Masahiro YamamotoRole of PLCbeta4 in CD8+ T cell signaling.

Shimon Sakaguchi

Induction of tumor immunity with long‐lasting memory by depleting clonally expanding
Tregs in tumor tissues.

Gunther Hartmann &Waldemar Kolanus

Closing Remarks

This symposium was supported by “Core-to-Core Program”
of Japan Society for the Promotion of Science.

83

IFReC ImmunoSeminar

To prevent the spread of COVID-19, the IFReC Colloquium, an
important event held every two months allowing researchersof IFReC to gather together, has been organized using an
online tool in FY 2021.

IFReC started a series of online seminars called the“ImmunoSeminar Series” in March, 2021. This seminar waswell received, and the series have been continued by inviting
world-class immunologists to speak at the online seminars
through the fiscal year.

Events & Outreach Activities

IFReC Colloquia

Speaker
Professor Laura Mackay(the Peter Doherty Institute for Infection and Immunity,
the University of Melbourne, AUS)

Title

'Functional heterogeneity oftissue-resident lymphocytes'
Date: December 21 (Tue.), 2021
Time: 16:00-17:00 (Osaka, Tokyo)
If you wish to attend this Zoom seminar, please acces to the following
registration site by 16:00 (JST) on December 16.
https://forms.gle/UGzkoEMnMQSKn1Bi8
You will receive a Zoom invitation e-mail a few days before the seminar.

IFReC
ImmunoSeminar
SeriesNo.

Date

Speaker

Title

Takahide Itokazu

50 May 26, 2021

(Molecular Neuroscience/
Neuro-Medical Science)

Plasticity and recovery from central nervous system damage

May 19, 2021

Yu Miyamoto(Immunology and Cell Biology)

Resident macrophages give rise to spatial heterogeneity of
immune responses in the liver

June 4, 2021

Mitsuhiro Iwaki

DNA-based force sensor for single-molecule mechanobiology

July 30, 2021

Identification of a lipid scramblase involved inglycosylphosphatidylinositol

Aug. 27, 2021

(Single molecule imaging)

51 July 29, 2021

Yicheng Wang
(Immunoglycobiology)

Kentaro Kajiwara(Oncogene Research)

52 Sept. 29, 2021

Shintaro Okumura(Aging Biology)

Weizhen Jia(Signal Transduction)

53 Nov. 25, 2021

Fumitaka Muramatsu(Signal Transduction)

Hyota Takamatsu

Identification of gut bacteria abundant in CRC patientsthat promote tumorigenesis
Indispensable role of Galectin-3 in promoting quiescence ofhematopoietic stem cells
Vascular endothelial cells create drug-resistant tumor
microenvironment through iron ion regulation by ceruloplasmin
Caspase-1–mediated secretion of mitochondrial DNA-rich
exosomes causes pathological inflammation in Behçet’s syndrome

Floris van Eerden

Mechanism of SARS-CoV-2 infection-enhancing antibodies

Masafumi Minoshima &Shahi Imam Reja
(Chemical Imaging Techniques)84

CDCP1 promotes compensatory renal growth by integrating
Src and Met signaling(Immunopathology)(Systems Immunology)

54 Mar. 17, 2022

Date

Oct. 15, 2021

Nov. 4, 2021

Dec. 20, 2021

March 11, 2022

Speaker
Shane Crotty(La Jolla Institute for Immunology,
USA)

Hao Wu(Harvard University, USA)

Gabriel Nuñez(University of Michigan, USA)

Akiko Iwasaki(Yale School of Medicine, USA)

Title
Adaptive immunity and immune memory to SARS-CoV-2 and
COVID-19

Structural deciphering of immune sensors

Host-Microbiota Interactions in Health and Disease

Immune responses to SARS-CoV-2

Zhijian Chen
(University of Texas Southwestern
Medical Center, USA)

Igniting an Immune Response with cGAS

Michel Nussenzweig

Human Immune Responses to SARS-CoV-2 Infection and
Vaccination(Rockefeller University, USA)

Laura Mackay(University of Melbourne, Australia)

Feng Shao(NIBS, Beijing, China)

Functional heterogeneity of tissue-resident lymphocytes

Pyroptosis in antibacterial and antitumor immunity

Development of Fluorescent Chemical Probes for Imaging pH
in Bone Tissue and Single Molecule Imaging

85

The 10th WPI Science Symposium for Students“Nano World toward the Future”● Date: January 11, 2022

December 18, 2021
● Date :● Organizers : NanoLSI (Kanazawa University), other WPI institutes and JSPS
Ishikawa Music Hall, Kanazawa● Venue :Osaka University established the Center for Infectious Disease Education and Research (CiDER) in 2021 as a contribution toconquering infectious diseases. To celebrate its launch, "CiDER Kick-Off Week" was held on january 10-12, 2022. On the second day,
"The 1st International Symposium of CiDER on Microbiology and Immunology," an online event mainly organized by IFReC, was held
where eminent world scientists gave talks.

Speaker
Akiko Iwasaki
Yale University, USA

Shane Crotty
La Jolla Institute for Immunology, USA

Yohei Yamauchi
University of Bristol, UK

Wataru Ise
CiDER/IFReC, Osaka University

James B. Wing
CiDER/IFReC, Osaka University

Yoshihiro Kawaoka
University of Tokyo, Japan

Hisashi Arase
CiDER/IFReC/RIMD, Osaka University

Yoshiharu Matsuura
CiDER/RIMD, Osaka University

86

Title

Events & Outreach Activities

The 1st International Symposium of CiDER on
Microbiology and Immunology

The 10th WPI Science Symposium had an attendance of over 300 high school students, mainly from the Super Science HighSchools in Ishikawa Prefecture, and was simultaneously streamed online. At IFReC’s booth, we introduced the immunology research
activities at Osaka University, and distributed our PR magazine “Imuneco” to attendees.

Immune responses to SARS-CoV-2
Understanding adaptive immunity and immune memory to SARS-CoV-2 and
COVID-19 vaccines
Cell biology of SARS-CoV-2 entry by Neuropilin-1
Regulation of plasma cell generation and survival for long-term protection from
pathogens
Age, sex and severity associations of regulatory T-cells in COVID-19

Addressing the threat of emerging viral infections

Neutralizing and infectivity-enhancing antibodies against SARS-CoV-2

Construction of reverse genetics and VLP vaccine for SARS-CoV-2

87

Events & Outreach Activities

Online Outreach Activities
From FY2020, IFReC implemented a new approach to holding its outreach activities by utilizing the web and online tools. The
experience gained from holding these virtual events is expected to be highly useful even after COVID-19 restrictions on activities arelifted.

Research Frontline Lecture for Educators“ Let's get to know the latest research at WPI! ”
June 19 & 26, and July 3, 2021
● Date :Organizers:
IFReC(Osaka Univ) and other five WPI institutes●● Speakers : Jun Hatazawa (IFReC), Naoto Shirahata (MANA), and others

Life Science Seminar for High School Students
July 30, 2021
● Date :● Organizers : Senri Life Science Foundation and IFReC (supported by Osaka Prefectural Board of Education)● Speakers : Kazuyo Moro (IFReC) and two scientists of Osaka University

88

Science Agora 2021 “Dialogue for Life”
Neo-Science “Information, AI, Big data for Cutting Edge Researches”
November 7, 2021
● Date :Organizer:
IFReC, ICReDD (Hokkaido University) and IRCN (University of Tokyo)●● Speakers : Naganari Ohkura (IFReC) and two scientists

SpringX Super School “Protecting our Lives from Infectious Diseases”● Organizer : © KNOWLEDGE CAPITAL and Center for Infectious Disease Education and Research (CiDER),
Osaka University● Speakers : Wataru Ise, Hisashi Arase, and Yukinori Okada (CiDER/IFReC)

89

Japanese Language Class
Japanese language classes are held for overseas researchers / students to alleviate any stress and inconvenience in their researchand in their daily lives that may be caused by the language barrier. We offer two classes, one is for beginners, and another is for preintermediate level students. In 2021, all classes were organized by the use of an online tool.

Message from Ms. Kaori Tajima, Japanese language teacher
Hi. I’m Tajima. I’m really excited to teach Japanese at IFReC, again. The class is focused on speaking. You are expected
to talk about various things like your hobbies, your experiences in Japan, your plans for the future and so on, using new
grammar and vocabulary that you learn in the class. I hope you enjoy learning Japanese as well as communicating withyour class mates.

■ Focus on Daily Conversion ■Learn practical conversations for use in various situations in daily life, such as when shopping or at a restaurant. In addition,
learning Japanese for use in your lab and meetings will broaden your communication skills.

■ Enjoy Talking with Other Students ■Use the Breakout Room to talk in pairs or groups, and you can not only learn Japanese, but also make new friends outside yourlaboratory or department by sharing fun and useful information. It's easy to stay motivated when you have fellow students to talkwith, and they can encourage you to continue learning.

90

Information

Information

Major Awards

Shizuo Akira & Shimon Sakaguchi

Hisashi Arase◆ The World's Most Influential Scientific Minds 2021

The Erwin von Bälz Prize 2021

Highly Cited Researchers are researchers whose theseswith large number of citations from all over the world. In
2021, about 6,600 researchers from 21 fields were selected
as "the most influential researchers in the world". The threeresearchers in the two fields were selected from Osaka
University, and two (Profs Akira and Sakaguchi) of themwere from IFReC.

The Erwin von Bälz Prize was established in 1964 by Nippon Boehringer
Ingelheim Co., Ltd. Arase was awarded First Prize for the theme "Regulation
Mechanisms for Viral Infections."

Taroh Kinoshita◆ Osamu Hayaishi Memorial Prize 2021
◆ Toyoichi Ohtawara Award 2021

Tadamitsu Kishimoto

Kinoshita has made considerable achievements in the research field of GPI(glycosylphosphatidylinositol)-anchored protein, a series of proteins anchored tothe cell membrane (commented by the Hayaishi Prize committees).

◆ A Clarivate Citation Laureate 2021
Since the papers by the laureate have been cited so often in the last two or
more decades that these scientists typically rank in the top 0.1% in their research
fields. The laureate are recognized to have written multiple high-impact articles
over many years.

James B. Wing
◆ Osaka University Prize 2021
The prize is given to the faculty members of Osaka University, who havemade remarkable contributions to the education, research, and organizational
management. Wing was awarded for "the studies for immune dynamics ofregulatory T cells using single cell analysis".

Shigekazu Nagata◆ Pioneer, Australasian Cell Death Society 2021
In the early 90th, his group in Osaka demonstrated that apoptosis could be
induced by a death factor (Fas ligand).

Ryu Okumura & Yasutaka Motomura◆ JSI Young Investigator Award 2021
Okumura (Lab Mucosal Immunology) and Motomura (Lab
Innate immune systems) were awarded for "Elucidation of themechanism of colonic homeostasis by Lypd8 expressed byintestinal epithelial cells" and "Elucidation of the pathogenesisof allergy by IL-4/IL-13", respectively.

92

93Original Support Programs for Young Researchers

IFReC is recruiting post-doctoral researchers for its Advanced Postdoc Program. This program offers three-year employment andfunding (3 million yen per year) for original research to promising young researchers. Selected applicants have access to continually
upgraded state-of-the-art facilities at IFReC for their research, including equipment for single-cell analysis.

To strengthen our international research network and our basis for international collaborative research, IFReC has established
two kinds of financial support programs for researchers. 1) “IFReC Kishimoto Foundation Fellowship,” which has been used to inviteinternational 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 practical skills and abilities of young researchers
in international collaborative research and to develop their network with researchers overseas. Since 2009, 132 researchers havereceived these grants.

Information

Advanced Postdoc Program at IFReC

Support Program for Young Researchers
17
26
25
EuropeMiddle East, Asia

Africa3

43

America6241

Oceania5
Original Project of IFReC
OverseasOsaka

FY2009-2021

70 recipientsz IFReC Kishimoto Fellowship
Osaka

Overseas

62 recipientsz Program for International
Circulation of Young Talented Researchers

Support for Paper Submission

Advanced Postdoc members as of March, 2022.

Due to the influence of COVID-19, it has been difficult for researchers to find opportunities to present their research results atoverseas conferences. This program aims to support the dissemination of research results by the researchers of IFReC. Since 2020,
over 40 young scientists have used this system.

Grant for Next Generation Principal Investigators
This program aims to foster the next generation of principal investigators at IFReC. In particular, challenging 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, andthree in FY2020 with the continuous research funding for three fiscal years. The grant is expected to generate excellent researchachievements, raise the international recognition of these PIs, and contribute to the acquisition of external research funding. The
final debriefing session for the program is held in 2022.

94

95

IFReC and its parent institution, the Research Institute for Microbial Diseases (RIMD) are located on the same
site, constituting a large research complex. The complex contains the Core Instrumentation Facility, the Animal
Resource Center and the Network Administration Office, all of which are jointly operated by IFReC and RIMD. The
Core Instrumentation Facility is equipped with various highly advanced instruments and skilled technicians provide
in-house services to IFReC and RIMD researchers. The Animal Resource Center consists of three buildings for specificpathogen-free (SPF) animals and the live immuno-imaging facility. With a largecapacity animal-breeding facility in
IFReC, researchers are able to choose animal rooms suitable for their experiment purpose. Using these common
facilities, IFReC researchers are able to effectively and smoothly carry out their experiments to promote their worldleading research at IFReC.

Information

Common Facilities (IFReC, RIMD, Animal Resource Center)

Composition & Finance■■ Composition
Number of IFReC Staff

As of Feb, 1st350
Researchers (JPN)

Researchers (Overseas)

Administrative and Support Staff

300
97

250
102

117
106

90

105

92

102

70

200

89

90

86
55

52

150

6

4

5

2

1

42

117

119

128

138

137

2011

2012

2013

2014

2015

24

50

48

54

50

47

43

44

130

124

124

117

2016

2017

2018

2019

43

42

38

100

3

55

54

53

16
8

129

142

162

2020

2021

2022

93
65

44

0
2008

2009

2010

Year

❶ IFReC Research Building❷ Integrated Life Science Building■■ Finance❸ Main Building, Research Institute for Microbial Diseases, RIMD
❹ South Building, Research Institute for Microbial Diseases, RIMD

Sources❺ Cutting-edge Research Building for Infectious Diseases

Expenditure❻ Animal Resource Center for Infectious Diseases
From the
University
13.3%

Animal Resource Center for Infectious Diseases

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

LAN system and servers (web, mail, mailing lists, etc.)

Main Grant
32.0%

JPY
4,324,056,73r9■ In vitro fertilization and embryo transplantation

Personnel
25.7%Management
Expenses Grant
12.7%

Individual
research
project
36.5%

JPY
4,324,056,739

Donation
9.5%■ Intracytoplasmic sperm injection■ Transgenic and knock-out animals■ Genome editing in experimental animals

Core Instrumentation Facility■ Basic and advanced instruments

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

96■ In-house service■ DNA sequencing, cell sorting, electron microscopy, mass

Grant-in-Aid
for Scientific
Research
5.8%

Contract Research
26.7%

Equipment
11.4%

Project
Activities
26.3%

Travel 0.1%

spectrometry and next-generation sequencing analysis■ Radio isotope facility

97

Information

Selected Articles

SLPI is a critical mediator that controls PTH-induced bone formation.
Morimoto A, Kikuta J, Nishikawa K, et al.

Sasai M, Ma JS, Okamoto M, et al.

Nat Commun. 12:2136 (2021).

J Exp Med. 218(7) : e20201763 (2021).

The research group of Masaru Ishii identified a serine
protease inhibitor, secretory leukocyte protease inhibitor(SLPI), as a critical mediator that is involved in the anabolicparathyroid hormone (PTH)-mediated shift to the
osteoblastic phase. Slpi is highly upregulated in osteoblasts
by PTH, while genetic ablation of Slpi severely impairs PTHinduced bone formation. Slpi induction in osteoblasts
enhances its differentiation, and increases osteoblast–
osteoclast contact, thereby suppressing osteoclastic
function. Intravital bone imaging reveals that thePTHmediated association between osteoblasts and osteoclasts is
disrupted in the absence of SLPI. These results demonstratethat SLPI regulates the communication between osteoblasts
and osteoclasts to promote PTH-induced bone anabolism.

Masahiro Yamamoto and his research group showed that
TCR signaling in CD8+ T cells is qualitatively different fromthat in CD4+ T cells, since CD8α ignites another cardinalsignaling cascade involving phospholipase C β4 (PLCβ4).
TCR-mediated responses were severely impaired in PLCβ4deficient CD8+ T cells, whereas those in CD4+ T cells wereintact. PLCβ4-deficient CD8+ T cells showed perturbed
activation of peripheral TCR signaling pathways downstreamof IP3 generation. PLCβ4-deficient mice exhibited defective
antiparasitic host defense and antitumor immune responses.
The authors concluded PLCβ4 differentiates TCR signalingin CD4+ and CD8+ T cells and selectively promotes CD8+ T
cell–dependent adaptive immunity.

Distinct Foxp3 enhancer elements coordinate development, maintenance and function ofregulatory T cells.

98

A novel role of PLCβ4 in selectively mediating TCR signaling in CD8+ but not CD4+ T cells.

An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies.

Kawakami R, Kitagawa Y, Chen KY, et al.

Liu Y, Soh WT, Kishikawa J, et al.

Immunity 54:947-961 (2021).

Cell 184:3452-3466.e18 (2021).

Regulatory T cells (Tregs) are one of the most significant
cells in the maintenance of immune tolerance. Tregs work
to maintain homeostasis of the body in various situations
such as prevention of autoimmune diseases, terminationof immune responses and damaging tissue repairing, bybraking excessive immune responses. The research group led
by Shimon Sakaguchi identified non-coding DNA sequences
indispensable for Treg generation. These DNA sequences are
distributed into the two separated regions near the Foxp3
gene locus, the master transcription factor of Tregs. Defectof these elements developed severe autoimmune-diseases.
Controlling the development and function of Tregs holds
promise for the new therapies for immunological diseases.

Antibodies against the receptor-binding domain (RBD) of
the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection.
However, the effects of antibodies against other spike
protein domains are largely unknown. The research groupled by Hisashi Arase found that infection with SARS-CoV-2
produces not only neutralizing antibodies that prevent
infection, but also infection-enhancing antibodies. They
demonstrated that the infection-enhancing antibodies
enhance the infectivity of SARS-CoV-2 by modulating theconformation of spike protein of SARS-CoV-2. Furthermore,
the infection-enhancing antibodies attenuated the ability ofneutralizing antibodies to prevent infection.

99

Information

The lysosomal Ragulator complex plays an essential role in leukocyte trafficking byactivating myosin II.
Nakatani T, Tsujimoto K, Park J, et al.

Tani H, Li B, Kusu T, et al.

Nat Commun. 12:3333 (2021).

Proc Natl Acad Sci U S A. 118:e2100594118 (2021).

Atsushi Kumanogoh and their research group showedthat the lysosomal Ragulator complex plays an essential rolein leukocyte migration by activating myosin II. In immotile
cells (upper), the Ragulator complex localized to lysosomes
is preferentially distributed in the perinuclear region (L), and
MPRIP anchors MLCP on myosin–actin bundles by binding
MYPT1, a subunit of MLCP, resulting in suppression of MLC
phosphorylation (R). In motile cells exposed to chemokines(lower), the lysosomes bearing the Ragulator complex moves
to the uropod (L), where the Ragulator 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 (R).

Hisako Kayama, Kiyoshi Takeda and their research group
showed that E-NTPD8 is highly expressed in large intestinal
epithelial cells and hydrolyzes microbiotaderived luminal
ATP. E-NTPD8 in colonic epithelial cells maintains the gut
homeostasis through hydrolysis of luminal ATP produced bycommensal bacteria (left). An increased level of luminal ATP
caused by lack of E-NTPD8 modulates neutrophil physiology,
such as prolonged survival, through P2X4-mediatedpromotion of glycolysis, which links to aggravation of colitis(right).

A sublethal ATP11A mutation causes aberrant phosphatidylcholine flipping in plasma
membranes.

100

The ATP-hydrolyzing ectoenzyme E-NTPD8 attenuates colitis through modulation of P2X4
receptor-dependent metabolism in myeloid cells.

Gut bacteria identified in colorectal cancer patients promote tumourigenesis via butyrate
secretion.

Segawa K, Kikuchi A, Noji T, et al.

Okumura S, Konishi Y, Narukawa M, et al.

J Clin Invest. 131:e148005 (2021).

Nat Commun. 12:5674 (2021).

ATP11A translocates phosphatidylserine (PtdSer), but notphosphatidylcholine (PtdCho), from the outer to inner leafletof plasma membranes, thereby maintaining the asymmetric
distribution of PtdSer. Shigekazu Nagata (Biochemistry &Immunology, IFReC), Katsumori Segawa (present: TMDU)
and the research group detected a de novo heterozygous
point mutation in ATP11A in a patient with developmental
delays and neurological deterioration. Mice carrying thecorresponding mutation died perinatally or soon after birthwith signs of neurological disorders. This mutation caused anamino acid substitution (Q84E) in the first transmembranesegment of ATP11A, and mutant ATP11A flipped PtdCho.

The research group of Eiji Hara showed that 12 faecal
bacterial taxa are enriched in CRC patients in two
independent cohort studies. Among them, 2Porphyromonas species are capable of inducing cellularsenescence, an oncogenic stress response, through thesecretion of the bacterial metabolite, butyrate. Notably, theinvasion of these bacteria is observed in the CRC tissues,coinciding with the elevation of butyrate levels and signs ofsenescence-associated inflammatory phenotypes. Moreover,although the administration of these bacteria into Apc14/+
mice accelerate the onset of colorectal tumours, this is not
the case when bacterial butyrate-synthesis genes are
disrupted. These results suggest a causal relationshipbetween Porphyromonas species overgrowth and colorectal
tumourigenesis which may be due to butyrate-induced
senescence.

101

Information

Glycan engineering of the SARS-CoV-2 receptor-binding domain elicits cross-neutralizing
antibodies for SARS-related viruses.
Shinnakasu R, Sakakibara S, Yamamoto H, et al.

Lu X, Hosono Y, Nagae M, et al.

J Exp Med. 218:e20211003 (2021).

J Exp Med. 218:e20211327 (2021).

In order to induce the antibodies predominantly against
the regions that are structurally conserved in RBD among
SARS-related viruses, the research group of Ryo Shinnakasu,
Tomohiro Kurosaki and Shuhei Sakakibara performed glycan
engineering to mask the dominant epitope on the RBD
head region which is a structurally non-conserved region.
When mice are immunized with this modified RBD vaccine,as expected, antibodies that recognize the core-RBD regionof not only SARS-CoV-2 but also other SARS-related viruses
such as SARS-CoV or WIV1-CoV are predominantly induced.
Furthermore, these induced antibodies showed a highprotective effect against various SARS-related viruses.

Follicular helper T (Tfh) cells are a subset of CD4+ T cells thatmediate the production of protective antibodies; however, the
SARS-CoV-2 epitopes activating Tfh cells are not well characterized.
Sho Yamasaki and the research group identified and crystallized

The tertiary structure of the human Xkr8-Basigin complex that scrambles phospholipids at
plasma membranes.

102

Identification of conserved SARS-CoV-2 spike epitopes that expand public cTfh clonotypes
in mild COVID-19 patients.

TCRs of public circulating Tfh (cTfh) clonotypes that are expandedin patients who have recovered from mild symptoms. Thesepublic clonotypes recognized the SARS-CoV-2 spike (S) epitopes
conserved across emerging variants.

TBK1 is an essential factor for germinal center formation during infection and vaccination.

Sakuragi T, Kanai R, Tsutsumi A, et al.

Lee MSJ, Inoue T, Ise W, et al.

Nat Struct Mol Biol. 28:825-834 (2021).

J Exp Med. 219:e20211336 (2022).

Xkr8–Basigin is a plasma membrane phospholipid
scramblase activated by kinases or caspases. The researchgroup of Takaharu Sakuragi and Shigekazu Nagata
combined cryo-EM and X-ray crystallography to investigate
its structure at an overall resolution of 3.8 Å. Its membranespanning region carrying 22 charged amino acids adopts
a cuboid-like structure stabilized by salt bridges betweenhydrophilic residues in transmembrane helices. Its mutation
to alanine made the Xkr8–Basigin complex constitutively
active, indicating that it plays a vital role in regulating its
scramblase activity. The structure of Xkr8–Basigin provides
insights into the molecular mechanisms underlying
phospholipid scrambling.

Michelle S. J. Lee (IMS, Univ Tokyo), Cevayir Coban (IMS,
Univ Tokyo/IFReC), and their research group identified an
unexpected role of Tank-binding kinase-1 (TBK1) as a crucial
B cell intrinsic factor for GC formation during infection andvaccination in mice. Wild type mice (top of the figure) andB cell intrinsic TBK1-deficient mice (bottom of the figure)responded differently to vaccination and malaria infection
due to distinct B cell fate decision. The horizontal axis
represents the stages of germinal center B cell development.
The vertical axis represents the expression level of B cellsignaling molecules. B cell receptor (BCR) and costimulatory
receptor (CD40) engagement activates CD40-BCR signaling
to initiate B cell activation. P stands for phosphorylation for
TBK1 activation, GC stands for germinal center.

103

Information

SARS-CoV-2 infection triggers paracrine senescence and leads to a sustained
senescenceassociated inflammatory response.
Tsuji S, Minami S, Hashimoto R, et al.

Uenaka M, Yamashita E, Kikuta J, et al.

Nat Aging 2:115-124 (2022).

Nat Commun. 13:1066 (2022).

Reports of "post-acute COVID-19 syndrome," in which
the inflammatory response persists even after SARSCoV-2 has disappeared, are increasing but the underlying
mechanisms of post-acute COVID-19 syndrome remain
unknown. The research group of Eiji Hara (IFReC/
RIMD/CiDER, Osaka University) show that SARS-CoV-2
infected cells trigger senescence-like cell-cycle arrestin neighboring uninfected cells in a paracrine manner
via virus-induced cytokine production. The sustained
infection-induced paracrine senescence described here
may be involved in the long-term inflammation caused
by SARS-CoV-2 infection.

The mechanism of the transition from bone formation
to bone resorption has been unknown. Using original
cutting-edge technology for in vivo bone imaging, Maki
Uenaka, Junichi Kikuta, Masaru Ishii and their researchgroup revealed that bone-forming cells (osteoblasts)
secrete micro particles (vesicles) outside the cells. These
extracellular vesicles secreted from osteoblasts suppress
the differentiation of osteoblasts and promote thedifferentiation of osteoclasts. The regulation of the vesicles
is expected to be applied to new therapeutic agents forbone diseases.

Selective targeting of multiple myeloma cells with a monoclonal antibody recognizing theubiquitous protein CD98 heavy chain.

104

Osteoblasts Secrete Extracellular Vesicles to Exchange Information.

Abrogation of self-tolerance by misfolded self-antigens complexed with MHC class II
molecules.

Hasegawa K, Ikeda S, Yaga M, et al.

Jin H, Kishida K, Aease N, et al.

Sci Trans Med. 14 (2022)

Sci Adv. 8:eabj9867 (2022).

Searching for cancer-specific surface antigens could
identify potential targets for cancer therapy that may
be missed by -omic approaches. Kana Hasegawa, Naoki
Hosen, and their research group screened monoclonal
antibody (mAb) clones against patient-derived multiple
myeloma (MM) cells, in the process identifying R8H283,
an mAb that recognized the CD98 heavy chain protein,which is part of an amino acid transporter. Despite theubiquitous presence of CD98 heavy chain in normal
cells, the mAb only bound to MM cells, and differing
glycosylation patterns between normal cells and MM
cells contributed to this selectivity. In MM xenograftmodels in mice, R8H283 injections prolonged survival,suggesting that the mAb should be further assessed for
utility in treating MM.

In Graves' disease, one of the autoimmune diseases,
autoantibodies against thyroid-stimulating hormone
receptors are produced. The research group of Hui Jin andHisashi Arase found that autoantibodies in patients with
Graves’ disease preferentially recognize thyroid-stimulating
hormone receptor (TSHR) complexed with MHC class II
molecules of Graves’ disease risk alleles. This suggests thatthe aberrant TSHR transported by MHC class II molecules is
the target of autoantibodies produced in Graves’ disease.

105

Information

Publications

33

Hikichi, Y; Motomura, Y; Takeuchi, O; Moro, K. Posttranscriptional
regulation of ILC2 homeostatic function via tristetraprolin.
Journal of Experimental Medicine 218, e20210181 (2021).

De Marco, M; Gauttier, V; Pengam, S; Mary, C; Ranieri, B; Basile,
A; Festa, M; Falco, A; Reppucci, F; Cammarota, AL; Acernese, F;
De Laurenzi, V; Sala, G; Brongo, S; Miyasaka, M; Shalapour, S;
Vanhove, B; Poirier, N; Iaccarino, R; Karin, M; Turco, MC; Rosati, A;
Marzullo, L. Concerted BAG3 and SIRP alpha blockade impairs
pancreatic tumor growth. Cell Death Discovery 8, 94 (2022).

34

Honjo, Y; Fujita, Y; Niwa, H; Yamashita, T. Increased expression ofNetrin-4 is associated with allodynia in a trigeminal neuropathic
pain model rats by infraorbital nerve injury. Plos One 16,
e0251013 (2021).

35

19Diez, D; Morte, B; Bernal, J. Single-Cell Transcriptome Profilingof Thyroid Hormone Effectors in the Human Fetal Neocortex:
Expression of SLCO1C1, DIO2, and THRB in Specific Cell Types.
Thyroid 31, 1577-1588 (2021).

Hosen, N; Yoshihara, S; Takamatsu, H; Ri, M; Nagata, Y; Kosugi, H;
Shimomura, Y; Hanamura, I; Fuji, S; Minauchi, K; Kuroda, J; Suzuki,
R; Nishimura, N; Uoshima, N; Nakamae, H; Kawano, Y; Mizuno,
I; Gomyo, H; Suzuki, K; Ozaki, S; Nakamura, S; Imai, Y; Kizaki, M;
Negoro, E; Handa, H; Iida, S. Expression of activated integrin
beta 7 in multiple myeloma patients. International Journal of
Hematology 114, 3-7 (2021).

36

20

Duval, R; Nicolas, G; Willemetz, A; Murakami, Y; Mikdar, M;
Vrignaud, C; Megahed, H; Cartron, JP; Masson, C; Wehbi, S; Koehl,
B; Hully, M; Siquier, K; Chemlay, N; Rotig, A; Lyonnet, S; Colin, Y;
Barcia, G; Cantagrel, V; Kim, CL; Hermine, O; Kinoshita, T; Peyrard,
T; Azouzi, S. Inherited glycosylphosphatidylinositol defects cause
the rare Emm-negative blood phenotype and developmental
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Information

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111

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