WPI Immunology Frontier Research Center FY 2015

World Premier International
Research Center

Osaka University

Immunology
Frontier
ResearchCenterAnnual Reportof IFReC
FY 2015

Edit : Research Planning & Management Office, IFReC
Published in June, 2016

Osaka University

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

Message from the Director

Contents
Message from the Director ・
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・ 1
Looking back on IFReC’s activities over the years ・
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The 1 Workshop for WPI Research Support Staff ・
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Japanese Class ・
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st

As the Director of the Immunology Frontier Re-

Although the governing structure will change,

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

our most important mission “Constructing aam very pleased to present the IFReC annual re-world-top immunology research center” remains

port for fiscal 2015.

the same. We will make unceasing efforts to de-Since its inception in 2007, IFReC has establishedvelop immunology research to ensure translation

itself as a Visible International Research Center ofto medical science.

Immunology with the support of many people

In FY2015, as an approach to nurturing young

including the WPI Program Director and the Pro-

researchers, the seventh International Sympo-

gram Officer.

sium of IFReC “Immunology at the Forefront” was

IFReC was created as a part of the WPI program,effectively organized in combination with thea national project led by the Japanese Ministry offifth NIF Winter School by IFReC and Singapore

Education, Culture, Sports, Science and Technol-

Immunology Network. The symposium and theogy. However, from FY2017, IFReC will create aschool pride themselves on offering not only pro-new mark in its history with a novel agreement

ductive educational content but also an oppor-

for academic-industry partnership. The gover-tunity for the young researchers to form a global

nance system will be an ambitious program with-

network.

out precedent.

We are committed to continuing contributions
to scientific advances through research and education and to evolving into a world top immunol-

Organization

Outreach Activities

Organization Chart ・
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Science Cafe ・
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・ 84

Committee and Advisory Board for IFReC ・
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・ 8

Super Science High School Student Fair 2015・
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・ 86

Administrative Office of IFReC ・
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The 5 t h Annual WPI Joint Symposium・
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・ 87

ogy research center.

AAAS 2016 Annual Meeting / IFReC on edX ・
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Laboratories
Immunology Groups ・
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Research Projects

Imaging Groups ・
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Support Programs for Fusion Researches ・
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Informatics Groups ・
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Young Scientist Support Program for Research Abroad・
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Shizuo Akira, MD/PhD

Units for Combined Research Fields ・
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Data
Symposia & Seminars

Common Facilities・
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The 7 th International Symposium of IFReC・
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Kishimoto Foundation Fellowships・
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IFReC Seminars ・
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Composition ・
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IFReC Colloquia ・
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Major Awards・
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・ 98

The 6 Kishimoto Foundation Lecture ・
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Finance ・
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・ 99

th

Director
WPI Immunology Frontier Research Center

Publications ・
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・100

EventsThe 5 th NIF Winter School on Advanced Immunology ・
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Access Map・
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Immunology Lecture Series ・
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Emergency Drill / Explanation Session for Preventing theMisuse of Funds ・
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・ 79

1

Looking back on IFReC’s activities over the years
Jun Sakanoue ( Research Planning and Management Office, IFReC )Since the establishment of IFReC in 2007, there have been
1090 science articles published by IFReC researchers (as ofMarch, 2016). About 10% of these articles have appeared in“High Impact Journals” such as Science, Nature, Cell and theiraffiliates (1). The impact factor of a journal is a measure reflecting the number of citations to recent articles published in thatjournal, and is used to reflect the relative importance of a journal within its field.
Here, I evaluate the quality of the articles by IFReC researchers using percentiles of top papers. A percentile defines a fraction or subset of papers in a research field that has received aminimum number of citations. For example, if the value of the

10% percentile is 20, a paper in the same research field in thesame year must receive at least 20 citations to belong to thatpercentile (2). The percentile in each research field gives a more
equitable evaluation compared to citation numbers, because
IFReC researchers have such diversity.
Table 1 shows the numbers of articles that appear in thepercentiles of top papers in the three major research fields for
IFReC; Immunology, Cell Biology, and Biochemistry & Molecular
Biology. Data for the whole of Osaka University is also shownfor comparison. Table 2 shows the percentage of these top papers among all papers.

Table 3 shows the academic contribution of IFReC to Osaka University in the three research fields.

Tabel 3 Contribution of IFReC to Osaka University

Tabel 1 Number of Top 1% & Top 10% Articles
As well as the number and percentage of top papers in Tables
1 and 2, the average citation number per article (Citation Impact) of IFReC is much higher than that of Osaka University as awhole or other departments at the university (outside of IFReC).
The academic contribution IFReC makes to Osaka University isreaffirmed through this objective analysis.

The percentage for international co-authored papers in thesethree research fields by Osaka University and IFReC were 29.5%
and 43.0%, respectively (3). It is believed that international
papers provide a high level of research. The high research level
attracts higher international cooperation, and consequently
improves the performance of internationalization in IFReC. The
maintenance of a high academic research level is an indispensable element for the “Internationalization of the Institute”, oneof the goals of the WPI project.

References(1) Annual Report of IFReC FY2014(2) InCites TM by Thomson Reuters

Tabel 2 Percentage of Top 1% & Top 10% Articles

The top papers by the whole of Osaka University appeared
at a high level except for the top 10% in Biochemistry & Molecular Biology (9.4%), whereas the 23.1% level produced inthis field by IFReC was more than double. Further, the value of
8.9% for the top 1% in Immunology represents an almost ninefold compilation of top papers in immunology fields at IFReC.

2(3) Web of ScienceTM by Thomson Reuters

All the top 1% papers are listed in the final table (P.4). The
highest score by the percentile analysis is recorded by the Daron Standley group (Katoh and Standley. Mol. Biol. Evol., 2013).
0.01% in a percentile means an unusually high value in thesubject area.

3All the Top 1% Articles by IFReC Researchers Since 2008
No.

ArticlePercentile
in Subject
Area

1

Katoh, Kazutaka & Standley, Daron M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution
30, 772-780 (2013).

0.01

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Ivanov, Ivaylo I. et al. Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria. Cell 139, 485-498 (2009).

0.01

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Wing, Kajsa et al. CTLA-4 control over Foxp3(+) regulatory T cell function. Science 322, 271-275 (2008).

0.02

4

Forrest, Alistair R. R. et al. A promoter-level mammalian expression atlas. Nature 507, 462-470 (2014).

0.02

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Takeuchi, Osamu & Akira, Shizuo. Pattern Recognition Receptors and Inflammation. Cell 140, 805-820 (2010).

0.03

6

Miyara, Makoto et al. Functional Delineation and Differentiation Dynamics of Human CD4(+) T Cells Expressing the FoxP3 Transcription Factor. Immunity 30, 899-911 (2009).

0.03

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Kawai, Taro &; Akira, Shizuo. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nature Immunology 11, 373-384 (2010).

0.04

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Kawai, Taro & Akira, Shizuo. Toll-like Receptors and Their Crosstalk with Other Innate Receptors in Infection and Immunity. Immunity 34, 637-650 (2011).

0.04

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Atarashi, Koji et al. T-reg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature 500, 232-236 (2013).

0.04

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Klionsky, Daniel J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445-544 (2012).

0.05

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Abbas, Abul K. et al. Regulatory T cells: recommendations to simplify the nomenclature. Nature Immunology 14, 307-308 (2013).

0.06

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Kato, Hiroki et al. Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5. Journal of Experimental Medicine 205, 1601-1610 (2008).

0.07

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Hibar, Derrek P. et al. Common genetic variants influence human subcortical brain structures. Nature 520, 224-229 (2015).

0.11

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Saitoh, Tatsuya et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1 beta production. Nature 456, 264-268 (2008).

0.15

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Atarashi, Koji et al. ATP drives lamina propria T(H)17 cell differentiation. Nature 455, 808-812 (2008).

0.15

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Kumar, Himanshu et al. Toll-like receptors and innate immunity. Biochemical and Biophysical Research Communications 388, 621-625 (2009).

0.20

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Misawa, Takuma et al. Microtubule-driven spatial arrangement of mitochondria promotes activation of the NLRP3 inflammasome. Nature Immunology 14, 454-460 (2013).

0.20

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Deretic, Vojo et al. Autophagy in infection, inflammation and immunity. Nature Reviews Immunology 13, 722-737 (2013).

0.22

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Satoh, Takashi et al. The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection. Nature Immunology 11, 936-944 (2010).

0.23

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Oka, Takafumi et al. Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure. Nature 485, 251-255 (2012).

0.23

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Uematsu, Satoshi et al. Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5. Nature Immunology 9, 769-776 (2008).

0.24

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Ohkura, Naganari et al. T Cell Receptor Stimulation-induced Epigenetic Changes and Foxp3 Expression are Independent and Complementary Events Required for Treg
Cell Development. Immunity 37, 785-799 (2012).

0.26

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Sakaguchi, Shimon et al. The plasticity and stability of regulatory T cells. Nature Reviews Immunology 13, 461-467 (2013).

0.29

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Sakaguchi, Shimon et al. FOXP3(+) regulatory T cells in the human immune system. Nature Reviews Immunology 10, 490-500 (2010).

0.33

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Nishikawa, Hiroyoshi & Sakaguchi, Shimon. Regulatory T cells in tumor immunity. International Journal of Cancer 127, 759-767 (2010).

0.35

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Saijo, Shinobu et al. Dectin-2 Recognition of alpha-Mannans and Induction of Th17 Cell Differentiation Is Essential for Host Defense against Candida albicans. Immunity
32, 681-691 (2010).

0.35

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Yang, Huan et al. A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release. PNAS 107, 11942-11947 (2010).

0.35

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Ueno, Masaki et al. Layer V cortical neurons require microglial support for survival during postnatal development. Nature Neuroscience 16, 543-551 (2013).

0.35

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Clarke, Michael et al. Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling. Genome Biology 14, R11 (2013).

0.36

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Satoh, Takashi et al. LGP2 is a positive regulator of RIG-I- and MDA5-mediated antiviral responses. PNAS 107, 1512-1517 (2010).

0.37

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Saitoh, Tatsuya et al. Neutrophil Extracellular Traps Mediate a Host Defense Response to Human Immunodeficiency Virus-1. Cell Host & Microbe 12, 109-116 (2012).

0.37

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Ishii, Ken J. et al. TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines. Nature 451, 725-729 (2008).

0.40

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Tokunaga, Fuminori et al. Involvement of linear polyubiquitylation of NEMO in NF-kappa B activation. Nature Cell Biology 11, 123-132 (2009).

0.41

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Dorner, Marcus et al. Completion of the entire hepatitis C virus life cycle in genetically humanized mice. Nature 501, 237-241 (2013).

0.41

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Matsunaga, Kohichi et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nature Cell Biology 11, 385-396 (2009).

0.44

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Lesina, Marina et al. Stat3/Socs3 Activation by IL-6 Transsignaling Promotes Progression of Pancreatic Intraepithelial Neoplasia and Development of Pancreatic Cancer.
Cancer Cell 19, 456-469 (2011).

0.48

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Oldenburg, Marina et al. TLR13 Recognizes Bacterial 23S rRNA Devoid of Erythromycin Resistance-Forming Modification. Science 337, 1111-1115 (2012).

0.51

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Schubert, Desiree et al. Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations. Nature Medicine 20, 1410-1416 (2014).

0.52

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Yanai, Hideyuki et al. HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses. Nature 462, 99-103 (2009).

0.55

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Marichal, Thomas et al. DNA released from dying host cells mediates aluminum adjuvant activity. Nature Medicine 17, 996-1002 (2011).

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Goto, Yoshiyuki et al. Innate lymphoid cells regulate intestinal epithelial cell glycosylation. Science 345, 1254009 (2014).

0.61

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Ishikawa, Eri et al. Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle. Journal of Experimental Medicine 206, 2879-2888 (2009).

0.63

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Yokosuka, Tadashi et al. Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase
SHP2. Journal of Experimental Medicine 209, 1201-1217 (2012).

0.67

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Ohkura, Naganari et al. Development and Maintenance of Regulatory T cells. Immunity 38, 414-423 (2013).

0.70

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Fukuda, Akihisa et al. Stat3 and MMP7 Contribute to Pancreatic Ductal Adenocarcinoma Initiation and Progression. Cancer Cell 19, 441-455 (2011).

0.73

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Nishikawa, Hiroyoshi & Sakaguchi, Shimon. Regulatory T cells in cancer immunotherapy. Current Opinion In Immunology 27, 42376 (2014).

0.77

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Wing, Kajsa & Sakaguchi, Shimon. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nature Immunology 11, 42564 (2010).

0.78

48

Yamasaki, Sho et al. Mincle is an ITAM-coupled activating receptor that senses damaged cells. Nature Immunology 9, 1179-1188 (2008).

0.79

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Kitano, Masahiro et al. BcI6 Protein Expression Shapes Pre-Germinal Center B Cell Dynamics and Follicular Helper T Cell Heterogeneity. Immunity 34, 961-972 (2011).

0.79

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Sugiyama, Daisuke et al. Anti-CCR4 mAb selectively depletes effector-type FoxP3(+)CD4(+) regulatory T cells, evoking antitumor immune responses in humans. PNAS 110,
17945-17950 (2013).

0.79

51

Satoh, Takashi et al. Critical role of Trib1 in differentiation of tissue-resident M2-like macrophages. Nature 495, 524-528 (2013).

0.80

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Nishikawa, Keizo et al. DNA methyltransferase 3a regulates osteoclast differentiation by coupling to an S-adenosylmethionine-producing metabolic pathway. Nature Medicine 21,281-287 (2015).

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Katoh, Kazutaka & Frith, Martin C. Adding unaligned sequences into an existing alignment using MAFFT and LAST. Bioinformatics 28, 3144-3146 (2012).

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Saitoh, Tatsuya et al. Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. PNAS 106, 20842-20846 (2009).

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Shichita, Takashi et al. Peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain. Nature Medicine 18, 911-917 (2012).

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Nguyen, Nam Trung et al. Aryl hydrocarbon receptor negatively regulates dendritic cell immunogenicity via a kynurenine-dependent mechanism. PNAS 107, 19961-19966 (2010).

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Haldar, Malay et al. Heme-Mediated SPI-C Induction Promotes Monocyte Differentiation into Iron-Recycling Macrophages. Cell 156, 1223-1234 (2014).

1.00

InCites dataset updated 2016-02-26.
TOP 1% Articles: 57 - 5.4 times as many as average (based on theoretical value)
TOP 10% Articles: 289 - 2.7 times as many as average (based on theoretical value)

4

Organization

Immunology Group

Organization Chart

MEXT
WPI Initiative
Program Committee

JSPS

WPI Working Group

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

International

Imaging Group

Advisory Board

Director

Research Groups

Deputy Directors

Management
Committee

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

Informatics Group
Board ofRepresentatives

Administrative
Director

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

Units for Combined Research Fields
Accounting Section

Quantitative Immunology .................................................................... Yutaro Kumagai
Shunsuke Teraguchi
Diego Diez
Next Generation Optical Immune Imaging ................................... Noriko Takegahara
Kazuaki Tokunaga
Immuno-Genomics ................................................................................ Alexis Vandenbon
Hiromasa Morikawa

Administrative Department

Research PlanningandCommon Facilities
Osaka University

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

Domestic RIKEN Center for Integrative Medical Sciences
President

Administrative Bureau

6

National Institutes of Biomedical Innovation, Health and Nutrition

Cooperative
Institutions
Overseas

Pohang University of Science and Technology, Korea
Convergent Research Consortium for Immunologic Disease,
Seoul, St Mary's Hospital, Catholic University of Korea
Indian Institute of Science Education and Research, India
Maurice Wilkins Centre, The University of Auckland, New Zealand7

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

WPI Program Committee
Program Director
Toshio Kuroki

International Scientific Advisory Board
As of FY2015

The International Scientific Advisory Board conducts evaluations on scientific achievements of IFReC PIs by reviewing their reports
or direct interviews.

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

As of FY2015

Jeffrey Ravetch

The Rockefeller University, USA

Immunology

Christopher Goodnow

Garvan Institute of Medical Research, Australia
Australian National University

Immunology

Richard Locksley

University of California, San Francisco

Immunology

Anne O'Garra

The Francis Crick Institute, UK

Immunology

Lewis L. Lanier

University of California, San Francisco , USA

Immunology

Yale E. Goldman

University of Pennsylvania, USA

David Westhead

University of Leeds, UK

Bioinformatics

Vladimir Brusic

Boston University, USA

Bioinformatics

Mo Jamshidi

University of Texas, USA

Bioinformatics

Philip Chen

University of Macau, China

Bioinformatics

Deputy Program Director
Akira Ukawa

Deputy Director, RIKEN Advanced Institute for Computational Science, Japan

Program Committee Members
Toshiaki Ikoma

Special Adviser, Canon Inc., Japan[Chairperson]
Hiroo Imura

Honorary Chairman, Foundation for Biomedical Research and Innovation, Japan
Former President, Kyoto University, Japan

Hiroto Ishida

President Emeritus, Kanazawa Gakuin University, Japan

Tsutomu Kimura

Advisor, National Institution for Academic Degrees and University Evaluation, Japan

Makoto Kobayashi

Director, Research Center for Science Systems, Japan Society for the Promotion of Science
Nobel Laureate in Physics (2008)

Kiyoshi Kurokawa

Adjunct Professor, National Graduate Institute for Policy Studies, Japan

Ryozo Nagai

President, Jichi Medical University, Japan

Michiharu Nakamura

Counselor to the President, JST, Japan

Ryoji Noyori

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

Shinichiro Ohgaki

President, Japan Water Research Center

Robert Aymar

Senior Advisor, Atomic Energy Authority, France

Rita Cowell

Distinguished Professor, University of Maryland, USA

Richard Dasher

Consulting Professor, Stanford University, USA

Ian Halliday

Professor Emeritus, The University of Edinburgh, UK

Chuan Poh LIM

Chairman, Agency for Science, Technology and Research, Singapore

Imaging

The Selected Nine World Premier International Research Centers

Tohoku University
Advanced Institute for Materials Research

National Institute for Materials Science
International Center for Materials Nanoarchitectonics

The University of Tokyo
Kavli Institute for the Physics andMathematics of the Universe

Kyoto University
Institute for Integrated Cell-Material Sciences

Working Group Leader and Assigned Members

As of FY2015

University of Tsukuba
International Institute forIntegrative Sleep Medicine

Osaka University[Program Officer ]Takehiko Sasazuki

Institute for Advanced Study, Kyushu University, Japan

Hiroshi Kiyono

Institute of Medical Science, The University of Tokyo, Japan

Nagahiro Minato

Kyoto University, Japan

Kazuhiko Yamamoto

The University of Tokyo, Japan

Günter J. Hämmerling

German Cancer Research Center DKFZ, Germany

Hisataka Kobayashi

National Cancer Institute, NIH, USA

Philippe Kourilsky

The Institute of Pasteur, France

Immunology Frontier
Research CenterTokyo Institute of Technology
Earth-Life Science Institute

Nagoya University
Institute of Transformative Bio-Molecules

8

Kyusyu University
International Institute for Carbon-Neutral Energy Research9Symposia & Seminars

Administrative Office of IFReC

General Affairs Section
Employment /acceptance of researchers and staff procedures
Social insurance / employment insurance
・ Management of work hours
・ Procedures related to patents
・ Issuing various certificates
・ Procedures related to international students
・ Housing arrangements for researchers and staff
・ Support for international researchers
・・

Accounting Section
Budget drafting / implementation / management
Purchasing procedures
・ Acceptance and implementation of third-party funding
・ Payment of payroll, travel expense and honorarium
・ Health insurance procedures・ Management of buildings and assets
・ R I (Radio Isotope) procedures・・

Research Planning & Management Office
Research promotion and support( Consultation for grants and patents, fusion research program, etc.)
・ Establishing research environments( Facility and safety management, research agreement, etc.)
・ Fostering young scientists( Winter School, Dual Mentor program, etc.)
・ Organizing scientific events( Symposia, colloquia, seminars, etc.)
・ Public relations( Publishing, website, outreach to citizens, etc.)
・ WPI evaluation issues( Progress report, Advisory Board meeting, etc.)
・

10

Laboratories

Host Defense

Shizuo Akira, MD/PhD

Professor

Shizuo Akira■

Associate Professor

Kazuhiko Maeda■

Assistant Professor

Takashi Satohtion of various viruses, such as SINV, Ebola virus, Marburg virus,
MLV, HIV-1 and HBV, indicating that ZAP targets both positivesense and negative-sense RNA viruses, retroviruses and DNA viruses. On the other hand, ZAP does not suppress the replicationof VSV, a negative-sense RNA virus. ZAP inhibits the proteintranslation of viral components and viral RNA splicing and destabilizes viral RNA. These findings raise important questions. How
does ZAP sense a wide variety of viruses? How does ZAP specifically identify its target viruses? How does ZAP distinguish the different modes of anti-viral actions? In future studies, we will address these questions to better understand the anti-viral innate
immune response.

Kenta MaruyamaMikaël Martino

The immune system serves to defend against pathogens. Evolutionally conserved pattern-recognition receptors (PRRs) play a
key role in the innate immune response to microbes ranging
from bacteria to fungi, protozoa and viruses. After sensing microbial components, PRRs stimulate production of inflammatory
factors such as cytokines/interferons by dendritic cells to inducean acquired immune response, which is mediated by T cells and B
cells, resulting in elimination of invading microbes. However, aberrant activation of the immune responses often causes massive
inflammation, leading to the development of autoimmune diseases in our body. Therefore, both activation and inactivation of
immune responses must be strictly controlled. To gain a deeper
understanding of the immune system, we have examined theregulatory mechanisms of innate and acquired immune responses, and novel PRRs capable of sensing microbial components. We
focused on the role of zinc-finger antiviral protein (ZAP) against
viral infection.

1. Zinc-finger antiviral protein mediates retinoic acidinducible gene I-like receptor-independent antiviral response to murine leukemia virus
ZAP acts as an RNA sensor and induces the degradation of murine leukemia retrovirus (MLV) transcripts by the exosome, an
RNA degradation system, on RNA granules (Lee et al, PNAS, 2013).
The loss of ZAP greatly enhances the replication efficiency of
MLV. ZAP localizes to RNA granules, where the processing-body(P-body) proteins assemble. ZAP induces the recruitment of the
MLV transcripts and exosome components to the RNA granules.
12■

Postdoctoral Fellow

4■

Research Assistant

7■

Visiting Scientist

1■

Support Staff

6

3. Suppression of NLRP3-inflammasome formation by
resveratrol (Figure 1)
We have clarified the molecular mechanism of colchicine, adrug for gout attack. In response to Nigericin, monosodium urate,or silica particles, NLRP3 forms the inflammasome with its adaptor protein ASC and mediates innate immune responses. NLRP3inflammasome inducers cause aberrant mitochondrial homeostasis to reduce the NAD+ level, which in turn inactivates the
NAD+-dependent α-tubulin deacetylase Sirtuin 2 (SIRT2), resulting in accumulation of acetylated α-tubulin. Accumulated acetylated α-tubulin mediates ASC-NLRP3 contact to promote NLRP3inflammasome activation. Colchicine blocks the proximity of ASC
and NLRP3 by disrupting tubulin structure. We also showed thatresveratrol also suppresses NLRP3-inflammasome formation by
inhibiting acetylation of α-tubulin, and may be therapeuticallyuseful for treatment of gout (Misawa et al. Int Immunol 2015).

The CCCH-type zinc-finger domains of ZAP, which are RNA-binding motifs, mediate its localization to RNA granules and MLV transcripts degradation by the exosome. Thus, ZAP is the cytosolic
RNA-sensing PRR that induces elimination of intracellular RNA
viruses including MLV.

2. Role of zinc-finger anti-viral protein in host defense
against Sindbis virus
Accumulating evidence indicates that type I interferon (IFN)
mediates the host protective response to RNA viruses. However,
the antiviral effector molecules involved in this response havenot been fully identified. Here, we show that zinc-finger antiviralprotein (ZAP), an IFN-inducible gene, plays a critical role in theelimination of Sindbis virus (SINV) in vitro and in vivo. The loss of
ZAP greatly enhances the replication of SINV but does not inhibit
type I IFN production in primary mouse embryonic fibroblasts(MEFs). ZAP binds and destabilizes SINV RNA, thereby suppressing the replication of SINV. Type I IFN fails to suppress SINV replication in ZAP-deficient MEFs, whereas the ectopic expression of
ZAP is sufficient to suppress the replication of SINV in MEFs lacking the expression of type I IFN and the IFN-inducible genes. ZAPdeficient mice are highly susceptible to SINV infection, although
they produce sufficient amounts of type I IFN. Therefore, ZAP isan RNA-sensing antiviral effector molecule that mediates thetype-I-IFN-dependent host defense against SINV (Kozaki et al, Int
Immunol 2015). Although we have clarified the importance of
ZAP in anti-viral responses, we still do not fully understand how
ZAP eliminates the viruses. Indeed, ZAP suppresses the replica-

Figure 1.
Effect of resveratrol on NLRP3-inflammasome activation

Recent Publications■ Martino MM, Maruyama

K, Kuhn GA, Satoh T, Takeuchi O, Muller R, Akira
S. Inhibition of IL-1R1/MyD88 signalling promotes mesenchymal stem
cell-driven tissue regeneration. Nat. Commun. 7, 11051 (2016).

■ Yokogawa M, Tsushima T, Noda

NN, Kumeta H, Enokizono Y, Yamashita K,
Standley DM, Takeuchi O, Akira S, Inagaki F. Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions.
Sci. Rep. 6, 22324 (2016).

■ Mino T, Murakawa Y, Fukao A, Vandenbon A, Wessels HH, Ori D, Uehata T,

Tartey S, Akira S, Suzuki Y, Vinuesa CG, Ohler U, Standley DM, Landthaler
M, Fujiwara T, Takeuchi O. Regnase-1 and Roquin regulate a common element in inflammatory mRNAs by spatiotemporally distinct mechanisms.
Cell 161, 1058-1073 (2015).
■ Kozaki T, Takahama M, Misawa T, Matsuura Y, Akira S, Saitoh T. Role of zinc-finger anti-viral protein in host defense against Sindbis virus. Int. Immunol. 27, 357-364 (2015).

■ Misawa T, Saitoh T, Kozaki T, Park S, Takahama M, Akira S. Resveratrol in-hibits the acetylated alpha-tubulin-mediated assembly of the NLRP3-inflammasome. Int. Immunol. 27, 425-434 (2015).
13

Immunoglycobiology

Taroh Kinoshita, PhD■

Professor

Taroh Kinoshita

Associate Professor

Yusuke Maeda

Therefore, different mutations in the same PIGY gene caused
very different symptoms and thus expanded the phenotype of
IGDs. Five individuals from three families with biallelic loss-offunction PIGG mutations showed intellectual disability, early-onset seizures and hypotonia.

To date, pathogenic mutations that cause IGDs were identified
in 14 of 27 genes involved in biosynthesis, transfer to proteinsand maturation of GPI anchors.

Yoshiko Murakami
Yuko Tashima■

Assistant Professor■

Postdoctoral Fellow

1■

Research Assistant2■

Support Staff

3

Noriyuki Kanzawa

Figure 1.
The N-acetylgalactosamine (GalNAc)-containing side-branch of GPI anchors. Some GPI-anchoredproteins, such as prion proteins, Thy-1 and renal dipeptidases, have a GalNAc side-branch linked
to the first mannose. The GalNAc side-brunch can be elongated by galactose and sialic acid.

Glycosylphosphatidylinositol (GPI) is a glycolipid that acts as amembrane anchor of 150 or more human cell surface proteins.
Our laboratory aims to clarify the entire process of GPI-anchoredprotein (GPI-AP) biosynthesis, functional roles of lipid and glycan
moieties of GPI-anchors, and molecular bases of diseases associated with GPI-anchor deficiencies. We reported several new findings in 2015.

1. Identification of PGAP4 gene involved in attachment of a side-branch to GPI
A number of mammalian GPI-APs, such as prion proteins, Thy-1
and renal dipeptidase, have N-acetylgalactosamine (GalNAc)
side-branch attached to the first mannose (Figure 1). This sidebranch can be elongated by galactose and sialic acid. Biological
functions of the GalNAc side-branch had been unknown primarily because genes involved in generation of the side-branch had
not been identified. Taking advantage of a monoclonal antibody
that recognizes the GalNAc side-branch, we identified a genenecessary for transfer of GalNAc to GPI. The gene termed PGAP4(for Post GPI Attachment to Proteins 4) encoded a Golgi-resident
membrane protein that most likely is GalNAc transferase itself. In
order to clarify the biological significance of the GalNAc sidebranch, we are generating PGAP4 knockout mice.

14

2. Demonstration that endosome-to-trans Golgi network (TGN) retrograde transport system is criticalfor post-Golgi anterograde transport of GPI-APs
In the ER-to-plasma membrane anterograde transport of GPIAPs, ER-to Golgi transport is mediated by COPII coated transport
vesicles whereas mechanisms of post-Golgi transport have been
unclear. Aiming to clarify post-Golgi transport of GPI-APs, we developed a system for genome-wide screening of genes involved
in ER-to-plasma membrane transport of GPI-APs and identified
components of the Golgi-associated retrograde protein (GARP)
complex, a tethering factor involved in endosome-to-TGN transport. In GARP-defective cells, post-Golgi anterograde transport of
not only GPI-APs but also transmembrane proteins was impaired.
We concluded that GARP-dependent endosome-to-TGN retrograde transport is required for recycling of factors critical to efficient post-Golgi anterograde transport of GPI-APs (Hirata T et al.,Mol Biol Cell, 2015).

3. Characterization of new mutations that cause inherited GPI deficiencies (IGDs)
We reported the first cases of inherited GPI deficiency caused
by mutations in PIGY and PIGG genes in collaborations with domestic and international research groups (Ilkovsky B et al., Hum
Mol Genet, 2015; Makrythanasis P et al., Am J Hum Genet, 2016).
Two affected individuals with homozygous hypomorphic PIGY
mutations suffered from severe multi-system disease and early
death. Two individuals with homozygous PIGY promoter mutations showed moderate developmental delay and microcephaly.

Figure 2.
Mutations in two genes PIGY and PIGG (shown in red) involved in GPI anchor biosynthesis were
found to be causal to inherited GPI deficiency.

Recent Publications■ Hirata T, Fujita M, Nakamura S, Gotoh K, Motooka D, Murakami Y, Maeda Y,■ Wang Y, Murakami Y, Yasui T, Wakana S, Kikutani H, Kinoshita T, Maeda Y.

Kinoshita T. Post-Golgi anterograde transport requires GARP-dependentendosome-to-TGN retrograde transport. Mol. Biol. Cell 26, 3071-3084(2015).

Significance of GPI-anchored protein enrichment in lipid rafts for the control of autoimmunity. J. Biol. Chem. 288, 25490-25499 (2013).
■ Kanzawa N, Shimozawa N, Wanders RJA, Ikeda K, Murakami Y, Waterham■ Theiler R, Fujita M, Nagae M, Yamaguchi Y, Maeda Y, Kinoshita T. The alphahelical region in p24γ2 subunit of p24 cargo receptor is pivotal for therecognition and transport of glycosylphosphatidylinositol-anchored proteins. J. Biol. Chem. 289, 16835-16843 (2014).

HR, Mukai S, Fujita M, Maeda Y, Taguchi R, Fujiki Y, Kinoshita T. Defective
lipid remodeling of GPI anchors in peroxisomal disorders, Zellweger syndrome, and rhizomelic chondrodysplasia punctata. J. Lipid Res. 53, 653663 (2012).

■ Murakami Y, Tawamie H, Maeda Y, Buttner C, Buchert R, Radwan F, Schaffer

S, Sticht H, Aigner M, Reis A, Kinoshita T, Jamra RA. Null mutation in
PGAP1 impairs GPI-anchor maturation in patients with intellectual disability and encephalopathy. PLoS Genet. 10, e1004320 (2014).
15

Immunopathology

Atsushi Kumanogoh, MD/PhD

Our research team is involved in two approaches, that is, basic
and clinical immunology. For the basic aspects of our projects,
our proposed 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 axon-guidance
molecules that function during neuronal development. However,cumulative evidence indicates that semaphorins also participate
in immune responses, both physiological and pathological, andthey are now considered to be potential diagnostic and/or therapeutic targets for a range of diseases. Beyond such basic implications, we are trying to apply the findings from this proposed
study into the diagnosis and therapy of human immunologicaldisorders, such as autoimmunity, allergy, immune deficiency,
cancer/metastasis, and neurodegenerative diseases. We focus
here on the involvement of Sema4A in functions of CD8-positie T
cell responses.

Sema4A and CD8-positive T cells
Sema4A, a class IV transmembrane semaphorin, plays crucial
roles in dendritic cells (DCs), macrophages, and T cells. Sema4A ishighly expressed in polarized T helper type 1 (Th1) cells and isimportant for helper T-cell differentiation. In Sema4A-deficient
mice, Th1 responses to heat-killed Propionibacterium acne—a
Th1-inducing bacterium, are impaired in vivo. In contrast, T helper type 2 (Th2) responses against Nippostrongylus brasiliensis—a
Th2-inducing intestinal nematode, are potentiated in these mice.
TIM-2 appears to be the functional receptor for Sema4A, and the16

Professor

Atsushi Kumanogoh■

Assistant Professor

Shohei Koyama■

Support Staff

5

association between TIM-2 and Sema4A in Th1 cells negatively
regulates Th2 cells. A recent study revealed a novel function of
Sema4A associated with maintenance of regulatory T (Treg) cells.
Sema4A directly interacts with the receptor neuropilin-1 (Nrp-1)
expressed by Treg cells, and this interaction potentiates Treg-cellfunction and survival at inflammatory sites. In this pathway,
Sema4A ligation of Nrp-1 restrains Akt phosphorylation in the cellbody and at the immunologic synapse by recruiting phosphataseand tensin homologue (PTEN). This in turn promotes nuclear localization of the transcription factor Foxo3a, which plays an important role in the development and programming of Treg cells.
This Sema4A/Nrp-1-dependent pathway is crucial for maintenance of immune homeostasis, and is involved in inflammatory
colitis and Treg-cell stability in tumor tissues in vivo. These findings suggest that Sema4A is a potential target for therapies
aimed at limiting Treg-cell–mediated tumor-induced tolerance
without inducing autoimmunity. With respect to DC, Sema4A derived from both DCs and T cells is important for T cell–mediatedimmunity. Soluble Sema4A proteins and anti-Sema4A mAb
bound to Sema4A on the surface of DCs enhance T-cell activation. Consistent with this, DCs derived from Sema4A-deficient
mice stimulate allogeneic T cells poorly compared to wild-type
DCs. By contrast, when CD4+ T cells from Sema4A-deficient or
wild-type mice are cultured with allogeneic DCs derived fromwild-type mice, there are no differences in mixed lymphocyte reactions. These results suggest that DC-derived Sema4A is directly
and critically involved in the activation of T cells reactive to alloantigens on DCs.

In this study, we show that a class IV semaphorin, Sema4A,
regulates CD8+ T cell activation and differentiation through activation of mTOR complex (mTORC) 1. Sema4A deficient CD8+ T
cells exhibited impairments in production of IFN-γ and TNF-α andinduction of the effector molecules granzyme B, perforin, and
FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8+ T cell responses were significantly impaired in Sema4A deficient mice. Furthermore,
Sema4A deficient CD8+ T cells exhibited reduced mTORC1 activi-

ty and elevated mTORC2 activity, suggesting that Sema4A is required for optimal activation of mTORC1 in CD8+ T cells. IFN-γ
production and mTORC1 activity in Sema4A deficient CD8+ T cells
were restored by administration of recombinant Sema4A protein.
In addition, we show that plexin B2 is a functional receptor of
Sema4A in CD8+ T cells. Collectively, these results not only demonstrate the role of Sema4A in CD8+ T cells, but also reveal a novel link between a semaphorin and mTOR signaling.

Figure 1.

Figure 2.

■ Ito D, Nojima S, Nishide M, Okuno T, Takamatsu H, Kang S, Kimura T, Yoshida■ Kumanogoh A, Kikutani H. Immunological functions of the neuropilins

Y, Morimoto K, Maeda Y, Hosokawa T, Toyofuku T, Ohshima J, Kamimura D,
Yamamoto M, Murakami M, Morii E, Rakugi H, Isaka Y, Kumanogoh A.
mTOR complex signaling through the SEMA4A-Plexin B2 axis is requiredfor optimal activation and differentiation of CD8+ T Cells. J. Immunol. 195,934 (2015).

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

Recent Publications■ Hayashi M, Nakashima T, Taniguchi M, Kodama T, Kumanogoh A, Takayanagi

H. Osteoprotection by Semaphorin 3A. Nature 485, 69-74 (2012).

■ Yoshida Y, Ogata A, Kang S, Ebina K, Shi K, Nojima S, Kimura T, Ito D,■ Takamatsu H, Takegahara N, Nakagawa Y, Tomura M, Taniguchi M, Friedel

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

RH, Rayburn H, Tessier-Lavigne M, Yoshida Y, Okuno T, Mizui M, Kang S,
Nojima S, Tsujimura T, Nakatsuji Y, Katayama I, Toyofuku T, Kikutani H,
Kumanogoh A. Semaphorins guide the entry of dendritic cells into thelymphatics by activating myosin II. Nat. Immunol. 11, 594-600 (2010).

■ Nojima S, Toyofuku T, Kamao H, Ishigami C, Kaneko J, Okuno T, Takamatsu

H, Ito D, Kang S, Kimura T, Yoshida Y, Morimoto K, Maeda Y, Ogata A, Ikawa
M, Morii E, Aozasa K, Takagi J, Takahashi M, Kumanogoh A. A point mutation in Semaphorin 4A associates with defective endosomal sorting andcauses retinal degeneration. Nat. Commun. 4, 1406 (2013).
17

ImmunochemistryD) Misfolded proteins complexed with MHC class II
molecules are targets for autoimmune diseases

Hisashi Arase, MD/PhD

Professor

Hisashi Arase■

Associate Professor

Tadahiro Suenaga■

Assistant Professor

Masako Kohyama

MHC class II allelic polymorphisms are associated with susceptibility to many autoimmune diseases. However, it has remained
unclear how MHC class II molecules are involved in autoimmune
disease susceptibility. We found that cellular misfolded autoantigens are rescued from protein degradation by MHC class II molecules (Jiang et al. Int. Immunol. 2013). Furthermore, we foundthat misfolded proteins complexed with MHC class II molecules

are targets for autoantibodies in autoimmune disease patients(Jin et al. Proc. Natl. Acad. Sci. USA. 2014; Tanimura et al. Blood.
2015). Autoantibody binding to misfolded proteins transported
to the cell surface by MHC class II molecules was strongly correlated with susceptibility to autoimmune disease. This suggested
that misfolded proteins, which normally would not be exposed tothe immune system, can be targets for autoantibodies as ‘neo
self’ antigens, which is involved in the pathogenicity of autoimmune diseases (Arase Adv. Immunol. 2016)(Figure 2).

Kouyuki Hirayasu

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

A) Interaction between immune receptors and herpesviruses
PILRα is one of the immune inhibitory receptors that are expressed on various immune cells. We previously found that both
PILRα and PILRβ recognize CD99 as a host ligand (Shiratori et al. J.
Exp. Med. 2004). In addition, we have identified PANP as a newligand for PILR (Kogure et al. Biochem. Biophys. Res. Commun.
2011). PILRα is a unique receptor that that has binding sites forboth sugar chain and protein structure (Wang et al. J. Immunol.
2008; Kuroki et al. Proc. Natl. Acad. Sci. USA. 2014). We found that
PILRα associates with glycoprotein B (gB), an envelope protein of
herpes simplex virus-1 (HSV-1), and the interaction between
PILRα and gB is involved in membrane fusion during HSV-1 infection (Satoh et al. Cell 2008; Wang et al. J. Virol. 2009). We further
analyzed host cell molecules that associate with HSV-1 gB andfound that non-muscle myosin heavy chain (NMHC-IIA) associates with gB and is involved in HSV-1 infection (Arii et al. Nature2010). We also found that Siglec-4 (MAG, myelin associated glycoprotein) associates with VZV gB and mediates VZV infection.
Because Siglec-4 is specifically expressed in neural tissues, Si18■

Postdoctoral Fellow

4■

Research Assistant3

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

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

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

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

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

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

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).

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

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

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

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

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

Immune Regulation

Tadamitsu Kishimoto, MD/PhD

Tadamitsu Kishimoto■

Associate Professor

Barry Ripley■

Assistant Professor

Kazuya Masuda■

Postdoctoral Fellow

5■

Research Assistant3■

Support Staff

2

A Summary of our Recent Research
1. Arid5a regulates naïve CD4+ T cell-fate through selective stabilization of Stat3 mRNA (published)Thus, certain therapeutic properties of Thalidomide can be separated from its harmful side effects.

We recently demonstrated that Arid5a in T cells regulates thehalf-life of Stat3 mRNA in an IL-6-dependent manner. Reductionof STAT3 level in Arid5a-deficient T cells led to the imbalance of
STATs (STAT1, 3, 5) activation under Th17-polarizing conditions,which contributed to the alteration of the character of inflammatory T cells into that of anti-inflammatory T cells. Thus, Arid5a
plays an important role in T cells through post-transcriptional
control of inflammatory gene expression.

3. Regulation of expression levels of Arid5a in macrophages through TLR4 signaling (ongoing study)

2. Immunomodulatory drugs inhibit TLR4-induced
type-1 interferon production independently of
Cereblon via suppression of the TRIF/IRF3 pathway(published)
Here we developed Cereblon-deficient mice using the CRISPRCas9 system. TLR-induced cytokine responses were unaffectedby Cereblon deficiency in vivo. Moreover, IMiD treatment inhibited cytokine production even in the absence of Cereblon. The
IMiD-induced suppression of cytokine production therefore occurs independently of Cereblon in mice. Further investigation
revealed that IMiDs are potent inhibitors of TLR-induced type-1
interferon production via suppression of the TRIF/IRF3 pathway.
These data suggest that IMiDs may prove effective in the treatment of disorders characterized by the ectopic production of
type-1 interferon. Significantly, these properties are mediatedseparately from thalidomide’s teratogenic receptor, Cereblon.
20

Professor

We recently found that Arid5a protein is rapidly increased andthen quickly degraded in LPS-treated macrophages. Interestingly,
Arid5a protein is highly phosphorylated and ubiquitinated whenits protein levels reach peak maximum levels in macrophages following LPS stimulation. Our goal is to define precisely the regulatory pathways and the key proteins that control expression levelsof Arid5a and with respect to Regnase-1 expression levels in macrophages.

tected from tissue injury after LPS induced shock. We recently
found that Arid5a in T cells regulates production of IFN-γ throughstabilization of Tbx21 mRNA. Consistent with the data, IFN-γ protein level in serum was significantly reduced in LPS-treated Arid5a KO mice. We are also generating macrophage and T cell specific Arid5a KO mice to examine which cells are important forArid5a to augment endotoxin shock.

6. The analysis of Arid5a function using Arid5a-transgenic mice (ongoing study).

Figure 1.
T cell-fate decision by Stat3 stabilizing Arid5a.

Figure 2.
The anti-inflammatory of immunomodulatory drugs (IMiDS) result from
Cereblon dependent and independent pathways.

We recently generated Arid5a-transgenic mice using DNA pronuclear injection. The phenotype of Arid5a-transgenic miceseemed to be normal; however, cytokine levels such as IL-6 andTNF-α in serum were significantly elevated in LPS-treated Arid5atransgenic mice compared to WT mice. Similarly, IL-6 level wasincreased in peritoneal macrophages following LPS stimulation.
These results suggest that Arid5a promotes production of inflammatory cytokines in macrophages following LPS treatment. Currently we are investigating the mechanism of how Arid5a drives
pro-inflammatory cytokine productions through posttranscriptional modulation.

4. Arid5a deficiency protects against the developmentof bleomycin-induced pulmonary fibrosis (ongoingstudy)We have observed that Arid5a KO mice are highly resistant tobleomycin-induced lung injury-mediated mortality. Immunohistological data of lung tissue suggests that Arid5a deficiency could
protect mice from bleomycin-induced lung fibrosis which indicates the important role of Arid5a in lung tissue fibrosis.

Recent Publications■ Chinen I, Nakahama T, Kimura A, Nguyen NT, Takemori H, Kumagai A,■ Masuda K, Ripley B, Nyati KK, Dubey PK, Zaman MM, Hanieh H, Higa M,

Kayama H, Takeda K, Lee S, Hanieh H, Ripley B, Millrine D, Dubey PK, Nyati KK,
Fujii-Kuriyama Y, Chowdhury K, Kishimoto T. The aryl hydrocarbon receptor/microRNA-212/132 axis in T cells regulates IL-10 production to maintain intestinal homeostasis. Int. Immunol. 27(8), 405-415 (2015).

Yamashita K, Standley DM, Mashima T, Katahira M, Okamoto T, Matsuura Y,
Takeuchi O, Kishimoto T. Arid5a regulates naive CD4+ T cell fate throughselective stabilization of Stat3 mRNA. J. Exp. Med. 213(4), 605-619 (2016).
■ Masuda K, Kishimoto T. CD5: A new partner for IL-6. Immunity 44 (4), 720–

5. Pathogenic role of Arid5a in endotoxin shock (ongoing study)
Interestingly, Arid5a KO mice are found to be completely resistant to lipopolysaccharide mediated shock. H&E staining of lung,
liver and spleen tissue suggested that Arid5a KO mice are pro-■ Kang S, Tanaka T, Kishimoto T. Therapeutic uses of anti-interleukin-6 re-

722 (2016).

ceptor antibody. Int. Immunol. 27, 21-29 (2015).
■ Millrine D, Miyata H, Tei M, Dubey P, Kishan N, Nakahama T, Gemechu Y,

Ripley B, Kishimoto T. Immunomodulatory drugs inhibit TLR4 induced
type-1 interferon production independently of Cereblon via suppressionof the TRIF/IRF3 pathway. Int. Immunol. (2016).
21

Mucosal Immunology

Kiyoshi Takeda, MD/PhD

Professor

Kiyoshi Takeda■

Associate Professor

Eiji Umemoto■

Assistant Professor

Hisako Kayama
Shih-Han Tsai

The intestine is a unique organ, inhabited by a large number ofcommensal microbes. The intestinal mucosa is protected fromcommensal microbes and pathogenic microorganisms by several
types of barriers. One of these barriers is the mucus layers produced by intestinal secretory cells. Mucus is a viscous fluid enriched in mucin glycoproteins and the mucus layers play an indispensable role in the prevention of enteric pathogen infection andintestinal inflammation. Epithelial cells in the large intestine, wheretremendous numbers of bacteria exist, are covered by thick mucus composed of two layers: the inner, firm mucus layer and outer, loose mucus layer. Many commensal bacteria are present in
the outer mucus layer, whereas none are present in the inner mucus layer. The segregation of commensal bacteria and colonicepithelia by the inner mucus layer is critically involved in thepathogenesis of intestinal inflammation. Indeed, several mousemodels of spontaneous colitis show the disappearance of thebacteria-free zone in the inner mucus layer. In the small intestine,a variety of cationic anti-microbial peptides including α, β-defensinthat are produced by Paneth cell, and regenerating islet-derived
3 (Reg3) family proteins that are induced by pattern recognition
receptor signaling, mediate the segregation of intestinal bacteria
and epithelial cells. However, the large intestine contains no professional cells that produce anti-microbial peptides, althoughmany bacteria are present. Thus, the mechanism by which mucus
prevents bacterial access to the epithelial surface of the large intestine is unknown.

22■

Postdoctoral Fellow

2■

Research Assistant5■

Support Staff

3

ministration induced severe intestinal inflammation with highmortality in Lypd8-/- mice. Oral administration of gentamicin,
which is active for flagellated bacteria such as Proteus, Escherichia
and Helicobacter, reduced the numbers of flagellated bacteria in
the intestine and restored the bacteria-free zone just above thecolonic epithelia in Lypd8-/- mice. Furthermore, gentamicin treatment ameliorated the intestinal inflammation in DSS-treated
Lypd8-/- mice. Thus, invasion of the epithelial layer by flagellated
bacteria such as Proteus, Escherichia and Helicobacter correlateswith the increased sensitivity to intestinal inflammation in Lypd8-/mice.

Lypd8 binds to bacterial flagella and thereby suppresses their motility
We next addressed the mechanisms by which Lypd8 inhibits
bacterial invasion of the epithelial layer. Flow cytometric analysis

revealed that Lypd8 binds to a subpopulation of the intestinalbacteria, and further showed that Lypd8 preferentially binds toflagellated bacteria such as Proteus and Escherichia. Scanningelectron microscopic analysis of Proteus mirabilis incubated withrecombinant Lypd8 showed that Lypd8 binds to flagella of P. mirabilis. We next investigated whether Lypd8 affects motility offlagellated bacteria. Lypd8 expressed on Caco-2 cells, human colonic epithelial cell line suppressed swarming motility of P. mirabilis. Furthermore, swimming motility of P. mirabilis and commensal E. coli was inhibited in semi-agar plate including
recombinant Lypd8 protein. Thus, highly glycosylated Lypd8 associated and suppressed the motility of P. mirabilis.
Taken together, these findings suggest that Lypd8 mediates
segregation of commensal flagellated bacteria from the intestinal epithelial layer in the large intestine, and thereby contributes
to the maintenance of gut homeostasis.

Ly6/Plaur domain containing 8 (Lypd8) segregates intestinal bacteria and colonic epithelia
Ly6/Plaur domain containing 8 (Lypd8) is a member of Ly6/
Plaur family proteins characterized by the presence of Ly6/Plaur
domain. We found that Lypd8 was a highly glycosylated GPI-anchored protein, highly and selectively expressed in epithelial cellsof the colon and constitutively secreted into the intestinal lumen.
We generated Lypd8-deficient mice to analyze the function of
Lypd8 in vivo. In wild-type mice, there is a bacteria-free space justabove the colonic epithelia, however this free space was not present, and many bacteria invaded the inner mucus layer and thecolonic epithelia in Lypd8-/- mice. We next examined which kindsof bacteria invaded the inner mucus layer and the colonic epithelia by analyzing bacterial DNA in the colonic tissue of Lypd8-/mice. Several bacterial genera were increased in Lypd8-/- colonictissues, particularly Proteus, Escherichia and Helicobacter weretremendously increased. Thus, intestinal bacteria such as Proteus,
Helicobacter and Escherichia, all of which are Gram-negative rod
bacteria possessing multiple flagella, penetrated the inner mucus
layer and were present adjacent to the epithelial cell layers of
Lypd8-/- mice.

Lypd8-deficient mice are highly susceptible to intestinal inflammation
Proteus and Helicobacter are involved in the pathogenesis of
inflammatory bowel diseases in both mice and humans. Therefore, we next analyzed the sensitivity to dextran sulfate sodium(DSS)-induced intestinal inflammation in Lypd8-/- mice. DSS ad-

Figure. Lypd8 maintains gut homeostasis by segregating intestinal bacteria and colonic epithelia
Lypd8 is a highly glycosylated GPI-anchored protein, and highly and selectively expressed in epithelial
cells on the uppermost layer of the intestinal gland and shed into the intestinal lumen. Lypd8 inhibits
bacterial invasion of the colonic mucosa by binding to the intestinal bacteria, and thereby regulates
the intestinal inflammation.

Recent Publications■ Okumura R, Kurakawa T, Nakano T, Kayama H, Kinoshita M, Motooka D,■ Ogino T, Nishimura J, Barman S, Kayama H, Uematsu S, Haraguchi N, Uemura

Gotoh K, Kimura T, Kamiyama N, Kusu T, Ueda Y, Wu H, Iijima H, Barman S,
Osawa H, Matsuno H, Nishimura J, Ohba Y, Nakamura S, Iida T, Yamamoto
M, Umemoto E, Sano K and Takeda K. Lypd8 promotes the segregation offlagellated microbiota and colonic epithelia. Nature 532, 117-121 (2016).

M, Hata T, Takemasa I, Mizushima T, Yamamoto H, Takeda K, Doki Y andMori M. Increased Th17-inducing activity of CD14(+) CD163(low) myeloid
cells in intestinal lamina propria of patients with Crohn's disease. Gastroenterol. 145, 1380-1391 (2013).

■ Tsai SH, Kinoshita M, Kusu T, Kayama H, Okumura R, Ikeda K, Shimada Y,■ Kayama H, Ueda Y, Sawa Y, Jeon SG, Ma JS, Okumura R, Kubo A, Ishii M,

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

Okazaki T, Murakami M, Yamamoto M, Yagita H and Takeda K. IntestinalCX3C chemokine receptor 1high (CX3CR1high) myeloid cells prevent T
cell-dependent colitis. Proc. Natl. Acad. Sci. USA. 109, 5010-5015 (2012).

■ Masahata K, Umemoto E, Kayama H, Kotani M, Nakamura S, Kurakawa T,

Kikuta J, Gotoh K, Motooka D, Sato S, Higuchi T, Baba Y, Kurosaki T,
Kinoshita M, Shimada Y, Kimura T, Okumura R, Takeda A, Tajima M, Yoshie
O, Fukuzawa M, Kiyono H, Fagarasan S, Iida T, Ishii M and Takeda K. Generation of colonic IgA-secreting cells in the cecal patch. Nat. Commun. 5,3704 (2014).
23

Molecular Immunology

Hitoshi Kikutani, MD/PhD

Professor

Hitoshi Kikutani■

Associate Professor

Teruhito Yasui■

Assistant Professor

Shuhei Sakakibara■

Postdoctoral Fellow

1■

Research Assistant2

1) Molecular mechanisms in immunopathology causedby host-pathogen interaction: Epstein-Barr virus(EBV)-encoded latent membrane proteins (LMP) 1
and 2a affect B cell survival, selection and differentiation
EBV infects memory B cells for persistent infection. Although it
has been shown that EBV-encoded LMP1 and 2a constitutively
activate the CD40 and BCR signals, respectively, effects of theseviral proteins on the humoral immune responses remains largely
unclear. In our laboratory, we generated conditional transgenic
mice for EBV LMP1 or 2a to evaluate their function in vivo.
Our conditional LMP2a Tg mice exhibited impaired antigenspecific antibody production after immunization. In the spleen of
LMP2a Tg mice, normal germinal center (GC) formation was observed whereas antigen-specific GC B cells were fewer at two
weeks after immunization (Figure 1). In addition, plasma cell differentiation was significantly accelerated in LMP2a Tg mice.
These results indicate that EBV LMP2a reduced the threshold forselection of high affinity B cells, which may contribute to the latent infection of EBV in memory B cells.

Unlike LMP2a, expression of LMP1 in B cells strongly inhibited
GC formation. Interestingly, GC formation and antibody response
were also impaired in chimera mice co-transferred with LMP1 Tg
and wild-type-derived bone marrow cells, suggesting that LMP1+
B cells provide inhibitory signals to neighboring wild type cells.
Thus, LMP1 may contribute to EBV infection by suppressing hosthumoral responses.

Figure 1. EBV LMP2A reduces the threshold for selection of high-affinity B cells.
(A) The conditional expression of LMP2a by the AID (activation-induced
cytidine deaminase) promoter-driven Cre did not alter GC size and structurein the spleen upon immunization. (B) Generation of antigen-binding B cells
was extremely impaired in the spleen of LMP2a Tg mice after NP-CGG/alum
immunization.

Figure 2. Crystal structure of the ligand-bound anti-DNA antibody,clone 71F12 derived from an acute SLE patient.
Fab of 71F12 and oligonucleotides (5’-TTTTT-3’) were co-crystalized (a)and (b). In the crystal, electron density of 3 thymines were determined.
71F12 interacted with the third thymine base (T3) through base-stacking
interaction, for which the conserved mutation Y33F we found in NGS
analysis was required (b).

2) Phylogenetic and structural bases for anti-nuclear
antibody generation in acute systemic lupus erythematosus

Performing deep sequencing for the immunoglobulin variable
region of representative clones, we found that there were many
sequences belonging to the ANA lineages, which share several
mutated nucleotides, in blood from acute patients. Phylogenetic
analysis demonstrated that autoreactive B cells underwent affinity maturation to self-antigens.

SLE is a refractory disease characterized by high-titer of serum
IgG autoantibodies that are reactive to nuclear antigens such as
DNA, histone, RNP and others. Despite extensive studies, themechanisms for development of autoreactive B cells remain to beelucidated.
Anti-nuclear antibodies (ANAs) are one of the diagnostic markers for SLE and are also considered a pathogenic factor for thisdisease. We isolated several monoclonal ANAs from acute SLE
patients and characterized their properties. We found that most
ANAs were antigen-specific and their reactivity to respective antigens was highly dependent on somatic hypermutation (SHM).

The crystal structure of the anti-DNA antibody 71F12 that exhibited high-affinity to both ds- and ssDNA, revealed that somatic
mutation contributed to rigid antigen recognition largely throughinteraction with a nucleobase, but not with ribose or phosphodiester of DNA. Thus, we proposed that at least a portion of highaffinity anti-ssDNA antibodies could target nucleobases of unfolded dsDNA. The structure analysis also supports affinity
maturation of anti-DNA lineage in SLE.

Recent Publications■ Minamitani T, Yasui T, Ma Y, Zhou H, Okuzaki D, Tsai CY, Sakakibara S,

Gewurz BE, Kieff E, Kikutani H. Evasion of affinity-based selection in germinal centers by Epstein-Barr virus LMP2A. Proc. Natl. Acad. Sci. USA.
112(37), 11612-11617 (2015).

24

25

Experimental Immunology

Shimon Sakaguchi, MD/PhD■

Professor

Shimon Sakaguchi

Assistant Professor

Wing James Badger

better prognosis than those with predominant FOXP3hi Treg-cell
infiltration. Development of such inflammatory FOXP3lo non-Treg
cells appeared to depend on tissue secretion of IL-12 and TGF-β,
and was correlated with tumor invasion of intestinal bacteria, especially Fusobacterium nucleatum. Our results indicate that functionally distinct subpopulations of tumor-infiltrating FOXP3+ T

cells contribute in opposing ways to determining CRC prognosis.
Depletion of FOXP3hi Treg cells from tumor tissues to augment
tumor immunity can be an effective treatment of CRCs and othercancers, while strategies to locally increase FOXP3lo non-Treg
cells could be tumor-suppressive and -preventive.

Norihisa Mikami
Noriko Sakaguchi
Kenji Ichiyama

This laboratory studies: (i) the cellular and molecular basis of
immunologic self-tolerance, in particular the roles of regulatory T
cells; (ii) the strategy for eliciting effective immune responses to
autologous tumor cells, or inducing immunologic tolerance toorgan transplants, by manipulating the mechanism of immunologic self-tolerance; and (iii) the cause and pathogenetic mechanism of systemic autoimmune diseases, such as rheumatoid arthritis, by utilizing an animal model established in our laboratory.
One aspect of immunologic self-tolerance (i.e., immunologicalunresponsiveness of the normal immune system to normal selfconstituents) is actively maintained through a T cell-mediateddominant control of self-reactive T cells by naturally occurring
regulatory CD4+ T cells (Tregs). Yet how Treg cells effectively control potentially hazardous self-reactive T cells in humans remains
an open question. In the previous year, we showed that Treg cells
were able to render self-reactive human CD8+ T cells anergic (i.e.,
hypo-proliferative and cytokine hypo-producing upon antigen
re-stimulation) in vitro, likely by controlling the co-stimulatory
function of antigen-presenting cells, in particular, the expressionof CD80 and CD86, via CTLA-4 expressed by Treg cells. This year,
in collaboration with Kajsa Wing’s group in Karolinska Institute,
Sweden, we have addressed the role of CTLA-4 in immune regulation by conditionally deleting CTLA-4 expression in Treg cells
for a limited period in adult mice (Klocke et al., PNAS, 2016). Suchmice spontaneously developed fatal systemic autoimmune disease. Together with our previous findings with congenital Tregspecific CTLA-4 deficiency in mice and CTLA-4 gene mutations inhumans (Wing et al., Science 2008; Yamaguchi et al., PNAS, 2013;
26■

Postdoctoral Fellow

3■

Research Assistant4■

Support Staff

6

Wing et al., Immunity, 2014; Schubert et al., Nature Med., 2014),
the results indicate that CTLA-4-mediated control of self-reactive
T cells by Treg cells is indispensable for maintaining self-tolerance, and can be a key target in controlling autoimmunity andtumor immunity.
In our study of FOXP3+ CD25+ CD4+ T cells in humans, we havepreviously shown that they are heterogeneous in function anddifferentiation status, including suppressive or non-suppressive
cells as well as resting or activated Treg cells (Miyara et al., Immunity, 2009). Our attempts to further differentiate suppressive
FOXP3+ T cells from non-suppressive FOXP3+ T cells have revealed
that CD15s (Sialyl Lewis x) is expressed by activated or effector
FOXP3+ Treg cells but not by non-suppressive FOXP3+ T cells, enabling reliable distinction of the two populations (Miyara et al.,
PNAS, 2015). This year we have also analyzed tumor-infiltrating
FOXP3+ T cells, in particular, how FOXP3+ T cell subpopulations
contribute to tumor immunity (Saito et al., Nat. Med., 2016). In
general, abundant Treg-cell infiltration into tumors is associatedwith poor clinical outcomes in various types of cancers. However,
the role of Treg cells is controversial in colorectal cancers (CRCs),in which FOXP3+ T-cell infiltration indicated better prognosis insome studies. We have shown that CRCs, which are commonly
infiltrated by suppression-competent FOXP3hi Treg cells, can beclassified into two types by the degree of additional infiltration of
FOXP3lo non-suppressive T cells. The latter, distinguished from
FOXP3+ Treg cells by being CD45RA– and unstable in FOXP3 expression, secreted inflammatory cytokines. Indeed, CRCs with
abundant infiltration of the FOXP3lo T cells showed significantly

Figure 1. FOXP3+ T-cell subsets in colorectal cancers (CRCs). (A) In half of CRCs, tumor-infiltrating
FOXP3+ T cells are predominantly effector or activated Treg cells, which are FOXP3high CD45RA- (Fr.
II); in the other half of CRCs, tumor-infiltrating FOXP3+ T cells contain FOXP3lowCD45RA- non-Treg
cells (Fr. III) in addition to Fr. II FOXP3high effector Treg cells. The former type CRCs, designated as
Type A, show poor prognosis when FOXP3 gene transcription by tumor tissues is high because thehigh expression is due to predominant presence of FOXP3high Treg cells. In contrast, Type B CRCs
show better prognosis when FOXP3 gene transcription by cancer tissues is high, because non-Treg
cells, which produce inflammatory cytokines, contribute to the high transcription. (B) Predominant
infiltration of FOXP3low non-Treg cells in Type B CRCs can be attributed to tumor infiltration ofintestinal bacteria, in particular, Fusobacteria.

Recent Publications■ Morikawa H, Ohkura N, Vandenbon A, Itoh M, Nago-Sato S, Kawaji H, Lassmann T,■ Wing JB, Ise W, Kurosaki T, & Sakaguchi S. Regulatory T-cells control anti-

Carninci P, Hayashizaki Y, Forrest A, Standley D, Date D, Sakaguchi S, & the
FANTOM consortium. Differential roles of epigenetic changes and Foxp3
expression in regulatory T cell-specific transcriptional regulation. Proc.
Natl. Acad. Sci. USA. 111, 5289-5294 (2014).

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

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

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

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

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

■ Ito Y, Hashimoto M, Hirota K, Ohkura N, Morikawa H, Nishikawa H, Tanaka A,

Furu M, Ito H, Fujii T, Nomura T, Yamazaki S, Morita A, Vignali DA, Kappler
JW, Matsuda S, Mimori T, Sakaguchi N, & Sakaguchi S. Detection of T-cell
responses to a ubiquitous cellular protein in autoimmune disease. Science 346, 363-368 (2014).
27

Cell Signaling

Takashi Saito, PhD■

T cells play critical roles in initiating and regulating immune
responses. Deregulation of T cell activation and function leads tovarious immune diseases. We have analyzed the molecular mechanism of T cell activation upon antigen recognition and the subsequent homeostasis and differentiation of effector T cells. After
we found that initial T cell activation is induced in TCR microclusters, which are generated by accumulation of signaling molecules
critical for activation, we analyzed the activation regulation in therelationship with other signals such as co-stimulation and innate
signaling and cytoskeletal regulation.

[1] Regulation of activation signal at immune synapse
T cell activation is induced through TCR microclusters, which
recruit TCR, kinase and adaptors and initiate activation signals.
During analyses of dynamic movement of cytoplasmic molecules
ZAP70 and SLP76, we found that SLP76 made clear clusters uponvery weak stimulation while ZAP70 did not, and that SLP76 clusters were generated by outside-in signal through LFA1. Each TCRMC was bordered by adhesion molecules; LFA-1, paxillin, Pyk2
and vinculin at initial phase. Because the centralized TCR-MC withsurrounded adhesion ring resembles the immune synapse in micro-scale, we named the structure “microsynapse”. TCR-MCs,
ZAP70, SLP76 and F-actin were detected at the center, and LFA1,
Paxillin, Pyk2 are in the outside ring of the microsynapse. Downmodulation of microsynapse resulted in the reduction of the sizeof TCR-MC and clustering of F-actin and SLP76, consequently, decreasing pErk, IL-2 production and cell growth. TCR activation,
particularly weak stimulation, was supported by the microsyn28

Professor

Takashi Saito

Postdoctoral Fellow

1

how the signaling events trigger T cells upon self-recognition has
not been analyzed. We investigated the mechanism of semi-activation and its status of T cells by self-recognition. We show that
self-recognition could trigger multiple signaling pathways downstream of TCR including enhanced phosphorylation of p38, Erk
and S6, and most remarkably the calcium-calcineurin-NFAT pathway was activated leading to the accumulation of NFAT in the

nuclei. Chip-Seq analysis shows that NFAT activated by self-recognition can bind to genes such as Egr that play a critical role in
the activation and function of T cells. Indeed, transgenic Egr expression rescued IL-2 response in T cells after loss of self-recognition. This may partly represent the status of semi-activation of T
cells upon self-recognition in vivo.

apse. The microsynapse is essential for the generation of mature
TCR-MCs and the following events of cell adhesion and T cell activation.

[2] Regulation of CD4 CTL development
We have cloned CRTAM as an early activation molecule predominantly expressed on CD8 T cells and NK cells upon activation. We demonstrated that CRTAM is an early activation marker
and an adhesion receptor, and is bound to its ligand Necl-2. We
found that a small proportion of CD4+T cells also express CRTAM.
CRTAM+CD4 T cells possess mRNA expression profiles of both
CD4+ and CD8+ T cells, that is, they express genes in cytotoxic T
cells (CTL) such as perforin, granzyme B, Eomesdermin and IFNγ.
CRTAM+CD4 T cells but not CRTAM-CD4 T cells exclusively secrete
IFNγ, and exhibit cytotoxicity, indicating that CRTAM+ CD4 T cells
confer generation of CD4+ CTLs. CRTAM induces the expressionof Eomes and CTL-related genes through intracellular signalingof CRTAM, which induce CTL lineage T cells. Thus, CRTAM is also
the first surface marker for CD4+CTL, and can be used for therapeutic targets for inflammatory diseases where CD4+ CTLs seems
to induce the diseases. (Figure 1)

Figure 1. CRTAM determines CD4+CTL lineage[A] CD4+CTLs are generated from CRTAM-expressing CD4+T cells. CRTAM+ but not CRTAMCD4+T cells develop CD4+T cells with CTL function, which may function in protecting infection
and cancer, and in inducing inflammation depending on circumstances.
[B] CRTAM signals induce development of CD4+CTL. Full-length CRTAM (FL)- but tail-less
CRTAM (TL)-expressing T cells induce CD4+CTL exhibiting cytotoxicity.

Recent Publications■ Hashimoto-Tane A, et al. Micro adhesion rings surrounding TCR microclu-[3] T cell activation regulation by self-recognition
It has been suggested that T cells recognize self-antigen. T cells
are semi-activated by self-recognition through continuous contact with APC presenting self-antigens, which subsequently enhance T cells responsiveness to foreign antigen, while T cells become anergic in the absence of the semi-activation. However,sters are essential for T cell activation. J. Exp. Med. (2016) in press.
■ Takeuchi A, et al. CRTAM determines the CD4+ cytotoxic T lymphocyte

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

■ Kong KF, et al. Protein kinase C-η controls CTLA-4-mediated regulatory T

cell function. Nat. Immunol. 15, 465–72 (2014).
■ Imanishi T, et al. Nucleic acid sensing by T cells initiates Th2 cell differen-

tiation. Nat. Commun. 5, 3566 (2015).

■ Hara H, et al. Clustering of CARMA1 through SH3-GUK domain interac-tions is required for its activation of NF-kB signaling. Nat. Commun. 6,5555 (2015).
29

Lymphocyte Differentiation

Tomohiro Kurosaki, MD/PhD■

Professor

Tomohiro Kurosaki

Associate Professor

Yoshihiro Baba

wide variety of genetic perturbations have shown that, overall,distortions in the affinity composition of GC B cells are indistinguishable from those in memory B cells, suggesting that memory
B cells are selected stochastically from among GC B cells. The alternative “instructive” model postulates that selection into thememory B cells is regulated by a large number of means, such as
extrinsic signals from T cells, including cytokines and cell-contact-dependent signals, and affinity-based signals through the
BCR.
To address the selection and generation mechanisms of memory B cells, we established a mouse model for marking GC B cells

and their progeny within a given time window. Using these mice,
we found that LZ GC B cells with BCRs of lower affinity had higher
expression of the transcriptional repressor Bach2 than those with
BCRs of higher affinity and were predisposed to enter the memory B cell pool. Moreover, haploinsufficiency of Bach2 resulted insuppression of the generation of memory B cells and Bach2 expression in GC cells was inversely correlated with the strength of
T cell help. Together, we propose a model in which weak help
from T cells maintains relatively high expression of Bach2 in LZ
GC B cells, which predisposes them to enter the memory B cellpool (Figure).

Wataru Ise■

Assistant Professor

Takeshi Inoue
Ryo Shinnakasu
Shinya Tanaka■

Research Assistant1■

Support Staff

3

Introduction
In the case of T cell-dependent humoral immune responses,
after initial T-B cell interactions, B cells undergo brisk proliferation
before entering the germinal center (GC) reaction. Once a GC isestablished in the B cell follicle, it develops a dark zone (DZ) and alight zone (LZ) and B cells migrate between these two zones. Proliferation and somatic hypermutation (SHM) occur in the DZ followed by B cell shuttling to the LZ, where they exit the cell cycle.
Because antigen-presenting follicular dendritic cells (FDCs) andfollicular helper T (TFH) cells reside in the LZ, GC B cells expressing
high-affinity BCRs in the LZ are selected in response to signals
provided by cognate TFH cells. Then, as cells transition from the LZ
to DZ state, proliferation is induced. Such iterative cycles of diversification and selection are thought to be a driving force for subsequent affinity maturation. With successful selection, the LZ GC
B cells, in addition to returning to the DZ, can enter into two terminal fates, memory B cells or plasma cells. Our laboratory has
focused on understanding the cellular and molecular basis underlying fate decisions of LZ GC cells (back to the DZ GC, differentiation into memory B cells, or plasma cells).

Regulation of GC B cell proliferation by transcription
factor Foxo1
Foxo1, forkhead box protein O1, is a transcriptional regulator
that localizes to the nucleus and controls gene expression. Phosphorylation by Akt causes Foxo1 translocation to the cytoplasm
where it can be degraded. Foxo1 is perhaps best known for its
ability to promote cellular quiescence, thus phosphatidyl inositol
30

3’OH kinase (PI3K)-mediated activation of Akt and subsequent
inactivation of Foxo1 is necessary for pre-B cell proliferation.
However, the role of Foxo1 in mature B cells, particularly GC B
cells, remained obscure. A strong hint that Foxo1 might play a
critical and unconventional role in controlling gene expression
within GC B cells has been provided by the discovery that GCderived lymphomas frequently carry mutations in Foxo1 that prevent its inactivation by Akt.
To address Foxo1 function in GC biology, we used inducible
gene knockout of Foxo1. We found that Foxo1 exerts a proliferative role, rather than promoting quiescence as previously
thought. Mechanistically, upon sufficient provision of T cells help,
Foxo1-/- LZ GC cells could not induce transcription factors, c-Myc
and BATF, both of which are essential for GC B cell proliferation.
Hence, Foxo1 is required for stimulating GC cell division, explaining the previous observations that GC-derived lymphoma frequently possesses a constitutively active form of Foxo1. Moreover, we found that the regulatory network through Foxo1 ishighly context-specific (anti-proliferative in pre-B cells versus
proliferative in GC B cells).

Selection mechanisms of GC cells into the memory
compartment
One key unresolved question about the cell-fate decisions
made in the GC is how memory B cells are selected and generated. Two models, stochastic and instructive, have been put forward to explain the memory-selection mechanism. Studies of a

Figure . Mechanism of selection of memory B cells and plasma cells from germinal center B cells
The affinity of B cell receptor for antigen and the expression level of Bach2 are inversely correlated
in germinal center B cells. Low affinity germinal center B cells, which receive weaker stimulationfrom antigen and antigen specific CD4 T cells, maintain a relatively high level of Bach2 expression
and tend to be induced to form memory B cells in the germinal center. On the other hand, highaffinity cells, which receive strong stimulation from antigen and antigen specific CD4 T cells, havedifficulty maintaining Bach2 expression at high levels and tend to be induced to form plasmablasts(plasma cells).

Recent Publications■ Shinnakasu R, Inoue T, Kometani K, Moriyama S, Adachi Y, Nakayama M,

Takahashi Y, Fukuyama H, Okada T, Kurosaki T. Regulated selection of germinal center cells into the memory B cell compartment. Nat. Immunol.
(2016) in press.
■ Inoue T, Morita M, Hijikata A, Fukuda-Yuzawa Y, Adachi S, Isono K, Ikawa T,

Kawamoto H, Koseki H, Natsume T, Fukao T, Ohara O, Yamamoto T, Kurosaki T. CNOT3 contributes to early B cell development by controlling Ighrearrangement and p53 mRNA stability. J Exp. Med. 212, 1465-1479(2015).

■ Kurosaki T, Kometani K, Ise W. Memory B cells. Nat. Rev. Immunol. 15, 149-

159 (2015).
■ Matsumoto M, Baba A, Yokota T, Nishikawa H, Ohkawa Y, Kayama H, Kallies A,

Nutt SL, Sakaguchi S, Takeda K, Kurosaki T, Baba Y. Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation.
Immunity 41, 1040-1051 (2014).

■ Uebelhart R, Hug E, Bach MP, Wossning T, Duhren-von Minden M, Horn

AHC, Kometani K, Kurosaki T, Sticht H, Reth M, Jumaa H. Responsivenessof B cells is regulated by the hinge region of IgD. Nat. Immunol. 16, 534543 (2015).
31

Malaria Immunology

Cevayir Coban, MD/PhD

In general, our group is interested in host-pathogen interactions. Our infection model is malaria caused by Plasmodium parasites. We have several reasons to study this pathogen. First of all,malaria disease continues to be a global burden by killing millions every year due to its complications such as cerebral malaria.
Yet there is no fully potent drug and/or vaccine against this disease (WHO Report, 2015). Second, Plasmodium parasites are sodiverse that each Plasmodium spp. causes different disease outcomes in humans as well as in mice and we still know very little
about the reasons.
Death from malaria occurs due to organ-specific immunopathology caused by parasites; however, detailed understanding of
this immunopathology is lacking. Therefore, our recent focus is tounderstand how immunopathology is caused by Plasmodium
parasites at the tissue and cell levels and transfer this information
into treatment modalities. For example, we recently delineatedthe role of Lipocalin-2, a host protein with multiple cellular functions including controlling iron metabolism, during malaria infection (Cell Host Microbe, 2012). We concluded that Lipocalin 2 isone of the key molecules of the host secreted against Plasmodium parasites.
One of the topics we investigate is the pathology of cerebral
malaria. Cerebral malaria is one of the deadliest complications of
P. falciparum infection in humans. Early diagnosis of cerebral malaria is not easy due to non-specific symptoms, but is very important to initiate effective adjunct therapies which can save lives.
Therefore, early, quick, and cheap diagnosis of cerebral malaria
has been the target of investigation in patients and in animal
32

Professor

Cevayir Coban■

Postdoctoral Fellow

2■

Research Assistant1■

Support Staff

2models. We’ve recently reported a new understanding of cerebral malaria pathogenesis by using cutting-edge imaging technologies such as ultra-high field MRI and multi-photon live imaging microscopy during experimental cerebral malaria. Deep
investigation of the brain by ultra-high field MRI showed that theolfactory bulb is physically and functionally damaged by Plasmodium parasites. Live multiphoton imaging of the olfactory bulb
confirmed that the complex trabecular small capillaries comprising the olfactory bulb show parasite accumulation and cell occlusion followed by microbleeding, events associated with high fever and cytokine storm. With these findings, we have provided
evidence that olfactory functional impairment (loss of smell)
could be a valuable early diagnosis marker for cerebral malaria(Cell Host Microbe, 2014).
The final goal of our research is the translation of our understanding of host-pathogen interactions into vaccines or drugs totreat disease. Given that there is still more to do for the development of potent vaccines against malaria, we focus on the adjuvants, because if rationally designed, adjuvants improve vaccine
efficacy (J Immunology, 2015; PNAS, 2014). “Rational adjuvants”
are the key for better and controllable immune responses. In recent years, we’ve developed a new adjuvant called synthetic hemozoin, a synthetic analog of Plasmodium-produced hemozoin,
and completed its preliminary GLP non-clinical safety and toxicology studies in several animals and infection models (Vaccine,2016). These studies performed according to GLP procedures inrats concluded that synthetic hemozoin is a safe adjuvant anddisplays very low autoimmune properties.

Figure.
Blood stage Plasmodium infection causes multiple and deadly complications, although easily treated if diagnosed early.
However, high prevalence of drug resistance and the lack of successful vaccines are the drawbacks to efforts in the eliminationof this disease. Therefore additional treatment modalities should be investigated to prevent and treat complications andprevent death caused by malaria. Our approach to this problem is to use cutting edge imaging technologies to understandimmunopathology caused by Plasmodium parasites.

Recent Publications■ Lee MSJ. et al. Current status of synthetic hemozoin adjuvant: A prelimi-

nary safety evaluation. Vaccine 19, 34(18), 2055-2061 (2016).

■ Kobiyama K. et al. Nonagonistic Dectin-1 ligand transforms CpG into a

multitask nanoparticulate TLR9 agonist. Proc. Natl. Acad. Sci. USA. 111,
3086-3091 (2014).

■ Onishi M. et al. Hydroxypropyl-β-Cyclodextrin spikes local inflammationthat induces Th2 cell and T follicular helper cell responses to the coadministered antigen. Journal of Immunology 194, 2673-2682 (2015).

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

to blood-stage malaria infection by reinforcing host iron metabolism. Cell
Host Microbe. 12, 705-716 (2012).

■ Zhao H. et al. Olfactory plays a key role in spatiotemporal pathogenesis of

cerebral malaria. Cell Host Microbe. 15, 551-563 (2014).
33

Vaccine Science

Ken J. Ishii, MD/PhD

Professor

Ken J. Ishii■

Associate Professor

Etsushi Kuroda■

Assistant Professor

Natsuko Kishishita
Takuya Yamamoto

The primary goal of our laboratory is to understand the immunological mechanisms of the intra- and inter-cellular signaling
pathways that mediate the immunogenicity of successful vaccines, as well as biological responses to adjuvants. Such knowledge will enable us to develop novel concepts, modalities andnext generation immuno-preventive and/or therapeutic agentsagainst infectious diseases, cancer and allergy as well as othernon-communicable diseases.

Vaccine science with old, but newly evolving adjuvant
researches
Our group and others have recently clarified that most successful vaccines, such as FLU and DNA vaccines, possess DNA and/orRNA, which appear to act as essential “built-in” adjuvants (Ishii KJ
et al Nature 2008, Koyama S et al, Science Trans. Med. 2010).
Recently, we reported that nucleic acids such as dsDNA or
RNA:DNA hybrid can be found in the cytosol of cancer cells suchas B cell lymphoma (Koo CX et al J. Biol. Chem. 2015, Shen YJ et al,
Cell Reports 2015), potentially activating innate and adaptive immunity to cancer cells.
In FY2015-2016, we continued to elucidate the mechanisms of,and to develop novel, vaccine adjuvants. We recently identified
that two STING ligands, DMXAA and another natural cyclic dinucleotide such as cGAMP, were unexpectedly allergic. The ligands function as type-2 inducing adjuvants to co-administeredantigens, and addition to other DNA-based adjuvants such asCpG DNA, abrogated STING-L-induced type-2 adjuvant effect,and synergized toward type-1 adjuvant effect (Tang CK et al PLoS
34■

Postdoctoral Fellow

2■

Research Assistant

4■

Visiting Scientist

6■

Support Staff

5

Fig. 1 Adjuvant activity of hydroxyapatite was dependent on its size irrespective to the shape. (Hayashi
M. et al Vaccine 2015)

One 2013, Temizoz B et al Eur. J. Immunol. 2015).
We coincidently found that hydroxypropyl-β-cyclodextrin (HPβ-CD), a common additive for many drugs, acts as a vaccine adjuvant. HP-β-CD induces host cell death derived dsDNA and local
inflammation, and HP-β-CD also induces Th2 cells and T follicularhelper cell responses to the co-administered antigen (Onishi M etal J. Immunol. 2015). HP-β-CD was also shown to be a potent mucosal adjuvant for intra-nasal Flu vaccination (Kusakabe T et al
Vaccine 2016).
We also developed novel adjuvants such as hydroxyapatite,natural biomaterial of bone (Hayashi M et al Vaccine 2015, Fig.1),
and non-aggregating Poly-A Tailed Immunostimulatory A/DType CpG Oligodeoxynucleotides Applicable for Clinical Use(Aoshi T et al J. Immunol. Res. 2015).
Finally, we have conducted various preclinical and clinicalstudies to develop humanized K-type CpG ODN (code name: K3)
and its 2nd generation CpG complexed with β-glucan (code name:
K3-SPG), some of which were published (Kobiyama K et al Eur. J.
Immunol. 2016)(Fig.2).

Fig. 2 Potent antigen-specific CD8+ T-cell proliferation by K3-SPG, a humanized CpG ODN complexedwith β-glucan. (Kobiyama K et al Euro. J. Immunol. 2016)

Recent Publications■ Kobiyama K, et al. Species-dependent role of type I IFNs and IL-12 in the■ Kobiyama K, et al. Nonagonistic Dectin-1 ligand transforms CpG into a

CTL response induced by humanized CpG complexed with β-glucan. Eur.
J. Immunol. 46, 1142-1151 (2016).

multitask nanoparticulate TLR9 agonist. Proc. Natl. Acad. Sci. USA. 111,
3086-3091 (2014).

■ Onishi M, et al. Hydroxypropyl-β-cyclodextrin spikes local inflammation■ Desmet C and Ishii KJ. Nucleic acid sensing at the interface between in-that induces Th2 cell and T follicular helper cell responses to the coadministered antigen. J. Immunol. 194, 2673-2682 (2015).

nate and adaptive immunity in vaccination Nat. Rev. Immunol. 12, 479491 (2012).

■ Temizoz B, et al. TLR9 and STING agonists synergistically induce innate

and adaptive type-II IFN. Eur. J. Immunol. 45, 1159-1169 (2015).
35

Immunoparasitology

Masahiro Yamamoto, PhD■

Professor

Masahiro Yamamoto

Assistant Professor

Miwa Sasai

RabGDIα-deficient mice exhibit enhanced IFN-γ-dependent clearance of T. gondii. Moreover, the enhanced clearance by RabGDIα
deficiency is accompanied by increased recruitment of Irga6 andGbp2 to the parasite. Notably, Gbp2 is required for Irga6 recruitment, which is suppressed by direct and specific interactions of
RabGDIα with Gbp2 through a lipid-binding pocket. Further-more, a high dose of type II T. gondii infection in RabGDIαdeficient mice results in increased resistance, which is characterized by a decreased parasite burden in the brain. Taken together,our data have indicated that RabGDIα play a negative role in the
Gbp2-Irga6 axis of IFN-γ-inducible GTPase-dependent cell-autonomous resistance to T. gondii.

Jisu Ma
Youngae Lee■

Interferon-γ (IFN-γ) is an important T-helper 1 (Th1) cytokine
that inhibits the survival and growth of a wide range of intracellular pathogens such as viruses, bacteria, and parasites. Stimulation of innate immune cells such as macrophages by IFN-γ up-regulates almost 2000 effector genes encoding various IFN-γ-inducible
proteins including immunity-related GTPases such as the MX proteins, p47 immunity-related GTPases (IRGs) and p65 guanylatebinding proteins (GBPs). MX proteins and GBPs have been shown
to restrict replication of viruses. Moreover, IRGs and GBPs playroles in host defense against vacuole-forming bacteria including
Salmonella, Chlamydia, Mycobacteria and Listeria by induction of
antibacterial responses involving autophagic effectors, inflammasome and phagocytic oxidases.
Not only viruses and bacteria but also the vacuolar parasite
Toxoplasma gondii is targeted by IFN-γ-inducible GTPases. IFN-γ
is critically required for suppression of T. gondii replication inside
PVs and cell-autonomous clearance. Nitric oxide that is produced
by inducible nitric oxide synthase (iNOS) in the infected cells
mainly inhibits the replication. On the other hand, T. gondii survival within infected cells is suppressed by cooperative action
between IRGs and GBPs. Indeed, various types of cells such as
macrophages, fibroblasts and astrocytes derived from mice lacking IRGs such as Irgm1 (also known as LRG-47), Irgm3 (IGTP), andIrga6 (IIGP1), or GBPs such as Gbp1, Gbp2, and a cluster of GBPs
on murine chromosome 3 (GBPchr3; Gbp1, Gbp2, Gbp3, Gbp5, andGbp7) were defective for IFN-γ-mediated intracellular killing of T.
gondii. After the formation of PVs, GBPs and a subfamily of IRG
members called GKS-IRGs such as Irga6, Irgb6 (TGTP) and Irgb10
36

Support Staff

1

are shown to accumulate of PV membrane (PVM) and oligomelizedependently on GTP-binding to destroy PV membrane integrity
and structure, resulting in cell-autonomous clearance by intracellular digestive pathways.
The regulatory mechanism of how IFN-γ-induced GTPases arerecruited to PVs has gradually been elucidated. In the absence ofessential autophagy-related proteins Atg3, Atg5, Atg7 and Atg16L1, and another subfamily of IRGs called GMS-IRGs such as
Irgm1 and Irgm3, the recruitment of IFN-γ-inducible GTPases andthe killing of T. gondii are severely impaired. Thus, Atg3/Atg5/
Atg7/Atg16L1 and Irgm1/Irgm3 are required for proper targetingof GKS-IRGs and GBPs to T. gondii PVM and play positive roles in
the cell-autonomous resistance to the pathogen. On the other
hand, the inhibitory mechanism for the IFN-γ-inducible GTPasedependent immunity remains unclear.
To explore the molecular mechanism to control the action of
IFN-γ-inducible GTPases, we have attempted to identify binding
partners of Gbp2, since a single deletion of Gbp2 in mice has beenshown to result in impaired in vitro and in vivo resistance to type
II T. gondii. In the present study, we identify RabGDIα as a Gbp2interacting protein. We have an interest in this protein for two
reasons: One is because RabGDIα has been shown to participate
in the regulation of Rab proteins, which like GBPs belong to another family of GTPases, and the other is because we demonstrate that overexpression of RabGDIα in cells impairs IFN-γinduced reduction of T. gondii numbers. We have tested whether
RabGDIα acts as a regulator of IFN-γ-inducible GTPases underphysiological condition. Macrophages and fibroblasts from

Figure. RabGDIα negatively regulates IFN-γ-inducible GTPase-dependent cell-autonomous
immunity to Toxoplasma gondii

Recent Publications■ Lee Y, Sasai M, Ma J, Sakaguchi N, Ohshima J, Bando H, Saitoh T, Akira S,

Yamamoto M. p62 plays a specific role in interferon-γ-induced presentation of a Toxoplasma vacuolar antigen. Cell Reports. 13, 223-233 (2015).
■ Ohshima J, Sasai M, Liu J, Yamashita K, Ma JS, Lee Y, Bando H, Howard JC,

Ebisu S, Hayashi M, Takeda K, Standley DM, Frickel EM, Yamamoto M.
RabGDIα is a negative regulator of interferon-γ-inducible GTPase-dependent cell-autonomous immunity to Toxoplasma gondii. Proc. Natl. Acad.
Sci. USA. 112, E4581-4590 (2015).
37

Biochemistry and Immunology

Shigekazu Nagata, PhD

Professor

Shigekazu Nagata■

Associate Professor

Jun Suzuki■

Assistant Professor

Katsumori Segawa

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

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

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

Toshihiro Fujii

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

Postdoctoral Fellow

2■

Research Assistant

2■

Visiting Scientist

1■

Support Staff

1

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

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

motility. Mol. Cell Biol. 36, 645-659 (2016).
■ Fujii T, Sakata A, Nishimura S, Eto K, Nagata S.

TMEM16F is required forphosphatidylserine exposure and microparticle release in activatedmouse platelets. Proc. Natl. Acad. Sci. USA 112, 12800-12805 (2015).

■ Segawa K, et al.

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

■ Suzuki J, Denning DP, Imanishi E, Horvitz HR, Nagata S. Xk-related protein

8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science 341, 403-406 (2013).

■ Segawa K, Nagata S. An apoptotic 'eat me' signal: phosphatidylserine ex-posure. Trends Cell Biol. 25, 649-650 (2015).
39

Single Molecule Imaging

Toshio Yanagida, PhD■

Professor

Toshio Yanagida

Associate Professor

Hideaki Fujita
Tomoyuki Yamaguchi
Yutaka Komai■

Postdoctoral Fellow

1■

Visiting Scientist

2■

Support Staff

1
Fig. 1 Counting T-cell activation upon engagement with APC in the presence of regulatory T cell. Rhod2
stained T-cells (red) were held with glass micro-needle and placed on APC and the fluorescence was monitored.
Regulatory T cell (blue) and conventional T cell (green) are also present on APC.

Measurement of single T cell activation by APC

Raman observation of T cell activation

In the common model of T cell activation, T cells are expected
to be activated by antigen-presenting cells (APC) on contact
when an appropriate peptide is presented. However, by counting
the number of activated T cells attached to APC by micro-pipette,
we previously found that only 22% became activated eventhough the antigen was presented by APC. On the other hand,
11% of T-cells showed activation even when antigen peptide wasabsent, indicating that activation of T-cell is not deterministic. To
understand how the immune system distinguishes between self
and non-self in such non-deterministic manner of T cells, we further investigated the effect of regulatory T cells (Fig. 1), and foundthat in the presence of regulatory T cells, activation of T cells was
suppressed in the absence of antigen peptide, whereas activation probability did not change in the presence of antigen peptide. This observation show that regulatory T cells act as a faucet
for the leaky immune system.

Using Raman spectroscopy, we succeeded in visualizing theactivation status of living T cells without labeling. Raman spectrafrom activated T cells largely differed compared to naïve T cells,
enabling the prediction of the activation status of T cells at thesingle cell level. Our analysis revealed that spectra of individual T
cells gradually change from the pattern of naïve T cells to that ofactivated T cells during the first 24 h of activation. The presented
results indicate that Raman spectrum enables the detection of
dynamic change in individual cell state scattered in heterogeneous populations.

Immune model predicting stochastic T cell activation
A numerical model to explain the adaptive immunity predicting stochastic T cell activation and suppression of self- responseby regulatory T cell was made and evaluated. Theoretical modeling showed that T cell association to APC is crucial to maintainstable unresponsiveness in addition to inhibition of activationsignaling when ligands for stimulation are limited and competedamong T cells. The optimized model to discriminate reactivity
under self and non-self conditions well reproduced the proliferation patterns of T cells under various conditions examined in in
vitro experiments.
40

Fig. 2 Pseudo colored Raman image of T cells activated for 12 h. Images are pseudo colored so that naïve stateis represented by blue whereas activated state is in red.

Recent Publications■ Morikawa TJ, Fujita H, Kitamura A, Horio T, Yamamoto J, Kinjo M, Sasaki A,■ Ichimura T, Chiu LD, Fujita K, Kawata S, Watanabe TM, Yanagida T, Fujita H.

Machiyama H, Yoshizawa K, Ichimura T, Imada K, Nagai T, Watanabe TM.
Dependence of fluorescent protein brightness on protein concentration
in solution and enhancement of it. Sci. Rep. 6, 22342 (2016).

Visualizing cell state transition using Raman spectroscopy. PLoS One. 9(1),e84478 (2014).
■ Watanabe TM, Imada K, Yoshizawa K, Nishiyama M, Kato C, Abe F, Morikawa TJ,■ Iwaki M, Iwane AH,

Ikezaki K, Yanagida T. Local heat activation of single
myosins based on optical trapping of gold nanoparticles. Nano Letters,
15, 2456-2461 (2015).

Kinoshita M, Fujita H, Yanagida T. Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure. PLoS One. 8(8), e73212(2013).

■ Fujita H, Esaki T, Masujima T, Hotta A, Kim SH, Noji H, Watanabe TM. Com-

prehensive chemical secretory measurement of single cells trapped in a
micro-droplet array with mass spectrometry. RSC Adv. 5, 16968-16971 (2015).
41

Biofunctional Imaging

Yoshichika Yoshioka, PhD■

Professor

Yoshichika Yoshioka

Assistant Professor

Chizuko Inui
Yuki Mori
Fuminori Sugihara

Our group has developed highly sensitive and specific in vivo
visualization techniques with magnetic resonance imaging (MRI)and magnetic resonance spectroscopy (MRS) to non-invasively
visualize the dynamic immune responses. The technique can beused to obtain images and spectra of the same mouse repeatedly
over time, and the precise information, which is obscured by individual differences, can be obtained. Technical developments andrefinements are important and necessary to obtain refined images and spectra (information).

Ultra high filed MRI and iron oxide nanoparticles
MRI in particular offers a significant advantage in imaging deep
regions with good spatial resolution and tissue contrast. A stronger magnetic field can be applied to increase the signal to noise
ratio (SNR) of in vivo MRI. In addition, super-paramagnetic
nanoparticles of iron oxide (SPIO), a contrast agent for MRI, improves MRI contrast to noise ratio (CNR) and detectability in thestronger magnetic fields by shortening the T2/T2* relaxation
times. Combining high magnetic-field strength with high-sensitivity radio-frequency (RF) coils and optimal contrast agents will
enable the visualization of immune cell dynamism and molecular
events in vivo in both animals and humans.
SPIO has been used in preclinical and clinical diagnosis with
MRI, particularly for liver, spleen, lymph nodes and bone marrow
because nanoparticles such as SPIO are taken nonspecifically bythese tissues and by macrophages. Many studies aimed at improving the unique physicochemical and biological properties of
SPIO have been conducted to visualize a specific biological event
42■

Postdoctoral Fellow

1■

Research Assistant

1■

Visiting Scientist

9■Support Staff

2

including inflammation by modifying the particle structure, size,
coating, and so on.
Our collaborator synthesized a new type of SPIO which showsa stealth property to macrophages. We thought that new immunological phenomena could be visualized by the new SPIO. Actually, the particle showed some characteristic contrasts in miceorgans and tissues. One being kidney tissue. Figure 1 shows theunique contrast pattern obtained by the new SPIO. Figure 1 (a)MRI was obtained in vivo before and after the new SPIO administration. The new SPIO produced low signal intensity regions in
the boundaries between cortex and outer medulla and between
outer medulla and inner medulla. Although these low intensity
regions are unique, the intensity in cortex regions were also low
compared to the images obtained before administration. Usingex vivo high resolution MRI (Figure 1 (b)) we could confirm thatthe dark spots by the new SPIO were found mostly in the boundaries between cortex and outer medulla and between outer medulla and inner medulla. Several small dark spots were found in
the cortex, which look like renal corpuscles. We examined the
SPIO distribution in the cortex histologically after ex vivo MRI. The
SPIO distributed in the lysosome of mesangial cells of glomerulus. These results indicate mesangial cells of control mice could
recognize some substance which macrophages do not recognize. Since mesangial cells are considered to play some immunological roles, the new SPIO could be used as a tool to assess theimmunological responses of the kidney and kidney disease regarding immune disorders.

Integration of MRI and MRS

Diverse information

MRS provides information non-invasively about metabolismof organs and tissues even at deep regions. We tried to obtain the
spectra of bone marrow in mice femurs. Bone marrow spectrafrom 0.7 x 0.7 x 0.7 mm3 regions could be obtained shown in Figure 2 (a, b). (a) and (b) are control and peroxisome proliferatoractivated receptor γ (PPARγ) agonist (rosiglitazone) treated mice,
respectively. Steatosis of bone marrow in the treated mice could
be visualized. Figure 2 (c, d) are high resolution MRI of these micefemurs. Decreases of trabecular and compact bones in the treated mouse femur could be assessed quantitatively by our highresolution MRI (Shiomi Y, et al. JBC 2015). The important point isthat the anatomical and metabolic information of mouse femurs
could be obtained simultaneously by ultra-high field magnetic
resonance.

MRI and MRS are non-invasive and provide diverse informationsuch as in Figures 1 and 2. The diverse information obtained bythese techniques will contribute to direct visualization of the immune responses in order to clarify how the integrated and dynamic immune system actually works in the body and how immune cells behave under pathological conditions in vivo.

Figure 1.
(a), Abdomen MRI of the same live mouse before and after the new
SPIO administration. Upper images were obtained before the SPIO
administration. The lower after the administration. Kidney showed
unique MRI contrast pattern. (b), ex vivo MRI of kidney after the new SPIO
administration. Several small round dark spots could be seen in the cortex
region. These are renal corpuscles. We found the new SPIO were taken
by mesangial cells of glomeruli. Mesangial cells and glomeruli could be
visualized by MRI.

Figure 2.
MRS and MRI of mice femurs. (a,b), bone marrow spectra from 0.7 x 0.7 x
0.7 mm3 regions in femurs. (a), control mouse. (b), peroxisome proliferatoractivated receptor γ agonist treated mouse. Steatosis of bone marrow in thetreated mice was found. (c, d), high resolution MRI of control and treatedmice femurs. Assessment of trabecular and compact bones could be done
quantitatively by high resolution MRI (Shiomi Y, et al. JBC 2015).

Recent Publications■ Arima Y, Kamimura D, Atsumi T, Harada M, Kawamoto T, Nishikawa N,■ Mori Y, Murakami M, Arima Y, Zhu D, Terayama Y, Komai Y, Nakatsuji Y,

Stofkova A, Ohki T, Higuchi K, Morimoto Y, Wieghofer P, Okada Y, Mori Y,
Sakoda S, Saika S, Yoshioka Y, Komuro I, Yamashita T, Hirano T, Prinz M,
Murakami M. A pain-mediated neural signal induces relapse in multiple
sclerosis models. eLife 4, e08733 (2015).

Kamimura D, Yoshioka Y. Early pathological alterations of lower lumber
cords detected by ultra-high field MRI in a mouse multiple sclerosis model. International Immunology 26, 93-101 (2014).
■ Satoh T, Kidoya H, Naito H, Yamamoto M, Takemura N, Nakagawa K,■ Mori Y, Chen T, Fujisawa T, Kobashi S, Ohno K, Yoshida S, Tago Y, Komai Y,

Hata Y, Yoshioka Y. From cartoon to real time MRI: in vivo monitoring ofphagocyte migration in mouse brain. Scientific Reports 4, 6997 (2014).

Yoshioka Y, Morii E, Takakura N, Takeuchi O, Akira S. Critical role of Trib1 indifferentiation of tissue-resident M2-like macrophages. Nature 495, 524528 (2013).

■ Zhao H, Aoshi T, Kawai S, Mori Y, Konishi A, Ozkan M, Fujita Y, Haseda Y,

Shimizu M, Kohyama M, Kobiyama K, Eto K, Nabekura J, Horii T, Ishino T,
Yuda M, Hemmi H, Kaisho T, Akira S, Kinoshita M, Tohyama K, Yoshioka Y,
Ishii KJ, Coban C. Olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria. Cell Host & Microbe. 15, 551-563 (2014).
43

Immunology and Cell Biology
3. Visualized macrophage dynamics and significanceof S100A8 in obese fat

Masaru Ishii, MD/PhD

Professor

Masaru Ishii■

Associate Professor

Keizo Nishikawa■

Assistant Professor

Junichi Kikuta
Hiroki Mizuno
Szandor Simmons
Takao Sudoh
Takahiro Matsui

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

1. Intravital bone imaging revealing osteoclast and osteoblast dynamics in vivo
By using intravital multiphoton microscopy, we have elaborated a novel imaging system for visualizing inside bones. By improving the bone imaging system, we succeeded in visualizing
the function of fully differentiated osteoclasts adhering to bone
surfaces in vivo (J Clin Invest 2013). This novel visualization identified two distinct mature osteoclast functional states; i.e., boneresorbing (R) osteoclasts firmly adhering to bones and devouring
the bone matrix by secreting acids, and non-resorbing (N) osteoclasts relatively loosely attached and wriggling along the bone
surface. Th17 cells, a bone destruction-prone T cell subset, express RANKL on their surface, although its functional role remains
elusive. This novel imaging system showed that RANKL-bearing
Th17 could stimulate osteoclastic bone destruction by contacting Nstate osteoclasts directly to convert them to the R-state, a criticalmechanism underlying bone erosion in arthritic joints.
Furthermore, in collaboration with Dr. Kikuchi in the frame of
IFReC, we have developed a new chemical probe for detecting
proton secretion in bone resorption by osteoclasts (Nat Chem
Biol, in press). By using this probe, we could grasp the real time44■

Postdoctoral Fellow

1■

Research Assistant

2■

Visiting Scientist

1■

Support Staff

3

Chronic low-grade inflammation of adipose tissue plays a crucial role for the pathophysiology of obesity. Infiltration of several
immune cells such as macrophages into adipose tissue was observed in obesity, although the initial factors triggering their migration have not been elucidated. By using intravital multiphoton imaging technique, we analyzed the detailed time-courses ofinflammatory processes in adipose tissues under high-fat andhigh-sucrose (HF/HS) diet. Mobility of macrophages was shown
to be activated just 5 days after HF/HS diet, when the distinct hypertrophy of adipocytes and the accumulation of macrophages
have not yet become prominent. Significant increase of S100A8
was detected in mature adipocyte fraction just 5 days after HF/HS
diet. Recombinant S100A8 stimulated chemotactic migration
both in vitro and in vivo, as well as induced pro-inflammatory
molecules both macrophage and adipocytes, such as TNF-α andCCL2. Finally, a neutralizing antibody targeting S100A8 efficiently
suppressed the HF/HS diet-induced initial inflammatory change,i.e., increased mobilization of adipose macrophages. In conclu-

sion, time-lapse intravital multiphoton imaging of adipose tissues first identified the very early event exhibiting increased mobility of macrophages, which may be triggered by increased
expression of S100A8 and resultant to progression of chronic inflammation in situ (Proc Natl Acad Sci USA 2015).

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

course of osteoclastic bone resorption which was finely regulated
by cell-cell contact with bone-forming osteoblasts and otherbone cell types.

2. DNA methylation regulates osteoclast differentiation
Metabolic reprogramming occurs in response to the cellularenvironment to mediate differentiation, but the fundamental
mechanisms linking metabolic processes to differentiation programs remain to be elucidated. During osteoclast differentiation,a shift toward more oxidative metabolic processes occurs. We
identified the de novo DNA methyltransferase 3a (Dnmt3a) as atranscription factor that couples these metabolic changes to osteoclast differentiation. We also found that RANKL induces thismetabolic shift towards oxidative metabolism, which is accompanied by an increase in S-adenosylmethionine (SAM) production.
We found that SAM-mediated DNA methylation by Dnmt3a regulates osteoclastogenesis via epigenetic repression of anti-osteoclastogenic genes. Dnmt3a-deficient osteoclast precursor cells
do not differentiate efficiently into osteoclasts and mice with anosteoclast-specific deficiency in Dnmt3a have elevated bonemass due to a smaller number of osteoclasts. Furthermore, inhibition of DNA methylation by theaflavin-3,3 -digallate abrogatedbone loss in models of osteoporosis. Thus, we revealed the role of
epigenetic processes in the regulation of cellular metabolism anddifferentiation, which may provide the molecular basis for a new
therapeutic strategy for a variety of bone disorders (Nat Med2015).

Figure . Intravital imaging for various immune systems
Immune cells are high dynamic and interconnecting 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 with advanced imaging techniques.

Recent Publications■ Maeda H, et al. Real-time intravital imaging of pH variation associated■ Kikuta J, Kawamura S, Okiji F, Shirazaki M, Sakai S, Saito H, Ishii M. Sphin-with cell osteoclast activity and motility using designed small molecular
probe. Nat. Chem. Biol. (2016) in press.

gosine-1-phosphate-mediated osteoclast precursor monocyte migration
is a critical point of control in antibone-resorptive action of active vitamin
D. Proc. Natl. Acad. Sci. USA, 110, 7009-7013 (2013).

■ Sekimoto R, et al. Visualized macrophage dynamics and significance of

S100A8 in obese fat. Proc. Natl. Acad. Sci. USA, 112, E2058-66 (2015).

■ Kikuta J, et al. Dynamic visualization of RANKL and Th17-mediated osteo-

clast function. J. Clin. Invest. 123, 866-873 (2013).
■ Nishikawa K, et al. Dnmt3a regulates osteoclast differentiation by cou-pling to an S-adenosyl methionine-producing metabolic pathway. Nat.
Med. 21, 281-287 (2015).
45

Nuclear Medicine

Jun Hatazawa, MD/PhD■

In the clinical imaging of nuclear medicine, glucose analog
tracer F-18 fluoro-deoxy-glucose (FDG) is widely used for the diagnosis of cancer. It is well known that cancer cells and inflammatory cells both utilize glucose in energy production. It is difficult
to differentiate between cancer and inflammation by means of
glucose analog radiotracer. We studied the usefulness of L-4-borono-2-18F-fluoro-phenylalanine (F-18 FBPA) as a tumor-specificprobe, in comparison to F-18 FDG and C-11 methionine (Met),
focusing on its transport selectivity for L-type amino acid transporter 1 (LAT1), an isoform highly up-regulated in cancers.
Cellular uptake and kinetic analyses were performed to evaluate the transport capacity and Km value. PET studies were performed in rat xenograft models of C6 glioma (n = 9) and in rat models of turpentine oil-induced subcutaneous inflammation (n = 9).
The kinetic parameters and uptake values on static PET images
were compared using the one-tissue compartment model (K1,k2) and maximum standardized uptake value (SUVmax).
The cellular analyses showed that the transport capacity by
LAT1 was comparable among FBPA, Met and BPA, whereas thatfor a non–cancer-type isoform LAT2 was significantly decreased
for FBPA as compared to Met. The Km value of F18-FBPA for LAT1
(196.8+/-11.4μM) was dramatically lower than that for LAT2
(2813.8 ± 574.5μM), suggesting the higher selectivity of F-18
FBPA for LAT1. K1 and k2 values were significantly smaller in F-18
FBPA PET (K1 = 0.04 ± 0.01 ml/ccm/min and k2 = 0.07 ± 0.01/min)as compared to C-11 Met PET (0.22 ± 0.09 and 0.52 ± 0.10, respectively) in inflammatory lesions. Static PET analysis based on the
SUVmax showed significantly higher accumulation of F-18 FDG in
46

Professor

Jun Hatazawa

Research Assistant2

the tumor and inflammatory lesions (7.2 ± 2.1 and 4.6 ± 0.63, respectively) as compared to both F18 FBPA (3.2 ± 0.40 and 1.9 ±0.19) and C-11 Met (3.4 ± 0.43 and 1.6 ± 0.11). No significant difference was observed between F-18 FBPA and C-11 Met in thestatic PET images. The study showed the utility of F-18 FBPA as atumor-specific probe of LAT1 with low accumulation in the inflammatory lesions.
Based on the experimental works, clinical studies were conducted after the approval of institutional review board to differentiate between brain tumor recurrence and radiation necrosis.
F-18 FBPA PET accurately diagnosed patients with recurrent brain
tumor and radiation necrosis with the threshold accumulation
value of 2.0 of SUVmax.

Figure 1. F-18 FBPA animal PET in rats transplanted with RCG-6 cancer cell(arrow)

Figure 2. F-18 FBPA animal PET in rats with inflammation induced by
subcutaneous oil injection (arrow)

Figure 3. F-18FBPA PET in recurrent brain tumor (left: CT, right: F-18 FBPA
PET)Figure 4. F-18FBPA PET in radiation necrosis (left: CT, right: F-18 FBPA PET)

Recent Publications■ Kainuma S, Miyagawa S, Fukushima S, Pearson J, Chen YC, Saito A, Harada A,■ Watanabe S, Kato H, Shimosegawa E, Hatazawa J. Genetic and Environ-

Shiozaki M, Iseoka H, Watabe T, Watabe H, Horitsugi G, Ishibashi M, Ikeda H,
Tsuchimochi H, Sonobe T, Fujii Y, Naito H, Umetani K, Shimizu T, Okano T,
Kobayashi E, Daimon T, Ueno T, Kuratani, T, Toda K, Takakura N, Hatazawa J,
Shirai M, Sawa Y. Cell-sheet Therapy with Omentopexy Promotes Arteriogenesis and Improves Coronary Circulation Physiology in Failing Heart.
Mol Ther. 23, 374-386 (2015).

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

■ Nakatani K, Watabe T, Masuda D, Imaizumi M, Shimosegawa E, Kobayashi T,

Sairyo M, Zhu Y, Okada T, Kawase R, Nakaoka H, Naito A, Ohama T, Koseki M,
Oka T, Akazawa H, Nishida M, Komuro I, Sakata Y, Hatazawa J, Yamashita S.
Myocardial energy provision is preserved by increased utilization of glucose and ketone bodies in CD36 knockout mice. Metabolism. 64, 11651174 (2015).
47

Chemical Imaging Techniques

Kazuya Kikuchi, PhD

Professor

Kazuya Kikuchi■

Associate Professor

Shin Mizukami■

Assistant Professor

Yuichiro Hori

the surface charges of the PYP-tag. Based on the structure of PYP,
three aspartic acids (D53, D71, D97) and one glutamic acid (E74)
exist on the same face as a reactive cysteine residue (C69). We
speculated that charge reversal at these residues would reduce
repulsive forces, and facilitate interaction between FCANB and
PYP-tags. To this end, a series of cationic PYP mutants were designed and created by point mutation of the acidic amino acids:
D53R, D71R, E74R, D97R (Fig. 2).

Time course fluorescence measurement of FCANB in the presence of PYP mutants revealed that all the mutants showed improved binding rates compared with that of the wild-type PYP.
The highly efficient molecular dynamics calculations used here
were sufficient to recapitulate the binding rates of large charged
ligands on charged proteins. The resulting AcFCANB/PYP 4R pairenabled no-wash imaging of intracellular proteins in a desirable
time frame (t1/2 < 30 min).

Yuko Kamikawa
Masafumi Minoshima■

Support Staff

1

Fig. 1(left) Chemical structure of pHocas for imaging of osteoclast activity (right) Two-photon time lapse imaging of bone tissue
after the injection of pHocas. Excited at 940 nm. Scale bars: 20 μm.

Real-time intravital imaging of pH variation associatedwith osteoclast activity
Intravital imaging by two-photon excitation microscopy(TPEM) has been widely utilized to visualize cell functions. However, small molecular probes (SMPs) commonly used for cell imaging cannot be simply applied to intravital imaging because of
the challenge of delivering them into target tissues, as well as
their undesirable physicochemical properties for TPEM imaging.
Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and fluorescenceswitch, and imaged osteoclast activity by injecting the SMP into
living mice.
We designed a new pH-activatable probe “pHocas” (pH-activatable fluorescent probe for osteoclast activity sensing), consisting of an amide-substituted BODIPY fluorophore with an electron-deficient character, which prevents its degradation duringlaser irradiation of TPEM. pHocas possesses an N,N-dialkyl-o-toluidine moiety that serves as pH-dependent fluorescence via photo-induced electron transfer (PeT), as well as two bisphosphonate
groups for bone-specific delivery (Fig. 1). The fluorescence emission of pHocas increased at lower pH and maintained under continuous light irradation, indicating that it can be used to visualize
pH variation in the acidic compartment created by active osteoclasts.
Next, we explored the utility of pHocas for intravital imaging ofosteoclast activity in living mice over a long period of time. After
subcutaneous injection of pHocas into transgenic mice expressing the red fluorescent protein tdTomato in osteoclasts. Time48

lapse imaging revealed the local appearance and disappearanceof fluorescence signals under the basal membrane of osteoclasts
accompanied by membrane fluctuations. Interestingly, we foundthat the motility of bone-resorbing osteoclasts, which is believed
to be static, fluctuates in a time-dependent manner. We suggestthat these bone-resorbing osteoclasts have a different function,in contrast to the static osteoclasts, which may be required to explore the signals that activate bone resorption by slightly moving
the bone surface.

Design of a protein tag and fluorogenic probe for livecell imaging of intracellular proteinsSpecific protein labeling by synthetic fluorescent probes andcomplementary protein tags is an emerging technology that enables conditional labeling at specific time points. We previously
developed protein-labeling systems using the Photoactive Yellow Protein (PYP) as a protein tag in combination with a fluorogenic probe, FCANB. FCANB has a triblock modular structure: hydroxy cinnamic acid acts as the PYP ligand, fluorescein the
fluorophore, and nitrobenzene the quencher moiety. Although
FCANB enhances its fluorescence upon reaction with the PYPtag, it is cell-impermeable and the labeling rate (t1/2~1 h) is not
sufficiently fast.
We re-designed the fluorogenic probe AcFCANB, as a membrane-permeable probe by incorporating two acetyl groups into
the fluorophore. Once incorporated inside the cells, AcFCANB is
rapidly digested by cellular innate esterases to recover the anionic FCANB. Next, to accelerate the labeling rate, we focused on

Fig. 2(left) Schematic illustration for the mechanism of labeling acceleration. (right) Structure of PYP showing the reactive C69
surrounded by the four acidic residues targeted for mutation.

Recent Publications■ Maeda H, Kowada T, Kikuta J, Furuya M, Shirazaki M, Mizukami S, Ishii M■ Hori Y, Hirayama S, Sato M and Kikuchi K. Redesign of Fluorogenic Label-and Kikuchi K. Real-time Intravital Imaging of pH Variation Associatedwith Osteoclast Activity, Nat. Chem. Biol. 12, (2016), in press.
DOI: 10.1038/nchembio. 2096

ing System to Improve Surface Charges, Brightness, and Binding Kinetics
for Imaging Functional Localization of Bromodomains, Angew. Chem. Int.
Ed. 54, 14368–14371 (2015).

■ Kamikawa Y, Hori Y, Yamashita K, Jin L, Hirayama S, Standley DM and Kiku-

chi K. Design of a Protein tag and Fluorogenic Probe with Modular Structure for Live-Cell Imaging of Intracellular Proteins. Chem. Sci. 7, 308–314(2016).
49

Biophotonics

Nicholas Isaac Smith, PhD■

Associate Professor

Nicholas Isaac Smith

Assistant Professor

Alison Hobro

In addition to main lab activities, Dr. Hobro is running a Kakenhi project “Detection and identification of virions in host bodily
fluids and immune cells” on the measurement by both Raman
and absorption spectroscopy aimed at identifying molecules ofinterest in a complex biomolecular background such as thosethat occur in plasma or inside a cell. Raman techniques can readily identify single types of molecules when measured in isolation,
but a major challenge is to find limits of detection among competing signals from other molecules. Dr. Pavillon is also running a

Kakenhi project, “Full-field holographic optical coherence tomography combined with Raman spectroscopy for in vivo microscopic imaging of biological tissue”, which is also a project where
we collaborate with the Photonics Advanced Research Center at
Osaka University. This project is based on simultaneous Ramanand phase measurements geared towards tissue imaging, using alonger wavelength broadband source to allow penetration of turbid samples such as tissue. These additional projects are ongoing.

Nicolas Pavillon

Figure 1. Multimodal label-free imaging of MEF (fibroblast) cells, from Pavillon et al 2015.
The black and white mode gives quantitative phase information while the colored image is constructed from Raman scattering bands at 2853cm-1 (red, lipids)
and 2973cm-1 (green, C-H stretching). The phase map is dominated by proteins.

The biophotonics lab uses and develops different optical techniques to study biological and immunological processes. Primarily label-free, we exploit the different physical interactions of lightwith the sample to image or measure cellular information. Raman
spectroscopic imaging is one of our main tools, and we develop
new imaging modes to go along with the Raman measurements.
In practice, this means illuminating the sample with a high intensity laser beam and measuring the wavelength shifts of the scattered light. By resonance with the endogenous molecules in aliving cell or tissue sample, we can observe the spatial and chemical distributions in the sample. In combination with this, we areinvestigating methods of using other wavelengths of light to simultaneously measure different features of cells or tissue. Our
targets are typically lymphocytes, macrophages and their responseto stimulation.
We combined quantitative phase imaging with Raman imaging, which are both label-free imaging modalities that provide
complimentary information on the cell response, where protein
distribution in the cell is imaged mainly by the phase mode, while
lipid and other components are more dominant in the Raman imaging mode (Pavillon et al 2013, Pavillon et al 2014). For the purposes of discriminating cell lines, we found that it is possible toimage the cells using the phase mode, allowing the Raman signals to be collected in an optimized mode instead of relying on
Raman for imaging. The outcome of this is that it is possible touse an optimized label-free approach to discriminate cells. Targets of this type of discrimination have been lymphocytes (Hobro
et al 2016), but can be of any cell type. We termed the new mode50

hybrid scanning and found that it behaves very well for applications such as label-free cell screening, with some high-throughput characteristics (Pavillon et al 2015).
We also extended our studies in using gold nanoparticles in acell to enhance the Raman signal. Using photoreduction, we
showed that it is possible to generate gold nanoparticles within acell (Smith et al 2014), which can then be used to enhance theRaman signal. Most of our work is label-free and uses only a laser
or other light source, the nanoparticle enhanced projects offerdifferent avenues to study a cell response.
In collaboration with the Coban lab, which is focused on malaria immunology, we have been studying the application of Raman scattering to diagnosis of malaria and then more recently tothe observation of uptake processes in macrophages. Upon exposure to the malaria byproduct hemozoin, macrophages engulf
and transport the nano-sized crystals. Using Raman imaging wewere able to observe this process and identify some cellularchanges occurring during uptake. In particular we found a transient lipid response that occurs during or soon after the initial
uptake (Hobro, et al 2015). The Raman measurement of lipids
formed or concentrated near the particles is consistent with theappearance of vesicles during uptake.
Through collaboration with our colleagues in the department
of Applied Physics, we also worked on a number of projects
aimed at extending the applications and capabilities of imaging
techniques. We have hosted a number of students from physics,
and believe that sharing students, projects, and ideas between
our labs enables innovative research.

Figure 2. Raman imaging of hemozoin uptake by macrophage cells, from Hobro et al 2015.
The black and white image is brightfield, while the green channel overlay represents the
distribution of hemozoin in the cells. The spectra on the right-hand side show the sensitivity ofthe measurement to the presence of hemozoin, represented by the strong peaks.

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

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

for label-free lymphocyte cell line discrimination. Analyst 2016 (in press).

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

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

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

■ Pavillon N, Hobro AJ and Smith NI. Cell optical density and molecularcomposition revealed by simultaneous multimodal label-free imaging.
Biophys. J. 105(5), 1123-1132 (2013).

■ Pavillon N and Smith NI. Maximizing throughput in label-free microspec-

troscopy with hybrid Raman imaging. J. Biomed. Opt. 20(1), 016007-1016007-10 (2015).
51

Immune Response Dynamics

Kazuhiro Suzuki, MD/PhD

During the last century, extensive studies were performed on
individual organ systems and yielded huge progress in biomedical science. However, because the organ systems in our body mutually communicate to cross-regulate their functions, it is nearly
impossible to precisely predict responses of one organ system
without knowing the states of other organ systems. Thus, the importance of studies that clarify interconnections among multiple
organ systems is being increasingly recognized. In this regard,
recent studies that have revealed the cellular and molecular basis
for connections between the nervous and immune systems represent the new trend. Studies during the last decade have demonstrated that peripheral nerves of the autonomic and somatosensory nervous systems regulate both the development andfunctions of immune cells through multiple modes of action (Ordovas-Montanes, et al. Trends immunol. 36: 578, 2015).
Adrenergic nerves constitute efferent pathways of the sympathetic nervous system and coordinate organ functions throughsecretion of a neurotransmitter, noradrenaline. In the 1980s,
comprehensive morphological studies mapped adrenergic nerve
fibers in both primary and secondary lymphoid organs (Felten, etal. J. Immunol. 135: 755s, 1985). In secondary lymphoid organs,including spleen and lymph nodes, the adrenergic nerve fibers
exhibit a uniform pattern of distribution, where they mainly supply T-cell zones but are absent from B-cell follicles. These findings
established an anatomical basis for connections of the sympathetic nervous system with the immune system. Pharmacologicalstudies of the same period demonstrated that noradrenaline
52

Associate Professor

Kazuhiro Suzuki■

Postdoctoral Fellow

1■

Research Assistant2■

Support Staff2some questions remain unsolved. First, the molecular mechanisms of the crosstalk between β2-adrenergic receptors and chemokine receptors are unclear. Second, it remains to be visualized
how adrenergic nerves interact with lymphocytes and influence
their migratory behaviors in lymph nodes. Finally, the physiological significance of this neural control mechanism for lymphocyte
recirculation through lymph nodes remains to be addressed. We
are currently working on these questions to reveal the whole picture of adrenergic control of lymphocyte trafficking. Our researchcontributes to comprehensive understanding of immune regulation by the nervous system.

bound to the surface of lymphocytes, which was almost exclusively mediated by β2-adrenergic receptors (Brodde, et al. Life Sci.
29: 2189, 1981), indicating that inputs from the sympathetic nervous system can directly act on immune cells. However, it was
unclear how lymphocyte behaviors are influenced by adrenergic
nerves that project to lymphoid organs.
After spending several hours in a lymph node, lymphocytes
exit from the lymph node into lymph, return to blood flow
through the thoracic duct, and travel to other lymphoid organs tocontinue antigen surveillance in the whole body. We recently
found that inputs from adrenergic nerves control lymphocyte
egress from lymph nodes through β2-adrenergic receptors (Nakai, et al. J. Exp. Med. 211: 2583, 2014). Activation of lymphocyte
β2-adrenergic receptors augments signals through CCR7 andCXCR4, chemokine receptors that promote lymph node retentionof lymphocytes, and consequently inhibit their lymph nodeegress. Additionally, in murine models of inflammatory diseases,
including multiple sclerosis and allergic dermatitis, we showedthat administration of a β2-adrenergic receptor agonist ameliorates the diseases, which is accompanied by reduced lymph nodeegress of pathogenic T lymphocytes and their recruitment to target organs (Suzuki and Nakai, Clin. Exp. Neuroimmunol. 7: 10-17,
2016).
Our findings have established a novel mechanism by which adrenergic nerves control the immune system and implied its therapeutic potential for inflammatory diseases (Figure). However,

Figure. Control of lymphocyte trafficking by adrenergic nerves
Activation of β2-adrenergic receptors expressed on lymphocytes inhibits their egress fromlymph nodes by enhancing retention-promoting signals mediated by chemokine receptors,
CCR7 and CXCR4.

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

■ Wang X, Cho B, Suzuki K, Xu Y, Green JA, An J and Cyster JG. Folliculardendritic cells help establish follicle identity and promote B cell retention
in germinal centers. J. Exp. Med. 208, 2497-2510 (2011).

■ 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).
■ Gray EE, Friend S, Suzuki K, Phan TG and Cyster JG. Subcapsular sinus mac-

■ Green JA, Suzuki K, Cho B, Willison D, Palmer D, Allen CDC, Schmidt TH, Xu

Y, Proia R, Coughlin SR and Cyster JG. The sphingosine 1-phosphate receptor S1P2 maintains the homeostasis of germinal center B cells and promotes niche confinement. Nat. Immunol. 12, 672-680 (2011).

rophage fragmentation and CD169+ bleb acquisition by closely associated IL-17-committed innate-like lymphocytes. PLoS One. 7, e38258 (2012).
53

Brain-Immune Interaction

Ben Seymour, MD/PhD

Ben Seymour■

Associate Professor

Aya Nakae■

Assistant Professor

Masaki Maruyama■

Postdoctoral Fellow

1■

Support Staff

1

Homeostasis and defense after injury

Neurogenic inflammation

A core question we aim to answer is how does the brain andimmune system cause changes to behavior after injury? Although
reduced motivation and pain may be adaptive, for instance allowing us to prioritize recuperation and recovery, they may also
underlie pathological states such as excessive fatigue and depression. We have been studying brain activity and behavior in ahuman model of thermal injury (i.e. burns), to see how our ability
to control the injury (by cooling it) influences both behavior andautonomic nervous system activity. We have found that the extent to which humans perceive their ability to find relief of thepain is tightly linked with efferent autonomic output, providing a
potentially important mechanism by which the brain can modulate peripheral loci involved in the inflammatory response. We’ve
also identified the pathway in the brain that underlies this – fromthe anterior cingulate cortex to the brainstem periaqueductal
grey (submitted), identified as a possible key link in the brain-immune pathway.

Following tissue injury, the local inflammatory response is coordinated by a population of small umyelinated nerve fibers
called c-fibers. Understanding the role of these fibers is critical tounderstand disorders such as psoriasis, which is dependent onneurogenic control. We have been studying a human model ofneurogenic inflammation involving topical application of the TRP
channel ligand capsaicin, which causes is vasoactive oedema,
hyper-sensitivity and pain. In particular, we have been using integrated brain imaging techniques (MEG and fMRI) to try and identify if the brain has an internal representation of peripheral inflammation. Our provisional results suggests that this is indeed
the case. If this is confirmed, this means that the brain has thecritical substrate from which it can make ‘intelligent’ responses tocontrol the inflammatory response.

Using insights from engineering and information science,
we’ve begun to use these findings to construct control theorymodels of homeostasis after injury, which aim to unite behavior
and immune system function within a single model. Following alarge translational award from Arthritis Research UK, we now aim
to use this to identify immune and brain markers of fatigue in inflammatory arthritis (rheumatoid arthritis patients), and probe
their modulation by biologic therapies.

54

Professor

pain. We have developed a surgical plantar flap model of injury,
which causes immediate and prolonged behavioural responses
associated with pain. We have subsequently probed this in various murine knock-out models, including MyD88 and TRIF, andshown that they reduce pain-related responses. This identifies
possible target pathways for understanding how peripheral immune mediators might modulate the development of chronic
pain.
With collaborators within IFReC, we have also initiated a neuroimaging program to identify regions of the brain that are involved in injury and pain, using manganese-enhanced MRI andfunctional MRI. We aim to apply these methods to identify the
immune control of brain activity associated with inflammationand pain in mice (in the plantar flap model) and the rat (in the
CFA inflammatory arthritis model).

Figure 1.

Figure 2.
Potential brain region underlying efferent control during injury.

■ Chassang S, Snowberg E, Seymour B, Bowles C. Accounting for behaviour■ Granovsky Y, Anand P, Nakae A, Nascimento O, Smith B, Sprecher E, Valls-

in treatment effects - New applications for blind trials. PLoS ONE 10(6),w0127227 66, (2015).

Solé, J. Normative data for Aδ contact heat evoked potentials in adult
population: a multicenter study. Pain 157, 1156-63 (2016).

■ Pauli W, Larsen T, Collette S, Tyszka J, Seymour B, O’Doherty J. Distinct■ Seymour B, Maruyama M, Martino BD. “When is a loss a loss? Excitatory

contributions of subregions of the human substantia nigra to appetitive
and aversive learning. Journal of Neuroscience. 35, 14220-33 68 (2015).

and inhibitory processes in loss-related decision-making.” Current Opinion in Behavioral Sciences 5, 122-127 (2015).

Recent Publications
We have also been developing objective behavioural andphysiological tests on the local inflammatory response as a toolfor identifying the cognitive influences on immune processes,with collaborators within IFReC and industry. This is being done
as a precursor to identify the potential psychological causes ofvaccine-induced pain. This is a major problem for the HPV vaccination program in Japan, with a large consequent mortality.

Rodent injury and neuroimaging
A further component of our lab aims to develop animal models
of injury that can be used to test more mechanistic questions
about the role of the immune system in peripheral injury and■ Zhang S, Mano H, Gowrishanker G, Robbins T, Seymour B. Dissociable

learning processes underlie human pain conditioning. Current Biology
26, 52-58 (2015).

■ Maruyama

M. Neural substrates for understanding math expressions.
Clinical Neuroscience 33, 936-938 (2015).

■ Yasuda Y, Hashimoto R, Nakae A, Kang H, Ohi K, Yamamori H, Fujimoto M,

Hagihira S, Takeda M. Sensory cognitive abnormalities of pain in autism
spectrum disorder: a case-control study. Ann Gen Psychiatry. 15, 8 (2016).
55

Information Systems

Yutaka Hata, PhD■

Professor

Yutaka Hata

Associate Professor

Syoji Kobashi
Shugo Yasuda
Shinichiro Shima■

Assistant Professor

Manabu Nii■

Support Staff

1

Figure 1. Macrophage tracking result
Each color shows individual macrophage trajectory.

(1) We first propose an automated single macrophage tracking
method in 11.7 T magnetic resonance (MR) images. The method
is based on motion artifacts and spatial/temporal noise reduction
algorithms for preprocessing, a background subtraction methodfor detection, and the Hungarian method for tracking. The method has been applied to a brain-stroke mouse model, and has provided trajectory of macrophages and numerical results of macrophages including numbers, densities, velocities, etc. Imaging andquantification of macrophage kinematics will strongly assist the
progress of immunology research. Figure 1 shows the tracking
imaging result.
(2) A new simulation method for swarming motile cells says thechemotactic bacteria is developed on the basis of the kinetic chemotaxis model. The Monte Carlo algorithm is employed to reproduce the run-and-tumble motions of cells interacting with thechemical cues in the local environment. The reaction-diffusion
equations to describe the spatial and temporal variations ofchemical cues are also numerically solved coupling with theMonte Carlo simulation. We apply the method to the traveling
population wave of bacteria generated by the chemotaxis anddivision of bacteria. We also carry out the analytical calculation of
the continuum model for this problem and compare the analytical results with the numerical results. It is found that the travelingwave solutions exist for the specific division rates. The numericalresults also indicate the possibility of an interesting instability
problem which will be analyzed theoretically.
(3) We proposed a 4D macrophage simulator based on evolving
cellular automata. The simulator can imitate and visualize macrophages' movement in 3D space with time progress. First, macrophages' data were extracted from some images observed by a
two-photon microscope. The extracted data were used as training data for learning and evolving cellular automata. The cellular
automata used in this research are stochastic cellular automata(SCA) in 3D space and can evolve by using genetic algorithms.
The results from the proposed simulator are similar to movements of real macrophages. Figure 2 shows the 4D simulator result.
(4) We studied several basic researches on computer engineering
on medical science.

Figure 2. Result of the 4D simulator
Red objects show macrophages and yellow objects show T-cells.

Recent Publications■ Tashita A, Kobashi S, Mori Y, Morimoto M, Aikawa S, Yoshioka Y, Hata Y.

■ Taniguchi Y, Nakajima H, Tsuchiya N, Tanaka J, Aita F, Hata Y. Prediction ofMacrophage tracking using the Hungarian algorithm in time lapse MR images. Proceeding of 7th International Conference on Emerging Trends inEngineering & Technology, 169-173 (2015).

Human Posture with Bayseian Inference. 2015 IEEE International Conference on Systems, Man, and Cybernetics, 2322-2327 (2015).
■ Sato K, Shima S. Various oscillation patterns in phase models with locally■ Morita K, Kobashi S, Wakata Y, Ando K, Ishikura R, Kamiura N. ICP based

attractive and globally repulsive couplings. Phys. Rev. E 92, 042922 (2015).

Neonatal Brain MRI Normalization Method. Proceeding of 2015 IEEE International Conference on Fuzzy Systems, online (2015).
■ Alam SB, Nakano R, Kobashi S, Kamiura N. Feature selection of manifold

learning using principal component analysis in brain MR image. Proceeding of 4th International Conference on Informatics, Electronics & Vision,
online (2015).
56

57

Systems Immunology

Daron M Standley, PhD

Professor

Daron M Standley■

Associate Professor

Kazutaka Katoh■

Assistant Professor

Kazuo Yamashita■

Postdoctoral Fellow

2■

Support Staff

4

have also recently found that different donors immunized withthe same vaccine can produce widely different antibodies at thesequence level, which are highly similar at the structural level,
and that the structurally-related antibody groups are more likely
to be functionally related (in preparation). Based on these findings, we subsequently showed that we could group anti-HIV antibody sequences according to their target epitope with high accuracy. First we developed a mathematical model that could
separate anti-HIV antibodies from other antibodies with > 95%
accuracy (Figure 3A), and showed that such separation would benearly impossible using only sequence information (Figure 3B).
We also found that we could predict epitope-MHC interactions
with high accuracy. For example, we could predict the correct
MHC type for 41 known cytomegalovirus epitopes with an accuracy of > 97%. Work is ongoing now to extend these predictions
to identify the correct TCR sequence from among thousands ofpossibilities. Both of these preliminary results are encouraging
with respect to our goal of leveraging high-throughput sequence
data to identify B and T cell populations associated with disease.

Figure 1. Docked model of the Regnase-1 NTD and PIN domains.
The figure shows the agreement between the docked model (center) andchemical shift perturbations on the NTD (left) and PIN (right) domains.

Our laboratory mainly focuses on two areas of research: Posttranscriptional regulation of immune responses and analysis ofhigh-throughput antibody and TCR sequence data. Below wesummarize our progress in these two areas.

Post-transcriptional regulation of immune responses
Post-transcriptional regulation can be mediated by RNA-binding proteins (RBPs) or by micro RNAs (miRNAs), both of which arecapable of either stabilizing or destabilizing messenger RNAs(mRNAs). Our lab has been investigating both types of regulators
in collaboration with experimental groups. In parallel, we havedeveloped novel in silico methods for such purposes.
Our interest in RBP-mediated post transcriptional regulation
began with our collaboration with the Akira Lab in the biochemical characterization of Regnase-1, an endoribonuclease that targets a number of mRNAs related to inflammatory responses(Matsushita et al., 2009). One of the open questions in this projectis how Regnase-1 recognizes its target mRNAs. It has now been
established that Regnase-1 recognizes a conserved stem-loop
structure and that its targets overlap with those of another posttranscriptional regulator, Roquin (Mino et al., 2015); however Regnase-1 and Roquin appear to utilize different motifs for target
RNA recognition. In vitro binding assays indicated that Regnase-1
RNase activity requires not only the RNase-containing PIN domain, but also the presence of an N-terminal domain (NTD)(Yokogawa et al., 2016). Using molecular docking, we predictedthat the NDT and PIN domains interact physically; resulting in stabilization of the RNase active site residues. The docked model58

was subsequently confirmed by NMR spectroscopy (Yokogawa etal., 2016) (Figure 1). As an extension of this work, we have developed an all-purpose tool, aaRNA, for the prediction of RNA binding residues (Li et al., 2014), and have applied it to a number ofRNA-binding proteins: Arid5a-Stat3 3’UTR interaction (Masuda etal., 2016), docking of ssDNA and anti-DNA antibody in SLE (withthe Kikutani Lab; in preparation) and interaction between Apobec3g and HIV RNA (with the Koyanagi Lab, Kyoto Univ; in preparation).
In order to comprehensively understand the functional role ofmiRNAs in the regulation of immune responses, we analyzed theintersection between miRNA targets and genes preferentially expressed in immune cells. We developed a consensus prediction
method using a random forest framework and applied it to bothhuman and mouse miRNAs. We observed modest, yet statisticallysignificant, differences in the targeting profiles of immune-related mRNAs as compared to other mRNAs (Figure 2A-C) and qualitative differences in the binding target profiles of circulating miRNAs compared to non-circulating miRNAs (Figure 2D-F) (submitted).

High-throughput antibody and TCR sequence data
One of the major goals of our lab is to discover immune-based
biomarkers and therapeutics from human antibody and TCR repertoires. This goal requires that we are able to identify the targetsof antibodies and TCRs from sequence data. Although this is avery ambitious goal, we have already made substantial progressin one aspect of the work: rapid and accurate 3D modeling of antibody structures from sequence (Yamashita et al., 2014). We

Figure 2. Circulating miRNAs preferentially regulate immune-related
mRNAs.
The upper panel (A-D) shows the distributions of human immune-related
and non-immune related mRNAs in terms of the number of targeting
miRNAs. Immune-related mRNAs had more miRNA interactions in terms ofall miRNAs (A), circulating miRNAs (B) and non-circulating miRNAs (C). Panel
D-F shows the distributions of human miRNAs in terms of predicted targets.
Circulating miRNAs targeted qualitatively more mRNAs regardless ofwhether all mRNAs (D), immune-related mRNAs (E) or non-immune related
mRNAs were considered (F).

Figure 3. Separation of anti-HIV (green) and other antibodies (red) using
structural information.
Figure 3A shows the successful separation of anti-HIV antibodies from otherantibodies using modeled structural information. Figure 3B shows that
separation using only sequence information is not possible.

Recent Publications■ Matsushita K. et al. Zc3h12a is an RNase essential for controlling immune

responses by regulating mRNA decay. Nature 458, 1185-1190 (2009).

■ Li S, Yamashita K, Amada KM & Standley DM. Quantifying sequence andstructural features of protein-RNA interactions. Nucleic. Acids Res. 42,
10086-10098 (2014).

■ Mino T. et al. Regnase-1 and Roquin regulate a common element in in-

flammatory mRNAs by spatiotemporally distinct mechanisms. Cell 161,
1058-1073 (2015).

■ Masuda K. et al. Arid5a regulates naive CD4+ T cell fate through selective■ Yokogawa M. et al. Structural basis for the regulation of enzymatic activity■ Yamashita K. et al. Kotai Antibody Builder: automated high-resolutionof Regnase-1 by domain-domain interactions. Sci. Rep. 6, 22324 (2016).

structural modeling of antibodies. Bioinformatics 30, 3279-3280 (2014).

stabilization of Stat3 mRNA. J Exp Med 213:605-619 (2016).

59

Quantitative Immunology
versible even after removing exposure to the harmful components (tobacco for COPD and silica for silicosis). Combining ATACseq and RNA-seq measurements of transcriptional activity in amouse model of silicosis, we study the regulatory pathways associated with irreversible inflammation.

Mathematical Modeling■

Associate Professor

Diego Diez■

Assistant Professor

Yutaro Kumagai
Shunsuke Teraguchi

The Quantitative Immunology Research Unit is a team of researchers with expertise in different scientific fields including immunology, bioinformatics and theoretical physics. Our aim is tounderstand how the immune system works by combining threedifferent but closely interconnected approaches; (1) quantitative
measurement of molecular dynamics, (2) integration of big datafrom multiple sources into network models, and (3) developmentof mathematical frameworks to understand the immune system’s
dynamics through the analysis of these massive datasets. These
approaches are combined in several projects that aim to get insight into specific questions related to the immune system. Someof these projects are described below to highlight specific topics.

■

Postdoctoral Fellow

1■

Research Assistant1■

Support Staff

2

The accurate quantification of immunological responses andthe integration of massive data open the door to approach immunology research from the theoretical perspective. We are developing novel mathematical frameworks for the quantitativedescription of the immune system.
A common obstacle for constructing dynamical models of cellular signaling is the biochemical determination of many parameters. To circumvent this problem, we have developed a mathe-signaling. However, because of limitations in biochemical assay
resolution, the details of this process are still poorly understoodfor most immune signaling pathways. To address this problem,
we are applying, in collaboration with RIKEN QBiC and other laboratories in IFReC, Total Internal Reflection Fluorescent Microscopy(TIRFM) to monitor the dynamics of single immune molecules.
We are using this technique to study TLR signaling, and have successfully monitored TLRs and their adaptors at the single molecule level. We developed a novel algorithm to quantify the diffusion dynamics of single molecules without bias, even under highmolecular density conditions. This highly quantitative technique
can be used to describe the dynamics of the immune system’s
signaling pathways.

Quantitative Approaches
Accurate quantification of biological responses is critical forunderstanding the dynamics of complex systems. Previously, wedeveloped a fluorescent protein reporter system for the quantitative monitoring of IFN-a6 (Kumagai et al., 2007). Now we are trying to increase the “dimensions” of the observation in two ways:
time and perturbation. Time lapse imaging of type I interferon
expression under microscope will be combined with multiple
fluorescent protein knock-in cells to monitor genes induced
upon antiviral responses. We are also developing automatedcomputational algorithms to extract important quantities to understand interferon regulation from such time lapse imaging
data.
Receptor molecule dynamics such as dimerization and clustering with downstream molecules is important for immune system
60

matical framework called Stochastic Binary Modeling (SBM),
which also allows us to represent the stochastic and heterogeneous nature of cell populations (Teraguchi et al., 2011). We havedeveloped a system to automatically identify the structure andparameters of the network of regulatory pathways from multidimensional data. Now we are applying this system to infer the
network dynamics of immune signaling.
In another attempt to utilize the power of mathematics in immunological research, we are developing a hierarchical model ofthe immune system. In this framework, we derive the macroscopic
dynamics of the immune system from the stochastic and heterogeneous behavior of the individual cells involved in antigen presentation. This framework allows us to address one of the mainquestions in immunology, “how the balance between protective
immunity and immune tolerance is achieved?”.

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

Data Integration
The accumulation of high-throughput (“omics”) datasets hasbrought biology into the big data era and the need for approaches that integrate, summarize and extract relevant informationthat reveals the relation between biological components. We aredeveloping methods that integrate measurements of transcription factor binding with transcriptome data from different experimental conditions, and with protein-protein interaction datafrom public databases, to obtain insight into signaling and gene
regulatory immune networks.
We apply these methods to study the mechanisms behind respiratory diseases, such as chronic obstructive pulmonary disease(COPD) and silicosis (Diez et al., 2015). A common feature of these
diseases is that inflammation and disease progression are irre-

Recent Publications■ Vandenbon A, Dinh V H, Mikami N, Kitagawa Y, Teraguchi S, Ohkura N, &■ Patil A, Kumagai Y, Liang KC, Suzuki Y. & Nakai K. Linking transcriptional

Sakaguchi S. Immuno-Navigator, a batch-corrected coexpression database, reveals cell type-specific gene networks in the immune system.
Proc. Nati. Acad. Sci. USA. 1604351113 (2016).

changes over time in stimulated dendritic cells to identify gene networks
activated during the innate immune response. PLoS computational biology 9, e1003323 (2013).

■ Bahrini I, Song J H, Diez D & Hanayama R. Neuronal exosomes facilitate■ Teraguchi S, Kumagai Y, Vandenbon A, Akira S. & Standley DM. Stochastic

synaptic pruning by up-regulating complement factors in microglia. Sci.
Rep. 5, 7989 (2015).

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

■ Diez D, Agusti A & Wheelock CE. Network analysis in the investigation ofchronic respiratory diseases. From basics to application. AJRCCM 190,
981-988, 201403-0421PP (2014).
61

Next Generation Optical Immune Imaging
could promote better adaptation to chronic injury or stress. A fullunderstanding of the mechanisms of polyploidization is necessary in order to reveal the physiological significance of osteoclast
polyploidization via cell fusion and incomplete cytokinesis.

Exploration of fusion master molecule(s)Polyploidy, in which a cell has more than the diploid complement of chromosomes, is a widespread physiological phenomenon observed especially in plants, fungi, and insects. Although it
is less common in mammals, polyploidization occurs in selected
tissues including the placenta, liver, heart, skeletal muscle andbone marrow during normal development and aging. Cell fusion
is one mechanism of generation of polyploidy. Myeloid cells suchas macrophages and osteoclasts have a pronounced potential forcell fusion during development. Especially for osteoclasts, polyploidization is necessary to acquire sufficient bone-resorbing activity. However, precise mechanism of cell fusion is not fully understood. Our aim is to try to make advances in understanding
the cellular and molecular mechanisms underlying polyploidization and cell fusion.

Identification of a novel mechanism for osteoclast polyploidization
During developmental programs, some mammalian somatic
cells obtain additional sets of chromosomes by various mechanisms, including endocycles, endomitosis, incomplete cytokinesis, and cell fusion. Endocycles, endomitosis, and incomplete cytokinesis are directly associated with the proliferative state of thecell. By contrast, cell fusion is entirely independent of cell proliferation. Osteoclasts are specialized polyploid cells that resorb
bone. Polyploidy is a hallmark of mature osteoclasts. Upon stimulation with receptor activator of nuclear factor kappa-B ligand(RANKL), myeloid precursors commit to becoming polyploid osteoclasts, largely via cell fusion. Although generation of polyploid
62■

Associate Professor

Noriko Takegahara■

Visiting Academic staff

Kazuaki Tokunaga

osteoclasts is thought to occur due to cell fusion, independentlyof cell proliferation, some reports have pointed out a relationshipbetween cell proliferation and osteoclast differentiation. These
reports prompted us to investigate whether cell-cycle progression has an impact on polyploidization during osteoclastogenesis, and if so, how, and to what extent, the cell cycle regulates thepolyploidization of osteoclasts. In 2015, we demonstrated that inaddition to cell fusion, incomplete cytokinesis also plays a role inosteoclast polyploidization. RANKL stimulation transiently increased basal proliferation and induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated
by incomplete cytokinesis had the potential to subsequently undergo cell fusion. In addition, nuclear polyploidy (i.e., they contained nuclei with more than the diploid complement of chromosomes [> 2N]) was also observed in osteoclasts in vivo, suggesting
the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Our findings reveal an unexpected
pattern of cell division and fusion during the generation of polyploid osteoclasts.
Although our findings reveal an importance of incomplete cytokinesis in the formation of polyploid osteoclasts, a normal
physiological process, the physiological significance of the phenomenon still remains unclear. This phenomenon may be justone aspect of the phenotype of terminally differentiated cells, ora consequence of stress response that preserves cell function. Alternatively, polyploid nuclei may create genetic diversity, which

Tetraploid cells generated by cell fusion or incomplete cytokinesis had the potential to subsequently undergo cell fusion, indicating that tetraploids should express molecules required for cellfusion. By isolating the tetraploid cells, we performed genescreening and identified transmembrane molecules of which ex-pression was detected in fusion competent cells but not in cells
that do not obtain permission of cell fusion. We continued to investigate the biological function of these molecules using molecular and cellular biological methods, genetic methods, andoptical methods such as flow cytometry and microscopy, andfound that overexpression of these molecules enhanced multinucleation while knocked down transmembrane molecules inhibited multinucleation of osteoclasts. To clarify mechanisms
underlying cell fusion, further analysis of transmembrane molecules is currently in progress.

Figure.
Diploid and polyploid nuclei are observed in osteoclasts in vivo. Representative micrographs offluorescence in situ hybridization (FISH).

Recent Publications■ Takegahara N, Kim H, Mizuno H, Sakaue-Sawano A, Miyawaki A, Tomura■ Hirosue A, Ishihara A, Tokunaga K, Watanabe T, Saitoh N, Chandra T, Narita

M, Kanagawa O, Ishii M, Choi Y. Involvement of receptor activator of nuclear factor-kB ligand (RANKL)-induced incomplete cytokinesis in thepolyploidization of osteoclasts. J. Biol. Chem. 291, 3439-54 (2016).

M, Shinohara M, Nakao M. Quantitative assessment of higher-order chromatin structure of the INK4/ARF locus in human senescent cells. Aging
Cell 11, 553–556 (2012).

■ Tokunaga K, Saitoh N, Goldberg IG, Sakamoto C, Yasuda Y, Yoshida Y, Ya-■ Kuwajima T, Yoshida Y, Takegahara N, Petros TJ, Kumanogoh A, Jessell TM,

manaka S, Nakao M. Computational image analysis of colony and nuclear
morphology to evaluate human induced pluripotent stem cells. Sci. Rep.
4, 6996 (2014).

Sakurai T, Mason C. Optic chiasm presentation of Semaphorin6D in thecontext of Plexin-A1 and Nr-CAM promotes retinal axon midline crossing.
Neuron 74, 676-90 (2012).

■ Kim H, Kim T, Jeong BC, Cho IT, Han D, Takegahara N, Negishi-Koga T,

Takayanagi H, Lee JH, Sul JY, Prasad V, Lee SH, Choi Y. Tmem64 modulates
calcium signaling during RANKL-mediated osteoclast differentiation. Cell
Metab. 17, 249-60 (2013).
63

Immuno-Genomics
Epigenetic changes in dendritic cells upon stimulation
by lipopolysaccharide

The ultimate goal of immunology is health care: The elucidation of causes of diseases and their treatment in human patients.
To achieve this goal, a complete understanding of the immune
network, the interactions and regulatory principles between cells
and between gene products is required. In the Immuno-Genomics Research Unit, our aim is to establish and apply methodologies for extracting the maximum amount of information possible
from limited experimental data, using integrative bioinformatics
approaches. Here, we briefly introduce some of the research projects we are involved in.

Development of Immuno-Navigator, a database forgene coexpression in the immune system
The huge amounts of data in public databases contain enormous potential for unraveling complex dynamics in cellular biology. However, traditional biology lacks the means to analyze
such big data. Moreover, it is hard to compare data of different
laboratories. Thus, the extraction of meaningful information frompublic data is very difficult in practice.
During the last year, we continued our work on Immuno-Navigator (http://sysimm.ifrec.osaka-u.ac.jp/immuno-navigator/), a
platform which allows researchers to use thousands of gene expression datasets for addressing key questions in immunology(Vandenbon et al., PNAS, 2016). The database at present contains
both human and mouse gene expression data, and functions for
exploratory analysis of this data. We added new functions allowing integrative study of coexpression data and experimental
64■

Assistant Professor

Alexis Vandenbon■

Visiting Academic Staff

Hiromasa Morikawa■

Research Assistant1

Several of our previous studies have revealed links between
transcription factors and epigenetic regulation, such as between
Irf4 and Jmjd3 (Satoh et al., Nature Immunology, 2010), and between Jdp2 and inhibition of histone acetylation at the Atf3 promoter (Maruyama et al., Immunity, 2012). More recently, we usedbioinformatics analyses to help understand the role of Akirin2 in
recruiting the SWI/SNF complex to NF-κB target promoters (Tartey et al., EMBO J., 2014). However, details about the control ofstimulus-induced epigenetic changes remain unknown.
In a collaborative study with Dr. Yutaro Kumagai of the Quanti-tative Immunology Unit, we are analyzing the temporal changes
in histone modifications in dendritic cells upon LPS stimulation,using ChIP-seq experiments. Focusing on promoters and enhancers of LPS-inducible genes, we observed that changes in histone
modifications follow a well-defined pattern, where some modifications appear early after stimulation, and others at later time
points. Integrative analysis of our data with transcription factor
binding data revealed that the binding of some transcription factors at promoters and enhancers coincides with induction times
of some histone modifications at these regions. We believe thisanalysis can help reveal interactions between transcription factors and histone modifiers and their role in controlling the response to immune stimuli.

data, such as ChIP-seq data. Using our platform, researchers can
explore information hidden in the data without concern for technical obstacles, and generate hypotheses regarding gene functions. Fig. 1 shows a conceptual overview of the usage of this database.

Identification of key factors for inducing functionallystable regulatory T cells
Regulatory T cells (Tregs) are essential for immune homeostasis and can suppress excessive immune reactions harmful to thehost. In previous studies, we investigated the role of Foxp3, Tregspecific gene expression profiles, and characteristic DNA hypomethylation in Tregs (Morikawa et al., PNAS, 2014).
In one of the applications of our Immuno-Navigator database,we predicted candidate regulators involved in the control ofgenes with enhanced expression in Tregs (Vandenbon et al.,
PNAS, 2016). Experimental validation showed that expression of
one candidate regulator, integrin β8 (Itgb8), in Tregs was indeed
associated with Treg-specific epigenetic features (see also Fig. 1).
Further applications of our analysis platform on genes with repressed expression in Tregs suggested several candidate regulators involved in this repression. We are currently conducting experiments to elucidate the roles of these regulators in theestablishment of functionally stable Treg cells.

Fig. 1 Conceptual overview of the usage of the Immuno-Navigator database.
We collected a large amount of publicly available gene expression data from human andmouse immune cells into our database. Using our database, researchers can easily inspect gene
expression and correlation of expression in different cell types. Using lists of genes of interest
as input, researchers can generate promising hypotheses, and perform validation experiments.

Recent Publications■ Vandenbon A, et al. Immuno-Navigator, a batch-corrected coexpression■ Yang X, et al. VEGF-B promotes cancer metastasis through a VEGF-A-inde-

database, reveals cell type-specific gene networks in the immune system.
Proc. Natl. Acad. Sci. USA 113, 2393-2402 (2016).

pendent mechanism and serves as a marker of poor prognosis for cancer
patients. Proc. Natl. Acad. Sci. USA 112, 2900-2909 (2015).

■ Mino T, et al. Regnase-1 and Roquin regulate a common element in in-■ Morikawa H, et al. Differential roles of epigenetic changes and Foxp3 ex-

flammatory mRNAs by spatiotemporally distinct mechanisms. Cell 161,
1058-1073 (2015).

pression in regulatory T cell-specific transcriptional regulation. Proc. Natl.
Acad. Sci. USA. 111, 5289–5294 (2014).

■ Onishi M, et al. Hydroxypropyl-β-cyclodextrin spikes local inflammationthat induces Th2 cell and T follicular helper cell responses to the coadministered antigen. J. Immunol. 194, 2673-2682 (2015).
65

Symposia & Seminars

Jan. 21
Speaker
Chyi-Song Hsieh
Washington University in St. Louis, USA

Leonie Taams
King’s College London, UK

Yenan Bryceson
Karolinska Institutet, Sweden

Masahiro Yamamoto
Osaka University, Japan

Rikinari Hanayama
Kanazawa University, Japan

Title

Symposia & Seminars

The 7th International Symposium of IFReC :Immunology at the Forefront

The development of the regulatory T cell receptor repertoire

Cellular and molecular mechanisms of immune regulation in rheumatoid arthritis

Natural killer cells remember

IFN-γ-inducible cell-autonomous immunity to intracellular pathogens

Mechanisms of hemophagocytosis and heterolysis by phagocytes

Jan. 22
Speaker
Christoph Scheiermann
Ludwig Maximilians University Munich, Germany

Kazuhiro Suzuki
Osaka University, Japan

Naoto Kawakami
Ludwig Maximilians University Munich, Germany

Masakatsu Yamashita
Ehime University, Japan

Date :

January 21-22, 2016

Venue :KNOWLEDGE THEATER, Grand Front Osaka

Koji Hase
Keio University, Japan

Keishi Fujio
This symposium provided a forum for the newest developments inwide - ranging areas of immunology. The symposium was effectively
organized in combination with the 5th NIF Winter School by IFReC andSingapore Immunology Network, and an opportunity for the young
researchers to form a global network.

University of Tokyo, Japan

Yoshihiro Baba
Osaka University, Japan

Tri Giang Phan
Garvan Institute, Australia

68

Title
Mechanisms mediating circadian leukocyte trafficking

Adrenergic control of lymphocyte recirculation through lymph nodes

In vivo imaging of autoimmunity in the Central Nervous System

Determination of T-cell fate by glutamine metabolism

Interkingdom interaction shapes the mucosal immune system

Suppression of B cell functions by regulatory cytokines

Regulatory function of B cells in autoimmune inflammation

Where and how memory cells are reactivated in the secondary antibody response in
the lymph node

69

Date

Speaker

Affiliation

Title

2015
May 19Masashi Watanabe

National Cancer Institute, NIH, USA

The role of tumor suppressor p53 in CD4 T cell function and homeostasis

Jun. 22

Bali Pulendran

Emory University School of Medicine, USA

Systems based approach to vaccine design

Serkan Göktuna

Bilkent University, Turkey

IKKe promotes intestinal tumorigenesis via
establishing a pro-inflammatory microenvironment

Tieu Lan Chau

University of Liege, Belgium

A role for APPL1 in TLR3/4-dependent TBK1 andIKKe activation in macrophages

Sep. 24

Scott I. Simon

University of California Davis, USA

Engineering the innate immune response to
cutaneous Staph-aureus infection

Sep. 30

Yihai Cao

Karolinska Institutet, Sweden

Angiogenesis in diseases

Oct. 9Chen Dong

Tsinghua University, China

Molecular mechanisms in T cell differentiation

Oct. 30

Eddy F. Y. Liew

University of Glasgow, UK

The role of IL-33 in infectious and inflammatory
diseaseNov. 6

Andreas Diefenbach

University of Mainz Medical Centre, Germany

Transcriptional control of cell fate decision in the
innate immune system

Nov. 16

Takeshi Egawa

Washington University, USA

A c-MYC-initiated transcriptional cascade in theregulation of lymphocyte clonal expansion inprotective immunity

Jan. 26

Jean Pieters

University of Basel, Switzerland

Regulation of pathogen survival and immune
responses through coronin 1-mediated activation ofcell surface signaling

Feb. 5

André VeilletteUniversity of Montréal/ McGill University, Canada

Receptors controlling NK cell functions

Feb. 19

Fijs W.B. Van Leeuwen

Leiden University Medical Center, The Netherlands

Hybrid fluorescence navigation surgery :from molecule to man

Symposia & Seminars

IFReC Seminars

Jul. 92016

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

70

71

Speaker

20

22nd

IFReC colloquia are a series of discussion meetings for IFReC members
held once every other month. At each colloquium, three speakers from
IFReC laboratories give talks about their latest research progress followed
by intensive discussion. After the colloquium, a small social gathering isheld to further the discussions in an informal setting. These events serve
as a platform to promote fusion researches among IFReC members.

Date :

Etsushi Kuroda (Vaccine Science)

Fine particle kills alveolar macrophage to release IL-1 alpha that induces
bronchus-associated lymphoid tissue in the lung

Hideaki Fujita (Single Molecule Imaging)

Predicting activation status of T-cellYuji Yoshida (Immunopathology)

Semaphorin 4D contributes to rheumatoid arthritis by inducing inflammatory
cytokine production: Pathogenic and therapeutic implications

Mikael Martino (Host Defense)

Inhibition of IL-1R1/MyD88 signaling promotes mesenchymal stem celldriven tissue regeneration

Kazuo Yamashita (Systems Immunology)

Modeling B cell repertoires at atomic resolution

Mohamed El Sherif Badr (Cell Signaling)

Mechanism and status of T cell activation by self-recognition

Ryo Shinnakasu (Lymphocyte Differentiation)

Selection of germinal center B cells into the memory compartment

Katsumori Segawa (Biochemistry and Immunology)

Caspase-mediated cleavage of phospholipid flippase for phosphatidylserine
exposure during apoptosis

Naganari Ohkura (Experimental Immunology)

The development of regulatory T cells

Yuichiro Hori (Chemical Imaging Techniques)

Fluorogenic probes for uncovering physiological function of N-glycan

Szandor Simmons (Immunology and Cell Biology)

Implications of Spns2-deficiency on S1P-driven lymphocytes egress, HEVintegrity and immunity

Yuki Mori (Biofunctional Imaging)

4D single cell tracking with MRI in living mouse brain

Yutaro Kumagai (Quantitative Immunology Unit)

Understanding type I interferon production by quantifying epigenomics andsingle cell gene expression

Fumiji Saito (Immunochemistry)

Immune evasion by Plasmodium falciparum via an inhibitory receptor

th

21st

23rd

Title

Symposia & Seminars

IFReC Colloquia

20 th Colloquium : April 15, 2015
21 st Colloquium : June 10, 2015

24th

22 nd Colloquium : August 5, 2015
23 rd Colloquium : December 9, 2015
24 th Colloquium : February 10, 2016
Venue : Taniguchi Memorial Hall, Osaka University

72

73

The 6th Kishimoto Foundation Lecture
Date :

June 29, 2015

Venue : Taniguchi Memorial Hall, Osaka University
Host :

Research Institute for Microbial Diseases & IFReC

EventsAkiko Iwasaki
Professor, Yale University School of Medicine
Investigator, Howard Hughes Medical Institute“Innate resistance to mucosal viral infection”

Ruslan Medzhitov
Professor, Yale University School of Medicine
Investigator, Howard Hughes Medical Institute“Inflammation, Homeostasis and Host Defense”

74Events

The 5th NIF Winter School on Advanced Immunology
Lecturer
Caetano Reis e Sousa
The Francis Click Institute, UK

Frederic Geissmann
Memorial Sloan-Kettering Cancer Center, USA

Florent Ginhoux
Singapore Immunology Network, Singapore

Eicke Latz
University of Bonn, Germany / UMass Medical School, USA

Marc K. Jenkins
University of Minnesota, USA

Dan R. Littman
New York University School of Medicine, USA

Alexander Y. Rudensky
Memorial Sloan Kettering Cancer Center, USA

Olaf Rötzschke
Singapore Immunology Network, Singapore

David Artis
Weill Cornell Medical College of Cornell University, USA

Lewis L. Lanier
University of California San Francisco, USA

The 5th NIF Winter School on Advanced Immunology was held with Singapore
Immunology Network (SIgN) in Japan on 17-22 January 2016. The program comprised a 4-day lecture course on Awaji Island and a 2-day international symposiumin Osaka. Forty-nine young researchers from around the world, selected from 191
applicants, learned cutting-edge immunology from 16 world-renowned seniorresearchers at the course and 13 up-and-coming researchers at the symposium.
The Winter School was highly evaluated by the participants and the lecturers as an
excellent opportunity for networking with young peers through active discussions
and communications as well as its high scientific level.

Shigekazu Nagata
IFReC, Osaka University, Japan

Tamotsu Yoshimori
Osaka University, Japan

January 17- 22, 2016

Venue : Awaji Yumebutai International Conference Center, Hyogo /KNOWLEDGE THEATER, Grand Front Osaka( The 7 th International Symposium of IFReC )76

Dendritic cell development and function
Resident and passengers: The architecture and functions of a layered myeloid
systemDendritic cell and macrophage ontogeny

Activation and regulation of inflammasomes in inflammatory diseases

Tackling the helper T cell repertoire

Mechanism of Th17 cell programming by the intestinal microbiota

Differentiation and function of regulatory T cells
Dissection of allergic reactions by systems biology in an allergen-saturated
tropical-urban environment
Immune regulation at barrier surfaces

Innate lymphoid cells and natural killer cells

Apoptosis and exposure of phosphatidylserine

Autophagy: The intracellular self-degradation system fighting against diseases

Erika L. Pearce
Max Planck Institute for Immunobiology andEpigenetics, Germany

Hiroshi Ohno

Date :Title

RIKEN Center for Integrative Medical Science, Japan

Cevayir Coban
IFReC, Osaka University, Japan

Lisa F.P. Ng
Singapore Immunology Network, Singapore

Metabolic interactions in the tumor microenvironment

Gut microbiota, host defense and immunity

Tissue-specific immunopathology during malaria
Cellular and molecular mechanism of alphavirus pathogenesis: Implications fordisease interventions

77

Emergency Drill
IFReC held a fire drill to raise all staff ’s awareness on their safety andsecurity in their work environment. The staff was reminded of theprocedures for evacuation and how to use fire extinguishers under the
instruction of a staff member from the Osaka University Department of
Safety and Hygiene.

The Immunology Lecture Series was initiated as staff development to provide fundamental knowledge of immunology to
IFReC research support staff such as technicians, secretaries
and administrative staff. A young researcher of IFReC is invited
as a speaker to give a talk about the basics up to the cuttingedge of their research in an easy-to-understand manner. The
lectures are open to all Osaka University members and heldin the evening so that the participants can attend after work.
During the lecture, participants can interact with the speaker,
asking questions in an informal setting. Initiated in December
2013, the series was held ten times until the end of FY2015 andthe questionnaire results from the participants show a highlevel of satisfaction throughout the series. The average numberof participants is 45. These events serve as effective measures
to develop IFReC support staff as members of a WPI center, topublicize IFReC in Osaka University as well as to give educational
value to the young speakers selected from IFReC researchers.
This event was proposed for “Osaka University’s Improvement

Venue :

Biken Hall, Osaka University

Date :

July 22, 2015

EventsImmunology Lecture Series

Idea Awards Contest” as an activity model. In this proposal, we
introduced IFReC “Immunology Lecture Series” to provide an
opportunity for young researchers to explain their research inplain words for support staff (secretaries, technical staff andadministrative staff ) of each department in Osaka University inorder to increase the understanding and motivation of them aswell as to improve the presentation skills of young researchers.
This proposal was admirably selected for the award.

Date :

November 11, 2015

Participants :All IFReC Staff

Explanation Session for Preventing the Misuse of Funds
IFReC conducted a seminar to communicate the necessity for compliance by ensuring the awareness of staff of the usage rules for competitive research funding and staff obligations when using the staff purchasing system.
The bilingual session was designed to ensure that all IFReC members
are fully informed of procedures and responsibilities.

Date :

February 10, 2016

Venue :

Taniguchi Memorial Hall,
Osaka University

Speaker : Masahiro Yamamoto ( Immunoparasitology)
Title :

Immune mechanism against parasites− How Toxoplasma hijacks a host body?

Date :March 17, 2016

Speaker : Fuminori Sugihara (Biofunctional Imaging)
Title :

Life phenomena observed− MRI as a research tool78

79

Events

The 1st Workshop for WPI Research Support Staff
DAY 1 ( May 18 )Opening remarks

Explanation of proceedings

Nobuo Sakaguchi(Administrative Director, IFReC )Akihiko Takagi(Research Planning and Management Office, IFReC)

Kavli-IPMU, The University of Tokyo
Research support at Kavli IPMU

IFReC, Osaka University
Towards a new style of research support- IFReC Research Planning and Management Office -iCeMS, Kyoto University
An outline of iCeMS

AIMR, Tohoku University
Research support at AIMR

MANA, National Institute for Materials Science
Omotenashi is our catchphrase -MANA Administration Division-Moderator
Akihiko Takagi (Research Planning and Management Office, IFReC )Panel Discussion
The Research Planning and Management Office at IFReC took initiative
to organize the first workshop for support staff of WPI institutes including administrative and technical staff, and others such as university research administrators. Forty-one participants from all nine WPI institutes
shared the successful achievements of each institute and discussed
support for globalization, promotion of interdisciplinary research, andsystem reformation in order to realize world-visible research institutes.

Date :

May 18 - 19, 2015

Venue :

Taniguchi Memorial Hall, Osaka University /The role, expectations and reality of research support in WPIPanelistsTomiyoshi Haruyama (Administrative Director, Kavli-IPMU)
Shinichi Sato (Deputy Administrative Director, AIMR)
Tomoko Narisawa (Technical Support Staff, ELSI)
Ayato Sato (Head of Research Promotion, ITbM)

DAY 2 ( May 19 )I 2 CNER, Kyushu University
Approaches by I 2 CNER Administration Division

IIIS, University of Tsukuba

IFReC Research Building (for IFReC introduction tour)

Solving the problem of sleep -Research support at IIIS

Participants : Research support staff at nine WPI centers;
ELSI, Tokyo Institute of Technology

Kavli-IPMU, iCeMS, AIMR, MANA, I 2 CNER, IIIS, ELSI, ITbM, and IFReC

Outline of research support at ELSI

ITbM, Nagoya University
Approaches by ITbM and future challenges

Closing remarks

Tour of IFReC

80

Nobuo Sakaguchi (Administrative Director, IFReC)① Prof. Akira's Laboratory, Integrated Life Science Bldg. 10F② Core Instrumentation Facility and Server room, IFReC Bldg. 4F③ MRI, IFReC Bldg. 3F④ Administrative Office, IFReC Bldg. 2F

81

Japanese Class
Japanese language classes are held for overseas researchers / students toalleviate any stress and inconvenience in research or daily life that may becaused by the language barrier.
We offer two lecture-style classes, "Class A: Beginner to Elementary" and"Class B: Pre-intermediate to Intermediate". Class members are expected tolearn hiragana, katakana, some kanji and basic phrases necessary for daily
conversation.

Outreach Activities

During the course, special events were arranged to give themembers an experience of Japanese culture.
In FY2015, international staff and Japanese volunteers enjoyed both traditional and trendy aspects of Japanese culture,with traditional “Tanabata”, and cooking trendy “Deco-Onigiri”(decorated rice ball). Participants learnt new vocabulary andpracticed speaking Japanese under the supervision of a pro-fessional Japanese teacher. It was also a good opportunity forparticipants to communicate with staff from other laboratories.
At the end of the course, the feedback received from participants indicated that most of them realized significant improvement in their Japanese proficiency.

June 30, 2015 Tanabata& Soumen

82

November 26, 2015 Onigiri

Outreach Activities

Science Cafe
The series of science cafes is a long- lasting IFReC outreachactivity to promote communication among researchers and thegeneral public. It also enhances people’s understanding of immunology researches and the researchers involved in them. In
FY2015, IFReC science cafes were held at Osaka University Suita

campus and at CAFE Lab. in the center of Osaka City. CAFE Lab.
at Grand Front Osaka attracts a lot of office workers, who canfeel free to stop by the venue after their work time. About 170
people in total participated in our science cafes.

Science Cafe on the Edge at 2015 Icho Festival

Science Cafe on the Edge 16[ Latest knowledge on gut immunity ][ Immunology research revealed by bioimaging techniques ]Date :

May 2, 2015

Date :

January 22, 2016

Venue :

Techno Alliance Building, Osaka University

Venue :CAFE Lab, Grand Front Osaka

Guest :

Kiyoshi Takeda (Graduate School of Medicine, Osaka University /Guest :

Naoto Kawakami (Ludwig-Maximilians University Munich,

Mucosal Immunology, IFReC)

Germany ),Masaru Ishii (Graduate School of Medicine, Osaka University /Immunology and Cell Biology, IFReC )84

85

The 5 th Annual WPI Joint Symposium
The annual WPI joint symposium is held to introduce WPI research achievements to mainly high school students, as well as
to provide them with a good opportunity to know about world
top-level science. It aims to encourage the students’ interest inscience.
The 5th WPI joint symposium entitled “Science to sense andfeel” was held in Kyoto and gathered about 400 participants.
The program included talks by WPI scientists, a special talk by

Super Science High Schools (SSH) are selected high schools
in Japan, which promote advanced math/science education as
well as collaborative researches with universities, and activities
to develop international perspectives.
The SSH Student Fair 2015 was held in Osaka and more than
200 schools, including several schools from overseas, held

Date :

August 5-6, 2015

Venue :INTEX Osaka

Host :

MEXT, JST

Support : Boards of Education(Osaka Prefecture and Osaka City)

booths with posters to present their researches. WPI institutes
held a collaborative booth and introduced the research activities of each institute using posters, booklets and demonstrations. Several WPI researchers including Akiko Nakai (Immune
Response Dynamics, IFReC ) gave short talks at "Researchers'mini-live talk".

the president of Kyoto University, panel discussion, and booth
exhibition.
Kazuo Yamashita (Systems Immunology, IFReC) gave a talktitled “Computer fighting against Viruses”, and joined the panel
discussion on laboratory notebooks. IFReC also opened a boothto introduce its research activities and held a demonstration
experiment to show stained immune cells in a mouse lung witha laser microscope, which attracted a lot of visitors.

Outreach Activities

Super Science High School Student Fair 2015

Date :
December 26, 2015
Venue : Clock Tower Centennial Hall, Kyoto University (Kyoto)
Host :
Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University
Cohost : WPI Institutes
Support : MEXT, JSPS, Boards of Education (Kyoto Prefecture and Kyoto City)

Speakers■ Koh Nagata (Assistant Professor, iCeMS, Kyoto University)■ Kazuo Yamashita (Assistant Professor, IFReC, Osaka University)■ Ken Takai (Principal Investigator, ELSI, Tokyo Institute of Technology)■ Jyuichi Yamagiwa (President, Kyoto University)

Navigator
Hejcik Pavel (Science Communicator, Miraikan - The National Museum of Emerging Science and Innovation)

Guest for panel discussion
Aya Ota (Freelance writer)

8687

Outreach Activities

AAAS 2016 Annual Meeting
The American Association for the Advancement of Science(AAAS) is the biggest international scientific society in theworld, and its mission is to “advance science and serve society”.
The AAAS 2016 Annual Meeting was held in the US capital andit offered a broad range of activities including lectures, symposia, seminars and exhibits, with the theme of "Global Science

Date :Engagement".
WPI institutes held a collaborative booth to introduce the WPI
program and the institutes’ activities using posters, booklets
and a demonstration experiment. More than 300 participants
visited the booth and gained interest in the WPI program andworld leading researches in Japan.

February 11-15, 2016

Venue : Washington Marriott Wardman Park (Washington D.C., USA)

Research Projects

IFReC on edX
Osaka University joined edX, one of the major Massive Open
Online Course (MOOC) platforms, founded by Massachusetts
Institute of Technology (MIT) and Harvard University. As thefirst course of OsakaUx (edX courses by Osaka University), eight
IFReC researchers including Director Akira provided lecturesfrom July to August 2015, with the support of the Teaching and
Learning Support Center of Osaka University. The course provided fundamental knowledge in immunology as well as someadvanced topics including cutting-edge research results. About
12,000 people enrolled in the course.
The second period of the program is scheduled to be distrib-

88

uted in FY2016. This time, IFReC researchers from bioinformatics and imaging fields will join the program.

89Research Projects

Support Programs for Fusion Researches
One of the goals of the WPI program is to generate novel research fields through fusion of existing research fields. IFReC
aims to create innovative immunology fields by combining
with imaging and bioinformatics technologies. In order to promote this challenge, we launched the following two programs.

WPI Mission
Science
Advancing to the highest
pinnacle globallyleading-edge research

Globalization
Establishing an international
research environment

ReformReforming researchorganizations

FusionCreating interdisciplinary
domains

Research Support Program for Combined Research Fields
was established in FY2009 to financially support research
projects, the members of which are researchers from different
groups / backgrounds. This program effectively encourages interaction and fusion among different groups. So far, 25 projects
have received financial support from IFReC and some of thestudies have been successfully published.
Dual Mentor Program focuses on graduate students oryoung post-doctoral fellows engaging in interdisciplinary projects under the supervision of two PIs from different disciplines.
This program was introduced as a platform to foster young pioneers in the fusion field and to further promote interdisciplinary research at IFReC.
Five Combined Research Program projects and one Dual Mentor Program project ended in September 2015 and 5 projects
are still in progress.

Evaluation Workshop for Research Support Program
All the project leaders presented their research results in front of IFReC
members. The IFReC PIs served as evaluators in the workshop. The resultof the evaluation, including scores and comments, was provided to theleaders. The projects which ended in 2015 were given awards according
to the score.

Date :

October 7, 2015

Venue :

Taniguchi Memorial Hall, Osaka University

Project Leader

Collaborators

Project Title

2012
Trilateral analysis of interferon-γ-mediated cellular innate immunity against toxoplasma gondii

Masahiro Yamamoto

D. Standley
E - M. Frickel

Masako Kohyama

C. Coban
F. Sugihara

Barry Ripley

D. Standley
G. Kurisu ( IPR )Fuminori Sugihara

R. Hanayama
M. Kohyama
S. Akira

K. Kikuchi
S. Satoh

Tomoyuki Yamaguchi

H. Fujita
T. Watanabe
C. Furusawa

S. Sakaguchi
H. Machiyama
S. EsakiDual Mentor Program
Takeshi Yoshida

R. Hanayama
D. Diez

K. Suzuki

Role of tissue macrophage in malaria infection, and their developmental control byparasite metabolite

K. Suzuki
T. AoshiRole of arid5A in the selective control of IL-6 mRNA stability and development of
TH17 cells
In vivo imaging of germinal center development in mouse spleen using MRI

Imaging analysis of immune activation and regulation

Visualizing the dynamics of exosomes during various immune responses in vivo

2013
Diego Diez

R. Hanayama

Yutaro Kumagai

J. Kozuka
N. Trost

S. Teraguchi

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

Naganari Ohkura

A. Vandenbon S. Nakamura
S. Yamazaki
S. KatoM. Hashimoto

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

Kazuhiro Suzuki

Y. Baba

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

Alexis Vandenbon

S. Sakaguchi
N. Ohkura

The dynamics of novel signaling networks of macrophages exposed to pathogens

H. Morikawa

Identification of key factors for inducing functionally stable regulatory T cells

Project leader from the groups of < Immunology>, < Imaging >, < Informatics >90

91

Young Scientist Support Program for Research Abroad
To strengthen our international research network and our basis for international collaborative research, IFReC has provided
financial support to IFReC young researchers who wish to participate in research activities at overseas institutions. The pro-gram aims to develop the practical skills and abilities of young
researchers in international collaborative research and to develop their network with researchers overseas. Four researchers
used this support program in FY2015.

Young Scientist Support Program for Research Abroad
Name
Chen Ting

Country
CanadaConferences attended
ISMRM (International Society for Magnetic Resonance Medicine)
1. PhysCell 2015
2. EMBO Meeting 2015
3. Visit to Chemistry department at Lancaster University

Alison Jane Hobro

Germany / UK

Szandor Simmons

Australia

Alison Jane Hobro

USA

SciX 2015

Mayuri Takana

USA

Keystonesyposia; Immunity in Skin Development, Homeostasis and Disease (B5)

4th European Congress of Immunology

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

Europe 15

North America 22

Asia 3Africa 1
Australia 2

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

92

Data

Data

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

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

Animal Resource Center for Infectious Diseases
・Specific pathogen-free (SPF) animal facility
・Sperm/ embryo freezing and preservation
・In vitro fertilization and embryo transplantation
・Intracytoplasmic sperm injection
・Transgenic and knock-out animals
・Genome editing in experimental animals

Live Immuno-Imaging Facility

IFReC–RIMD Research Complex at Suita Campus of Osaka University

・SPF animal facility with high-performance 11.7T MRI & two- photon microscope

Network Administration Office・Provision and maintenance of network infrastructures :LAN system and servers (web, mail, mailing lists etc.)162Core Instrumentation Facility
・Basic and advanced instruments

3・In-house service :5

DNA sequencing, cell sorting, electron microscopy, mass spectrometry and next
generation DNA sequencing analysis
・Radio isotope facility
・DNA chip center

4
Members of the Core Instrumentation Facility

Photo : S. Higashiyama

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

9495

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

Composition

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

Number of IFReC Staff
300

28
Administrative Staff
Research Support Staff
Researchers

250

Name (initials)

Nationality

Host researcher

PeriodSpecially Appointed Researcher

P. D.

India

KishimotoNov. 1, 2012 - Oct. 31, 2015

UK

Kishimoto

Y. R.

China

K. Ishii

Apr. 1, 2014 - Mar. 31, 2017

Specially Appointed Researcher

J. H.

China

Arase

May 1, 2014 - Apr. 30, 2015

Specially Appointed Researcher

K. R.

Slovenia

Akira

Nov. 16, 2014 - Nov. 15, 2015

Specially Appointed Researcher

L. H.

TaiwanCoban

Dec. 16, 2014 - Jul. 31, 2015

Specially Appointed Researcher

M. B.

Egypt

Saito

Apr. 1, 2015 - Dec. 31, 2015

Specially Appointed Researcher

T. N.

Germany

Seymour

Apr. 1, 2015 - Mar. 31, 2017

Research Fellow

H. H.

JordanKishimotoJun. 8, 2015 - Aug. 6, 2015

15
23

China

Yamamoto

13
3

P. L.

Australia

Coban

Oct. 1, 2015 - Sep. 30, 2016

Specially Appointed Researcher

D. H.

Australia

Sakaguchi

Oct. 16, 2015 - Oct. 15, 2016

T. T.

Vietnam

Kishimoto
KishimotoMar.2008

173

183

186
179

127

Mar.2009

Mar.2010

Mar.2011

Mar.2012

32%

31%

Mar.2013

Mar.2014

30%

26%

Mar.2015

Mar.2016

Number of Researchers
200
From Overseas
Japanese

55
31%

54

48

23%
42

54

31%

42
40

27%

Nov. 2, 2015 - Jan. 28, 2016
Nov. 2, 2015 - Jan. 28, 2016

171

0

100
Vietnam77

52

Jul.1, 2015 - Dec. 28, 2015

Specially Appointed Researcher

Research Fellow

28

89

150

N. N.

82

135

Dec. 1, 2013 - Mar. 31, 2017

Specially Appointed Researcher

Research Fellow

89

31

150

50

Y. C.

74

45

100

Research Fellow

29
25

21

Position of recipient

D. M.

24

28

63

200

FY2015 Kishimoto Fellowship Recipients

Specially Appointed Researcher

Data

Kishimoto Foundation Fellowships

24
50

117

15%
8

119

128

138

137
87

93
65

44
0
Mar.2008

96

Mar.2009

Mar.2010

Mar.2011

Mar.2012

Mar.2013

Mar.2014

Mar.2015

Mar.2016

97

Shimon Sakaguchi

Data

Major Awards

Finance
Thomson Reuters Citation Laureate

Breakdown of total expenditure at IFReC in FY2015

Sakaguchi was elected as a Thomson Reuters Citation Laureate
2015. The Laureates are cited so frequently in the previous two
or more decades that these scientists typically rank in the top
0.1% in their research fields. The laureates are recognized to havewritten multiple high-impact articles over many years.

Individual Research
Project
Personnel

23.7%

32.5%
Total Expenditure

3,506,446,426( JPY )
Sakaguchi at the Thomson Reuters Citation Laureate
Award Ceremony (June, 2016)

Equipment

23.4%

Project
Activities

20.0%

Six IFReC Researchers The World's Most Influential Scientific Minds 2015
TravelHighly Cited Researchers are researchers with papers that have a large number of citations
from all over the world as selected by Thomson Reuters. In 2015, some 3,000 researchers from
21 fields were selected as being some of the most influential researchers in the world.
Seven selections were from IFReC for a total of six researchers (Director Akira was selected fortwo categories).
Biology & Biochemistry
・Shizuo Akira, Host Defense, IFReC
Immunology
・Shizuo Akira, Host Defense, IFReC
・Shimon Sakaguchi, Experimental Immunology, IFReC・Ken Ishii, Vaccine Science, IFReC
・Kiyoshi Takeda, Mucosal Immunology, IFReC
・Masahiro Yamamoto, Immunoparasitology, IFReC・Hiroaki Hemmi, Immune Regulation, IFReC (~2015)

0.4%

Travel0.7%
Equipment

Grant-in-Aid for
Scientific Research

5.1%

20.6%

From the
University

20.2%

Project
Activities

WPI Grant

36.2%

Other Sources

1,285,680,000

Contract
Research

2,220,766,426( JPY )

17.9%

( JPY )

Personnel

58.0%

Taroh Kinoshita

International Glycoconjugate Organization Award

DonationManagement
Expenses Grant

17.2%

24.1%

Kinoshita was awarded the International Glycoconjugate Organization (IGO) Award 2015 andpresented an award lecture at XXIII International Symposium on Glycoconjugates held in Split,
Croatia on September 15, 2015. The title for the award lecture was "Glycosylphosphatidylinositol-anchored proteins: biosynthesis, transport, shedding and deficiencies".

98

99

Data

Publications
1

Adachi, Y; Onodera, T; Yamada, Y; Daio, R; Tsuiji, M; Inoue, T;
Kobayashi, K; Kurosaki, T; Ato, M; Takahashi, Y. Distinct germinal center selection at local sites shapes memory B cell
response to viral escape. Journal of Experimental Medicine
212, 1709-1723 (2015).

2

Arase, N; Arase, H. Cellular misfolded proteins rescued fromdegradation by MHC class II molecules are possible targets
for autoimmune diseases. Journal of Biochemistry 158, 367372 (2015).

3

4

5

Arima, Y; Kamimura, D; Atsumi, T; Harada, M; Kawamoto, T;
Nishikawa, N; Stofkova, A; Ohki, T; Higuchi, K; Morimoto, Y;
Wieghofer, P; Okada, Y; Mori, Y; Sakoda, S; Saika, S; Yoshioka, Y; Komuro, I; Yamashita, T; Hirano, T; Prinz, M; Murakami,
M. A pain-mediated neural signal induces relapse in murine
autoimmune encephalomyelitis, a multiple sclerosis model.
Elife 4, e08733 (2015).
Arita, Y; Nakaoka, Y; Otsuki, M; Higuchi, K; Hashimoto-Kataoka, T; Yasui, T; Masaki, T; Ohtani, T; Kishimoto, T; YamauchiTakihara, K; Komuro, I; Sakata, Y. Cytokine storm after cessation of tocilizumab in a patient with refractory Takayasu
arteritis. International Journal of Cardiology 187, 319-321(2015).

6

Baba, Y. Store-operated Calcium Entry into B Cells Regulates Autoimmune Inflammation. Journal of PharmaceuticalSociety of Japan 136, 473-478 (2016).

7

Baba, Y; Matsumoto, M; Kurosaki, T. Signals controlling thedevelopment and activity of regulatory B-lineage cells. International Immunology 27, 487-493 (2015).

8

Bando, K; Smith, NI; Ando, J; Fujita, K; Kawata, S. Analysis ofdynamic SERS spectra measured with a nanoparticle during intracellular transportation in 3D. Journal of Optics 17,114023 (2015).

9

Bharti, D; Kumar, A; Mahla, RS; Kumar, S; Ingle, H; Yadav,
T; Mishra, A; Raut, AA; Kumar, H. Low prevalence of CCR5Delta 32, CCR2-64I and SDF1-3 ' A alleles in the Baiga and
Gond tribes of Central India. Springerplus 4, 451 (2015).

10

Bosch, DGM; Boonstra, FN; Kinoshita, T; Jhangiani, S; de Ligt,
J; Cremers, FPM; Lupski, JR; Murakami, Y; de Vries, BBA. Cerebral visual impairment and intellectual disability caused
by PGAP1 variants. European Journal of Human Genetics
23, 1689-1693 (2015).

11

100

Arato, T; Daimon, T; Heike, Y; Ishii, K; Itho, K; Kageyama,
S; Kawakami, Y; Nakayama, E; Ozawa, K; Sato, N; Shiku, H;
Takeuchi, M; Tani, K; Tamada, K; Ueda, R; Yamaguchi, Y;
Yamanaka, T; Yamaue, H; Yasukawa, M; Heike, Y; Iguchi, T;
Kageyama, S; Kitano, S; Miyahara, Y; Nagata, Y; Noguchi, M;
Terashima, T; Yamaguchi, Y; Arato, T; Asano, T; Asonuma,
M; Ikeda, H; Kakimi, K; Takesako, K; Tanaka, M; Amakasu, K;
Arato, T; Yamada, A; Harada, N; Aoshi, T; Harada, N; Ishii, K;
Kuroda, E; Kobiyama, K; Muraoka, D; Yamada, A; Arato, T;
Harada, N; Ikeda, H; Kageyama, S; Kawakami, Y; Kitano, S;
Tamada, K; Terashima, T; Ueda, R; Yamazaki, N; Daimon, T;
Kuchiba, A; Takeuchi, M; Tanaka, S; Michimae, H; Yamada,
T; Yamanaka, T. 2015 Guidance on cancer immunotherapy
development in early-phase clinical studies. Cancer Science
106, 1761-1771 (2015).

Briquez, PS; Hubbell, JA; Martino, MM. Extracellular MatrixInspired Growth Factor Delivery Systems for Skin Wound
Healing. Advances In Wound Care 4, 479-489 (2015).

12

13

14

15

Chinen, I; Nakahama, T; Kimura, A; Nguyen, NT; Takemori, H;
Kumagai, A; Kayama, H; Takeda, K; Lee, S; Hanieh, H; Ripley,
B; Millrine, D; D ubey, PK; Nyati, KK; Fujii-Kuriyama, Y; Chowdhury, K; Kishimoto, T. The aryl hydrocarbon receptor/microRNA-212/132 axis in T cells regulates IL-10 production tomaintain intestinal homeostasis. International Immunology
27, 405-415 (2015).

24

Gyobu, S; Miyata, H; Ikawa, M; Yamazaki, D; Takeshima, H;
Suzuki, J; Nagata, S. A Role of TMEM16E Carrying a Scrambling Domain in Sperm Motility. Molecular and Cellular Biology 36, 645-659 (2016).

25

Halder, SK; Matsunaga, H; Ishii, KJ; Ueda, H. Prothymosin-alpha preconditioning activates TLR4-TRIF signaling to induce
protection of ischemic retina. Journal of Neurochemistry
135, 1161-1177 (2015).

Chiu, LD; Palonpon, AF; Smith, NI; Kawata, S; Sodeoka, M; Fujita, K. Dual-polarization Raman spectral imaging to extract
overlapping molecular fingerprints of living cells. Journal ofBiophotonics 8, 546-554 (2015).
Daniels, NJ; Hyde, E; Ghosh, S; Seo, K; Price, KM; Hoshino, K;
Kaisho, T; Okada, T; Ronchese, F. Antigen-specific cytotoxic
T lymphocytes target airway CD103(+) and CD11b(+) dendritic cells to suppress allergic inflammation. Mucosal Immunology 9, 229-239 (2016).
David, BG; Okamoto, K; Kakizuka, T; Ichimura, T; Watanabe,
TM; Fujita, H. Gene dynamics of core transcription factors for
pluripotency in embryonic stem cells. Journal of Bioscienceand Bioengineering 119, 406-409 (2015).

16

Eickhoff, S; Brewitz, A; Gerner, MY; Klauschen, F; Komander, K;
Hemmi, H; Garbi, N; Kaisho, T; Germain, RN; Kastenmuller, W.
Robust Anti-viral Immunity Requires Multiple Distinct T CellDendritic Cell Interactions. Cell 162, 1322-1337 (2015).

17

Flores-Langarica, A; Bobat, S; Marshall, JL; Yam-Puc, JC; Cook,
CN; Serre, K; Kingsley, RA; Flores-Romo, L; Uematsu, S; Akira,S; Henderson, IR; Toellner, KM; Cunningham, AF. Soluble
flagellin coimmunization attenuates Th1 priming to Salmonella and clearance by modulating dendritic cell activationand cytokine production. European Journal of Immunology
45, 2299-2311 (2015).

18

Fujii, T; Sakata, A; Nishimura, S; Eto, K; Nagata, S. TMEM16F
is required for phosphatidylserine exposure and microparticle release in activated mouse platelets. PNAS 112, 1280012805 (2015).

19

Fujiwara, I; Murakami, Y; Niihori, T; Kanno, J; Hakoda, A; Sakamoto, O; Okamoto, N; Funayama, R; Nagashima, T; Nakayama, K; Kinoshita, T; Kure, S; Matsubara, Y; Aoki, Y. Mutations
in PIGL in a Patient with Mabry Syndrome. American Journalof Medical Genetics Part A 167, 777-785 (2015).

20

Fujiwara, S; Yoshioka, Y; Matsuda, T; Nishimoto, H; Murakami, T; Ogawa, A; Ogasawara, K; Beppu, T. Brain temperature
measured by H-1-magnetic resonance spectroscopy inacute and subacute carbon monoxide poisoning. Neuroradiology 58, 27-32 (2016).

21

Fukasaka, M; Asari, D; Kiyotoh, E; Okazaki, A; Gomi, Y; Tanimoto, T; Takeuchi, O; Akira, S; Hori, M. A Lipopolysaccharide
from Pantoea Agglomerans Is a Promising Adjuvant for Sublingual Vaccines to Induce Systemic and Mucosal Immune
Responses in Mice via TLR4 Pathway. Plos One 10, UNSP
e0126849 (2015).

22

Fukushima, N; Ishii, S; Tsujiuchi, T; Kagawa, N; Katoh, K.
Comparative analyses of lysophosphatidic acid receptormediated signaling. Cellular and Molecular Life Sciences 72,
2377-2394 (2015).

23

Goritzka, M; Makris, S; Kausar, F; Durant, LR; Pereira, C; Kumagai, Y; Culley, FJ; Mack, M; Akira, S; Johansson, C. Alveolar
macrophage-derived type I interferons orchestrate innate
immunity to RSV through recruitment of antiviral monocytes. Journal of Experimental Medicine 212, 699-714 (2015).

26

Hasegawa, K; Tanaka, S; Fujiki, F; Morimoto, S; Nakajima,
H; Tatsumi, N; Nakata, J; Takashima, S; Nishida, S; Tsuboi,
A; Oka, Y; Oji, Y; Kumanogoh, A; Sugiyama, H; Hosen, N. An
Immunocompetent Mouse Model for MLL/AF9 Leukemia
Reveals the Potential of Spontaneous Cytotoxic T-Cell Response to an Antigen Expressed in Leukemia Cells. Plos One
10, e0144594 (2015).

27

Hasegawa, K; Tanaka, S; Fujiki, F; Morimoto, S; Nakano, K;
Kinoshita, H; Okumura, A; Fujioka, Y; Urakawa, R; Nakajima,
H; Tatsumi, N; Nakata, J; Takashima, S; Nishida, S; Tsuboi, A;
Oka, Y; Oji, Y; Miyoshi, E; Hirata, T; Kumanogoh, A; Sugiyama,
H; Hosen, N. Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-MediatedCytolysis. Plos One 11, e0152326 (2016).

28

29

Hashimoto, N; Tsuboi, A; Kagawa, N; Chiba, Y; Izumoto, S;
Kinoshita, M; Kijima, N; Oka, Y; Morimoto, S; Nakajima, H;
Morita, S; Sakamoto, J; Nishida, S; Hosen, N; Oji, Y; Arita, N;
Yoshimine, T; Sugiyama, H. Wilms tumor 1 peptide vaccination combined with temozolomide against newly diagnosed
glioblastoma: safety and impact on immunological response.
Cancer Immunology Immunotherapy 64, 707-716 (2015).
Hashimoto-Kataoka, T; Hosen, N; Sonobe, T; Arita, Y; Yasui, T;
Masaki, T; Minami, M; Inagaki, T; Miyagawa, S; Sawa, Y; Murakami, M; Kumanogoh, A; Yamauchi-Takihara, K; Okumura,
M; Kishimoto, T; Komuro, I; Shirai, M; Sakata, Y; Nakaoka, Y.
Interleukin-6/interleukin-21 signaling axis is critical in thepathogenesis of pulmonary arterial hypertension. PNAS 112,
E2677-2686 (2015).

30

Hatzihristidis, T; Desai, N; Hutchins, AP; Meng, TC; Tremblay,
ML; Miranda-Saavedra, D. A Drosophila-centric view of protein tyrosine phosphatases. Febs Letters 589, 951-966 (2015).

31

Hayashi, M; Aoshi, T; Kogai, Y; Nomi, D; Haseda, Y; Kuroda, E;
Kobiyama, K; Ishii, KJ. Optimization of physiological properties of hydroxyapatite as a vaccine adjuvant. Vaccine 34,
306-312 (2016).

32

Hibar, DP et al. Common genetic variants influence humansubcortical brain structures. Nature 520, 224-U216 (2015).

33

Hirai, T; Yoshioka, Y; Takahashi, H; Ichihashi, K; Udaka, A;
Mori, T; Nishijima, N; Yoshida, T; Nagano, K; Kamada, H; Tsunoda, S; Takagi, T; Ishii, KJ; Nabeshi, H; Yoshikawa, T; Higashisaka, K; Tsutsumi, Y. Cutaneous exposure to agglomeratesof silica nanoparticles and allergen results i n IgE-biased
immune response and increased sensitivity to anaphylaxis
in mice. Particle and Fibre Toxicology 12, 16 (2015).

34

Hirata, T; Fujita, M; Nakamura, S; Gotoh, K; Motooka, D; Murakami, Y; Maeda, Y; Kinoshita, T. Post-Golgi anterograde
transport requires GARP-dependent endosome-to-TGN retrograde transport. Molecular Biology Of The Cell 26, 30713084 (2015).

35

Hirayasu, K; Arase, H. Functional and genetic diversity ofleukocyte immunoglobulin-like receptor and implication for
disease associations. Journal of Human Genetics 60, 703-708(2015).

36

Huehn, J; Floess, S; Ohkura, N; Sakaguchi, S. Comment on"Cutting Edge: Epigenetic Regulation of Foxp3 Defines a
Stable Population of CD4(+) Regulatory T Cells in Tumors
from Mice and Humans". Journal of Immunology 194,33533-33533 (2015).

37

Ichimura, T; Chiu, LD; Fujita, K; Machiyama, H; Kawata, S;
Watanabe, TM; Fujita, H. Visualizing the appearance and disappearance of the attractor of differentiation using Raman
spectral imaging. Scientific Reports 5, 11358 (2015).

38

Ichiyama, K; Chen, TT; Wang, XH; Yan, XW; Kim, BS; Tanaka, S;
Ndiaye-Lobry, D; Deng, YH; Zou, YL; Zheng, P; Tian, Q; Aifantis, I; Wei, L; Dong, C. The Methylcytosine Dioxygenase Tet2
Promotes DNA Demethylation and Activation of Cytokine
Gene Expression in T Cells. Immunity 42, 613-626 (2015).

39

Ikeda, T; Ong, EBB; Watanabe, N; Sakaguchi, N; Maeda, K;
Koito, A. Creation of chimeric human/rabbit APOBEC1 with
HIV-1 restriction and DNA mutation activities. Scientific Reports 6, 19035 (2016).

40

Ilkovski, B; Pagnamenta, AT; O'Grady, GL; Kinoshita, T;
Howard, MF; Lek, M; Thomas, B; Turner, A; Christodoulou,
J; Sillence, D; Knight, SJL; Popitsch, N; Keays, DA; Anzilotti,
C; Goriely, A; Waddell, LB; Brilot, F; North, KN; Kanzawa, N;
Macarthur, DG; Taylor, JC; Kini, U; Murakami, Y; Clarke, NF.
Mutations in PIGY: expanding the phenotype of inheritedglycosylphosphatidylinositol deficiencies. Human Molecular
Genetics 24, 6146-6159 (2015).

41

Ingle, H; Kumar, S; Raut, AA; Mishra, A; Kulkarni, DD; Kameyama, T; Takaoka, A; Akira, S; Kumar, H. The microRNA miR-485
targets host and influenza virus transcripts to regulate antiviral immunity and restrict viral replication. Science Signaling 8, ra126 (2015).

42

Inoue, T; Morita, M; Hijikata, A; Fukuda-Yuzawa, Y; Adachi,
S; Isono, K; Ikawa, T; Kawamoto, H; Koseki, H; Natsume, T;
Fukao, T; Ohara, O; Yamamoto, T; Kurosaki, T. CNOT3 contributes to early B cell development by controlling Igh rearrangement and p53 mRNA stability. Journal of Experimental
Medicine 212, 1465-1479 (2015).

43Ise, W. Development and function of follicular helper T cells.
Bioscience Biotechnology and Biochemistry 80, 1-6 (2016).

44

Ito, D; Nojima, S; Nishide, M; Okuno, T; Takamatsu, H; Kang,
SJ; Kimura, T; Yoshida, Y; Morimoto, K; Maeda, Y; Hosokawa,
T; Toyofuku, T; Ohshima, J; Kamimura, D; Yamamoto, M; Murakami, M; Morii, E; Rakugi, H; Isaka, Y; Kumanogoh, A. mTOR
Complex Signaling through the SEMA4A-Plexin B2 Axis
Is Required for Optimal Activation and Differentiation of
CD8(+) T Cells. Journal of Immunology 195, 934-943 (2015).

45

Jin, T; Tsuboi, S; Komatsuzaki, A; Imamura, Y; Muranaka, Y;
Sakata, T; Yasuda, H. Enhancement of aqueous stability andfluorescence brightness of indocyanine green using small
calix[4]arene micelles for near-infrared fluorescence imaging. Medchemcomm 7, 623-631 (2016).

46

Jung, Y; Wen, T; Mingler, MK; Caldwell, JM; Wang, YH; Chaplin, DD; Lee, EH; Jang, MH; Woo, SY; Seoh, JY; Miyasaka, M;
Rothenberg, ME. IL-1 beta in eosinophil-mediated small
intestinal homeostasis and IgA production. Mucosal Immunology 8, 930-942 (2015).

47

Kaileh, M; Vazquez, E; MacFarlane, AW; Campbell, K; Kurosaki, T; Siebenlist, U; Sen, R. mTOR-Dependent and Independent Survival Signaling by PI3K in B Lymphocytes. Plos One
11, e0146955 (2016).

101

Data
48

49

50

51

52

Kamimura, D; Atsumi, T; Stofkova, A; Nishikawa, N; Ohki, T;
Suzuki, H; Katsunuma, K; Jiang, JJ; Bando, H; Meng, J; Sabharwal, L; Ogura, H; Hirano, T; Arima, Y; Murakami, M. Naive
T Cell Homeostasis Regulated by Stress Responses and TCR
Signaling. Frontiers in Immunology 6, UNSP 638 (2015).
Kamimura, D; Katsunuma, K; Arima, Y; Atsumi, T; Jiang, JJ;
Bando, H; Meng, J; Sabharwal, L; Stofkova, A; Nishikawa,
N; Suzuki, H; Ogura, H; Ueda, N; Tsuruoka, M; Harada, M;
Kobayashi, J; Hasegawa, T; Yoshida, H; Koseki, H; Miura, I;
Wakana, S; Nishida, K; Kitamura, H; Fukada, T; Hirano, T; Murakami, M. KDEL receptor 1 regulates T-cell homeostasis via
PP1 that is a key phosphatase for ISR. Nature Communications 6, 7474 (2015).
Kaneshiro, J; Watanabe, TM; Fujita, H; Ichimura, T. Full control of polarization state with a pair of electro-optic modulators for polarization-resolved optical microscopy. Applied
Optics 55, 1082-1089 (2016).
Kashiwagi, I; Morita, R; Schichita, T; Komai, K; Saeki, K; Matsumoto, M; Takeda, K; Nomura, M; Hayashi, A; Kanai, T; Yoshimura, A. Smad2 and Smad3 Inversely Regulate TGF-beta
Autoinduction in Clostridium butyricum-Activated Dendritic
Cells. Immunity 43, 65-79 (2015).

53

Kashiwagi, Y; Rokugawa, T; Yamada, T; Obata, A; Watabe,
H; Yoshioka, Y; Abe, K. Pharmacological MRI Response toa Selective Dopamine Transporter Inhibitor, GBR12909, in
Awake and Anesthetized Rats. Synapse 69, 203-212 (2015).

54

Kawamura, A; Miyagawa, S; Fukushima, S; Kawamura, T;
Kashiyama, N; Ito, E; Watabe, T; Masuda, S; Toda, K; Hatazawa, J; Morii, E; Sawa, Y. Teratocarcinomas Arising from Allogeneic Induced Pluripotent Stem Cell-Derived Cardiac
Tissue Constructs Provoked Host Immune Rejection in Mice.
Scientific Reports 6, 19464 (2016).

55

102

Kamimura, D; Arima, Y; Tsuruoka, M; Jiang, JJ; Bando, H;
Meng, J; Sabharwal, L; Stofkova, A; Nishikawa, N; Higuchi,
K; Ogura, H; Atsumi, T; Murakami, M. Strong TCR-mediatedsignals suppress integrated stress responses induced by
KDELR1 deficiency in naive T cells. International Immunology 28, 117-126 (2016).

Kayama, H; Takeda, K. Functions of innate immune cells
and commensal bacteria in gut homeostasis. Journal of Biochemistry 159, 141-149 (2016).

56

Kikuchi, K. F-19 MRI Probes with Tunable Switches and Highly Sensitive F-19 MRI Nanoprobes. Bulletin of the Chemical
Society of Japan 88, 518-521 (2015).

57

Kinoshita, T. Glycosylphosphatidylinositol (GPI) Anchors:
Biochemistry and Cell Biology: Introduction to a Thematic
Review Series. Journal of Lipid Research 57, 4-5 (2016).

58

Kinoshita, T; Fujita, M. Biosynthesis of GPI-anchored proteins: special emphasis on GPI lipid remodeling. Journal of
Lipid Research 57, 6-24 (2016).

59

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Mizuguchi, Y; Lee, BW; Okada, T; Tomonaga, T; Myojo, T;
Yamamoto, K; Kitajima, S; Horie, M; Kuroda, E; Morimoto,Y. Pulmonary toxicity of well-dispersed titanium dioxide
nanoparticles following intratracheal instillation. Journal of
Nanoparticle Research 17, 241 (2015).

144

Young, SAM; Miyata, H; Satouh, Y; Kato, H; Nozawa, K;
Isotani, A; Aitken, RJ; Baker, MA; Ikawa, M. CRISPR/Cas9Mediated Rapid Generation of Multiple Mouse Lines Identified Ccdc63 as Essential for Spermiogenesis. International
Journal of Molecular Sciences 16, 24732-24750 (2015).

145

Zhang, SY; Mano, H; Ganesh, G; Robbins, T; Seymour, B. Dissociable Learning Processes Underlie Human Pain Conditioning. Current Biology 26, 52-58 (2016).

The articles were published between April, 2015 and March, 2016. The data were acquired using Web of Science Core CollectionTM on June 1,
2016, and sorted by alphabetical order of the first authors.

105

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Access Map

Access Map

Graduate School of Medicine
Handaiguchi

Ibaraki Mihogaoka

Senri Gate

Osaka Monorail
Handai-byoin-mae Station
Handai Honbumae

Graduate School of Engineering

Hankyu Railway
Kita-Senri Station

Main Gate

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

Graduate School of Frontier Biosciences

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

Kita Fire
Station

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

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