enter rontier FY 2017 mmunology of IFReC World Premier International Premier World Center Research University Osaka I F Research C Annual Report 3.6

WPI Immunology Frontier Research Center FY2017 Osaka University Osaka University IFReC), (IFReC), Research Center Research Planning & Management Office, IFReC Copyright : Immunology Frontier Edit : Published in June, 2018 Message from the Director

As the Director of the Immunology Frontier Re- tious program without precedent. The research- search Center (WPI-IFReC) at Osaka University, I ers at IFReC have been making an increased ef- am very pleased to present the IFReC annual re- fort to make this innovative attempt successful in port for fiscal 2017. Japan. Since its inception in 2007, IFReC has estab- Although the governing structure has changed, lished itself as a Visible International Research our most important mission “Constructing a CONTENTS Center of Immunology with the support of many world-top immunology research center” remains people including the WPI Program Director and the same. We will make unceasing efforts to de- Message from the Director ・・・・・・1 Super Science High School Students Fair / Students Visit ・・・・73 the Program Officer. velop immunology research to ensure translation WPI Science Symposium ・・・・・・74 In the April of 2017, IFReC became one of the to medical science. Organization AAAS 2018 Annual Meeting ・・・・・・75 first members of the “WPI Academy”. Further- We are committed to continuing contributions Organization Chart ・・・・・・4 Naturejobs Career Expo ・・・・・・76 more, IFReC created a new mark in its history to scientific advances through research and edu- Committee and Advisory Board for IFReC ・・・・・・6 with a novel academic-industry partnership cation and evolvement as a world top immunol- Administrative O ce of IFReC 8 Research Projects ・・・・・・agreement. This governance system is an ambi- ogy research center. Advanced Postdoc System ・・・・・・78 Laboratories Young Scientist Support Program for Research Abroad ・・・・・・79 Immunology Group ・・・・・・10 Imaging Group・・・・・・46 Data Informatics Group ・・・・・・58 Common Facilities・・・・・・82 Unit for Combined Research Fields ・・・・・・60 Kishimoto Foundation Fellowship ・・・・・・83 Major Awards ・・・・・・84 Events & Outreach Activities Composition ・・・・・・86 The 7 th NIF Winter School on Advanced Immunology ・・・・・・64 Finance ・・・・・・87

The 9 th International Symposium of IFReC Selected Articles・・・・・・88 - Immunology at the Forefront・・・・・・66 Publications ・・・・・・92 IFReC Seminars・・・・・・68 IFReC Colloquia ・・・・・・70 Access Map ・・・・・・98 Shizuo Akira, MD, PhD Science Café・・・・・・72 Director WPI Immunology Frontier Research Center

1 Organization As of March, 2018

Organization Chart Immunology Group Host Defense ...... Shizuo Akira Immunoglycobiology ...... Taroh Kinoshita Immunopathology ...... Atsushi Kumanogoh Immunochemistry ...... Hisashi Arase Immune Regulation ...... Tadamitsu Kishimoto Mucosal Immunology ...... Kiyoshi Takeda MEXT Immune Regulation ...... Hitoshi Kikutani Experimental Immunology ...... Shimon Sakaguchi Osaka university Cell Signaling ...... Takashi Saito WPI Initiative International Advanced Program Committee Lymphocyte Di erentiation ...... Tomohiro Kurosaki Research Institute(IARI) Lymphocyte Development ...... Fritz Melchers Malaria Immunology ...... Cevayir Coban JSPS Vaccine Science ...... Ken J. Ishii Immunoparasitology ...... Masahiro Yamamoto Biochemistry and Immunology ...... Shigekazu Nagata Molecular Neuroscience ...... Toshihide Yamashita Molecular Immunology ...... Sho Yamasaki Statistical Immunology ...... Yukinori Okada Stem Cell Biology and Developmental Immunology ...... Takashi Nagasawa

Imaging Group DirectorDirector Research GroupsGroups Single Molecule Imaging ...... Toshio Yanagida / Ben Seymour Immunology and Cell Biology ...... Masaru Ishii Nuclear Medicine ...... Jun Hatazawa Deputy DirectorsDirectors MManagementanagement Chemical Imaging Techniques ...... Kazuya Kikuchi CommitteeCommittee Biophotonics ...... Nicholas Isaac Smith Immune Response Dynamics ...... Kazuhiro Suzuki BoardBoard of RepresentativesRepresentatives Informatics Group AdministrativeAdministrative InternationalInternational Systems Immunology ...... Daron M Standley DirectorDirector Scienti cScienti c AdvisoryAdvisory BoardBoard

GGeneraleneral AAairsairs SectiSectionon AccountinAccountingg SectioSectionn Unit for Combined Research Fields Quantitative Immunology ...... Yutaro Kumagai Administrative DepartmentDepartment Diego Diez

Joint Research Chair of Innovative Drug Discovery in Immunology

ResearchResearch PPlanninglanning Chugai-pharm ...... Kunihiro Hattori aandnd Ryusuke Omiya ManaManagementgement OOcece Junichi Hata

Core Instrumentation Facility Common Facilities Animal Resource Center for Infectious Diseases Network Administration Oce

Domestic Institute for Frontier Life and Medical Sciences, Kyoto University Cooperative RIKEN Center for Integrative Medical Sciences Institutions National Institute of Biomedical Innovation, Health and Nutrition Overseas Indian Institute of Science Education and Research, India

4 5 Committee and Advisory Board for IFReC

World Premier International Research Center Initiative (WPI) WPI Academy

Program Director As of Mar. 2018 In FY 2017, MEXT established the WPI Academy to be the vanguard in internationalizing and further renovating Japan’s research environment. The WPI Academy is a much-anticipated upgrade of WPI institutes, and is expected to position Japan as a hub at the Akira Ukawa Deputy Director, RIKEN Advanced Institute for Computational Science, Japan pinnacle of international researcher circulation. Deputy Program Director The five WPI centers including IFReC are regarded to have achieved “world-premier status”, and thus became the initial members of the WPI Academy. Minoru Yoshida Chief Scientist, Chemical Genetics Laboratory, RIKEN, Japan In the decade ahead, the research institutes of WPI and WPI Academy will work together to hold public relations and outreach ac- tivities. Program Committee Members Toshiaki Ikoma Special Adviser, Canon Inc., Japan Academy Director Shinichiro Ohgaki President, Japan Water Research Center, Japan Toshio Kuroki Special Advisor, Research Center for Science Systems, JSPS, Japan

Maki Kawai Director General, Institute for Molecular Science, National Institutes of Natural Sciences, Japan Academy Officer for IFReC Kiyoshi Kurokawa Professor Emeritus, National Graduate Institute for Policy Studies, Japan Takehiko Sasazuki University Professor, Institute for Advanced Study, Kyushu University, Japan

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

Norihiko Suzuki Chair of the Board/President, Akita International University, Japan International Scientific Advisory Board Ryozo Nagai President, Jichi Medical University, Japan As of Mar. 2018 Jeffrey Ravetch The Rockefeller University, USA Immunology Michiharu Nakamura Counselor to the President, Japan Science and Technology Agency (JST), Japan Christopher Goodnow Australian National University, Australia Immunology 〈Chairperson〉 Director-General, Center for Research and Development Strategy, JST, Japan Ryoji Noyori Nobel Laureate in Chemistry (2001) Richard Locksley University of California, San Francisco, USA Immunology

Michinari Hamaguchi President, JST, Japan Lewis L. Lanier University of California, San Francisco, USA Immunology

Hiroshi Matsumoto President, RIKEN, Japan Anne O'Garra The Francis Crick Institute, UK Immunology

Rita Colwell Distinguished Professor, University of Maryland, USA Kiyoshi Takatsu Toyama Prefectural Institute for Pharmaceutical Research, Japan Immunology Kazuo Sugamura Miyagi Cancer Center Research Institute, Japan Immunology Richard Dasher Consulting Professor, Stanford University, USA Yale Goldman University of Pennsylvania, USA Imaging Victor Joseph Dzau President, National Academy of Medicine, USA Yasuyoshi Watanabe Center for Life Science Technologies, RIKEN, Japan Imaging Director, Max Planck Institute for Solid State Research, Germany Klaus von Klitzing Nobel laureate in Physics (1985) Masamitsu Iino Nihon University School of Medicine, Japan Imaging

Chuan Poh Lim Chairman, Agency for Science, Technology and Research, Singapore Akinori Kidera Yokohama City University, Japan Informatics

Harriet Wallberg Former president, Karolinska Institutet, Sweden

Jean Zinn-Justin Scientific adviser, IRFU/CEA, France

6 7 Administrative Office of IFReC

General Affairs Section

・ Employment /acceptance of researchers and staff procedures ・ Social insurance (part-time)/ employment insurance ・ Management of work hours ・ Procedures related to patents ・ Issuing various certificates ・ Procedures related to international students ・ Support for international researchers

Accounting Section

・ Budget drafting / implementation / management ・ Purchasing procedures ・ Acceptance and implementation of third-party funding ・ Payment of payroll, travel expense and honorarium ・ Health insurance procedures ・ Management of buildings and assets ・ RI (Radio Isotope) procedures Laboratories Research Planning and Management Office

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

As of April, 2018

8 10 ent patternsingeneexpression. recognize differentmicrobialcomponents,andgiverisetodiffer ruses, andplayamajorroleininnateimmunity.IndividualTLRs ing organismsrangingfrombacteriatofungi,protozoaandvi of microorganisms.Toll-likereceptors(TLRs)arecapablesens recognition receptors(PRRs),whichrecognizespecificstructures pathogens. Innate immune responses are initiated by pattern lutionally conservedhostdefensemechanismagainstvarious progenitor involvedinfibrosis,SatM Identification ofan atypicalmonocyteandcommitted Host Defense regulated by CCAAT/enhancer binding protein beta (C/EBPβ), atypical monocytes (SatM),sharegranulocytecharacteristics, are monocytes, whichwetermed segregated-nucleus-containing macrophage subsetinthatCeacam1+Msr1+Ly6C-F4/80-Mac1+ monocytes andmacrophages.Recently, wehaveidentifiedanew The developmentoffibrosisis associatedwithactivationof esis ispoorlyunderstood,andthere arefeweffectivetherapies. is alife-threateningdiseaseofunknownaetiology.Itspathogen development of various diseases. Among these diseases, fibrosis meostasis ofperipheralorgans,keyimmunologicalprocessesand functions. Itisthoughtthatthesecellsplayacrucialroleinho and macrophagescompriseavarietyofsubsetswithdiverse anti-inflammatory reactions,andtissueremodeling.Monocytes the contrary,M2macrophagesareassociatedwithresponsesto are pro-inflammatoryandhaveacentralroleinhostdefense.On Macrophages consist of at least two subgroups. M1 macrophages Macrophages consistofatleasttwosubgroups.M1macrophages We arefocusedontheinnateimmunesystem,whichisanevo ------protein Regnase-1inimmune system Regulation ofmRNAstability byCCCH-typezinc-finger like 2(Batf2)intumor-associatedmacrophages Role ofbasicleucinezippertranscriptionfactorATF- logical roleofSatMandrelatedsubsets. and arecriticalforfibrosis.Wefurtherinvestigatingphysio zinc fingerproteins. Conventional Regnase-1 knockoutmicede checkpoint blockersandTLRagonists. may be an important target in anticancer treatment with immune tivation andaccumulationwithinthetumor.Therefore,BATF2 sociated macrophages,whicheventuallyinducesCD8+T-cellac- tumor effectthroughtheup-regulationofIL-12p40intumor-as p50/p65 andpromotesIL-12p40expression.BATF2hasananti ligands. Additionally,wefoundthatBATF2interactswithNF-kB Batf2-deficient macrophagesfollowingtheirstimulationbyTLR showed that theIL-12p40expressionwassignificantlylowerin of Batf2-deficientmicecomparedwithWTmice.Invitrostudies p40+ macrophagesandactivatedCD8+Tcellswithinthetumors Subcutaneous inoculatedtumormodelproducedfewerIL-12 per transcriptionfactorATF-like2(Batf2)hasanantitumoreffect. production ofvariouscytokines.Wefoundthatbasicleucinezip cells playanimportantroleinthecancerprogressionthrough mors isespeciallyimportantforcancerprognosis.Innateimmune gently needed,andtheaccumulationofCD8+Tcellswithintu Regnase-1, alsoknown asZc3h12a,isamemberof CCCH-type The development of effective treatments against cancers is ur- Shizuo Akira,MD/PhD Support Staff Support Visiting Scientist Research Assistant Postdoctoral Fellow Assistant Professor Associate Professor Professor 8 5 8 2 TanakaHiroki Takashi Satoh Kazuhiko Maeda Shizuo Akira ------● ● ● volved in control of the mRNA stability of T cell effector genes. T cellactivation.Consistently,theproteaseactivityofMalt1isin- by Malt1proteaseafterTcellreceptorstimulationandenhances and c-Rel,Ox40,IL-2inCD4 age oftheir3'UTRssuchasIL-6andIL-12p40inmacrophages nuclease activity anddestabilizesaset of mRNAs through cleav splenomegaly and lymphadenopathy. Regnase-1 has the endo- veloped spontaneousautoimmunediseasesaccompaniedby Recent Publications E7331 (2017). tion intumor-associated macrophages. Proc. Natl. Acad. USA.114, Sci. H.etal. AntitumorKanemaru effectofBatf2 through IL-12 p40up-regula 30, 69-78(2018). inexperimental autoimmuneactivation encephalomyelitis. Immunol. Int. etal. Schlafen-8 K. is essential forNakagawa lymphatic endothelial cell (2018) inpress. viaphospho-STAT3lates NF-κBactivity Immunol. Int. dephosphorylation. H.etal. BATF2Kanemaru activates DUSP2geneexpression andupregu The morphologyofSatM. Figure 1. + Tcells.Regnase-1itselfiscleaved - - - mune-related inflammatorydiseases. immune system and to develop an effective treatment for im- achieve thegoalofacomprehensiveunderstandinginnate and tissue-specificRegnase-1deficientmice.Wearestudyingto nase-1 inimmuneandnon-immunecellsbyusingbothmutant ings, wepromotetheunderstandingofpreciousroleReg critical formodulationofTcellactivation.Basedonthesefind This indicatesthatdynamiccontrolofRegnase-1expressionis ● ● proteins in immunecells. Immnunol.29,149-155(2017). Int. by CCCH-type S. RegulationofmRNAstability K&Akira Maeda zincfinger B cells. Immunol.29,211-220(2017). Int. somatic hypermutationby AID-induced GANPduring center ingerminal Eid MMA. etal. of immunoglobulin variableregion Integrity is supported Concept ofdisorderspecificmacrophages. Figure 2. - - 11 Immunology 12 studies onGPIbiosynthesisanddeficiency. teins (GPI-APs).In2017,wemadethefollowingprogressin cies ofglycosylphosphatidylinositol(GPI)andGPI-anchoredpro Studies onGPI-APbiosynthesis Immunoglycobiology teins. Wefound that N-glycanattachedtonascent GPI-APregu tol deacylation)that occurssoonafterattachment ofGPItopro and studied regulation of the first GPI remodeling reaction (Inosi- inserted (Hirata,T.etal.,Nat.Commun., 2018). domain closetotheGolgimembrane wherethesubstrateGPIis ure). WeproposeTM2andTM3act tolocalizeaglycosyltransferase syltransferase domainasshowninthe3Dstructuralmodel(Fig membrane domains(TM2andTM3)insertionintoatypicalglyco and stemregion)intheC-terminalportionhastandemtrans characteristics in theN-terminal part (short cytoplasmic part, TM1 membrane passproteins.PGAP4hastypeIIprotein (TMs) whereasmanyotherglycosyltransferasesaretypeIIsingle glycosyltransferases: PGAP4hasthreetransmembranedomains (Figure). PGAP4hasanovelstructureamongmanyGolgi-resident ring afterfattyacidremodelingmediatedbyPGAP3andPGAP2 This isthe17thstepinGPI-APbiosynthesispathwayoccur PGAP4 transfersN-acetylgalactosamineside-chaintoGPI-anchor. resident N-acetylgalactosaminetransferaseandtermeditPGAP4. side-chain modificationhavebeenunclear.WeidentifiedaGolgi- side-chain glycan. Biosynthesis and biological functions of the We collaboratedwithDr.Fujitain JiangnanUniversity,China, GPI-anchors insomemammalianGPI-APsaremodifiedbya We havebeenstudyingbiosynthesis,functionsanddeficien ------effective measures toamelioratesymptomsofIGDs. and progressin development ofbetterdiagnostics ofIGDsand these studiescontributestoour deeper understandingofIGDs biosynthesis andmaturationof GPI.Knowledgegainedfrom genic mutationsthatcauseIGDswere foundin18of27genesfor mutant gene(Pagnamenta,A.T.et al.,Hum.Mutat.,2018).Patho we determinedfunctionsofPIGH proteinsgeneratedfromthe identified thefirstcaseofIGDcausedbymutationsinPIGHgene, In collaborationwithDr.PagnamentaandcolleaguesinUK,who function ofGPAA1(Nguyen,T.T.M.etal.,Am.J.Hum.Genet.,2017). GPI-APs. Wefoundthepatients’mutationscausepartiallossesof transamidase thattransfersGPItoprecursorproteinsgenerate ties causedbythosemutations.GPAA1encodesasubunitofGPI tations inGPAA1gene,wecharacterizedfunctionalabnormali group inCanada,whoidentifiedthefirstcasesofIGDhavingmu J. etal.,Hum.Mutat.,2017).IncollaborationswithDr.Campeau types from infantile lethalityto mild learning difficulties (Tanigawa, phenotype correlationsofPIGOdeficiencywithvariablepheno ing withIGDcausedbyPIGOmutations,weclarifiedgenotype- Studies oninheritedGPIdeficiencies(IGDs) J. CellBiol.,2017). AP foldingbeforeexitingtheendoplasmicreticulum(Liu,Y.etal, sults indicatethatN-glycanparticipatesinqualitycontrolofGPI- glycan andGPI,facilitatesGPI-inositoldeacylation.Thesere chaperone calnexinassociateswithGPI-APs,dependentuponN- lates GPI-inositoldeacylation.Mechanistically,amolecular In collaborationswithcliniciansandmedicalgeneticistswork Yoshiko Murakami,MD/PhD Taroh Kinoshita,PhD Support Staff Support Visiting Scientist Research Assistant Professor 4 3 1 Yoshiko Murakami Taroh Kinoshita ------● ● ● Recent Publications embryos by sheddingCRIPTO.embryos J. Cell Biol. 215, 705-718(2016). A GPI processing phospholipase A2, PGAP6, modulates Nodal signaling in E, Kajikawa Takada Y, M,HamadaH,Maeda Ikawa Saito K, Y &Kinoshita T. Lee G-H,Fujita M, Takaoka Murakami K, Y, Fujihara Y, K, N,Murakami Kanzawa to difficulties. mildlearning Mutat. Hum. 38,805-815(2017). from with phenotypes infantilevariable lethality tions of PIGO deficiency Kinoshita T, Wakamatsu N&Murakami Y. Niihori K, Hayasaka T, Aoki Y, Nabatame S, Hayakawa M, Natsume J, Ozono K, Kidokoro H,Muramatsu Y, D, S,Motooka E, Nakamura Nomura Nishi N, Tanigawa N,Fukushi S,Okamoto J, H,Mizuno D, Mimatsu Tominaga K, membrane regions inthecatalytic domain.Nat. Commun. 9,405(2018). transferaseof aGolgi GPI-N-acetylgalactosamine withtandemtrans- Murakami Y, Maeda Y, Fujita M, Yamaguchi Y &Kinoshita T. Identification Hirata D,T, Motooka S,Saito K, Nakamura SK, Mishra Takada Y, N, Kanzawa G EtN o P P N G M l (right). domains (TM2andTM3)insertionintoaglycosyltransferasedomainasshowninthe3Dstructuralmodel (short cytoplasmicpart,TM1andstemregion)intheC-terminalportionhastandemtransmembrane biosynthesis pathway(left).PGAP4hastypeIImembraneproteincharacteristicsintheN-terminalpart PGAP4 transfers N-acetylgalactosamine side-chain toGPI-anchor at the17thstepinGPI-anchoredprotein Figure. P g - l a u a EtN P i n c c n o G e r o s t F e ( y A a s 1 a m l e m g P 5 t a o t i ) 3 y n l d a a e e c EtN P P t l c o i P n i s d P EtN Phenotype-genotype correla- Phenotype-genotype g a

G m ( 1 A i n 6 P e ) 2 P EtN P E P t N P EtN P U E S m S t n G a h o ( s t i a u d 1 a d A n r e 7 t i a u o f - P ) i t r l c c e a a h a d m 4 t e c a t i i d n o i h EtN n P P c e n a

p P h i n EtN a h ● ● i o n s 3084 (2015). dent endosome-to-TGN retrograde Mol. Biol. Cell. transport. 26,3071- & Kinoshita T. Post-Golgi anterograde requires transport - GARP-depen Hirata T, D, Fujita Motooka S,Gotoh Murakami K, M,Nakamura Y, Maeda Y 98,615-626(2016). Genet. withseizures disability andhypotonia.cause intellectual Am. J. Hum. Kinoshita T, Antonarakis SE&Murakami Y. Pathogenic variants inPIGG DA, N,Parry Matsumoto JohnsonCA, N,Hamamy E, H,Sheridan Roberts Miyatake S, M,IssaMY, Nakashima GuipponiM,Letourneau A,Logan C, P,Makrythanasis Santoni F, K, H,Nakamura M,Saitsu M,Zaki Kato p h a t e P P P G 1 A - a P l k 4 y l - 2 - a D c y TM3 l H o TM2 EDD r P d I i a c TM1 y l P I stem 13 Immunology 14 Immunopathology rophage activation, butpromotedpro-inflammatory cytokine in macrophages was refractory to Th2 cytokine–induced M2 mac- regulation offattyaciduptakeand metabolism.LossofSema6D ma6D expressionplaysanessential roleinM2polarizationvia exposure toexogenousstimuli,mTORC activity–dependentSe- rophage activation state. In this study,wefound that following However, itremainsunclearhow metabolic statusregulatesmac get ofrapamycincomplex)pathwayinmacrophagepolarization. ing evidencepointstoacriticalroleofmTORC(mechanistictar alternative M2stateshasdistinctmetabolicrequirements.Emerg- mune andmetabolicsystems. on thefunctionsofSemaphorin6D(Sema6D)inneuronal,im cer/metastasis, andneurodegenerativediseases.Wefocushere orders, such asautoimmunity,allergy,immunedeficiency,can study intothediagnosis/therapyforhumanimmunologicaldis tions, wearetryingtoapplythefindingsfromthisproposed peutic targetsforarangeofdiseases.Beyondsuchbasicimplica they arenowconsideredtobepotentialdiagnosticand/orthera in immuneresponses,bothphysiologicalandpathological, cumulative evidence indicates that semaphorins also participate molecules that function during neuronal development. However, tors. Semaphorinswereoriginallyidentifiedasaxon-guidance mune guidancemolecules’suchassemaphorinsandtheirrecep gratory behaviorinvivobysolubleandmembrane-bound‘im proposed studyistheregulationofimmunecellmotilityandmi and clinicalimmunology.Asbasicaspectsofourprojects, Polarization ofmacrophagesintothepro-inflammatoryM1or Our researchteamisinvolvedintwoapproaches,thatis,basic ------programming withimplicationsformacrophagehomeostasis. polarization, andprovidemechanisticinsightsintometabolicre role for Sema6D reverse signaling in modulating macrophage vivo andinvitro.Collectively,thesefindingshighlightacrucial CX3CR1 axis governstheanti-inflammatoryfunctionofintestinalresident ing. Furthermore, we found that the Sema6D–PPARγ signaling Sema6D contributes to PPARγ expression through reverse signal- sociation withthetyrosinekinasec-AblviaSH3domainof metabolism. Specifically, we found that upon IL-4 stimulation,as but alsoforuptakeoffattyacidandreprogrammingrelated indispensable notonlyforoptimalM2macrophagepolarization, production. Moreover,Sema6D-mediatedPPARγexpressionis Atsushi Kumanogoh,MD/PhD Support Staff Support Assistant Professor Professor hi macrophagesandpreventsdevelopmentofcolitisin 5 Shohei Koyama Atsushi Kumanogoh - - ● ● ● Recent Publications ic target for CAR T cell therapy. Nat. Med. 23,1436-1443 (2017). activated conformation ofintegrin β7isa novel multiplemyeloma-specif J,Nishimura Takeda Oji K, Y, Sugiyama H, Takagi JandKumanogoh A. The Nakao T, Baba R, Ozawa M, Uchibori Hino K, Y, Terakura S, Wada E, N, Morii S, Nishida Tsuboi A,Oka Y, M,Ichihara H,Aoyama Manabe Y, Mugitani A, Watanabe K, Yoshida S,Fujiki Morimoto M,Satoh F, K, J, H,Nakata Nakajima Hosen N,Matsunaga Y, H,Nakamura Matsuno Hasegawa K, Y, M, Makita 19-31 (2018). sponses andautoimmune rheumaticdiseases. Nat. Rev. Rheumatol. 14, MandKumanogoh A. Nishide The role inimmunere ofsemaphorins tion. Nat. Immunol.(2018)inpress. controls polariza macrophage lipidmetabolismandanti-inflammatory Takagi J, Toyofuku 6D reverseT andKumanogoh A. Semaphorin signaling M,HayamaNojima S,Nishide Y, Kinehara Y, Kato Y, Nakatani T, Shimogori T, S,Nakanishi Kang Y, Kioi Y, D, Okuzaki Kimura T, Takamatsu H,Koyama S, properties onCX3CR1 fully M2activation.ThisSema6D-PPARγaxis signaling confersanti-inflammatory fatty acid uptake and metabolic reprogramming, which are essential events for that inturnactivatesc-AblkinasetoPPARγexpression.Activatedmediates During IL-4inducedM2activation,mTORC1isactivatedandupregulatesSema6D, Figure. hi intestinalmacrophageandpreventdevelopmentofcolitis. - - - ● kine and amino-acid signals. andamino-acid Nat. Commun.kine 7,13130(2016). Polarization ofM2macrophages requires Lamtor1 that integrates cyto Maeda Y, Takamatsu D, H,Okuzaki Yamada MandKumanogoh A. M,Okada Hosokawa T, Hayama Y, Mitsui Y, M, H,Nishide N,Sarashina-Kida Sakurai Kimura T,Nada S, Takegahara N,Okuno T, D, S,Ito NojimaS,Kang K, Morimoto - 15 Immunology 16 get forautoantibodies. that theseproteinsareinvolvedinautoimmunediseasesasatar of misfoldedproteinstransportedtothecellsurfacerevealed transport cellularmisfoldedproteinstothecellsurface.Analyses that MHCclassIImoleculesfunctionasmolecularchaperonsto gens andvariouspairedreceptors.Inaddition,wehavefound Host pathogeninteractionmediatedbypairedreceptor Immunochemistry infection (Suenagaetal. associates withvaricellazostervirus(VZV)gBandmediatesVZV (MAG, myelinassociatedglycoprotein), one ofpairedreceptors, (Satoh etal.Cell2008;WangJ.Virol.2009).Similarly,Siglec-4 and gBisinvolvedinmembranefusionduringHSV-1infection pes simplexvirus-1(HSV-1),andtheinteractionbetweenPILRα associates withglycoproteinB(gB),anenvelopeproteinofher Kohyama etal. (Wang etal.Nat.Immunol.2012 plays animportantroleintheregulationofimmuneresponse pressed onvariousimmunecells.WehaveshownthatPILRα receptors. PILRαisoneofpairedinhibitoryreceptorsthatareex cleaved bymicrobial proteasesbutnotnormal immunoglobu- activating LILRA2 recognizesabnormalimmunoglobulins receptors areinvolvedinviralinfection. et al.J.Biol.Chem.2015).Thesefindingssuggested thatpaired

Paired receptorsarecomposedwithactivatingandinhibitory We havebeenworkingontheinteractionsbetweenpatho LILR is another type of paired receptor family. We found that Eur. J.Immunol Proc. Natl.Acad.Sci.USA . 2016).WealsofoundthatPILRα ; Kishidaetal.Int.Immunol . 2010;Suenaga . 2015; - - - - cers (Araseeta.Br.J.Dermatol HLA classIIcomplexinthepatients withrefractorycutaneousul 2015). Inaddition,wecoulddetect autoantibodiesagainstβ2GPI/ (Jin etal. are targetsforautoantibodiesinautoimmunediseasepatients that misfoldedproteinscomplexedwithMHCclassIImolecules ecules (Jiang et al. gens arerescuedfromproteindegradationbyMHCclassIImol disease susceptibility.Wefoundthatcellularmisfoldedautoanti unclear howMHCclassIImoleculesareinvolvedinautoimmune tibility tomanyautoimmunediseases.However,ithasremained ported tothecell surfacebyMHCclassIImolecules wasstrongly matol. 2017).Autoantibody bindingtomisfoldedproteins trans- bodies inANCA-associatedvasculitis (Hiwaetal. myeloperoxidase/HLA classIIcomplex isatargetforautoanti cules aretargetsforautoimmunediseases Misfolded proteinscomplexedwithMHCclassIImole et al.Nature2017). role in immune evasion by ings suggest that binding of RIFIN to LILRB1 plays an important pression ofRIFINswasassociatedwithseveremalaria.Thesefind ry LILRB1anddownregulateimmuneresponse.Furthermore,ex the multigenefamilyof ology 2016).Ontheotherhand,wefoundthatRIFINs,productsof normalities inmicrobialinfection(Hirayasuetal.NatureMicrobi- lins. LILRA2seemstobeasensordetectimmunoglobulinab MHC classIIallelicpolymorphismsareassociatedwithsuscep Hisashi Arase,MD/PhD Research Assistant Postdoctoral Fellow Assistant Professor Associate Professor Professor Proc. Natl.Acad.Sci.USA Int. Immunol. 2013). Furthermore, we found Plasmodium falciparum Plasmodium falciparum (Figure 1. Saito . 2017).Similarly,wealsofoundthat . 2014;Tanimuraetal.Blood. 3 4 HirayasuKouyuki Masako Kohyama Tadahiro Suenaga AraseHisashi , bindtoinhibito Arthritis. Rheu------of autoimmunediseases(Figure2). ies as ‘neo self’ antigens, which are involved in the pathogenicity exposed to the immune system, can be targets for autoantibod- gested thatmisfoldedproteins,whichnormallywouldnotbe correlated withsusceptibilitytoautoimmunedisease.Thissug ● ● ● parasites (Satioetal.Nature2016). which mayleadtoinefficientdevelopmentofimmunityagainstmalaria inhibitory receptors, thus facilitating escape from host immune systems, infected erythrocytes.IndividualRIFINsmayhaveevolvedtotargethost Plasmodium falciparuminducestheexpressionofRIFINsonsurface inhibitory receptors. Figure 1. Immune evasion of Plasmodium falciparum by RIFINvia Recent Publications Nat. Microbiol. 1,1-7(2016). is an innate immune sensor for microbially cleaved immunoglobulins. Chibana H,Nagai Nakamura Y, KatayamaI,Colonna M,Arase H.LILRA2 Saito F,Hirayasu K, Suenaga T, Arase K, N,Oikawa ShidaK, Yamaoka T, Murota H, Polyangiitis. Rheumatol. 69,2069-2080(2017). Arthritis. HLA ClassIIComplexes Recognized by Autoantibodies inMicroscopic Saito F, Terao C,Atsumi T, H,Mimori Iwatani T, Arase H.Myeloperoxidase/ Kohyama Arase N,JinH,Hirayasu R,Ohmura K, M,Suenaga K, Hiwa T, 552, 101-105(2017). receptors.sion ofPlasmodium falciparumby RIFINviainhibitory Nature. Suenaga T, Colonna M, Takagi J, Lavstsen T, Horii T, eva Arase- H. Immune Palacpac S, NMQ, S,Iwanaga Itagaki Takashima E, Tsuboi T, Kohyama M, Saito F, Satoh Hirayasu K, T, Wang CW, Lusingu J, Arimori T, ShidaK, - Arase etal.Br.J.Dermatol.2017;HiwaArthritisRheumatol2017). response toself-antigens(Jiangetal. self’ antigensbytheimmunesystem,whichmightinitiateaberrant to thecellsurfacebyMHCclassIImoleculesmayberecognizedas‘neo- transported tooutsidethecells.Therefore,misfoldedproteins Cellular misfoldedproteinsaregenerallydegradedinthecellsandnot II moleculesaretargetsforautoantibodies. Figure 2.MisfoldedproteinstransportedtothecellsurfacebyMHCclass Acad. Sci.USA ● ● tion. Nat. Immunol.14,34-40(2012). ing inflammation is regulated by PILRαviamodulation ofintegrin activa Wang J, Shiratori M,Arase J, H.Neutrophil I, Uehori Ikawa infiltration dur USA. 111,3787-3792(2014). susceptibility.sociated withrheumatoid arthritis Proc. Natl. Acad. Sci. Lanier LLandArase H.Autoantibodies to IgG/HLA-DR complexes are as Takasugi Ohmura K, K, Yoshikawa H, Saito T, Atsumi T, Sasazuki T, Katayama I, ShiK, S,EbinaK, Toyama-SorimachiMatsuoka N, Yasuda S,Horita T, R, Hiwa KohyamaJin H,Arase N,Hirayasu K, M,Suenaga T, Saito F, Tanimura K, . 2014;Tanimuraetal. Blood 2015,Arase Int. Immunol.2013;Jinetal. Adv. Immunol.2016; Proc. Natl. - - - 17 Immunology 18 transcriptional regulation andinflammatorydisease control. These findingssuggest thatArid5aplaysimportant rolesinpost- encephalomyelitis (EAE),andbleomycin-induced lunginjury. are highly resistant to endotoxic shock, experimental autoimmune Introduction Immune Regulation T cell-fateregulation.Moreover,Arid5a-deficient (Arid5a stabilizes eitherStat3orT-bet mRNAs inTcellsand is involved in lizes Il6mRNAbybindingtoits3'-UTR. Additionally,Arid5aalso macrophages in response to lipopolysaccharide (LPS) and stabi- binds tothe3'UTRofIL-6mRNA.Arid5aishighlyexpressedin RNA-binding protein,AT-richinteractivedomain5a(Arid5a),which leads tosustainedIL-6production.Previously,weidentifiedan regulated byRNAbindingproteins,thedysfunctionofwhich tivated macrophagesunderTLRsignaling.IL-6mRNAistightly overproduction ofIL-6,wefocusedonIL-6geneexpressioninac (CRS) Topursue themolecularmechanismsof syndrome release systemic inflammatoryresponsessyndrome(SIRS)andcytokine the sera of patients hasalsobeenresponsiblefortheinductionof ic immunedisorders.Inaddition,ahighlevelofIL-6expressionin manized anti-IL-6receptorantibody,whenusedforthosechron ic diseases, hasbeensuccessfullyprovenbytocilizumab,ahu- arthritis (JIA).ThesignificanceofIL-6inthepathogenesischron diseases suchasrheumatoidarthritis(RA)andjuvenileidiopathic tent productionofIL-6leadstodevelopmentvariouschronic body againstinvasionofpathogens.Ontheotherhand,persis hematopoietic cellsandvascularendothelialtoprotectthe of acuteimmuneresponses,aswellactivationlymphocytes, Interleukin-6 (IL-6)isanimportantcytokineintheearlyphase −/− ) mice - - - - cay element (ADE)-like stem-loop (SL) in the 3 Arid5a stabilized OX40 mRNA by recognizing the alternative de- els andrepressedIL-17production uponOX40ligation.Notably, immunity remainunknown. balance ofIL-6mRNAexpression. Arid5a byNF-κBandMAPKsignalingisrequiredtomaintainthe phorylation increasedIL-6expression.Thus,theregulationof through theE3ubiquitinligase,WWP1.InhibitionofArid5aphos phosphorylates Arid5a, leading to the degradation of Arid5a phosphatase-1. During the late phase of TLR4-stimulation, p38 to theArid5amRNA3'-UTRdestabilizeit,whichinvolvesMAPK tion. Subsequently, AU-rich element RNA-binding protein 1 binds lization oftheIL-6mRNAduringearlyphaseTLR4stimula vate Arid5ageneexpression,increasingIL-6expressionviastabi NF-κB andanNF-κB-triggeredIL-6-positivefeedbackloopacti bilization ofIL-6mRNAhasremainedunclear.Weidentifiedthat tion, however,themechanismsofArid5aproteinduringsta sion inCD4 mice showedresistance toEAEbyeitherdecreased OX40expres lished) recognizing astem-loopstructureinits3'-UTR(pub Arid5a stabilizes OX40 mRNA in murine CD4+ T cells by mRNA-stabilizing proteinArid5a(published) TLR4-induced NF-κBandMAPKsignalingregulateIL-6 Deficiency ofArid5ainTcellsdecreased OX40expressionlev The rolesofArid5ainTh17cellsanditsassociationwithauto Arid5a mRNAexpressionisinducedinresponsetoLPSstimula Tadamitsu Kishimoto,MD/PhD Support Staff Support Visiting Scientist Research Assistant Postdoctoral Fellow Assistant Professor Associate Professor Professor + Tcellsorreduction ofCD4 3 3 3 1 Sujin Kang Shigeru Hashimoto Tadamitsu Kishimoto + CD45 + Tcellnumbersin '-UTR. Arid5a −/− ------

● ● ● (ongoing) Analysis ofanti-inflammatoryeffectslenalidomide (published) subcellular localizationofRNA-bindingproteinArid5a Regulation ofinflammatoryresponsesbydynamic cells playsimportantrolesinthepathogenesisofEAE. the CNS.Thus,ourfindingssuggestthatArid5a-OX40axisinTh17 mation throughachangeintheArid5a:Regnase-1ratio(Figure1). tance of the nucleocytoplasmic localization ofArid5a in inflam maintenance 1. Taken together, our results suggest the impor regulated byanuclearexportreceptor,chromosomalregion ciated with up-frameshift protein 1, and its nuclear export was cytoplasmic Arid5alevelsincreased.Moreover,wasasso way. Onstimulation,nuclearArid5alevelsdecreasedwhereas ed intothenucleusviaaclassicalimportin-α/β1-mediatedpath function in the immune response. In addition, Arid5a was import- and subcellularlocalizationofArid5amightbeimportantforits not inanunstimulatedcondition,indicatingthatthedynamics by LPSstimulationinArid5aoverexpressing-transgenicmice,but remained unclear.WefoundthatIL-6wassubstantiallyproduced how itssubcellularlocalizationimpactsimmuneregulationhas Recent Publications (2017). mRNA stabilizing protein Nucleic Arid5a. Acids Res. 45(5), 2687-2703 et al. KK. Nyati TLR4-induced NF-κB and MAPK signaling regulate the IL-6 munol. 48(4),593-604(2018). sup> T cells by recognizing astem-loop in its 3'UTR.Eur. structure J. Im Hanieh H.etal. stabilizes Arid5a OX40 CD4^{+Lenalidomide isanimmunomodulatorydrug(IMiD)havinga Although Arid5aplaysimportantrolesinimmuneregulation, Model ofinflammatoryresponsebyArid5a/Regnase-1/UPF1. Figure. ------tally dependentofcereblon. of cereblonwhereasits effect onIKZF1/3andinterleukin-2isto peutic opportunitiesagainstinflammatorydiseasesindependent less pronounced.Weconcludethatlenalidomideoffersthera fect inDSSmodelofcolitis,whiletheeffectpomalidomidewas manized micelenalidomideshowedasignificanttherapeuticef dextran sulfate sodium (DSS) induced colitis. In both WT and hu tionally, weassessedthepotentialtherapeuticeffectsofIMiDsin humanized cereblonmicebutnotinthewildtypegroup.Addi dation ofIKZF1/3resultedinanincreaseintereukin-2among degree ofIKZF1/3proteindegradation.Furthermore,IMiDdegra cytes fromhumanizedcereblonmicerespondedwithahigher wild type mice, which were resistant to Lenalidomide, T lympho- (IKZF1/3), apotentialtargetsubstrateoflenalidomide.Unlike degradative effectstowardsIkarosfamilyzincfingerprotein1/3 logical setup.Inourcurrentstudy,weinvestigatedpotentialIMiD to unravelcomplexIMiD mechanisms of action inmousephysio manized cereblonmice,therebyprovidinganovelanimalmodel therapeutic effects.Toovercomethisdifficulty,wegeneratedhu strate receptorforIMiDsaction—isresistanttosomeofIMiDs’ in murinediseasemodelsbecausecereblon—thesub es. However,itisdifficulttostudythemechanismofIMiDaction therapeutic actioninseveralautoimmune/inflammatorydiseas ● ● stabilization ofStat3 mRNA.J. ExpMed. 213(4),605-19(2016). etal. regulates Arid5a K. Masuda naive CD4+ T cell fate through selective 20,113(38),10625-30 (2016). regulates Proc. 1IFNproduction. TLR-induced type Natl. Acad. USA. Sci. D.Millrine etal. isalenalidomidetarget Rabex-5 molecule that negatively ------19 Immunology 20 expressed inthe innateimmunecellssuppresses IL-23produc inhibiting with c-JUNpreventedc-JUN/ATF-2 complexformation,thereby gan pathology,despitereduced levels ofparasitemia.InBatf2 was abrogatedin IL-6, wasincreased.TheT.cruzi-inducedenhancedTh17response innate immunecells, more highlyproducedbyCD4 type mice.Inthiscontext,Batf2 BATF2 Regulation ofprotozoa-inducedimmuneresponseby Mucosal Immunology livers of BATF2 in manner. Therefore,weanalyzedtherolesofIFN-γ-inducible duced inMфandDCsaTLR-independentIFN-γ-dependent demonstrated. WefoundthattranscriptionfactorBATF2isin dent hostdefenseagainst cruzi. Inaddition,themechanismsunderlyingTLR-indepen responses andthuscontributetotheresistanceofhost tor moleculepathwaysinitiatebothinnateand adaptiveimmune poorly understood.TheToll-likereceptor(TLR)–MyD88/TRIFadap regulation oftheIL-23/IL-17axisduring IL-17 production, but the molecular mechanisms underlying the chronic tissueinflammation.IL-23contributesto T. cruziinfection,inappropriateIL-17responsesareimplicatedin panosoma cruzi.AlthoughIL-17confersprotectionagainstlethal caused byinfectionwiththeintracellularprotozoanparasite Chagas’ disease,characterizedbychroniccardiomyopathy,is T. cruzi-infected T. cruzi-infected innate immune cells. In the spleens and expression. Taken together, IFN-γ-inducible BATF2 Il23a expression.Taken together,IFN-γ-inducibleBATF2 Batf2 T. cruzi-inducedproductionofIL-23,but not –/– Batf2 Il23a T. cruziinfectionhaverecentlybeen + –/– –/– Tcellscomparedtothoseofwild- –/– mice.TheinteractionofBATF2 mice, IL-17, but not IFN-γ, was miceshowedseveremulti-or T. cruziinfectionare T. cruzi-specific Try- –/– T. - - - - -}

ing T.cruziinfection(Fig.1). mer, resulting in the inhibition of excessive Th17 responses dur- tion by preventing the formation of the c-JUN/ATF-2 heterodi are tightlyregulatedbyinnatemyeloidcells,suchasCD103 cluding Crohn’sdiseaseandulcerativecolitis.Therefore,Teffcells plicated inthepathogenesisofinflammatoryboweldiseases stimuli. However,anexcessofpro-inflammatorycytokinesisim nal mucosalisconstantlyexposedtopotentiallypathogenic late in intestinal tissue toprovide immune surveillance, as intesti and CX trations. Wefoundthatthexenobiotictransporter,Mdr1,isex acids (CBAs)intheileumarepresentathighmicro-molarconcen is lessclear.Mucosa-associatedtaurine-/glycine-conjugatedbile whether intestinal T cells interface with host-derived metabolites the ileum.Inaddition,CD103 pressed inmouseTeffcellsatsteady-state,whereitishighest mation Intestinal metabolitesthatregulatemucosalinflam Rag1 cells duringCBAs exposure.BlockingilealCBAreabsorption in ry cytokines expression and then enforced homeostasis in Teff tine invivo,Mdr1mitigatedoxidative stressandpro-inflammato cells-transferred sal dysfunctionandinducedCrohn’s disease-likeileitisinnaïveT lated Mdr1intheileum,butthose lackingMdr1displayedmuco induced Mdr1expressioninTeffcells.Wild-typecellsupregu Pro-inflammatory CD4 Kiyoshi Takeda,MD/PhD –/– Support Staff Support Visiting Scientist Assistant Professor Associate Professor Professor micelackingan apical sodium-dependentbileacid trans 3 CR1 + phagocytes, and microbiota in the intestine. But Rag1 –/– mice.Bothinvitroandthesmall intes + Teffector(Teff)cellscontinuallycircu + dendriticcellsinthesmallintestine 3 1 Murakami Mari OkumuraRyu Hisako Kayama Eiji Umemoto Kiyoshi Takeda + DCs ------regulates intestinalimmunehomeostasis(Fig.2). dinated interaction between Teff cells and CBAs in the ileum MDR1loss offunction.Together,theseresultssuggestthatcoor more, asubsetofilealCrohn’sdiseasepatientsexhibited deficient Teffcellhomeostasisandattenuatedileitis.Further porter Asbt, which is expressed in epithelial cells, restored Mdr1- ● ● ● infection. prevention ofthec-JUN/ATF-2heterodimerformationduring mediated multi-organpathologybysuppressingIL-23productionthrough Transcription factorBATF2inducedbyIFN-γinMфandDCspreventsTh17- Figure 1. Recent Publications intestine. Arthritis Rheum. 68,2646-2661(2016). Rheum. intestine. Arthritis development of autoreactive utesvia activation to arthritis T cells in the Iida T, Saeki Y, S& Kumanogoh A,Sakaguchi Takeda contrib Dysbiosis K. M,FurutaMatsushita Y, S, H,Nakamura N,Kayama M,Sakaguchi Narazaki Maeda Y, Kurakawa T, D, Umemoto E, Motooka Ito Y, HirotaK, Gotoh K, cruzi infection.J. Exp. Med. 214,1313-1331(2017). Th17 responses by suppressing expression Il23a during Trypanosoma M& M,Ikawa Murakami Takeda BATF2 K. inhibitsimmunopathological D, H,Okuzaki S,Kayama KogaR,KobayashiKitada M,Arima Y, Kumanogoh A, 1196 (2017). 47,1182- adaptation to bileacidreabsorption intheileum.Immunity Takeda Sundrud, MS: K, The permits xenobiotic Mdr1 transporter T cell SB, Rodriguez-PalaciosRG, A,Cominelli F,Koralov DW, Abreu MT, Hommes AP, Beck evitt K, OikonomopoulosA,Lacey PN,Martinez GJ, Izard T, Lorenz Cao W, SY, A,SunKim H,ChenML,Delmas Kayama ES,McK Rangarajan T. cruzi - - - -

CD4 Figure 2. CD103 ileum. driven oxidativestressandpro-inflammatorycytokinesexpressioninthe ● ● 42, 279-293(2015). pendent chronic allergic responses by basophilsandmastcells. Immunity & Takeda E-NPP3(CD203c) negatively regulates Ectoenzyme K. ATP-de Takeda A, Yoshikawa S, Kurashima Y, Sato S, Umemoto H E, Kiyono H, Karasuyama Tsai M,Kusu SH,Kinoshita T, Shimada K, H,Okumura R,Ikeda Kayama Y, ellated microbiota andcolonic epithelia.Nature 532,117-121(2016). & Umemoto E, K, Sano Takeda Lypd8 K. promotes thesegregation offlag- J, H,Nishimura Osawwa H,Matsuno Ohba Y, S,Iida Nakamura T, Yamamoto M, Kimura Gotoh K, T, N,Kusu Kamiyama T, Ueda Y, Wu S, H,Barman H,Iijima Okumura R,Kurakawa T, Nakano T, D, M, Motoka H,Kinoshita Kayama + Teffector(Teff)cellsintheileumupregulatesMdr1expressiona + DCs-dependentmechanism.Mdr1protectsTeffcellsfrombileacid- - 21 Immunology 22 may bepresentinareleaseddsDNA uponcelldeath. antibodies target“loosened”single-stranded segments,which proposed models.Wespeculatethathighaffinityanti-dsDNA ner thatisonlycompatiblewithssDNAunlikepreviously body recognizesthymine-containingoligonucleotidesinaman teristic stackinginteraction.Thestructurerevealedthatthisanti nucleobases are rigidly grabbedby the Fabthrough the charac matic hypermutation(SHM)totheantigenrecognition,inwhich The structuralanalysisclearlydemonstratedacontributionofso ignated as71F12)wassolved(ProteinDataBank[PDB]ID:5GKR). the crystallographyofligand-boundFabonemAbclone(des Junichi Takagi(InstituteofProteinResearch,OsakaUniv), Prof. KD in surface plasmon resonance (SPR). In collaboration with mAbs obtainedfromSLEmodelmice,theyexhibitednano-molar from acuteSLEsubjects.Unlikepreviouslyreportedanti-DNA isolated high-affinityanti-DNAmonoclonalantibodies(mAbs) structural studies of anti-DNA antibodies. In the current study, we bodies exertpathogenicity.Thismaybeduetoalackofprecise widely accepted. However, it is still unclear how anti-DNA anti- sis anditscontributiontoclinicalsymptomsinSLEhasbeen rived fromSLE pathogenic anti-dsDNAantibody-producing cellsde- Characterization oflow-affinity progenitorBcellsfor antibody insystemiclupuserythematosus(SLE) Structural basisforantigenrecognitionofanti-DNA Immune Regulation Our previousstudy hasdemonstratedthatSLEANA aregener- Anti-DNA antibodyistheestablishedcriterionforSLEdiagno ------with nasalpolyposis (CRSwNP) Tracking allergy-relatedIgE inchronicrhinosinusitis ing. AlthoughG9gl KI heavychainalleleswereinactivatedviaaberrantreceptoredit gotes, about60%ofBcellsexpressedendogenousBCRandtheir blood andlymphoidorgansoftheKImice.InG9glheterozy clones. ThenumberofG9gl-expressingBcellswasreducedin IgH andLchainsderivedfromoneoftheSLEanti-DNAantibody directed knock-in(KI)mouseline,G9gl,whichcarriesunmutated producing cells. To address these questions, we generated a site- tation todifferentiateintohighaffinityanti-dsDNAantibody- what conditionstheyundergoclonalexpansionandaffinitymu DNA precursor B cells escape from tolerance checkpoints and in Now, thefindingsraisequestionsofhowlow-affinityanti-ss to both ss- and dsDNA by only one or two mutations (Figure 2). in whichlow-affinityanti-ssDNABcellscanacquirehigh-affinity ated through rigorous clonal expansion and affinity maturation, ger self-reactivegerminalcenter reaction. during earlyBcelldevelopmentand retainthecapabilitytotrig derived anti-DNABcellscanescape fromtolerancecheckpoint mation in spleen.Takentogether,low affinity precursors ofSLE- ies suchasanti-DNAIgGwithspontaneousgerminalcenterfor A fractionofG9glmiceexhibitedhightiterserumautoantibod could inducevigorousactivationandproliferationinG9glBcells. ciently toLPS,anti-CD40orBCRcrosslinking.Moreover,ssDNA BCR, theywerenotfunctionallyanergicandcouldrespondeffi CRSwNP ischaracterized byeosinophilicinflammation andna Hitoshi Kikutani,MD/PhD Support Staff Support Research Assistant Postdoctoral Fellow Assistant Professor Professor + Bcellsdisplayedreducedlevelsofsurface 1 1 1 Shuhei Sakakibara Hitoshi Kikutani ------nasal cavity,suchas peared tobespecificbacteriawhichnormallyinhabitthesino monoclonal IgEantibodies.ThemajorityofisolatedmAbsap- produced inNPsbyusingantibodycloningandexpressionof IgE-producing BcellsinNPs.WefirstdeterminedantigensofIgE B cells.Wesoughttounderstandthedevelopmentpathwayof high concentrationofIgEpresumablyoriginatedfrominfiltrating sal polyposis.Nasalpolyps(NPs)ofCRSwNPpatientscontaina ● F33 oftheheavychainlocatesatantigenrecognitionsite. heavy chain)andseveralhydrogenbonds.Itshouldbenotedthatmutated The Fabbindstothyminesthroughastackinginteraction(W101ofthe Figure 1.X-raycrystallographyofanti-DNAantibody71F12. Recent Publications 16428 (2017). clear antibodies inacute 7(1), systemic Rep Sci lupus erythematosus. S.etal. Clonalevolution andantigen recognitionSakakibara ofanti-nu Streptococcus pyogenes.Next,tocompre strongly connectedwithIgGandIgAlineages. clone distributionacrossimmunoglobulin isotypes.LargeIgEcloneswere Deep sequencinganalysisofexpressedBCRrepertoiresunraveledthe immunoglobulins. Figure 3.ClonalconnectivitybetweenisotypesinNP-associated - - - found inIgGorIgA (Figure 3).ItshowedthatmajorclonallineagesofIgEwereoften sequencing ofNP-associatedBCRrepertoireswasperformed hend thedevelopmentpathwayofantigen-specificIgE,deep cells involvedinIgEproductionremainstobeelucidated. essary class switching to IgE takes place concomitantly. Immune tective immuneresponseagainstnasalbacteria,inwhichunnec switch toIgEinNPs.Takentogether,CRSwNPisderivedfrompro and dsDNAathighaffinity. SHM, high-affinityanti-DNAclonesacquirebindingcapabilitytobothss Unmutated low-affinityanti-ssDNAprecursorscannotbindtodsDNA.After Working model of generation process of anti-dsDNA antibodies in SLE. Figure 2. 1 clonallineages,indicativeofsequentialclass - - 23 Immunology 24 Experimental Immunology them inaunique positionofneedingtobalancethe IL-2depen Since Tfrareatype ofTregbutmustalsoexpress BCL6thisputs eration isdependentonIL-2(Sakaguchi etal.,J.Immnol,1995). pha chain(CD25)andbroadlyspeaking Tregsurvivalandprolif fied Tregsonthebasisoftheirexpression oftheIL-2receptoral transcription factorBCL6.Onthe otherhand,weinitiallyidenti hibited byIL-2whichsuppresses expression ofthecanonicalTfh cells thatprovidesupporttoGCB-cells.Tfhareknownbein through amultistepprocess,culminating in GC-localizedGC-Tfh ter formation(Wingetal.,PNAS,2017).Tfhcellsdifferentiate plored which type of Tfr cells controls Tfh cells and germinal cen- 2013; Wingetal.,Immunity2014).Thisyear,wehavefurtherex center (GC)development(WingandSakaguchi,Int.Immunol. essential forT follicular helper(Tfh) cell formationandgerminal cells suppressedBcellexpressionofCD80andCD86,whichwas and showedthatCTLA-4-expressingTfollicularregulatory(Tfr) studied howTregcellscontrolledhumoralimmuneresponses cal self-toleranceandhomeostasis.Inthepreviousyears,we Foxp3, areactively engaged in the maintenance of immunologi thritis, byutilizingananimalmodelestablishedinourlaboratory. nism ofsystemicautoimmunediseases,suchasrheumatoidar logic self-tolerance;and(iii)thecausepathogeneticmecha organ transplants,bymanipulatingthemechanismofimmuno autologous tumorcells,orinducingimmunologictoleranceto cells; (ii)thestrategyforelicitingeffectiveimmuneresponsesto immunologic self-tolerance,inparticulartherolesofregulatoryT Treg cells,whichspecificallyexpressthetranscriptionfactor This laboratorystudies:(i)thecellularandmolecularbasisof ------population withinbothmurineandhumanPD1 development withCD25 Tfr cells.DownregulationofCD25appearstobeamarker in vitroco-culture. WefoundthatcirculatingCD45RO CTLA-4, allowingthemtosuppress bothT-cellsandB-cellsduring signature andexpresskeyTreg suppressive moleculessuchas expression ofFoxp3,maintaina characteristicTregepigenetic of flexibilityin their phenotype. Despite thistheyretainstable tant betweenTfh and effector Tregsdisplayingan extremelevel have shiftedtheirgeneexpressionsignaturetoapointequidis around days3-5whereasCD25 tion ofmorehighlydifferentiatedCD25 tion, CD25 demonstrates that,inkeepingwiththeirgerminalenterlocaliza analysis ofchemokinereceptorandcelladhesionmolecules center residentTfrlackCD25expression.Accordingly,detailed follicle andneartheT-BborderexpressCD25,almostallgerminal nated micerevealsthatwhilethemajorityofTfrresidentin analysis ofTfrinthespleensanddraininglymphnodesvacci and coincidewithpeakgerminalcenterformation.Microscopic to protein vaccination CD25 man bloodalsodownregulate CD25,whileintonsils afractionof vestigating thisissuewehavefoundanovelCD25 dent TregidentifywithIL-2inhibitedBCL6expression.Whilein that whileCD25 characterization byflowcytometryandRNA-sequencingshows localization intheT-cellzonessuchasCCR7andPSGL-1.Further while reducingexpressionofmoleculesresponsiblyformaintain Shimon Sakaguchi,MD/PhD Support Staff Support Research Assistant Postdoctoral Fellow Assistant Professor Associate Professor Professor - Tfr expresssignificantlyincreasedlevelsofCXCR5, + Tfr aremoresimilartoeffectorTregs,CD25 + Tfr forminginitiallybeforelaterforma + Tfr formation occurs earlier at - Tfr formlaterataroundday6-7 5 4 3 Kenji Ichiyama Wing James Badger Shimon Sakaguchi - Tfr. Duringtheresponse + CXCR5 - negative sub- + Tfrinhu + Foxp3 - Tfr + ------

tion, CD25 tiation pathwaygeneratingamatureGClocalizedsubpopula BCL6 ● ● ● and eTregsarebothhighlyactivatedbuthavedistincteffector ously describedBLIMP-1dependenteffectorTregs(eTregs).Tfr tigating inmoredetail. human bloodalsoexpressCXCR5apointthatwearenowinves pression. Surprisingly,asignificantfractionofCD45RA Recent Publications the prognosis ofcolorectal cancers. Nat. Med. 22,679-684(2016). subpopulation ofCD25 Inoue T, Kurosaki T, S.Adistinct Crotty S, Coban C,Ohkura N, Sakaguchi Wing JB, Kitagawa Y, Locci M,HumeH, Tay C,Morita T, Kidani Y, K, Matsuda & Sakaguchi S. & Sakaguchi Two FOXP3 Takiguchi S, Tanoue T, Suda W, M,Doki Mori H,HattoriY, M,HondaK, Morita Hamaguchi M,Ohkura N,Sato J, E, NagaseH,Nishimura Yamamoto H, Saito T, H, Nishikawa Wada H,Nagano Y, Sugiyama D, Maeda Atarashi K, Y, establishment.dent super-enhancer Nat. Immunol.18,173-183(2017). S.Guidance ofregulatory Sakaguchi T cell development by Satb1-depen Yasuda D, Motooka S,KondoM, Nakamura K, Taniuchi I,Kohwi-Shigematsu T, Kitagawa Y, Ohkura N,Kidani Y, R,Vandenbon Kawakami A,HirotaK, nal centers. Proc. Natl. Acad. USA.114,E6400-E6409(2017). Sci. We suggestthat,similartoTfhcells,Tfrcellsfollowadifferen hi CXCR5 - Tfr cells.Thisrepresentsasignificantsplitfromprevi hi Foxp3 + Tfr could be found that lacked CD25 ex- - T-follicular cells regulatory localizes inthegermi + CD4 + T-cell control subpopulations distinctly center. AdaptedfromWingetal.Clin.Immunol.andAllergology.2018. Upon activationNaïveCD25 Figure. or earlyfollicularresidentCD25 BLIMP1 positive effector Tregs in the T-cell zone or non-lymphoid tissues and CXCR5expression,allowingtheseCD25 expression causing the loss of BLIMP1 expression and higher level BCL6 + Tregsin + TregsbifurcateintoeitherIL-2dependent + Tfr. ThesecanthemdownregulateCD25 ------the T-BborderandCD25 ure 1).FromthesefindingswealsoinferthatCD25 markers (PD1andICOSforTfr),CD103KLRG1eTregs(Fig eases. and also treatment of autoantibody-dependent autoimmune dis effective controlofvaccine-inducedhumoralimmuneresponses of theTfhformationprocess.Thesefindingscanbeexploitedfor have differentrolesinthecontrolofTfhresponsesateachphase ● ● CD8 Wing JB, Adeegbe D, of self-reactive S.Detection Katayama I,Sakaguchi Maeda Y, H, Sugiyama D, Nishikawa HaD, Hamaguchi M, Saito T, M, Nishioka es viathecoreceptor Immunity. 41,1013-1025(2014). CTLA-4. gen-specific expansion of Tfh cell numberand humoral immune respons Wing JB, Ise W, Kurosaki T, S.Regulatory &Sakaguchi T-cells control anti 346, 1536-1540(2014). - Tfr totravelthegerminal + T cells withananergic inhealthy phenotype individuals. Science. - Tfr locatedinthegerminalcentermay + Tfr locatedat - - - - 25 Immunology 26 ing proliferation of Tcellsaswellanti-tumorimmunity (Figure). response, STINGactivationinTcells canbeutilizedformodulat vation thatSTINGinTcellscontributes toanti-tumorimmune much higherthaninnateimmune cells.Togetherwithourobser manner. TheleveloftypeI-IFNproduced ineffectorTcellswas production oftypeI-interferon (IFN),inmTORC1-dependent analyzed. STINGactivationinTcellsinducedgrowtharrestand expressed inTcells,thefunctionofSTINGcellactivationwas major intracellularDNAsensorininnateimmunecells,ishighly nucleic acid recognition in T cells. Since we foundthat STING, a innate signals.WehavealreadyanalyzedthefunctionofTLRsand Sts-2 phosphataseasafeedbackregulationofactivation. TCR signalsbyrecruitingseveralnegativeregulatorsincluding cient micerevealedthatCIN85functionsinnegativelyregulating adaptor moleculeCIN85.AnalysisofTcell-specificCIN85defi TCR-MC anddownstreamsignalmoleculessuchasa naling molecules.Wehaveanalyzedfunctionanddynamicsof signaling clustersgeneratedbyrecruitingTCRandproximalsig particularly fromasignalingperspective. lar mechanismofTcellactivation,differentiationandhomeostasis, eases andcancer.Ourlaboratoryaimstodeterminethemolecu rancy ofTcellfunctionresultsininfectiousandautoimmunedis T cells,whichprotectagainstinfectionandoncogenesis.Aber mune responses,induceactivationandgeneratevariouseffector Cell Signaling One ofthefinal aims ofourdiverseapproachesis toelucidate We havealso investigated the regulation of T cell activationby T cellactivationisinducedthroughTCR-microclusters(MC),the T cellsplaycentralrolesinimmuneregulation.Theyinitiateim ------ally analyzedinthesemousemodels. and imaginganalysis.Theonsetofautoimmunitywillbeeventu phosphatases inTcellactivationbyanalyzingassociatedproteins deficient miceofthesephosphatases,andthefunction autoimmune diseases.WegeneratedandanalyzedTcell-specific (PTPN22, PTPN2),whosedeficiencyisrelatedtotheinductionof analyzed theregulationofTcellfunctionbyphosphatases hibit/prevent autoimmunityandallergicinflammation.Wehave rant Tcellfunctioninordertobeablemodulatethemand the mechanismsofinducingautoimmunediseasesthroughaber- Takashi Saito,PhD Professor Takashi Saito - - ● ● ● Recent Publications Differentiation and Function. Front.Immunol. in 8,194(2017). Takeuchi A & Saito T. a Cytotoxic CD4 CTL, Subset of CD4+ T Cells, Their naling Microclusters. Mol. Biol. Methods 1584,51-64(2017). Hashimoto-Tane A, Yokosuka T &Saito T. ofSig- Analyzing theDynamics 136 (2017). Adjuvant ofCo-administered Property Vaccines. 15,127- EBioMedicine Hayashi Potentiates M.etal. Advax, Microparticle, Inulin aDelta In-built and protectionfromviralinfection. cells producehighleveloftype-IIFN,whichmaycontributetoanti-tumorimmunity produces type-IIFNfromTcells.EffectorcellssuchasTh1andactivatedCD8 cGAMP) induces growtharrest ofTcellsbyinhibiting mTORC1andsimultaneously Stimulation ofTcellsthroughTCR(antigen)andSTING(STINGligandsuchas cells. Figure. STINGstimulationinducesgrowtharrestofandtypeI-IFNproductioninT ● ● lineage. J. Exp. Med. 213,123-138(2016). Takeuchi A. et al. CRTAM determines the CD4+ cytotoxic T lymphocyte ters are essential for T cell J. activation. Exp. Med. 213,1609-1625(2016). Hashimoto-Tane A.etal. surrounding adhesion-ring Micro TCR microclus - 27 Immunology 28 Tet3, onBcelltolerance Action mechanismsofDNAdemethylases,Tet2and Lymphocyte Differentiation cells express low levels of CD86 (B7.2), whereas ablation of Tet2/ transgene model system,wedemonstratedthat self-tolerantB erating. Mechanistically,byemploying thehenegglysozymeIg positive feed-forwardloopbetween activatedBandTcellsisop tivation ofCD4 CD20 oranti-CD4depletionantibodies amelioratedaberrantac autoantibody production,andlupus nephritis.Treatmentofanti- (using CD19-Cre)resultedinspontaneoushyperTcellactivity, pathway. (5hmc) and other intermediate products in the DNA metylation 5-methylcytosine (5mC)inDNAto5-hydroxymethylcytosine ered toactasDNAdemetylasebycatalyzingtheconversionof translocation proteins (Tet1, Tet2, and Tet3) were recently discov look for the roles of Tet proteins in B cell tolerance. Ten-eleven autoimmune diseasesisalteredDNAmetylation,promptingusto previously observedepigeneticabnormalitiesassociatedwith contribution of epigenetic factors has been proposed. One of the tal settings. In addition to the B cell signaling components, the ance, promotingautoimmunityintheappropriateenvironmen modest alterationsinBcellsignalingthresholdscanbreaktoler mation andregulatecellulardifferentiationproliferation. and DNAmetylation,influencethetransmissionofgeneticinfor We foundthatspecificdeletionofbothTet2andTet3inBcells As Bcellintrinsictolerancemechanisms,itisknownthateven The epigeneticmodifications,suchaschromatinmodification + TcellsorBcells,respectively,suggesting thata ------chromatin-modified manner. peripheral B cell tolerance by suppressing CD86 expression in a gether, ourdatademonstratethatTet2/Tet3contributesto by repressingtranscriptionofCD86inself-tolerantBcells.To action. We also found that Tet2 and Tet3 recruited HDAC2, there fate fromperipheraltolerancetoproliferationthroughT-Binter Tet3 renderedupregulationofCD86,therebyswitchingtheBcell ment andfunction Roles ofaSrc-familyregulator CskinBcelldevelop antigen inducedimmuneresponses Roles of DNAdemetylases,Tet2and Tet3, in foreign- vation isnegatively regulatedbyC-terminalSrc Kinase(Csk). transduction by a diversesetofsurfacereceptors, andtheiracti- transcription ofBcl6andIRF4. of Bcl6andIRF4.WearenowanalyzinghowTet2/Tet3regulates our datademonstratetheimportanceofTet2/Tet3ininduction critical forGCBcellorplasmadifferentiation,respectively, cells. BecauseatranscriptionfactorBcl6orIRF4isknowntobe could notdifferentiateintogerminalcenter(GC)Bcellsorplasma in the absenceof Tet2 and Tet3,whereas the deficient Bcells cells. Wefoundthatinitialproliferationtookplacenormallyeven Tet3 inimmuneresponses,weacutelydeletedTet2/Tet3B per-activation. Therefore,toaddressthefunctionofTet2and resulted inbreakingBcelltoleranceandtheresultantThy The Srcfamilykinases(SFKs)play keyrolesinregulatingsignal As mentionedabove,chronicdepletionofTet2/Tet3inBcells Tomohiro Kurosaki,MD/PhD Support Staff Support Research Assistant Assistant Professor Associate Professor Professor 3 2 Ryo Shinnakasu Takeshi Inoue Wataru Ise Tomohiro Kurosaki - - - - - ● ● ● and function. SFK activitybyCskplaysimportantrolesinBcelldevelopment with TDantigen.Theseresultssuggestedthattheregulationof paired GCandmemoryBcellgenerationuponimmunization cells, inmutantspleen.Inaddition,themiceshowedim decrease ofmatureBcells,mostnotablymarginalzone(MZ) in mutantmice,andflowcytometricanalysisshowedsignificant nation ofmultipletissuesrevealedthesmallersplenicwhitepulp lyzed Csk kinase-dead knock-inmutantmice.Histological exami in vivoremainsunclear.Todefinethis,wegeneratedandana However, thephysiologicalsignificanceofSFKregulationbyCsk Te Te A) Recent Publications (2018). Convey Enhanced Protective Immunity. Immunity. 48(4),716-729.e8. T Cells Lose Differentiate KLRG1, intoMemory All T Cell Lineages, and Kluger Y, SM, Kurosaki Kaech T, Okada T, Flavell Effector KLRG1+ CD8+RA. Zhao J, Lietzenmayer M,KroehlingL, Takumi Inoue A,KometaniK, T, Herndler-Brandstetter D, R,Plajer IshigameH,Shinnakasu V, Stecher C, Center Germinal Recycling Cell Fate. Immunity. 48(4),702-715.e4.(2018). nal Center BCell into Strength Plasma Interaction RegulatesCell Entry or Yamashita Okada K, T, Suzuki K, Kurosaki T. T Follicular HelperCell-Germi Ise W, Fujii A,KometaniK, Ito ShiroguchiTakeda K, K, E, Kawakami K, Bcells andtheirreactivation. Rev. Immunol. ry 283(1):138-149. (2018). Inoue T, R,Phan I,Shinnakasu Moran TG, Kurosaki T. Generation ofmemo t2/T t2/T chronic Ag(auto) chronic Ag(auto) et et 3 3 +/+ о / о Te t2/T et 3 +/+ acute Ag CD86. (B)Tet2 and Tet3recruit HDAC to the CD86locus in the chronic state, thereby dampening transcriptionof expression, therebychangingtheBcellfatefromperipheraltolerancetoproliferation. But intheabsenceofTet2andTet3,suchchronicantigenstimulationcannot downregulateCD86 Therefore, TcellactivationandsubsequentBproliferationstop(self-tolerantstate). When thisantigenstimulationcontinueslikeauto-antigenstimulation,suchCD86upregulationceases. which inturn,evokescytokineproductionbyTcells,therebyBcellproliferation(upper panel). (A)In thepresenceofTet2andTet3,acuteantigenstimulationinducesaco-stimulatorymolecule,CD86, Figure. Tet2andTet3contributetoperipheraltoleranceofBcells. B CD86 CD28 CD4 T - - (breaking (Self-toler - - - B) Te Te ● ● to ant) t2/T t2/T 17(7), 861-869(2016). minal center Nat. Bcell Immunol. compartment. cells into thememory Takahashi Y, Fukuyama H,Okada T, Kurosaki T. ofger Regulated selection R, Inoue Shinnakasu T, S,Adachi Moriyama KometaniK, Y, M, Nakayama to T cell help. J. Exp. Med. 214(4),1181-1198(2017). Foxo1tion factor controls center germinal Bcell proliferation inresponse Inoue T, R,Ise Shinnakasu W, C,Egawa Kawai T, Kurosaki T. The transcrip ler ant) et et 3 3 о +/+ / о Te Te t2/3 t2/3 HD HD AC AC ʹ Ac Ac Ac OFF ON CD86 CD86 - - 29 Immunology 30 experience long-term ‘hidden’pathologies.Using well-estab- malaria (CellHostMicrobe,2014). of smell)couldbeavaluableearly diagnosismarkerforcerebral we providedevidencethatolfactory functionalimpairment(loss sociated withhighfeverandcytokine storm.Withthesefindings, lation andcellocclusionfollowed bymicro-bleeding,eventsas capillaries comprisingtheolfactorybulbshowparasiteaccumu the olfactorybulbconfirmedthatcomplextrabecularsmall damaged by MRI showedthattheolfactorybulbisphysicallyandfunctionally malaria model.Deepinvestigationofthebrainbyultra-highfield thology ofcerebralmalariabyusinganexperimental Malaria Immunology International Immunology,2018). bone tissueenvironment(NatureReviewsImmunology,2018; cal growthretardationinchildrenandadetrimentaleffectonthe long-term ‘hidden’pathologiessuchasBurkittlymphoma,physi illness caused by incomplete immunity to malaria may cause from mildsymptomswithlowparasitemia.Hence,the‘chronic’ patients developonlypartialimmunityovertheyearsandsuffer our lab.However,afteraninitialsystemicinfection,mostmalaria laria, hasnotbeenfullyclarifiedandisextensivelyinvestigatedin sudden andlife-threateningcomplicationssuchascerebralma- lion peopleeveryyear.Whyandhowmalariainfectioncauses laria isstillanimportantdiseaseandcontinuestokillhalf-amil mechanisms involvedinthehostand There isincreasing compellingevidencethatmalaria survivors In theabovementionedcontext,wehaveinvestigatedpa Research inourlaboratoryfocusesontheelucidationof Plasmodium parasites. Live multiphoton imaging of Plasmodium parasites.Ma ------malaria. Ourstrategy istofocusontheadjuvants,because ifratio of host-Plasmodium interactionsinto vaccinesordrugstotreat rent anti-malarials. of noveladjuncttherapiestobe usedincombinationwithcur standing thesepathologiesmight becriticalforthedevelopment of certainorgans(NatureReviews Immunology,2018).Under the bloodandotherhosttissuesthatoftenresultindysfunction mainly viathemanipulationofinter-tissueinteractionsbetween tissue specificimmunopathologyduringbloodstageofinfection, that systemicinfectionbyPlasmodiumparasitescauseslocal,but malarial treatment(Figure1,ScienceImmunology,2017). and thepotentialbenefitsofcouplingbonetherapywithanti- results highlight the risk of bonelossinmalaria-infectedpatients cidol, avitaminD3analog,couldpreventtheboneloss.These did notcauseboneloss.Importantly,supplementationofalfacal- tion thatproduceslittlehemozoin,amajor fection withamutantparasiteimpairedhemoglobindiges stimulation ofosteoclastogenesisfavoringboneresorption.In precursors, leadingtoincreasedRANKLexpressionandover- pendent inflammatory responses in osteoclast and osteoblast modium productsinthebonemarrownicheinducesMyD88-de presses bonehomeostasis,butsustainedaccumulationof retardation inyoungmice.Acutemalariainfectionseverelysup nificant and long term bone loss in adult mice, and growth modium infection, werecently showed that infection causes sig lished mousemodelsmimickingvariousaspectsofhuman The finalgoalofourresearchisto translateourunderstanding These recentstudiesfromourlabhaveledustohypothesize Cevayir Coban,MD Support Staff Support Visiting Scientist Research Assistant Postdoctoral Fellow Assistant Professor Professor 1 2 1 2 SueJann LEE Michelle Cevayir Coban by-product, Plasmodium by-product, Plas- Plas------preliminary GLPnon-clinicalsafetyandtoxicologystudiesinsev analog ofPlasmodium-producedhemozoin,andcompletedits veloped anewadjuvantcalledsynthetichemozoin, nally designed, adjuvants improve vaccine efficacy. We havede eral animalsandinfectionmodels(Vaccine,2016). ● ● ● Recent Publications Cerebral Cell 15(5),551-63 (2014). Malaria. HostMicrobe Plays inSpatiotemporal aKeyRole Zhao Hetal. Pathogenesis Olfactory of pii: eaam8093(2017). in thebonemarrow andpromote chronic boneloss. 2(12), Immunol. Sci. Coban persist C.Plasmodium products SM,Kuroda Khan S,IshiiKJ, E, Akira Lee Fujita MSJ, K, Maruyama Y, S,Horii KonishiA,Lelliott PM, Itagaki T, LinJW, infection. Nat.laria Rev. (2018). Immunol., doi:10.1038/nri.2017.138 Coban C,Lee MSJ, immunopathologyma IshiiKJ. during Tissue-specific skews boneremodelingtowardresorptionandeventuallyleadsto loss. RANKL synergisticallyinduceosteoclastogenesis.TherobustactivationofOCs afterclearanceofparasites upregulate RANKLexpressiononosteoblasts.Theinflammatorycytokines(IL-1α, IL-1β,IL-6andTNFα) immature andmatureosteoclastsosteoblasts. abundantly in the bone marrow for long periods leading to MyD88-dependent chronic inflammation in infection, althoughparasitesareclearedsystemically,parasiteproductsremainandgraduallyaccumulate disrupt thesignalinginosteoclastogenesis.Duringconvalescenceandchronicphaseof such as IL-4,IL-10, IL-13andIFNγreleased from activatedTcells in responseto infected andnon-infectedredbloodcellsstronglyinhibitsbothosteoclastsosteoblasts.Cytokines During acute malaria infection, heme is one of the factors released abundantly due to hemolysis of infection causeschronicillnessessuchasbonelossorgrowthretardation. immunity andsufferfrommildsymptoms.Recentevidencesuggeststhattheincompleterecovery Although bloodstage after thecureofmalariadisease(ScienceImmunology,2017). Figure. Malariainduceschronicbonelossduetosustainedaccumulationof Naive Plasmodium infectioncausesdeadlycomplications,mostpeopledeveloppartial - - - Plasmodium product-inducedinflammatoryresponses Convalescent/chronic phase ● ● and Microbe 7(1),50-61(2010). and Microbe modium falciparum Involves Hemozoin Malarial and Host TLR9. Cell Host Coban of C et al. Immunogenicity Whole Parasite Vaccines Against Plas 12(5),705-16(2012).Host Microbe to infectionby blood-stagemalaria reinforcing hostiron metabolism. Cell Zhao Hetal. Lipocalin2bolsters innate andadaptive immuneresponses Malaria-infected Plasmodium productseven Plasmodium Plasmodium infection,

- 31 Immunology 32 in NonhumanPrimates ing ImmunotherapyToBolsterMemoryCD8(+)TCells An Antigen-Free,PlasmacytoidDendriticCell-Target Vaccine Science cells toproduce both IFN-αandIL-12/23p40invitro andinvivo. primate model. K3-SPG activated monkey plasmacytoid dendritic monoimmunotherapeutic potential ofK3-SPGtoanonhuman established tumorsinmicevivo. Inthisstudyweextendthe shown toactasapotentmonoimmunotherapeutic agentagainst K3-SPG, a nonagonistic Dectin-1 ligand.K3-SPGwaspreviously humanized CpGDNA(K3)complexed withschizophyllan(SPG), als. Werecentlydevelopedasecond-generationTLR9agonist, cer Ag(s);however,sofartheyhavelackedefficacyinclinicaltri monotherapeutic agentsevenwithoutcoadministrationofcan- acid-based compoundshavebeenshowntoactasanticancer strong Ag-specificCTLresponsesasavaccineadjuvant.Nucleic pounds haveattractedattentionduetotheirabilityelicit an areaofactiveresearch.Particularly,nucleicacid-basedcom CD8+ Tlymphocytesbyvaccinationorimmunotherapyinvivois non-communicable diseases. against infectious diseases, cancer and allergy as well as other next generationimmuno-preventiveand/ortherapeuticagents edge willenableustodevelopnovelconcepts,modalitiesand cines, aswellbiologicalresponsestoadjuvants.Suchknowl pathways thatmediatetheimmunogenicityofsuccessfulvac- nological mechanisms of the intra- and inter-cellular signaling Thepriming,boosting,andrestorationofmemorycytotoxic The primary goal of our laboratory is to understand the immu- - - - - riasis Essential RoleofCARD14inMurineExperimentalPso notherapeutic agentforchronicinfectiousdiseasesandcancer. boosting capabilities,highlightingitspotentialasamonoimmu show thatK3-SPGhaspotentAg-specificmemoryCTLresponse- cytidylic acid.Therefore,evenintheabsenceofspecificAgs,we lower thanthoseinducedbythesamedoseofpolyinosinic-poly matory serumcytokines and circulating neutrophil influx were duced mild systemic inflammation,however,levels ofproinflam or polyinosinic-polycytidylicacid.Injectionof2mgK3-SPGin This increase did not occur inmacaques injected with freeCpGK3 including Ag-specificmemoryCTLs,incynomolgusmacaques. the frequenciesofactivated memory CD8+Tcells in circulation, A singleinjections.c.ori.v.withK3-SPGsignificantlyincreased which wasdependent onbothTLR7andTLR9but independent skin Langerinhigh Langerhans cells as a potent producerofIL-23, ed byIL-23.Celltype-specificanalysis ofthesemiceidentified vealed notonlytheirsimilarity,but alsodistinctgenesetstarget IMQ-cream-treated Card14-/-mice andTlr7-/-Tlr9-/-micere mice. Comparisonoftheearlygene signatureoftheskinbetween the psoriasiformskininflammation wasabrogatedinCard14-/- (IMQ) creamorrecombinantIL-23injection.Inallmodelstested, experimental modelsofpsoriasisinducedbyeitherimiquimod unclear. Inthisstudy,weexaminedtheroleofCARD14inmurine munological roleinthepathogenesisofpsoriasisvivoremains recently identifiedasapsoriasis-susceptibilitygene,butitsim Caspase recruitmentdomainfamilymember14(CARD14)was Ken J.ISHII,MD/PhD Support Staff Support Visiting Scientist Research Assistant Postdoctoral Fellow Assistant Professor Associate Professor Professor 2 6 4 4 Natsuko Kishishita Norifumi Iijima Etsushi Kuroda Ken J. ISHII ------● ● ● Cell Responses Vesicles viaDNAVaccinationResultsinRobustCD8+T CD63-Mediated AntigenDeliveryintoExtracellular get forpsoriasis. indicate thatCARD14isessentialandapotentialtherapeutictar trating throughthedermistoinflamedepidermis.Thesedata trolling thenumberofVγ4+TcellsproducingIL-17orIL-22infil required forIMQ-inducedpsoriasiformskininflammation,con gested thatCARD14inradio-sensitivehematopoieticcellswas 23, notTLR7orTLR9.Instead,abonemarrowchimerastudysug of CARD14,suggestingthatCARD14isactingdownstreamIL- OVA) producedOVA-carryingEVs.Immunizationswiththepuri- with plasmid DNAencoding an OVA Ag fused to CD63 (pCD63- EVs wouldimprovevaccineimmunogenicity.Invitrotransfection er aDNAvaccineencodinganAgfusedtoCD63deliveredinto various cellular membranes,including EVs, we examined wheth tems. ByfocusingonCD63,atetraspaninproteinexpressed such as extracellular vesicles (EVs), targetnatural Ag delivery sys mulations, electroporation,andgeneticadjuvants,butafew, immunogenicity areboostervaccinations,alternatevaccinefor improvement. AmongthestrategiesforimprovingDNAvaccine their ability to induce Ag-specificCD8+Tcell responses requires and cellularimmuneresponsestotheencodedAg.However, Recent Publications Experimental Psoriasis.”J. Immunol. 200(1),71-81(2018). IshiiKJ. K, Kabashima K, inMurine “Essential ofCARD14 Role Mizuguchi Tanaka Honda M, Kobiyama K, T, Uchio-Yamada Natsume-Kitatani Y, K, human Primates. J. Immunol. 200(6),2067-2075(2018). Cell-Targeting Immunotherapy To CD8(+) Bolster Memory T Cells inNon K, Mizuguchi Yasutomi Y, An Antigen-Free, IshiiKJ. Dendritic Plasmacytoid Masuta Y, Yamamoto T, Natsume-Kitatani Y, Kanuma T, E, Kobiyama K, Moriishi nucleic acidsensors. Curr OpinPharmacol. 2018Jun2;41:104-113. Temizoz B, Combination Kuroda andinducibleadjuvants E, targeting IshiiKJ. DNA vaccines are attractive immunogens for priming humoral DNA ( in vivo Dendritic cellsormuscle encoding antigen) CD63 of plasmidDNA va transfection ccination antig Plasmid DNA en Exosome-targeting DNA vaccine CD8 Tcells APCs ------Extracellular Naïve trition (NIBIOHN)accompanyingactiveexchangesofresearchers. with NationalInstituteofBiomedicalInnovation,HealthandNu- development vaccines against these diseases together closely style-related diseases.Wewillcontinueinnovativeresearchand such ascancer,allergy,Alzheimer'sdisease,andmanyotherlife work ofinfectiousdiseasesbutincludeabroadrange making itapotentiallyusefulcancervaccine. for eliciting strong CD8+ T cell responses to the encoded Ag, cate thatEVAgdeliveryviaDNAvaccinationoffersanewstrategy Future prospect compared with the control DNA vaccinations. These results indi ered DNAvaccinationsignificantlyinhibitedtumorgrowth el toevaluatepCD63-OVAasatherapeuticvaccine,theEV-deliv- mic reticulum.Whenweusedamousetumorimplantationmod CD8+ Tcellresponsesthancalnexindeliveryintotheendoplas larly thosefromCD8+T cells. CD63deliveryintoEVsledtobetter pCD63-OVA inducedpotentAg-specificTcellresponses,particu endoplasmic reticulumfailedtodoso.Vaccinatingmicewith tein aloneoracalnexin-OVAfusionproteindeliveredintothe from cellstransfectedwithcontrolplasmidsencodingOVApro CD4+ andCD8+Tcells,whereasimmunizationwithEVspurified fied OVA-carryingEVsprimednaivemicetoinduceOVA-specific ● ● “EXOSOME-TARGETED DNA Pate Kanuma Tetal (WO2017175253) 127-136 (2017). built Adjuvant ofCo-administered Property Vaccines. EBioMedicine. 15, PotentiatesPetrovsky Advax, Microparticle, Inulin aDelta N,IshiiKJ. In- Standley DM, Yamada H,Honda-Okubo Y, Hara H,Saito T, Takai T, Coban C, Hayashi M,Aoshi T, Haseda Y, Kobiyama K, Wijaya N,Igarashi E, Nakatsu Y, Cell Responses. J. Immunol.198(12),4707-4715(2017). into Extracellular Vesicles via DNA Vaccination CD8(+) in Robust Results T CD63-Mediated Antigen Kuroda Delivery Ozasa E, K, JounaiN,IshiiKJ. Kanuma T, Yamamoto T, E, Masuta Moriishi Kobiyama K, Y, Kusakabe T, Vaccine targetdiseasesarenownotonlyrestrictedtoaframe T cells nt Ve pending sicles (EV)/exosomecontains target antigen Th1 J. Immunol. PC T/ JP2016/001900 2017 VA CCINE” CD63 ------33 Immunology 34 Immunoparasitology ration andfunctions ofosteoclastsareassociated withLC3.Al other essentialcanonical autophagyregulator.Inaddition, matu such as Atg5, Atg7 and Atg16L1, it does not require Atg14, an- bacteria. AlthoughLAPrequiresthe LC3conjugationmachinery volved in autoimmunity and host defense against fungus and cess isknownasLC3-associated phagocytosis(LAP)andisin LC3 isdetectedonsinglemembranous phagosome,whosepro Atg8 ineukaryoticautophagyhavebeenestablishedsofar. (xenophagy) inhumancells.Thus,conservedcanonicalrolesof more, LC3C plays an important role in anti-bacterial autophagy Atg8 subfamiliesdifferentiallyparticipateinautophagy.Further gosomal maturation,suggestingthatbothLC3andGabarap contrast, Gabarapsubfamilymembersarerequiredforautopha important role in the elongation of phagophore membrane. In autophagy activityassessment tophagosomal membrane,whichisawidelyacceptedmarkerfor manner andinduceaccumulationofthelipidatedLC3onau crease LC3lipidationinanAtg3/Atg5/Atg7/Atg16L1-dependent as Gabarap2). Autophagic stimuli, such as nutrient starvation, in LC3a, LC3b,LC3C,Gabarap,Gabarapl1andGate-16(alsoknown five Atg8orthologuesconsistingofLC3andGabarapsubfamilies: Atg8, whichisessentialforautophagy,mammalsharboratleast autophagy-related (Atg)proteins.Althoughyeasthasonlyone ble membranousautophagosomeandautolysosomeinvolving aged cytoplasmiccomponentsorunwantedpathogensindou otic cellularsystemtosustainhomeostasisdegradeold/dam Noncanonical functionsofAtg8havebeenrecentlyreported. Macroautophagy (herein, autophagy) is a fundamental eukary- 7 . LC3subfamilymembersplayan ------with LC3aswellIFN-inducibleGTPases nonical autophagy.Inparticular, ventional usageoftheAtg8conjugationsystemotherthanca- Atg16L1 butnotAtg9a,Atg14andULK1/2,indicatingtheuncon extensively studiedandshowntorequireAtg3,Atg5,Atg7 life-threatening toxoplasmosisinhumanandanimals,hasbeen Toxoplasma gondii,anobligatoryprotozoanpathogenthatcauses lation inducesrecruitmentofIFN-inducibleGTPasestoPCV tory responses.ThemechanismbywhichtypeIIIFN(IFN-γ)stimu death ofvacuolarpathogensandsubsequentvariousinflamma cumulated onPCVmembranes,leadingtothedisruption, ed GTPasescalledIFN-inducibleGTPases.Theseareac ates IFNs,whichfurtherstimulateproductionofimmunity-relat infection byvacuolarpathogens,thehostimmunesystemgener mic innatereceptorsand allows efficient proliferation.Against oles (PCVs),whichprotectthemicrobefromsensingbycytoplas Many intracellularpathogenscreatepathogen-containingvacu cell-autonomous hostdefensestimulatedbyinterferons(IFNs). with autophagicactivity. lapidated LC3,thematurationofosteoclastsisnotassociated secretory functionsinboneresorptionrequiresAtg5,Atg7and though formationoftheruffledborderbyosteoclastsandtheir members and found that, although LC3 subfamily members are induced responsearenotfullyunderstood. tion systemandthebiologicalsignificance ofLC3intheIFN-γ- function. However,physiological relevanceoftheAtg8conjuga An unconventional Atg8 conjugation system is also reported in Here wehaveanalyzed micelackingindividual Atg8family Masahiro Yamamoto,PhD Postdoctoral Fellow Assistant Professor Associate Professor Professor T. gondiiPCVishighlydecorated 1 Youngae Lee Jisu Ma Sasai Miwa Masahiro Yamamoto 26 , suggestingthemajor ------● taining theintracellularpathogen. es throughoutthecytosolandsubsequentlytovacuolecon involves Arf1activationanddistributionofIFN-γ-inducedGTPas- against vacuolarpathogens.ThemechanismofactionGate-16 ticipates inIFN-inducibleGTPase-dependenthostdefense unexpectedly dispensable, Gate-16 uniquely and critically par Recent Publications Pase-mediated hostdefense. Nat. Immunol.18,899-910(2017). sential role for autophagy GABARAP proteins ininterferon-inducible GT Saitoh T, A,Standley DM, S,Iwasaki Akira Yoshimori T, Yamamoto M.Es- S,Kawabata JS,Nakamura N,Ma M,Sakaguchi T,Sasai BandoH,Lee Y, GABARAP subfamilyofmammalianATG8playsacriticalroleinIFN-γ-induced cell-intrinsic immunity. Figure. - - - 35 Immunology 36 Biochemistry and Immunology branes. branes, andisrequired fortheirflippaseactivity at plasmamem for ATP11AandATP11Ctoescort themfromERtoplasmamem destroyed duringapoptosis.The CDC50Aworksasachaperone tion sitesinthemiddleofmolecules, andtheirflippaseactivityis cytes. ATP11AandATP11Ccontain twoorthreecaspase-recogni expressed invariouscellsincluding lymphocytesandhepato expressed inbrains,whileATP11AandATP11Careubiquitously membrane. Among these three flippases, ATP8A2 is specifically translocate PtdSerfromoutertoinnerleafletsoftheplasma ATP11C) andtheirsubunitCDC50Aasflippasesthatactively rocytes, exosomes,andenvelopedvirus. served inactivatedlymphocytes,capacitatedsperm,agederyth platelets forbloodclottingfactor.ThePtdSer-exposureisalsoob me” signalforapoptoticcells,andasascaffoldontheactivated sure. ThePtdSer,thusexposedtothecellsurface,worksasan“eat and outerleafletsofplasmamembranes,leadingtoPtdSer-expo that non-specificallyscramblephospholipidsbetweentheinner rical distributionofphospholipidsisdisruptedbyascramblase(s) cells undergoapoptosis,orplateletsareactivated,theasymmet and phosphatidylethanolaminefromoutertoinnerleaflets.When ATP-dependent phospholipidflippases,thattranslocatesPtdSer asymmetrical distributionofphospholipidsismaintainedbyan ma membranes, is exclusively localized in the inner leaflet. This (PtdSer), oneofmostabundantphospholipidsineukaryoticplas tributed betweeninnerandouterleaflets.Phosphatidylserine We identifiedthreeP4-typeATPases(ATP8A2,ATP11Aand Phospholipids inplasmamembranesareasymmetricallydis------Human andmouseplateletsexpressonlyTMEM16FasaCa er membersarespecificallyexpressedinthebrainsorintestines. bilayers. TMEM16F canscramblephospholipidsbetweentwomembrane localized, we recently showed that a single dimeric molecule of membrane bilayersinwhichphospholipidsareasymmetrically phages forengulfment. Xkr4andXkr9arerathertissue-specifical ure 2). Xkr9 scramblases,toquicklyand irreversibly exposePtdSer(Fig and ATP11Cflippases,cleaves andactivatesXkr4,Xkr6, apoptosis, caspasecleavesand irreversibly inactivatesATP11A cleaved bycaspaseduringapoptosis. Thus,whencellsundergo caspase-recognition siteintheC-terminaltailregion,andare bling phospholipidsduringapoptosis.Thesememberscontaina supposed to carry 10 transmembrane segments support scram- function mutationinTMEM16F. a rarecongenitalbleedingdisorder,werefoundtocarrylossof bin forbloodclotting.Infact,humanpatientsofScottsyndrome, expose PtdSer,leadingtothereducedabilityproducethrom pendent scramblase.TheTMEM16F-/-activatedplateletscannot Ca family, thatcontain10transmembranesegments,functionas Five members(TMEM16C,16D,16F,16Gand16J)oftheTMEM16 non-specific scramblingofphospholipidsatplasmamembranes. TMEM16F isubiquitouslyexpressedinvariouscells,whileoth- The PtdSer on the dead cell’s surface is recognized by macro- Three members (Xkr4, Xkr8 and Xkr9) of the Xkr family that are There aretwofamiliesofmembraneproteinsthatsupport 2+ -dependent scramblases(Figure1).Inamicroarraysystemof Shigekazu Nagata,PhD Support Staff Support Visiting Scientist Research Assistant Postdoctoral Fellow Associate Professor Professor 1 2 3 4 SegawaKatsumori Nagata Shigekazu 2+ -de- - - - ease. Itispossiblethatunengulfeddeadcellsundergosecondary develop systemiclupuserythematosus-typeautoimmunedis- efficiently engulfedbymacrophages.TheXkr8-nullfemalemice dent manner. Thus, Xkr8-null thymocytes and neutrophils are not they undergoapoptosis,exposePtdSerinanXkr8-depen cytes, lymphocytes,andneutrophilsexpressonlyXkr8,when Xkr8 isubiquitouslyexpressedinvarioustissues.Mousethymo ly expressed in the brains and intestines, respectively. Whereas, reduces their flipping activity. When theCa TMEM16F toscramblephospholipids,whileitinactivatesP4-ATPasesand platelets, theintracellularCa PtdSer-exposing cellsfrombeingengulfedbymacrophages. ● ● ● Ca Figure 1.ThemolecularmechanismforPtdSerexposureincellswithhigh Ca A flippase comprised of P4-ATPase (ATP11A or ATP11C) and CDC50A, and a concentration ofATPandCa to thecellsurfaceinthisprocess,andlikelydependsonintracellular resume flippingPtdSerandPtdEtn.Thus,isonlytransientlyexposed normal level,TMEM16Fstopsscramblingphospholipids,whileP4-ATPases Recent Publications Sci. USA115, 2132-2137(2018). Sci. phospholipidscramblase. Proc. acaspase-dependent Natl.of Xkr8, Acad. MandNagataKawano S.Lupus-like autoimmune disease causedby alack 3066-3071 (2018). phospholipid scrambling by TMEM16F. Proc. Natl. Acad. USA 115, Sci. Watanabe R,Sakuragi T, analysis of NojiHandNagata S.Single-molecule nol. 36,489-517(2018). Nagata S.Apoptosis andclearance ofapoptotic cells. Annu. Rev. Immu 2+ 2+ -dependent scramblase(TMEM16F)isschematicallyshown.Inactivated -concentration. 2+ . TheconstantflippingofPtdSerpreventsthe 2+ concentrationincreasesandactivates 2+ concentration returns to - - - vate theimmunesystem. necrosis, andintracellularmaterialsreleasedfromdeadcellsacti the flippasesandscramblases. membranes, andalsothephysiologicalpathologicalrolesof and scramblasesflipscramblephospholipidsatplasma recognized bymacrophagesforengulfment. is an irreversible process, and the PtdSer exposed on the cell surface is while thesamecaspasescleaveandinactivateATP11AATP11C.This of the caspase-cascade cleaves Xkr8 to activate its scramblase activity, When cellsundergoapoptosis,caspase3or7inthedownstream (ATP11A/ATP11C associatedwithCDC50A)areschematicallyshown. A caspase-dependentphospholipidscramblaseofXkr8,andflippase Figure 2.ThePtdSer-exposureinapoptoticcells. ● ● Nat. Rev. Immunol.17, 333-340 (2017). Nagata Sand Tanaka M.Programmed cell death and theimmunesystem. (2018). lipid flippases to theplasmamembrane. J. Biol. Chem. 293,2172-2182 quired for complex forming withandchaperoning afunctional phospho KurataSegawa SandNagata K, S. The CDC50Aextracellular domainisre We arestudying the molecular mechanism of how flippases - - - 37 Immunology 38 Molecular Neuroscience seen-before trend inLifeSciences. as theirsignificancethatmaypotentially createanewandnever- to elucidatethemechanismsbehind aseriesofreactionsaswell sequent recoveryprocessasa“scrap-and-build” strategy,weaim the neuralnetworksandbiological reactionsduringthesub covery ofneuralnetworksarescarce. Byperceivingdisordersin involvement oftheentirebiologicalsystemindisordersandre- in abiologicalsystem;further,studiesfromtheperspectiveof comprehensive analysisoftheassociationbetweeneachsystem. the dynamicsofentirebiologicalsystemandbyconductinga disorders andtheirfunctionalrecoveryprocesswithrespectto disorders within the central nervous system by observing such volved intheoperationoflivingorganismswithneuralnetwork lar system.Additionally,weaimtoelucidatetheprinciplesin ciations amongthenervoussystem,immuneandvascu study is to elucidate thecontrol mechanism exerted by the asso a seriesofprocesses(Fig.2).Particularly,theultimategoalthis the spatiotemporaldynamicsinthosebiologicalsystemscontrol tems (Fig.1).Further,wehaveanalyzedthemechanismbywhich from theperspectiveoffunctionalnetworkbiologicalsys central nervoussystemandthesubsequentrestorationprocess system. Wehaveanalyzeddisordersoftheneuralnetworksin ous biologicalsystems,suchastheimmunesystemandvascular cause spatiotemporal changes in thenervoussystem andin vari lar diseases,cerebrospinaltrauma,andencephalomyelitis,often We perceivethecentralnervoussystemasasingleorganwith- Disorders ofthecentralnervoussystem,suchascerebrovascu------Toshihide Yamashita,MD/PhD Support Staff Support Research Assistant Postdoctoral Fellow Assistant Professor Associate Professor Professor 4 3 3 Ken Matoba Hiroshi Tsujioka Shogo Tanabe Yuki Fujita MuramatsuRieko Toshihide Yamashita ● ● ● Recent Publications neuronal inthebrain. networks J. Exp. Med. 214,1431-1452 (2017). Miyakawa T, Shirahige K& Yamashita T. Cohesin regulates formation of Fujita Y, R,Katou Nakato K, Masuda Y, Tanaka T, M, Nakayama Takao K, 127, 3496-3509(2017). central system remyelination nervous by peripheral FGF21.J. Clin.Invest. H, Yoshida H& N, Mochizuki M, Konishi M,Itoh Yamashita T. Promotion of Kuroda M, Muramatsu N,Koyama R,Maedera Y, HamaguchiM,Fujishima brainduring development. Nat. Neurosci. (2018)inpress. Tanabe S & Yamashita T. promote lymphocytes oligodendrogenesis B-1a Biological systemsthatregulaterewiringofneuralnetworkafterCNSinjury. Figure 2. The mechanismofspontaneousfunctionalrecovery. Figure 1. ● ● 213, 2949-2966(2016). contributes to spinalsensitization inchronic painviaUnc5B. J. Exp. Med. Kuwabara S. & Yamashita T. interneuron-derived horn Netrin-4 Dorsal Hayano Y, Takasu Koyama K, Y, Asaki K, Minami T, Ogawa K, K, Kitada (2017). of miR-484/protocadherin-19 signaling. Mol. Psychiatry 22,364-374 16p13.11 microduplication through causeshyperactivity dysregulation Fujitani M,ZhangS,Fujiki R,Fujihara Y & Yamashita T. Achromosome 39 Immunology 40 abinomannan (LAM). This interaction triggered unique adjuvant ognizes mycobacteria throughahydrophiliclipoglycan, lipoal addition, wefoundthatanother CLR Dectin-2(Clec4n)alsorec TDM wereabrogatedinbothMincle- andMCL-deficientmice.In sion. Indeed,innateandacquired immuneresponsesinducedby ognizes mycobacterial cord factorand promotes Mincleexpres identified asaMincleligand.Another CLR,MCL(Clec4d),alsorec cord factor(alsocalledtrehalose-6,6’- bacterium tuberculosis.Awidely-knownmycobacterialadjuvant, (Mincle/Clec4e) isanFcRγ-coupledactivatingreceptorfor pathogens. Wefoundthatmacrophage-inducibleC-typelectin recently identifiedaspatternrecognitionreceptors(PRRs)for various immune responses.Amongthem,ITAM-coupledCLRs are teins thatshareacommonstructuralmotifandareinvolvedin C-type lectinreceptors Immune sensingofpathogensanddamaged-selfvia Molecular Immunology related toautoimmunediseases. sponses inducedby self-peptides, and 3) innate T cell subsets mune sensingofinsultsviaC-typelectinreceptors,2)Tcellre ulation, ourlaboratoryisfocusingonthefollowingareas;1)im remains elusive. To illustratethe molecular basisbehind this reg discriminate diverse insults to elicit suitable immune responses sis. However,themolecularmechanismbywhichthesereceptors marily sensedbyimmunereceptorstomaintaintissuehomeosta insults causedbyinfectionsandstresses,mostofwhicharepri C-type lectinreceptors(CLRs)comprisealargefamilyofpro Our bodiesarecontinuouslyexposedtoexternalandinternal dimycolate, TDM),was Myco------cient intheβ-GlcCer–degrading enzyme,further deletion ofthe temic inflammation. In a Gaucher model in which mice are defi- leads toGaucherdisease,adisorder characterizedmainlybysys responses viaMincleonmyeloid cells. Accumulationofβ-GlcCer (GlcCer). β-GlcCer induced inflammatory and acquiredimmune tants fromdamagedcellsand identified β-glucosylceramide ligands derivedfromdamaged cells, wefractionatedsuperna that Minclerecognizesdamagedcells.Toelucidateendogenous by PRRsisnotwelldocumented.Wehavepreviouslyreported tion receptors(PRRs).However,endogenousligandrecognition for thedevelopmentofanoveladjuvant. spectrum ofpathogens,andthusCLRscouldbepotentialtargets light onCLRsasanemergingimmunereceptorfamilyforwide purified ligands augmented T cell responses.Thesefindings shed ligands possessedadjuvantactivityinmice,astheinjectionof were identifiedasMincleligandsfromthesepathogens.These glycolipids, glycosylglycerolipidsandglycosyldiacylglycerols negative bacteriathatlackTDM.InsteadofTDM,severalnovel bacteria, Minclealsorecognizedpathogenicfungiandgram- ing receptorsformycobacteria(Figure1).Inadditiontomyco- CLRs clusteredonthesamechromosomearefoundtobeactivat Th1 protectiveimmunityagainstmycobacteria.Thus,thesefour pressed in monocyte-derived inflammatory cells and triggered inositol mannosidesofmycobacteria.DCARwasselectivelyex same genecluster,wasidentifiedasareceptorforphosphatidyl- MCL axis. Furthermore, DCAR (Clec4b1), a CLR located in the activities thataredistinctfromthoseinducedbyTDM-Mincle/ Sensing tissuedamageisacrucialfunctionofpatternrecogni Sho Yamasaki,PhD Support Staff Support Research Assistant Assistant Professor Professor 2 1 Chihiro Motozono Eri Ishikawa Sho Yamasaki - - - - - an immunostimulatoryfactoruponcelldamage(Figure2). suggest thatβ-GlcCerisanendogenousMincleligandandactsas Mincle geneattenuatedinflammatoryresponses.Theseresults ● ● ● glycolipids. based activation motifs (ITAMs) by directly recognizing multivalent through CLRs.CLRstriggersignalcascadesviaimmune-receptortyrosine- receptors. Mycobacterialcomponentsinitiatehostimmuneresponses Protective immunityagainstmycobacteriainitiatedthroughC-typelectin Figure 1. Recent Publications cytes through phosphorylation ofSHP-1. through Nat. phosphorylation cytes Commun. 7,12756(2016). Yamasaki S. Protein D regulates kinase ofCD4+thymo positive selection E, KosakoH, Ishikawa Yasuda T, Kurosaki Ohmuraya K, M,Araki T, Saito T, 45,1245-57 (2016). infection. Immunity during phosphatidyl-inositol mannosidestobacterial promote a Th1 response Miyamoto T, Yamasaki receptor lectin recognizes S.C-type DCAR myco Ohmuraya M, Yamamoto T, G, Umemura M, Mastuzaki Yoshikai Y, Yano I, Noguchi N,Chuma Y, Kiyohara K, H,Matsuo Tanaka H,Nakagawa Y, Sakuma T, Toyonaga K, Torigoe S, Motomura Y, Kamichi T, Hayashi JM, Morita YS, activity.stimulatory Proc. Natl. Acad. USA.114,E3285-94(2017). Sci. ligandpossessingimmuno isan endogenousMincle β-glucosylceramide T, Miyamoto T, Bamba Yamaji T, Yamasaki S.Intracellular metabolite Nagata M,Izumi Y, S,OmahdiZ, E,R,Iwai KiyotakeR,Doi Ishikawa - - - injury. generating disorderssuchasinflammatorydiseases,autoimmuneortissue the overreactionofanystepmayinduceharmfulresponsestoourbody, procedures arepreciselycontrolledinahealthyimmunesystem;however, Mincle functionsinhostdefenseanddeadtissueclearance.These myeloid cellscouldalsosecretesomeeffectors.Throughtheseresponses, and consequentlyactivateadaptiveimmunity.Ontheotherhand,activated the secretionofcytokinesaswellantigenpresentationbyMHCclassII, damaged self.Myeloidcellsareactivateduponligandbinding,leadingto expressed onmyeloidcellscansenseligandscomefrompathogensand Sensing pathogensanddamagedtissuebyCLRs.MinclerelatedCLRs Figure 2. ● ● rial cordrial factor. 38,1050-62(2013). Immunity an FcRγ-coupled receptor that mediates ofmycobacte theadjuvanticity Suyama M,Iwakura Ono K, Y, Yoshikai Y, Yamasaki S.C-Type MCLis lectin Miyake Y, Toyonaga D, Mori S,Hoshino K, Kakuta Y, A, Oyamada Yamada H, 41,402-13(2014). Immunity ria. lipoarabinomannan ofmycobacte receptordirect for mannose-capped Tanaka M, Yoneyama K, M,Oh-hora M,Akashi Yamasaki isa S.Dectin-2 Yonekawa S,Hoshino A,Saijo Y, Miyake Y, H, M,Inoue E, Suzukawa Ishikawa - - 41 Immunology 42 est large-scalediseasegenomeandmulti-layeromicsdata. laboratory istodevelopsuchmethodsandapplythemthelat diverse biological and immunological resources. The goal of our methodology tointegratelarge-scalehumangenomedatawith diseases. However,littleisknownregardinghowtodevelop comprehensive catalogues of genetic risk loci of immune-related nome dataofmillionssubjects,andsuccessfullyidentified genome sequencing technologies have provided the human ge- tistical and bioinformatics approaches. Recent developments in human geneticvariationsonimmune-relateddiseases,usingsta tistical immunologyisaresearchfieldthatevaluatescausalityof pact ontheriskforawiderangeofimmune-relateddiseases.Sta plex traitsusingtrans-layermulti-omicsanalysis Identification ofcausalimmunecellsonhumancom- Goal ofourlaboratory Statistical Immunology diseases suchas rheumatoidarthritisandGraves’ disease.We GWAS resultsof 30humandiseasesincludingimmune-related which halfwerenovelfindings. Wefurtherobtainedadditional 1,400 geneticvariantsassociated withthesephenotypes,of blood pressureandechocardiography). Ourstudyidentified tions, kidneyfunctions,andelectrolytes), andclinicaltests(e.g., chemical measurements(blood sugar, serumlipids,liverfunc red bloodcells,whitecellsubtypes,andplatelets),bio ments, including hematological measurements (e.g., counts of (GWAS) of>160,000Japanesesubjectson58clinicalmeasure- The geneticbackgroundsofindividualshaveasubstantialim We conductedlarge-scalegenome-wideassociationstudies ------human diseasegenomestudy In silicodrugrepositioningbasedonthelarge-scale et al.NatGenet,2018). ed diseasesbyintegratinggeneticandepigeneticdata(KanaiM tracted thebiologicalpathophysiologyofhumanimmune-relat- causes autoimmunethyroiditis.Our the originalfeatureofregulatoryTcellsinmicethatdepletion ease andautoimmunethyroiditis.Thisfindingshouldsupport relevant humandiseasestotheregulatoryTcellswasGraves’dis plex humantraitsandcelltypes(Figure1).Forexample,themost Our studysuccessfullyelucidatedahiddennetworkamongcom correlation amongthetraitsaccordingtoeachofcelltypes. disequilibrium scoreregression(LDSC)”whichquantifiesgenetic adopted thenewlydevelopedstatisticalmethodnamed“linkage promoter andenhancerinformationfrom>200celltypes.We sults ofhumancomplextraitswiththeepigeneticsignature conducted trans-layeromicsanalysistointegratetheGWASre drug classification curatedbyWHOAnatomical Therapeutic developed amethod toadditionallyintegratethe diseaseand ic studies on stroke comprising 520,000 individuals, we newly tis). Throughinternationalcollaboration withtrans-ethnicgenet sitioning ofCDK4/6inhibitorsfor treatment ofrheumatoidarthri connecting genetic information with drug databases (e.g., repo - od to screen novel candidate drugs for drug repositioning by pharmaceutical field.Wepreviouslydevelopedastatisticalmeth novel drugdiscoveryisoneofthemostfeaturedtopicsin Utilization oflarge-scalehumandiseasegeneticstudiesfor Yukinori Okada,MD/PhD Support Staff Support Visiting Scientist Research Assistant Professor in silicostudysuccessfullyex 1 1 1 Yukinori Okada ------discovery (RainerMetal.NatGenet2018). utilization ofgeneticriskvariantsshouldcontributetonoveldrug cation (Figure2).Thisresultisconsideredasevidencethat tithrombotic therapyasthemoststronglyassociatedclinicalindi tioning pipeline successfully identified a strong network with an ● ● ● variants onstroketothenewlydeveloped Chemical (ATC) Classification System. Application of genetic risk Recent Publications riasis vulgaris inJapanese. vulgaris riasis J. Invest. Dermatol. 138,542-548(2018). J.Hirata et al. Variants at and HLA-DQB1 HLA-A, confer HLA-C, of pso risk 400 (2018). ulation to links cell complex types human diseases. Nat. 50, 390- Genet. M.etal. analysis Genetic ofquantitativeKanai traits intheJapanese pop stroke subtypes. Nat. doi:10.1038/s41588-018-0058-3(2018). Genet. identifies 32lociassociatedstudy of520,000subjects withstroke and R.etal.Malik associationTyrosine genome-wide Multiancestry kinases genetic variantswithstrokeriskanddrugscurrentlyusedforantithrombotictherapy. Application of Figure 2. human autoimmunethyroiditis). analysis. The most relevant human disease with regulatory T cells was Graves’ disease (GD; Network amonghumancomplextraitsandcelltypesidentifiedbytrans-layeromics Figure 1. ᵱᶒᶐᶍᶉᶃ ᶐᶑᵓᵔᵑᵗᵑᵓᵎᵔ ᶐᶑᵔᵖᵐᵓᵒᵓᵒ ᶐᶑᵕᵑᵎᵒᵖᵒᵏ ᶐᶇᶑᶉᴾᵱᵬᵮ in silico in silicodrugreposi ᵢᶐᶓᶅᴾᶒᵿᶐᶅᶃᶒᴾᶅᶃᶌᶃ ᵱᶒᶐᶍᶉᶃᴾᶐᶇᶑᶉᴾᶅᶃᶌᶃ drugrepositioningmethodidentifiednetworkbetweenhuman ᵮᵢᵣᵑᵟ ᵤᵥᵟ ᵮᵪᵥ ᵄ - - - - - ᵲᶃᶌᶃᶁᶒᶃᶎᶊᵿᶑᶃ ᵱᶒᶐᶃᶎᶒᶍᶉᶇᶌᵿᶑᶃ ᵟᶌᶇᶑᶒᶐᶃᶎᶊᵿᶑᶃ ᵫᶃᶊᵿᶅᵿᶑᶒᶐᵿᶌ ᵰᶃᶒᶃᶎᶊᵿᶑᶃ ᵡᶇᶊᶍᶑᶒᵿᶘᶍᶊ ᵟᶊᶒᶃᶎᶊᵿᶑᶃ ● ● ᵢᶐᶓᶅ risk of rheumatoid arthritis. Nat. 49,1120-1125(2017). Genet. ofrheumatoidrisk arthritis. et al. Polygenic K. Ishigaki burdens on cell-specific pathways the underlie 1467 (2017). for body massindex intheJapanesepopulation. Nat. 49,1458- Genet. association studyidentifies 112newloci M.etal. Genome-wide Akiyama ᵫᶍᶑᶒᴾᶑᶒᶐᶍᶌᶅᶊᶗᴾᵿᶑᶑᶍᶁᶇᵿᶒᶃᶂ ᶁᶊᶇᶌᶇᶁᵿᶊᴾᶇᶌᶂᶇᶁᵿᶒᶇᶍᶌ ᵟᶌᶒᶇᶒᶆᶐᶍᶋᶀᶍᶒᶇᶁ ᶒᶆᶃᶐᵿᶎᶗ 43 Immunology 44 Stem CellBiologyandDevelopmentalImmunology phohematopoiesis duringhomeostasisanddiseases. signaling andCARcellsinthespatiotemporal regulationoflym et al.Nature2014).Wearestudying therolesofCXCL12-CXCR4 and maintenance of niches for HSCs and immunecells (Omatsu ipogenic processes in CAR cell progenitors, and development CXCL12 andSCFexpression was essentialforinhibitingad erentially expressedinCARcellsthemarrow,enhancing thermore, wefoundthatthetranscriptionfactorFoxc1waspref (niche) forHSCsandBcells(Omatsuatal.Immunity2010).Fur ducer ofCXCL12andSCF,creatingthespecialmicroenvironment CAR cellsareadipo-osteogenicprogenitorsandthemajorpro bone marrow(Sugiyamaetal.Immunity2006)andindicatedthat high levels,termedCXCL12-abundantreticular(CAR)cellswithin identified apopulationofreticularcellsexpressingCXCL12at diogenesis (Tachibanaetal.Nature1998).Subsequently,we Nat. Rev.Immunol.2006)aswellvascularformationandcar- cells, plasmacytoiddendriticcells(pDCs)andNK(Nagasawa. Immunity 2006),anddevelopmentofimmunecells,includingB maintenance ofapoolHSCsinbonemarrow(Sugiyamaetal. (HSCs)(Nagasawa etal.Nature1996;AraImmunity2003), tial forcolonizationofbonemarrowbyhematopoieticstemcells 1994) andfoundthatCXCL12itsreceptorCXCR4areessen stimulates thegrowthofBcellprecursors(Nagasawaetal.PNAS isolated achemokine,CXCL12(SDF-1/PBSF)asmoleculethat vironments knownasnicheswithinbonemarrowcavities.We cells, including immune cells, are maintained by special microen Hematopoietic stemcells(HSCs),whichgiverisetoallblood ------Genes Dev.2018). blast differentiation,andtomaintain spacesforHSCs(Seikeetal. Ebf3 thatisrequiredtocreateHSC niches,toinhibittheirosteo in theabsenceofEbf1/Ebf3.Thus,HSCcellularnichesexpress bone-producing cellswithreducedHSC-nichefactorexpression marrow cavities in early adulthood. CAR cells differentiate into sively moreosteosclerotic,leadingtothecompleteocclusionof in infantmarrow.Subsequently,themutantsbecomeprogres- their nichefunctionwasmarkedlyimpairedwithdepletedHSCs exhibiting anormal morphology wereabundantlypresentbut in agedmice(Figure1).InlackingEbf1andEbf3,CARcells paired andbonemarrowwasosteoscleroticwithincreased was deletedinCARcells,HSC-nichefunctionseverelyim cytes inadultbonemarrow,usinglineage-tracing.WhenEbf3 enchymal stemcells,whichgiverisetoallosteoblastsandadipo in CAR cellsand that Ebf3-expressing cells are self-renewing mes find that the transcription factorEbf3 ispreferentially expressed cells tomaintainHSCnichesandmarrowcavities.Recently,we remained unclearhowosteogenesisispreventedinmostCAR niches, giverisetoosteoblastsinbonemarrow.However,ithad faces. CARcells,whichareamajorcellularcomponentofHSC the bonemarrowcavity Hematopoietic stemcellniche-specificEbf3maintains Bone marrowisthetissuefillingspacebetweenbonesur Takashi Nagasawa,MD/PhD Support Staff Support Assistant Professor Associate Professor Professor 1 Yoshiki Omatsu Tatsuki Sugiyama Takashi Nagasawa - - - - - ● ● ● Recent Publications 286-298 (2018). Receptor andCXCR4. 48(2), Interleukin-7 sponses by Inducing Immunity Drive OscillationsGlucocorticoids Diurnal in T Cell andRe Distribution H, Yamada H,Hara T, Yoshikai, Y, Nagasawa T, K. G,Ikuta Schütz Shimba A, Cui G, Tani-Ichi F, S, Ogawa M, Abe S, Okazaki S, Miyachi Kitano 2124-2131 (2017). poietic stem cells homeostasis. remain during Blood empty 129(15), Shimoto M,Sugiyama T andNagasawa T. Numerous nichesfor hemato 359-372 (2018). nich-specific Ebf3maintains the bonemarrow cavity.Dev.Genes 32(5-6), M,Omatsu Seike Y, Watanabe H,KondohGandNagasawa T. Stem cell were preferentiallyandabundantlyexpressedinCARcellswithinthebonemarrow. The transcriptionfactorsFoxc1andEbf3,HSCnicheCXCL12 SCF Figure 2.ThedevelopmentandfunctionsofCARcells. Femurs inagedLepr-Cre;EBF3 Figure 1.AprogressionofocclusionthemarrowcavitiesinabsenceEbf3 filled withbone(Seikeetal.GenesDev.2018). Seike f/f , micerevealedthatthebonemarrow spaces were M. - - et al. 1mm , GenesDev ● mation. Nat. Cell Biol. 19(8),915-927,(2017). forDll4 andNotch signalling couples sprouting angiogenesis andartery Ferrara N,Borggrefe T andAdams RH. Park H,EhlingM,BiljesD, SF, Rocha LangenUH,Stehling M,Nagasawa T, Pitulescu I,GiaimoBD, ME, Schmidt Antoine T, Berkenfeld F, Ferrante F, 2018 1mm - 45 Immunology 46 Single MoleculeImaging(Yanagida–SeymourGroup) havior ('cognitiveneuroimmunology'). ter peripheralinjury,toprobethe normativebasisforillnessbe analyses usecomputationalmodels ofhowbehaviorchangesaf acterize differencesbetween brain states.Hypothesis-driven of graphtheoryandothernetwork topologicalanalysestochar gate classifiablechangesincomplexbrainnetworks,andtheuse ods utilize machine learning and deep learning tools to interro- the complexbrainchangesininflammation.Data-drivenmeth bine data-driven and hypothesis-drivenanalysestounderstand clinical modelsofpainandinflammation.Inparticular,wecom mation, usingbrainimaginginrodent,humanpre-clinicaland these symptomstodevelopandpersistafterperipheralinflam to identifytheadaptivechangesthatoccurinbrainallow limitation totheclinicalefficacyofthesedrugs.Ourresearchaims modifying drugssuchasimmunologicsrepresentsaprimary tory arthritis.Theirrelativeresistancetotreatmentbydisease two dominantsymptomsofimmunediseasessuchasinflamma tions. body thatsupportphysiologicalandpathophysiologicalfunc translational insightintothemulti-systeminteractionsin cies, with new intelligent approaches to data analysis, to provide Our goalistointegrateimagingmethodsacrosslevelsandspe opportunity tointerrogatebiologicalprocessatmultiplelevels. We know from our previous research that inducing changes in A particularinterestofourlabispainandfatigue,whicharethe Recent advancesinimagingmethodologiesprovideaunique ------modulate painand fatigueasanadjuncttoimmune modulators. sponse totreatment,andinnovate newtreatmentsthatcould disease, potentiallyprovidingaway topredictsymptomaticre derstand howpainandfatiguedevelop inchronicinflammatory research suggeststhatpgACCmay representanewtargettoun bined molecularandbrainimaging methods.Insummary,this thritis) androdentmodels(CFAjointinjection)[5],usingcom this ininflammatoryarthritis-bothhumans(rheumatoidar tion. Wehavenowstartedalargetranslationalprojecttoaddress pain todevelopinthetransitionfromacutechronicinflamma the injuredstate,reflectingaprocessthatcouldinprinciplecause provides amechanisticlinkbetweenthemodulationofpainand havioral responses[2].Thereasonthisissignificantbecauseit signals, andcontroloutgoingautonomictooptimizebe flamed skin:specifically,itappearstomodulateincomingsensory when peopletrytoengageinbehaviorprotectpotentially- healthy humans,wehaveshownthatpgACCmodulatespain significant asinanotherstudy,usingamodelofheatinjury region - thepregenualanteriorcingulate cortex (pgACC).This is ticular, thesechangescorrelatewithactivityinaspecificbrain identifies changes in the brain modular architecture [1]. In par- chronic musculoskeletalbackpain,usinganewmethodthat terize substantial reorganization of the sensorimotor cortices in brain afterinjury.Now,wehavebeenabletoidentifyandcharac if significant sensorimotor reorganization actually occurs in the lowing nerveinjuryinhumanpatients[5],butwehaven'tknown the functionofsensorimotorcortexcanmodulatepainfol Ben Seymour,MD/PhD Toshio Yanagida,PhD Support Staff Support Visiting Scientist Professor 1 1 Ben Seymour Ben Toshio Yanagida ------● ● ● musculoskeletal backpain. Sensorimotor brainregionsshowingmodularreorganizationinchronic Figure 1. Recent Publications Brain andNeuroscience Advances (2018)inpress. is associated withsuppression ofbehaviour inarat model ofchronic pain. Yoshioka Y, Morioka Y, B. Seymour Anterior cingulate connectivity cortex M, de laMora L, Sprenger C, Koda K, Morris Yamada T, H, Kashiwagi Mano Y, (2018). Control of Tonic Pain by Active Learning. Relief eLife 2018;7:e31949 H,LeeZhang S,Mano M, Yoshida W, Robbins T, KawatoB. M,Seymour The pain: Amulticenter study. Wellcome OpenResearch 3:19(2018). sification ofbrain andcharacterization changesinchronic network back Voon, Yoshida W, Lee M, Yanagida T, B. Kawato M,Seymour M,Rosa Clas H, Kotecha MatsubaraMano G, Leibnitz K, T, A,Shenker N, Shibata M, Nakae joint inflammationinrodents. Structural imagesshowingbrainchangesinanteriorcingulatecortexafter Figure 3. - behavioral 'resilience'afterinjury. Localization ofpregenualanteriorcingulatecortex(pgACC),whichsupports Figure 2. ● ● 13209 (2016). controlsbrain plasticity paininphantom limbpatients. Nat. Commun. 7, M, Hirata Yoshimine T, Kamitani Y, &Saitoh Y. sensorimotor Induced Yanagisawa T, Fukuma H,Shimizu Kishima B, R,Seymour HosomiK, T, Yokoi H, 13788 (2016). caused by interplay between RNA polymerases on DNA. Nat. Commun. 7, M, Fujita Iwaki K, Yanagida T. Transcriptional burstingisintrinsically 47 Imaging 48 a spatiotemporalmanner(seetheFigure). abled us to visualize the dynamic nature of different cell types in niques. Recentadvancesinopticalimagingtechnologyhaveen tissues andorgansinvivo,bymeansofadvancedimagingtech mental principle controlling cellular dynamics in various kinds of in vivo Intravital boneimagingrevealingcelldynamics Immunology andCellBiology spatiotemporally limitedareas.ApH-sensingfluorescence probe cell-to-cell contact wasdetectedbetweenmOBs andmOCsin distributed mainlyinasegregated fashion,althoughsomedirect osteoclasts (mOCs),andfoundthat themOBsandmOCswere communication betweenmature osteoblasts(mOBs)andmature cretion inboneresorptionbyosteoclasts (NatChemBiol,2016). have developedanewchemicalprobefordetectingprotonse vivo, incollaborationwithDr.KikuchitheframeofIFReC,we order tofurtheranalyzetheactualeventofboneresorptionin tively looselyattachedandwrigglingalongthebonesurface.In trix bysecretingacids,andnon-resorbing(N)osteoclastsrela- teoclasts firmlyadheringtobonesanddevouringthebonema mature osteoclastfunctional states; i.e.,bone-resorbing(R)os (J ClinInvest2013).Thisnovelvisualizationidentifiedtwodistinct fully differentiatedosteoclastsadheringtobonesurfacesinvivo bones. Wepreviouslysucceededinvisualizingthefunctionof elaborated thenovelimagingsystemforvisualizinginside Furthermore, wecoulddetectan invivomodeofdynamic By usingintravitalmultiphotonmicroscopy,wehaveoriginally The majoraimofourlaboratoryistounderstandthefunda ------(Ann RheumDis,2018). modes ofactiondrugs,optimizing theusageofdrugregimens different efficacies.Theseresults enableustograspthereal apeutic time-pointsduringosteoclasticbonedestruction,with ing revealedthatvariousbiologicDMARDsactedatspecificther being suppressedduringosteoclastmaturation.Intravitalimag Ig) wereprominentlyexpressedonlyinosteoclastprecursorcells, ment withtheseresults,CD80/86(thetargetmoleculesofCTLA4- sors, eliminatingtheirfirmattachmenttobonesurfaces.Inagree no actiononmatureosteoclastsbutmobilizedosteoclastprecur bone-resorbing osteoclaststonon-resorbingcells.CTLA4-Ighad and anti-TNFα mAbs affected mature osteoclasts and switched agents usedinclinicfortreatingrheumatoidarthritis,anti-IL-6R macological actionsofvariousdrugs.Forexample,thebiologic ogy turnedouttobeapowerfultoolfordissectinginvivophar secting human immunology Application ofintravitalimaging techniquesfordis ing ofbonehomeostasisinvivo(NatCommun,2018). between mOBsandmOCs,thuscontributingtoourunderstand ing techniquestorevealspatiotemporalintercellularinteractions rect cell–cell contact. This study is the first to use intravital imag- mOBs could inhibit the bone resorption activity of mOCs by di mOCs contactingmOBswerenon-resorptive,suggestingthat sorption whentheywerenotincontactwithmOBs,whereas we developed revealed that mOCs secreted protons for bone re- Moreover, wehaveshown that ourintravitalimagingtechnol Intravital imaging withmulti-photonmicroscopy isanun Masaru Ishii,MD/PhD Support Staff Support Visiting Scientist Research Assistant Assistant Professor Associate Professor Professor 2 1 2 Yutaka Uchida Szandor Simmons Takahiro Matsui Takao Sudo Mizuno Hiroki Junichi Kikuta Keizo Nishikawa Ishii Masaru ------● ● ● samples. By collaborating with companies (supported by AMED) limited inanimal models andmaynotbeforanalyzinghuman system dynamicsinvivo.However,theapplicationiscurrently intact tissuesandorgansthususefulforstudyingimmune doubtedly powerfultoolfordissectinglivecellulardynamicsin Recent Publications Nat. Chem.Biol. 12,579-85(2016). usingdesigned smallmolecular probe. andmotility cell osteoclast activity H.etal.Maeda intravital Real-time imaging ofpHvariation associated with 300 (2018). osteoclasts dynamically controls invivo. theirfunctions Nat. Commun. 9, Furuya between mature M.etal. contact cell-cell Direct osteoblasts and (2018) inpress. logic DMARDs inhibiting osteoclastic bone resorption. Dis. Ann. Rheum. Matsuura Y. etal.vivo In visualization ofdifferent ofbio modesofaction vivo behaviorswithadvancedimagingtechniques. principle controlling the dynamic nature of immune cells by visualizing in organs, byforminga‘soft-wired’network.Weareelucidatingthebasic Immune cellsarehighdynamicandinterconnectingvarioustissues Figure. Intravitalimagingforvariousimmunesystems. - able todissecthumanimmunologyinfuture. be usedfordifferentialdiagnosis(SciRep,2017),andwewill alizing non-labelledhumannormalandcancertissues,whichcan tissues andorgansinvivo.Currently,wehavesucceededvisu we aredevelopinganewmicroscopysystemforapplyinghuman ● ● 281-287 (2015). metabolic pathway.to methionine-producing an S-adenosyl Nat. Med. 21, etal. Dnmt3a regulates K. Nishikawa osteoclast differentiation by coupling (2016). ing cholesteatoma-induced Mol. Cell bonedestruction. Biol. 6,1610-20 fibroblasts induces localosteoclast differentiation: amechanismunderly Iwamoto Y. etal. Intercellular communication and between keratinocytes - - 49 Imaging 50 nal lymph nodes. tive findings,suchasnon-specific accumulationinthemediasti cells, butalsoininflammatorywhichmayleadtofalse-posi ever, theglucosetransporterisup-regulatednotonlyincancer tracer used worldwide for the evaluation of cancer patients. How- 18 Nuclear Medicine acid transporter1(LAT1) selectivity forLAT1 andtheusefulnessof evaluated the in-vitro cellular uptake of advantage to use it as a reliable tracer for cancer diagnosis. We uptake inmostnormalorgans,except thekidney,whichisagreat specific tracerin ratxenograftandinflammation models.Asare cancer therapy. lung cancer,suggestingpossibleapplicationofLAT1inhibitorsto of LAT1wasassociatedwithanantitumoreffectinnon-smallcell while beingexpressedatlowerlevelsinnormaltissues.Inhibition transporter which is strongly expressed in many cancercells prove personalizedprecisionmedicine. therapy againstcancersuchaslowperfusionandhypoxiatoim portant toevaluatetheresistantfactorsforchemo-radiation nostic imagingandtargetedalphaorbetatherapy.Itisalsoim tive targeting is essential using the same molecule for both diag is nowfocusingattentionontheoncologyfieldandtumorselec proper managementandtreatmentofthepatients.Theranostics using molecularimagingtechniquesandeventuallyleadtothe F-FBPA asatumor-specificprobeofL-typeamino L-typeaminoacidtransporter1(LAT1)isthemajor The aimofourlabistovisualizeandquantifyin-vivodynamics 18 F-fluorodeoxyglucose ( 18 F-FBPA was shown to have low physiological 18 18 F-FBPA focusing onits F-FDG) isamajorPET 18 F-FBPA asatumor------therapy andchemotherapyforcancer.Althoughitisdetectedby FBPA for LAT1 and in-vivo 18 18 flow andoxygenmetabolism The relationshipamongintratumoralhypoxia,blood tumoral hypoxic regions showed decreased blood flow with in in-vivo molecular PETimagingtechnique,weshowed thatintra factors to define the severity of intratumoral hypoxia. Using the Oxygen extractionandvasculardensity wereconsideredaskey served inoxygenextractionfraction andtumoralbloodvolume. moderate andseverehypoxia,significant differenceswereob registered imagesof tabolism inhypoxicregionsusingtheratxenograftmodel.Co- ied. Weevaluatedtheintratumoralperfusionandoxygenme intratumoral hypoxiaandoxygenmetabolismhasnotbeenstud sult, thecellularanalysesrevealedhigherselectivityof vealed thatthereweresignificantnegativecorrelationsbetween suggesting anecroticregion(Figure2).Quantitativeanalysisre was noaccumulationinthecentralareainsideFMISOuptake, cated asliningtheinsideofhighbloodflowareaandthere that intratumoral hypoxic regions with FMISO uptakes were lo (Figure 1). We could demonstrate the utility of in theglioma tumor andlowuptakeintheinflammatorylesion matory lesions. mor-specific probeofLAT1withlowaccumulationintheinflam- F-FMISO SUVandtumoralblood flow.Incomparisonbetween F-fluoromisonidazole (FMISO)PET,therelationshipbetween Intratumoralhypoxiaisoneoftheresistantfactorsinradio Jun Hatazawa,MD/PhD Support Staff Support Research Assistant Assistant Professor Associate Professor Professor 18 F-FMISO andtumoralbloodflowshowed 18 F-FBPA PET showed the high uptake 2 2 Tadashi Watabe Kato Hiroki Jun Hatazawa 18 F-FBPA as a tu- 18 F------● ● mor cellsinhypoxicregions. strategy tonormalizethe blood flow for oxygen-avid active tu creased oxygenextraction,suggestingtheneedforatreatment Recent Publications lation PET. Res. 7,16(2017). EJNMMI tumoral hypoxia: (18)F-fluoromisonidazole gasesinha- and(15)O-labelled Watabe T. etal. Quantitative evaluation ofoxygen metabolismintheintra- Res. 7,91(2017). tion algorithms. EJNMMI ofreconstruc scannerandimpact microPET evaluation usinga3D-mode Horitsugi G.etal. Oxygen-15 labeled CO2, O2,andCO insmallanimals: PET and fusionimage.Pimonidazolestainingcorrespondedto surrounded bybloodflowpreservedregions. in theinflammatorylesionsfor high uptakesinthetumorsonPETimages.Incontrast,lowwereobserved Comparison among pimonidazole staining, Figure 2. 18 Static PETimagesof Figure 1. F-FDG PETremainedhighintheinflammatorylesionsaswell. 18 F-FDG, 11C-Methionine(Met),and 18 F-FBPA and11C-MetPET,whereastheuptakevaluesof - - 18 F-FMISO PET, tumoral blood flow (TBF), ● nine PET. Eur. J. Nucl. Med. Mol. 44,321-331(2017). Imaging 1(LAT1):transporter acomparison studywith(18)F-FDGand(11)C-Methio Watabe T. et al. (18)F-FBPA as a tumor-specific probe of amino acid L-type 18 F-FBPA. Alltracersshowed 18 F-FMISO uptakeregions, - 51 Imaging 52 molecular dynamics single-molecule imagingofimmunereceptor-adaptor Intracellular protein-labelingprobesformulticolor Chemical ImagingTechniques carboxyl astheNIR fluorophores,whichpossess excellent bright and application to multicolorSMI.WeselectedSiR-Me andSiR- cell-permeable BL-tag ligand for intracellular protein-labeling interfere SMIduetothesignificant backgroundsignals. fluorescence, theintrinsichigh hydrophobicity ofthedyescan applications suchastheirlowspectraloverlapwithcellularauto- Although NIRfluorophoreshavemanyadvantagesinbiological ing, whichspecificallybindstoamutantβ-lactamasetag(BL-tag). veloped near-infrared(NIR)fluorescentprobesforprotein-label fluorescence intensitieswithinacell.Toaddressthisissue,wede both FPstodetermineconditionsunderwhichdisplaysimilar challenging, as it requiresoptimizationoftheexpressionlevels of expression levels.Dual-colorSMIusingtwoFPsisparticularly over, FPshavecommonlimitationsinstrictlycontrollingtheir plications islimitedbecauseoftheirpoorphotostability.More ability touseFPsforSMIandotherphoton-intensiveimagingap (FPs) arewidelyusedasfluorescencereporters.However,the actions inlivingcells.Geneticallyencodedfluorescentproteins quently, aboutdynamicandkineticparametersofbiologicalre- lar dynamicsandinteractionsinthecellularcontext,conse several seconds,andprovidesdirectinformationaboutmolecu- hundreds orthousandsofindividualmoleculesoveraperiod Here, wedevelopedNIRfluorescent probesconjugatedwitha Multicolor single-moleculeimaging(SMI)enablestrackingof ------the BL-tagprotein-labeling systemwasusedincombination with cence microscopy (TIRFM).ToanalyzetheTLR4-TIRAP dynamics, appropriate depth for SMI using total internal reflection fluores - the dynamicinteractionsbetween TLR4andTIRAPoccuratthe izes intheplasmamembrane,where itinteractswithTLR4.Thus, TIRAP was selected as the target adaptor protein because it local- molecular dynamicsuponinnate immune responseinlivingcells. RAP) uponlipopolysaccharide(LPS)stimulationtoanalyzetheir leukin-1 receptor(TIR)domain-containingadaptorprotein(TI tor interactionbetweenToll-likereceptor4(TLR4)andToll-inter- probe. ments incombinationwithaHaloTag-basedred-fluorescent ed SiRcB4asanNIR-fluorescentprobeformulticolorSMIexperi permeability andtheminimizednon-specificbinding,weselect SiRcB notpossessingahydrophiliclinker.Consideringthecell was detecteduponlabelingwithSiRcB( number of fluorescent spots with higher diffusion coefficients membrane. Trackinganalysisrevealedthatasignificantlylarger for SMIoftheBL-taganchoredtoinnerleafletplasma its capabilitiesforintracellularSMI,weusedSiRcB( in astepwisemanner:SiRcB( the distancebetweenfluorescentdyeandBL-tagligand drophilic oligoethylenoxy linker, increasing hydrophilicity and novel bacampicillin-basedlabelingprobesbyintroducingahy the BL-tagligandforintracellularprotein-labeling.Inaddition, ness, photo-stability,andcellpermeability, bacampicillin as In themulticolorSMIstudy,wefocusedonreceptor-adap Kazuya Kikuchi,PhD Support Staff Support Research Assistant Postdoctoral Fellow Assistant Professor Associate Professor Professor n) ( = 2,4,6)(Figure1a).Toevaluate 1 2 1 Minoshima Masafumi Yuichiro Hori Kikuchi Kazuya n) (=2,4,6)thanwith n) (=2,4,6) - - - - - lacking TLR4 mutant (∆TIR) revealed that the relative ratio of by theirinteraction.AnotherSMIexperimentusingTIR-domain result indicatesthatthemobilityofbothproteinswasdecreased TLR4 andTIRAPuponligandstimulationbyLPS(Figure1e-f).This namics showedanincreaseinthepopulationofslow-moving protein dynamicsbystatisticalanalysis.Analysisofdy stimulation with10μg/mLLPS,andquantitativelyanalyzedthe color SMIinlivingcellsexpressingBL-TIRAPandTLR4-Halo,after of TLR4(TLR4-Halo),respectively(Figure1d). tached totheN-terminusofTIRAP(BL-TIRAP)andC-terminus ● ● ● TIRAP expressingcells,uponLPSstimulation(10μg/mL).N>140,000(5cells). diffusion ofTLR4-Halo (f),and BL-TIRAP (g)in TLR4-Halo and BL-TIRAP expressing cells, andTLR4(∆TIR)-Halo (h),and BL-TIRAP(i) inTLR4(∆TIR)-Halo and BL- labeling ofTLR4 and TIRAP (d), and TLR4(∆TIR)-HaloBL-TIRAP (e) with theHaloTag-TMRligand and SiRcB4,respectively. (f–i) Transientresponse of the TLR4-Halo andBL-TIRAP.Thecellswereincubatedwith50pMHaloTag-TMR Ligand and100pMSiRcB4for30minat37°C.Scalebar:5μm.(d,e)Fluorescent for 30min.Imageswereacquiredbeforeandafter10minstimulationwith μg/mLofLPS.Scalebar:20μm.(c)MulticolorSMIHEK293Tcellsexpressing cells expressingTLR4-HaloandBL-TIRAPbyconfocalmicroscopy.The were incubatedwith100nMHaloTag-TMRLigandand500SiRcB4at37ºC (a) Structuresofbacampicillin-basedprobes.SiRcB2,SiRcB4,andSiRcB6containdi-,tetra-,hexa-ethyleneglycol,respectively.(b)ImagingHEK293T Figure. a commercialHaloTagsystem.BL-tagandHalo-Tagwereat Recent Publications Mg Matsui Y, ofIntracellular Imaging Ratiometric K. S&Kikuchi Mizukami (2018). ofDNAMethylation. Imaging J. Am.Live-Cell Chem.Soc. 140, 1686-1690 Synthetic-Molecule/Protein Probe Hybrid withFluorogenic Switch for Hori Y, K. M,UmenoSuetake I&Kikuchi A,Nishiura N,Nishida Otomura dynamics. J. Am. Chem.Soc. 139,17397-17404(2017). imaging ofimmunereceptor-adaptorcolor single-molecule molecular Intracellular protein-labeling K. probes S&Kikuchi for multi Mizukami J,R,KumagaiSato R,Kozuka UedaM,Mishima Y, Yoshimura M, A,Minoshima rescent Turn-on Probe. Chem.Lett. 47,23-26(2018). We usedSiRcB4andtheHaloTag-TMRligandtoperformmulti 2+ Dynamics Using Fluorescent aRed Dynamics Turn-off Probe andaGreen Fluo - - - - - calized atthecytoplasmicmembrane. vestigation of the immune response of downstream adaptors lo analysis ofotherreceptorsinresponsetovariousPAMPsand the firsttime.Thissystemmayalsobeusefulforquantitative TIRAP interactionsinlivingcellsatthesingle-moleculelevelfor SMI systemenabledquantitativeanalysisofLPS-inducedTLR4- system fordetectingprotein-proteininteractions.Ourmulticolor stimulation, which demonstrates the reliability of our imaging with thecurrentmodelofTLR4dynamicsinresponsetoligand main isessentialforchangingitsmobility.Thisresultconsistent act withTLR4(∆TIR),bindingofTIRAPtoTLR4throughtheTIRdo stimulation (Figure1g-i),indicatingthatsinceTIRAPdidnotinter- slow-moving TIRAP molecules did not change even after ligand ● ● (2017). Interference fromInterference Ca tion ofLong-term Mg Matsui Y, Funato Y, K. S&Kikuchi Visualiza H,Mizukami H,Miki Imamura Fluorescent Probes for Visualizing Intracellular Mg Matsui Y, Selective Highly K. Tridentate S&Kikuchi Mizukami KK, Sadhu table Fluorescent Probe. 8,8255-8264(2017). Chem.Sci. 2+ 2+ Fluctuation. Chem.Commun. 53,10644-10647 Dynamics inApoptotic Cells Dynamics withaNovel Targe 2+ Dynamics without Dynamics - - - - - 53 Imaging 54 sight forotherstudies thatdorequirefixation. Figure 1shows ascertain the changesthat occur during fixation toprovide in knowledge orlabelling,itispossible touseRamanimaging measures themolecularcontents ofthecellwithouta-priori sive, non-fixedmethods.Nevertheless, sinceRamanimaging permeabilization isrequired.Our labinterestsareinnon-inva performed inafullylabel-freeopticalmeasurement. (Hobro etal2016),wheretheidentificationofacelllinecouldbe tering todiscriminatebetweennumerouslymphocytecelllines markers. Alongthisdirection,wesuccessfullyusedRamanscat stream eventsthatoccurbeforetheexpressionofsurface or activation. In essence, the process may then measure the up- terize more of the cell features that emergeduring differentiation self isremoved,andanotherthatitbecomespossibletocharac- advantages. Oneisthatthevariabilityinlabellingprocessit ize lymphocytetypewithoutlabelling,itprovidesseveral velop tools to optically measure the features that best character- ling theproteinsexpressedonsurface.Ifwecaninsteadde- the discriminationofcelltypeisusuallyperformedbylabel without any modification of the cells of interest.ForTand B cells, means thatimmunecelltypeandfunctioncanbecharacterized diversity. The development of label-free single cell analytic tools with recentincreasedawarenessoftheimportancecell-to-cell In particular,single-celllevelanalysisisamainaim,coinciding ods whichcanbeusedtohelpunderstandtheimmuneresponse. Biophotonics In manytypesofimmunofluorescence imaging,fixationand Our researchisaimedatdevelopinglabel-freeimagingmeth ------tion withthesignals ofinducibleNOSinthecell,which isknown ed thesemodalities bycomparingtheRamanand phase informa generate featurevectorsthatrelate totheactivation.Wevalidat data, aswellthequantitative phase,couldbothbeusedto formation onchangesincellular composition. MeasuredRaman In additiontothis,theRamanmodality givesusquantitativein activation stateofasinglecellusing onlythephaseinformation. served tochangesufficientlysothatitispossibleevaluatethe the cell.Followingstimulation,macrophagemorphologywasob ing modeprovidesthespatialdistributionofrefractiveindexin stimulated by lipopolysaccharides.The quantitative phase imag modes toevaluatethechangesoccurringinmacrophageswhen imaging, we studied the potential for both of these label-free tivation. Using Raman spectroscopy as well as quantitative phase different label-freetechniquesforcharacterizingmacrophageac biochemical changes in the cells,we also exploredtheuse of two cell (Figure1).SeeHobroetal2017fordetails. nents, showingthattheprocessreducescomponentsin the Ramanimagegeneratedfromfirsttwoprincipalcompo structure and contents, actually show a reduction in intensity in paraformaldehyde, althoughassumedtomaintainoverallcell results showthatevencommoncross-linkingfixativessuchas directly comparableintermsofcomponentstotheothers.The ing andprincipalcomponentanalysis(PCA)sothateachcellis individual cellswasperformedusingline-scanningRamanimag data fromthisproject.TheRamanmappingofcomposition As wellascharacterizingphenotypeorforstudyinglarge-scale Nicholas IsaacSmith,PhD Assistant Professor Associate Professor Nicolas Pavillon Alison Hobro IsaacSmith Nicholas ------using progesterone, the samples comprised four different classes ments onthesamesinglecells.Whenactivationwasinhibited gave highlycorrelatedindicatorsofactivationinpairedmeasure- ments basedoncompositionandmorphology,respectively, different physicalfeaturesofthecell.Theindependentmeasure phase modalitiescoulddetectactivation,theyarebasedonvery tending theworkusingprimary-culturedmurinemacrophages. project. Whilethisstudyinitiallyusedcelllines,wearenowex phase measurements.SeePavillonetal2018fordetailsofthe using a fluorescence iNOS signal, compared to the Raman and tion ofLPS-exposedmacrophagesagainstcontrolcellsisshown, fluorescent measurements. See Figure 2, where the discrimina- activated) was foundtobesuperiorin some casestothe tion. Infact,theseparationoftwoclasses(activatedvsnon- bel-free methodscouldprovideanaccurateestimationofactiva ● ● ● al. 2017fororiginalfigureanddetails. generated fromthefirsttwospectralprincipalcomponents.SeeHobroet changes canbemadebetweenthegroupsofcells.Thecolorchannelsare analysis was performed on the whole dataset sothat comparison of then fixed with paraformaldehyde at 4 degrees C. The principal component changes occurringduringfixation.CulturedMEFcellswereimagedlive, Use ofRamanimagingandprincipalcomponentanalysistoevaluate Figure 1. to becorrelatedwithactivation.Theresultsshowedthatthela Recent Publications py". Opt. Express 24(26),pp. 30038-300052(2016). N. Pavillon andN. I.Smith,"Compressed sensinglaser scanningmicrosco mun. (2018)inpress. gens, microorganisms, andtheirinteractions withhostsystems". Opt. Com A. J. Hobro andN.I.Smith,"Vibrational spectroscopic imaging ofpatho ing". Proc. Natl. Acad. USA115(12),pp. Sci. E2676-E2685(2018). resolution through machinelearn macrophage withsingle-cell activation N. Pavillon, A.J. Hobro, andN.I.Smith,"Noninvasive S.Akira of detection An interestingfindingwasthatalthoughboththeRamanand ------activation. that the label-free methods are highly effective at estimating macrophage and LPS-exposed cells are in red. The separation of the two groups shows the Raman,phase,andiNOSsignalmeasurements.Controlcellsareinblack, phase measurements for the macrophage state, and comparisons between cells in iNOS. Panels B, C, D show the combined predictions of Raman and protocols inimmunology.PanelAshowsacomparisonbetweendifferent labelled foriNOS,allowingustocompareourmethodsmoretypical denotes morphology measured by phase imaging), and then immuno- first measuredlive,withoutmodificationinRamanandphase(“morph” Evaluating the changes in Raw264 cells under LPS stimulation. Cells were Figure 2. smaller targetssuchasRNA,virii,andblood/serumsamples. to thespectroscopicanalysisofstructureandcomposition we alsoaimedtoexpandtheapplicationsofthesetechnologies with othertechniques. be highlyuseful,providinginsightsthataredifficulttoachieve illustrates howamultimodalapproachtosingle-cellanalysiscan cient downstream effects to exhibit in the cell morphology. This in the overall composition of the cell, and that there are insuffi dicating thatthecellularchangesduetoprogesteronearemostly guish betweencontrolcellsandprogesteroneexposedcells,in contrast, thephaseimagingmodalitycouldnotreliablydistin ence inmolecularcompositionofthecelltobediscriminated.In progesterone on resting macrophages creates a sufficient differ- cessfully distinguishallfourclasses,implyingthattheeffectsof progesterone-exposed. TheRamanmeasurementscouldsuc of cells:control,LPS-exposed,progesterone-exposed,andLPS+ ● ● 3756-3764 (2016). tool for cell label-free linediscrimination". lymphocyte Analyst 141, pp. A. J. Hobro, Y. spectroscopy and as N. I. a Smith, Kumagai,"Raman S. Akira trosc. 91,pp. 31-45(2017). imaging". bycompositional Raman cellular changesobserved Vib. Spec A. J. Hobro andN.I.Smith,"An evaluation offixation methods:spatial and As wellasthemaintargetsoflabel-freeimagingandanalysis, - - - - - 55 Imaging 56 to otherlymphoid organstocontinueantigensurveillance. We cytes exitfromthe LNintolymph,returntoblood flow, andtravel and adaptiveimmuneresponses. role ofadrenergicnervesinthecontrol oflymphocytetrafficking among lymphoidorgans.Therefore, wehavebeenstudyingthe the inputs from adrenergic nerves affect lymphocyte migration supply of adrenergic nerves. However, it had been unclear how tors (ARs).Likeothervitalorgans,lymphoidorgansreceivearich cellular responsesthroughα thetic nervous system and produce noradrenaline that induces Immune ResponseDynamics cells throughmultiplemodesofaction. tems regulateboththedevelopmentandfunctionsofimmune onstrated thattheautonomicandsomatosensorynervoussys resent thenewtrend.Studiesduringlastdecadehavedem- for connectionsbetweenthenervousandimmunesystemsrep recent studies thathave revealed the cellular and molecular basis organ systemshasbeenincreasinglyrecognized.Inthisregard, portance ofstudiesthatclarifyinterconnectionsamongmultiple without knowingthestatesofotherorgansystems.Thus,im impossible to precisely predict responses of one organ system tually communicatetocross-regulatetheirfunctions,itisnearly cal science.However,becausetheorgansystemsinourbodymu individual organsystemsandyieldedhugeprogresstobiomedi After spendingseveralhoursin a lymphnode(LN),lympho- Adrenergic nervesconstitutetheefferentarcofsympa During the last century, extensive studies were performed on 1 , α 2 , β 1 , β 2 andβ 3 adrenergicrecep ------phocyte egressfromLNsthroughβ demonstrated thatinputsfromadrenergicnervescontrollym cyte β immune responseswasdependent onβ those inducedinthedaytime.The diurnalvariationofhumoral companied by more robust humoral immune responses than mulation oflymphocytesinLNsduringthenighttimewasac lymphocytes andanincreaseoftheirnumbersinLNs.Theaccu nerve activityinLNswasfoundtocauserestrictedLNegressof ward thenighttimeinmice.Thesurgeofadrenergic dents. Indeed, the noradrenalinecontentin LNs was elevated to the daytimeinhumans,whereasitreachesapeakatnightro- noradrenaline release from adrenergic nerves increases during that issynchronizedwiththerest-activitycycleofspecies.The tory sites(Nakaietal.,2014). egress ofpathogenicTcellsandtheirrecruitmenttoinflamma ameliorates thediseases,whichisaccompaniedbyreducedLN pendent differences inthemagnitudeofadaptive immunere of adaptiveimmune responses (Suzuki et al., 2016).The time-de control of lymphocyte trafficking contributes to the daily rhythm rosis andallergicdermatitis,weshowedthatactivationofβ murine modelsofinflammatorydiseases,includingmultiplescle and consequentlyinhibitstheirLNegress(Fig.1).Additionally,in chemokine receptors that promote LNretentionoflymphocytes, LNs wasstopped.Thesefindings suggest thattheβ nals and was diminishedwhen lymphocyte recirculation through The activityofadrenergicnervesdisplaysacircadianrhythm Kazuhiro Suzuki,MD/PhD Support Staff Support Research Assistant Assistant Professor Professor 2 ARs enhancestheresponsivenessofCCR7andCXCR4, 2 ARs. Activationoflympho 2 2 Nakai Akiko Kazuhiro Suzuki 2 AR-mediated neuralsig- 2 AR-mediated 2 ARs ------● ● ● solved. First,themolecularmechanismofcrosstalkbetween by thenervoussystem.However,somequestionsremainun cur (Fig.2). defense whenencounterswithpathogensaremorelikelytooc sponses may have evolved to maximize the efficiency of host Recent Publications lymph nodes. J. Exp. Med. 213,2567-2574 (2016). adaptive immuneresponse recirculation by lymphocyte diurnal through Hayano K, Suzuki Y, A,Furuta Nakai FandNodaM.Adrenergic control ofthe by adrenergicmunity Clin.Exp. nerves. Neuroimmunol. 8,15-22(2017). A.Control andadaptive im- trafficking KandNakai oflymphocyte Suzuki waves. J. Allergy Clin.Immunol. (2018) in press. modulation by shock A. Immune neuronal electric K and Nakai Suzuki Our findingshaveaddedanovellayerofimmuneregulation Figure 1.ControloflymphocyteegressfromLNsthroughβ Activation oflymphocyteβ retention inLNs,andinhibitslymphocyteegressfromLNs. responses. during theperiodoflymphocyteaccumulationinLNsenhancesadaptive immune restricted, whichleadstoanincreaseoflymphocytenumbersinLNs.Immunization During theperiodofhighadrenergicnerveactivity,lymphocyteegressfrom LNsis Figure 2.Diurnalcontrolofadaptiveimmunitybyadrenergicnerves. 2 ARs enhancessignalsthroughCCR7andCXCR4,chemokinereceptorsthatpromotelymphocyte - - 2 ARs. β and influence their behaviors inLNs. Third,theeffectof β be visualizedhowadrenergicnervesinteractwithlymphocytes gic controlofadaptiveimmuneresponses. questions infuturestudiestorevealthewholepictureofadrener activity, is poorly understood. We are going to address these vation onotherlymphocytefunctions,includingtheirmetabolic ● ● 2 ARs andchemokinereceptorsisunclear.Second,itremainsto 2583-2598 (2014). egress from lymphnodesthrough β A,Hayano Nakai Y, Furuta F, Control K. oflymphocyte Noda M.andSuzuki immunol. 7,10-17(2016). A.Autonomic KandNakai controlSuzuki ofinflammation. Clin.Exp. Neuro 2 -adrenergic receptors. J. Exp. Med. 211, 2 AR acti- - - 57 Imaging 58 Protein-nucleotide interactions Functional analysisofBCRandTCRrepertoires Systems Immunology et al.2017). sociated withacutesystemiclupus erythematosus(Sakakibara,S. proach tothesuccessfulmodeling ofanti-nuclearantibodiesas challenge ofantigenbinding.For BCRs,wehaveappliedthisap with anaverageerrorofunder2Å.Wearecurrentlypursuingthe cal run,10,000sequencescanbemodeledinunder30minutes is thefastestandmostaccuratetoolavailable(Figure1).Inatypi (Schritt, D.andLi,S.etal.).Toourknowledge,RepertoireBuilder structure from sequence accurately in ahigh-throughputmanner web-based tool(RepertoireBuilder)formodelingBCRandTCR diction ofBCRorTCRstructurefromsequence.Wedevelopeda as identificationofthetargetantigen.In2017,wefocusedonpre methods. wet immunologygroupsinordertofine-tuneourbioinformatics and 3Dstructuralmodeling.Wealsocollaborateintensivelywith ing methodsformultiplesequencealignment,machinelearning this end,wedevelopbioinformaticsmethodsinourlab,includ bution tobasicandappliedimmunologyresearchatIFReC.To Both ofthesethemeshavethepotentialtomakeastrongcontri ceptor (TCR)repertoiresandprotein-nucleotideinteractions. themes: functionalanalysisofBcellreceptor(BCR)andTre In the second research direction, we have improved a web- Functional analysisofBCRandTCRrepertoirescanbedefined In 2017,ourlabcontinuedtopursuetwomajorresearch ------Structural modeling Multiple sequencealignment specificity. teract withRegnase-1inordertounderstandthebasisfortarget RNA-binding proteins,withemphasisonthoseproteinsthatin (Figure 2).WearecurrentlyapplyingaaRNAtoalargerrangeof et al)andfortheanti-nuclearantibodyprojectmentionedabove peat interactions(Nyati,K.K.etal.2017),APOBEC3G-RNA(Izumi et al.2016),Arid5a-OX40(HHanieh2017),AUF1-AU-richre docking hasbeenvalidatedinthecaseofRegnase-1(Yokogawa ing thenucleotidesontoproteins.Suchflexiblenucleotide Li, S.etal.2014)andhaveextendedthistooltoDNAfordock based toolforpredictingRNAbindingsitesonproteins(aaRNA; al. 2017). sis oftheautophagy-related8(Atg8) proteinfamily(Sasai,M.et K. etal.2017).Wealsocarriedout sequenceandstructuralanaly added toouronlineserviceinresponse touserdemands(Katoh, nificantly (Nakamuraetal,2018).Othernewfeatureshavebeen gramming techniques,thecalculationspeedwasimprovedsig was impracticalforlarge-scaledatasets.Byusingparallelpro this method was well demonstrated in previous benchmarks but tended tolarge-scalesequencedata(Figure3).Theaccuracyof ample, a MAFFT option for high-quality alignment has been ex- of theMAFFTmultiplesequencealignmentsoftware.Asoneex We supportedstructural modelingforseveralwet labsin2017. We arecontinuouslyimprovingtheperformanceandusability Daron MStandley,PhD Support Staff Support Research Assistant Postdoctoral Fellow Assistant Professor Associate Professor Professor 2 2 2 Floris VanEerden Li Songling Shunsuke Teraguchi Katoh Kazutaka Daron MStandley ------● ● ● Rag GTPasecomplex(RYonehara,etal.2017). the complexandnovelstructuralarrangementofRagulator- inhibitors ofHCV(Hirano,J.etal2017).Wealsohelpedtomodel Using both 3Dmodeling and molecular docking we modeled SPP bining models.B,X-raycrystallographyconfirmedmodel1. A, Dockingbeforex-raystructuredeterminedrevealedtwopossibleDNA Figure 2.ssDNAdockedtoAnti-nuclearantibodymodel. A. Docking Recent Publications (2017). clear antibodies inacute systemic Rep. Sci. lupus erythematosus. 7,16428 S.etal. Clonalevolution andantigen recognitionSakakibara ofanti-nu (2017). on-inducible GTPase-mediated hostdefense. Nat. Immunol.18,899-910 M.etal.Sasai Essential role for autophagy GABARAP proteins ininterfer GTPase complex. Nat. commun. 8,1625(2017). Yonehara R.etal. basisfor Structural theassemblyofRagulator-Rag reve Mode 2 aled twopossiblebindingmodes Flexible DNADocking Mode 1 BCR BCR T T Significantly higherror (compar CR CR indicates statisticalsignificance. TCR CDRalpha-3 regionsforRepertoire Builderandthreepopular methods.Thebackground color The tableshowsroot-meansquaredeviationsofBCRmodels,CDRH3regions,TCRand Figure 1.RepertoireBuilderBCRandTCRmodelssignificantlymoreaccuratethanother methods. B3 H3 Error (Root-mean square deviation)of B. Mode1confirmedbyX-r eetieBuilder Repertoire 1.89 4.20 2.04 3.17 ay cry ed to sta bd ule isPro Pigs Builder Abody 1.99 4.67 NA NA llograph Re pertoire Builder): - - y more accuratelythanalternativemethods. Over a wide-range of data sizes, new options of MAFFT consistently perform Figure 3.ImprovementsinMAFFT. ● ● (2017). andprotozoa.of HCV Proc. Natl. Acad. USA.114,E10782-E10791 Sci. J.Hirano etal. ofSPPinhibitors Characterization suppressing propagation (2017). IL-6 mRNAstabilizing protein Nucl. Arid5a. Acids Res. 45, 2687-2703 et al. KK. Nyati and MAPK signalingTLR4-induced regulate NF-kappaB the

2.06 4.76 NA NA TC Alignment Accur -log(p) >5 R andBCRmodels 2.06 4.94 2.32 Lyra 3.75 -log(p) >10 ac y 59 Informatics 60 Quantitative approaches Quantitative Immunology data. derstand interferonregulationfrom suchtimelapseimaging computational algorithmstoextract importantquantitiestoun upon antiviral responses. We are also developing automated fluorescent proteinknock-incells tomonitorgenesinduced feron expression under microscope is combined with multiple ways: timeandperturbation.TimelapseimagingoftypeIinter are tryingtoincreasethe“dimension”ofobservationintwo quantitative monitoringofIFN-a6(Kumagaietal.2007).Nowwe have developedafluorescentproteinreportersystemforthe understanding thedynamicsofcomplexsystems.Previously,we low tohighlightspecifictopics. to theimmunesystem.Someoftheseprojectsaredescribedbe eral projectsthataimtogetinsightintospecificquestionsrelated these massivedatasets.Theseapproachesarecombinedinsev- derstand theimmunesystem’sdynamicsthroughanalysisof models, and (3) development of mathematical frameworks to un- integration of“bigdata”frommultiplesourcesintonetwork molecular dynamicsatsinglecellandmoleculelevel,(2) interconnected approaches;(1)quantitativemeasurementof immune systemworksbycombiningthreedifferentbutclosely munology andbioinformatics.Ouraimistounderstandhowthe searchers withexpertiseindifferentscientificfieldsincludingim Receptor molecule dynamicssuchasdimerization andcluster Accurate quantificationofbiologicalresponsesiscriticalfor The QuantitativeImmunologyResearchUnitisateamofre------ways. describe thedynamicsofimmunesystem’ssignalingpath agai, in press). Thishighlyquantitative technique can be used to under highmoleculardensityconditions(TeraguchiandKum tify thediffusiondynamicsofsinglemoleculeswithoutbias,even level (Satoetal,2017).Wedevelopedanovelalgorithmtoquan fully monitoredTLRsandtheiradaptorsatthesinglemolecular We areusingthistechniquetostudyTLRsignaling,andsuccess (TIRFM) tomonitorthedynamicsofsingleimmunemolecules. ratories inIFReC,TotalInternalReflectionFluorescentMicroscopy we areapplying,incollaborationwithRIKENQBiCandotherlabo- for mostimmunesignalingpathways.Toaddressthisproblem, resolution, thedetailsofthisprocessarestillpoorlyunderstood signaling. However,becauseof limitations in biochemical assay ing withdownstreammoleculesisimportantforimmunesystem Data integration chronic obstructive pulmonarydisease(COPD)and silicosis(Diez to study the mechanisms behind respiratory diseases, such as and generegulatoryimmunenetworks. Weapplythesemethods tion datafrompublicdatabases, to obtaininsightintosignaling ent experimentalconditions,and withprotein-proteininterac transcription factor binding with transcriptome data from differ - tion betweenbiologicalcomponents. summarize andextractrelevantinformationthatrevealstherela into the big data era and the need for approaches that integrate, We aredevelopingmethodsthat integratemeasurementsof High-throughput (“omics”)technologieshavebroughtbiology Support Staff Support Research Assistant Assistant Professor Associate Professor 2 1 Yutaro Kumagai Diez Diego ------validated. trolling RNAdegradationduringtheimmuneresponseisbeing 2016). Wealsoidentifiednovelmotifsforwhichtheirroleincon the RNA-bindingproteinsRoquinandRegnase-1(Kumagaietal. including thestem-loopstructurecontainingmotifsboundby quences of each cluster. We identified known structural motifs, and structuremotifidentificationisappliedtothe3’UTRse terns withsimilardegradationkinetics.Then,denovosequence degradation kinetics by clusteringthe time course data into pat after LPSstimulation.Transcriptomicdataisusedtodetermine rithm with RNA degradation time course in mouse dendritic cells network dynamicsofimmunesignaling. dimensional data.Nowweareapplyingthissystemtoinferthe parameters ofthenetworkregulatorypathwaysfrommulti- developed asystemtoautomaticallyidentifythestructureand neous natureofcellpopulations(Teraguchietal.2011).Wehave which alsoallowsustorepresentthestochasticandheteroge matical frameworkcalledStochasticBinaryModeling(SBM), eters. Tocircumventthisproblem,wehavedevelopedamathe- lular signalingisthebiochemicaldeterminationofmanyparam ● ● ● Mathematical modeling flammation. study theregulatorypathwaysassociatedwithirreversiblein ments oftranscriptionalactivityinamousemodelsilicosis,we ca forsilicosis).CombiningATAC-seqandRNA-seqmeasure exposure totheharmfulcomponents(tobaccoforCOPDandsili- tion anddiseaseprogressionareirreversibleevenafterremoving et al.2015).Acommonfeatureofthesediseasesisthatinflamma description oftheimmunesystem. developing novelmathematicalframeworksforthequantitative immunology researchfromthetheoreticalperspective.Weare the integration of massive data open the door to approach the Recent Publications 016-3325-7 (2016). motifsstructure in mRNAs. 17, 1032, doi:10.1186/s12864- BMC Genomics sequence andsecondary stimulation facilitates identification ofprimary patternsmap ofRNAdegradation cells LPS indendritic after kinetics Kumagai Y, Vandenbon A, Teraguchi S&Suzuki S,Akira Y. Genome-wide J.Dynamics. Am. Chem.Soc. 139(48),17397-17404(2017). Receptor-Adaptor ofImmune Imaging Molecular color Single-Molecule Intracellular Protein-Labeling K. S&Kikuchi ProbesMizukami for Multi J,R,KumagaiSato R,Kozuka UedaM,Mishima Y, Yoshimura M, A,Minoshima (Suppl. 1),15(2018). molecular measurement withoutexplicit tracking. BMCSyst. Biol. 12 Teraguchi S&Kumagai Y. Estimation ofdiffusion constants from single We areintegratinganoveldenovoRNAmotifsearchalgo A commonobstacleforconstructingdynamicalmodelsofcel The accuratequantificationofimmunologicalresponsesand ------to severalimmunedatasets. regulators asaproxyfortheiractivitiesweapplythesemethods ferent typesofrealnetworks.Usingtheexpressionlevel of theseassumptionsusingsimulatednetworksresemblingdif expression level of their target genes. We validate the feasibility tors. Furthermore,regulatorscontributeinanadditivewaytothe of eachgenedependsontheactivityasmallnumberregula linear regressionmethods.Weassumethattheexpressionlevel sion leveloftheirtargetgenes.Wemodeltheseprocessesusing regulators, includingtranscriptionfactors,determinetheexpres nants ofcellidentityandfunction.Inthisprocessthousands ● ● Care Med. 190,981-988,doi:10.1164/rccm.201403-0421PP (2014). chronic respiratory diseases. From basics to application. Am. J. Respir. Crit. Diez D, Agusti A& Wheelock CE. analysis Network intheinvestigation of 5,7989,doi:10.1038/srep07989entific (2015). reports synaptic pruningby complement up-regulating inmicroglia.- factors Sci JH,DiezD&Hanayama I,Song Bahrini R.Neuronal exosomes facilitate Transcriptional regulatorynetworksareimportantdetermi- quantitative, integrativeandtheoreticalapproaches. The QuantitativeImmunologyResearchUnitcombines Figure. - - - 61 Units for Combined Research Fields Events & Outreach Activities The 7th NIF Winter School on Advanced Immunology

Lecturer Title

Jinmiao Chen Constructing Cell Lineages From Single-Cell Transcriptomes (SIgN, Singapore)

James Di Santo Activities & Outreach Events Developmental Options and Functional Plasticity of Human Innate Lymphoid Cells (Institut Pasteur, France)

Markus Feuerer (The University of Regensburg / German Cancer Research Specialization of regulatory T cells in tissues Center, Germany)

Florent Ginhoux Human Dendritic Cells: From Development to Functions (SIgN, Singapore)

Kenya HONDA Modulation of the immune system by the gut microbiota (Keio University, Japan)

Takashi Nagasawa Microenvironmental niches for hematopoietic stem cells and immune cells (IFReC, Osaka University, Japan)

Lai Guan Ng Watching The Great Leukocyte Migration (SIgN, Singapore)

Yukinori Okada Statistical genetics, disease biology, and drug discovery (IFReC, Osaka University, Japan)

Hai Qi T-B cell interactions and the germinal center reaction (Tsinghua University, China) The 7th NIF Winter School on Advanced Immunology was held with Singapore Immunology Network (SIgN) at The Awaji Yumebutai International Conference Center in Japan on 21-26 Jan- Laurent Rénia Immune-mediated Pathologies During Malaria Infection uary 2018. The program comprised a four-day lecture course at Awaji Island and a one-day in- (SIgN, Singapore) ternational symposium at Suita Campus of Osaka University. Fifty-four young researchers from Brigitta Stockinger around the world, selected from 213 applicants, learned cutting-edge immunology from 15 Environmental influences on intestinal homeostasis and inflammation world-renowned senior researchers at the course and three up-and-coming researchers at the (The Francis Crick Institute, UK) symposium. Three young researchers from companies collaborating with IFReC also attended Georg Stoecklin Translation regulation during macrophage activation and joined in the discussions. The Winter School was highly evaluated by the participants and (Heidelberg University, Germany) the lecturers as an excellent opportunity for networking with young peers through active dis- cussions and communications as well as its high scientific level. Hiroshi Takayanagi Osteoimmunology and autoimmunity (The University of Tokyo, Japan)

Henrique Veiga-Femandes ■ Date : January 21- 26, 2018 Innate Lymphoid Cells and the Second Brain (Champalimaud Center for the Unknown, Portugal) ■ Venue : Awaji Yumebutai International Conference Center, Hyogo Icho-Kaikan, Suita Campus, Osaka University, (The 9th International Symposium of IFReC) Toshihide Yamashita The spatiotemporal dynamics in the biological systems regulate the neuronal (IFReC, Osaka University, Japan) network in the central nervous system

64 65 The 9th International Symposium of IFReC - Immunology at the Forefront

Speaker Title

Opening remarks by Director Shizuo Akira Events & Outreach Activities & Outreach Events Takashi Shichita The resolution of sterile inflammation after ischemic stroke (Tokyo Metropolitan Institute of Medical Science, Japan)

Daniel Mucida Tissue adaptation: implications for gut tolerance and immunity (Rockefeller University, USA)

Etsushi Kuroda (Osaka University/ National Institutes of Biomedical Innova- Mechanisms of allergic inflammation induced by inhaled fine particles tion, Health and Nutrition, Japan)

Michelle Linterman CXCL13 recruits B cells to the lung and initiates tertiary germinal centre formation (University of Cambridge, UK)

Thomas Korn Modes and outcome of IL-6 signaling into T cells (Technische Universität München, Germany)

Naganari Ohkura Significance of the regulatory T cell-specific epigenetics in autoimmune disease (Osaka University, Japan) susceptibility

Takao Hashiguchi Molecular dissection of virus entry and its inhibition by antibodies/inhibitors (Kyushu University, Japan)

Cevayir Coban Host-Plasmodium interactions at tissue level (Osaka University, Japan)

Closing Remarks by Deputy Director Kiyoshi Takeda

IFReC invited many talented scientists who are future leaders in various research fields as speakers. It was an excellent opportunity to share ideas and expertise in order to con- tribute to further development in Immunology.

■ Date : January 26, 2018 ■ Venue : Icho kaikan, Suita campus, Osaka University

66 67 IFReC Seminars

2017 Special Lecture

Jul. 28 Osamu Nagayama CEO, Chairman of Chugai Pharmaceutical Co., Ltd. Events & Outreach Activities & Outreach Events

Date Speaker Affiliation Title

Lost in translation: of the importance of protein Sep. 4 Christophe J. Desmet University of Liège / FRS-FNRS, Belgium translation for normal function of hematopoietic stem and progenitor cells

Sep. 4 Andrea Reboldi University of Massachusetts, USA Mechanisms controlling intestinal IgA production

Meirav Pevsner Sep. 4 Weizmann Institute of Science, Israel Host-microbiome interactions in health and disease -Fischer

Regulatory B cells in health: what goes wrong in Oct. 3 Claudia Mauri University College London, UK patients with autoimmunity?

University of Melbourne/Walter and Eliza Hall Molecular regulation of tissue-resident regulatory T Oct. 20 Axel Kallies Institute of Medical Research, Australia cell development

Fine tuning IL-2: superenhancers, STAT5 tetrameriza- Oct. 26 Warren J. Leonard NHLBI, NIH, USA tion, and partial agonists

Mechanisms underlying the fine-tuning of CD4+ IFReC holds seminars throughout the year with speakers from a variety of disciplines includ- Nov. 7 Hidehiro Yamane NIAID, NIH, USA T cell positioning during T follicular helper cell dif- ing immunology, imaging and informatics with the aim of promoting collaborative research, ferentiation as well as to inspire and educate the next generation of scientists. Since its establishment, IFReC has held more than a hundred seminars, which have served as a forum for effective interaction between researchers beyond national borders and academic disciplines. This program has certainly contributed to IFReC’s mission of promoting interna- tionalization and interdisciplinary research. On July 20, 2017, a seminar by Mr. Osamu Nagayama (CEO and Chairman of Chugai Phar- maceutical Co.) was held in commemoration of the Comprehensive Collaboration Agreement between Osaka University and Chugai Pharmaceutical Co., Ltd.

68 69 IFReC Colloquia Speaker Title Etsushi Kuroda Cyclic GMP-AMP exacerbates asthma via IL-33 (Vaccine Science)

Toshihide Yamashita The spatiotemporal dynamics in the biological systems regulate the (Molecular Neuroscience) neuronal network in the central nervous system

Shogo Tanabe B-1 cells promote oligodendrogenesis during brain development (Molecular Neuroscience) 30th

Hiroshi Tsujioka Interleukin-11 induces and maintains progenitor cells of various tissues Activities & Outreach Events (Molecular Neuroscience) during tail regeneration of Xenopus laevis tadpoles

Yukinori Okada Statistical genetics, disease biology, and drug discovery (Statistical Immunology)

Sayuri Gyobu Mechanism and physiological role of phospholipid scrambling by (Biochemistry and Immunology) TMEM16 family

Keiichi Namba Recent progress of electron cryomicroscopy for structural analysis of (Graduate School of Frontier Biosciences / Riken QBiC) protein assemblies at atomic level

Sho Yamasaki Immune recognition of pathogens and damaged-self via ITAM receptors (Molecular Immunology) 31st Songling Li Quantifying structural and functional convergence in B cell receptor (Systems Immunology) repertoires

Takashi Nagasawa Microenvironmental niches for hematopoietic stem cells and immune (Stem Cell Biology and Developmental Immunology) cells

Takashi Saito Regulation of T cell activation and function by innate signals-STING (Cell Signaling) activation induces growth arrest and IFN production in T cells-

Yuichiro Hori Development of Chemical Tool for Imaging DNA Methylation (Chemical Imaging Techniques) 32nd Naoki Hosen The activated conformation of integrin β7 as a target for multiple (Immunopathology) myeloma–specific chimeric antigen receptor T-cell therapy

Wataru Ise Selection of germinal center B cells into the plasma cell compartment IFReC colloquia are a series of discussion meetings for IFReC members held once every other month since FY2011. At (Lymphocyte Differentiation) each colloquium, three speakers from IFReC laboratories give talks about their latest research progress followed by in- Szandor Simmons High-endothelial cell-derived sphingosine-1-phosphate regulates tensive discussion. After the colloquium, a small social gathering is held to further the discussions and encourage the ex- (Immunology and Cell Biology) dendritic cell localization and vascular integrity in the lymph node change among IFReC members in an informal setting. These events serve as a platform to promote fusion researches and Yoshiko Murakami Autoinflammation-Paroxysmal nocturnal hemoglobinuria (PNH) deepen understanding of researches conducted in IFReC. 33 rd (Immunoglycobiology) syndrome

Eiji Umemoto Identification of bacteria-induced intestinal metabolites that influence th ■ Date : 30 Colloquium: April 19, 2017 (Mucosal Immunology) immune responses st 31 Colloquium: June 21, 2017 Hisashi Kanemaru The antitumor effect of BATF2 through IL-12 p40 up-regulation in nd 32 Colloquium: August 30, 2017 (Host Defense) tumor-associated macrophages 33rd Colloquium: October 11, 2017 Patrick Michael Lelliott th 34 th A novel technique to quantify extracellular trap release 34 Colloquium: December 27, 2017 (Malaria Immunology) 35th Colloquium: February 21, 2018 Kazuki Kishida ■ Venue : Taniguchi Memorial Hall, Osaka University Antigen-specific immune regulation mediated by TCR-like antibodies (Immunochemistry)

Nicolas Pavillon Label-free monitoring of activation and inhibition of macrophage (Biophotonics) responses

Jisu Ma A family member of phospholipase C is specifically involved in CD8 (Immunoparasitology) T cell-mediated immune responses 35 th Diego Diez Inferring gene regulatory networks from omics data (Quantitative Immunology Research Unit)

Akiko Nakai The COMMD3/8 complex dictates the specificity of GRK recruitment to (Immune Response Dynamics) chemoattractant receptors

70 71 Science Café Super Science High School Student Fair

The series of science Cafés is a long-term IFReC outreach ac- the two science cafés organized by IFReC in FY2017, a total of tivity to promote communication between researchers and the about 150 participants enjoyed novel topics in immunology in Super Science High Schools (SSH) are selected high schools by MEXT in Japan, which general public. It also enhances people’s understanding of im- a relaxing atmosphere. promote advanced math/ science education as well as collaborative researches with uni- munology researches and the researchers involved in them. In versities, and activities to develop international perspectives. In the SSH Student Fair 2017 held in Kobe, more than 200 schools, including several schools from overseas, held booths with posters to present their researches. WPI insti-

tutes held a collaborative booth and introduced the research activities of each institute Activities & Outreach Events using posters, booklets and demonstrations. “Science Café on the Edge” at 2017 Icho Festival

■ Date : August 9-10, 2017 ■ Speaker : Takashi Satoh (Assistant Professor, Host Defense, IFReC/RIMD) ■ Venue : Kobe International Exhibition Hall ■ Date : April 30, 2017 ■ Host : MEXT, JST ■ Venue : Techno Alliance Hall, Suita Campus, Osaka University ■ Support : Boards of Education (Kobe prefecture and Kobe city)

Students Visit and Facility tour

“Science Café on the Edge” as part of “Lab Café” During the summer vacation, IFReC welcomed 30 SSH students from Nara Prefecture. They heard a lecture by Dr. Yutaro by Osaka University Kumagai (Quantitative Immunology Research Unit), and visited Akira Lab and the Core Instrumental Facility to try some experiments and to talk with researchers. We also welcomed 55 junior high-school students and their parents. They also enjoyed the experiments and a tour of ■ Speaker : Jun Sakanoue (Associate Professor, RPMO, IFReC) the RIMD History Museum. ■ Date : March 9, 2018 ■ Venue : Art Area B1 at Naniwabashi station (Keihan Electric Railway Co.) ■ Date : August 3, 2017 (Junior High School) August 11, 2017 (High School)

72 73 WPI Science Symposium AAAS 2018 Annual Meeting Events & Outreach Activities & Outreach Events

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

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

The American Association for the Advancement of Science (AAAS) is the biggest international scientific society in the world and its mission is to “advance science and serve society”. The AAAS 2018 Annual Meeting was held in Austin, and it offered a broad range of activities including lectures, symposia, seminars and exhibits, with the theme of "Serving soci- ety through science policy ". WPI institutes held a collaborative booth to introduce the WPI program and the institutes’ activities using posters, booklets and a demonstration experiment. More than 400 participants visited the booth and gained interest in the WPI program and world leading researches in Japan.

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

74 75 Naturejobs Career Expo

Research Projects

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

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

76 Advanced Postdoc System Young Scientist Support Program for Research Abroad

For IFReC, fostering the education of young researchers is a IFReC has, therefore, established the Advanced Postdoc To strengthen our international research network and our ba- in international collaborative research and to develop their net- responsibility as a global research institution. It is also vital for system this fiscal year. It offers outstanding young researchers sis for international collaborative research, IFReC has provided work with researchers overseas. 4 researchers used this support

the institution to continually incorporate the original ideas of in the fields of immunology and cell biology opportunities to financial support to young researchers who wish to participate program in FY2017. Since the start of this program, IFReC has Projects Research young researchers and to promote international brain circula- work with field-leading researchers in IFReC as well as to con- in research activities at overseas institutions. The program aims provided support for 53 visits overseas by young researchers. tion in order to further develop IFReC’s research. Around the duct their own research and publish under their own merit. to develop the practical skills and abilities of young researchers world, leading research institutions are in fierce competition to IFReC has selected three excellent postdocs for three years discover excellent young researchers. In order to employ young under this system out of 78 applicants. They were assigned to researchers who are expected to be internationally active at IF- laboratories in IFReC with an international standard level salary Young Scientist Support Program for Research Abroad ReC and to strengthen IFReC’s function as a hub of internation- and research funds (3 million yen per year) to conduct original al brain circulation, it is necessary to improve the conditions for research. Name Country Conference Attended researchers in Japan, to an international standard. Rouaa Beshr USA Society of Nuclear Medicine and Molecular Imaging (SNMMI)

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

LIM Ee Lyn Switzerland World Immune Regulation Meeting XII

Taba Fargol France 17th Annual Congress of the European Society for Photodynamic Therapy

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

EU 15 America 15

Asia 2 Africa 1 Australia 3

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

78 79 Data Common Facilities Kishimoto Foundation Fellowship

IFReC and its parent institution, the Research Institute for RIMD researchers. The Animal Resource Center consists of IFReC launched the Kishimoto Foundation Fellowship pro- airfare to Japan. Microbial Diseases (RIMD) are located on the same site, con- three buildings for specific pathogen-free (SPF) animals and gram for researchers in various fields of immunology in 2010. The Kishimoto Foundation was established in 2008 in honor Data stituting a large research complex. The complex contains the the live immuno-imaging facility. With a large-capacity animal- The program is supported by the Kishimoto Foundation and of Tadamitsu Kishimoto, who, during the 1980s and 90s, elu- Core Instrumentation Facility, the Animal Resource Center breeding facility in IFReC, researchers are able to choose animal designed to support overseas researchers in order to promote cidated the function of interleukin-6 (IL-6), a key molecule for and the Network Administration Office, all of which are jointly rooms suitable for their experiment purpose. and develop immunological research and international ex- stimulating immune responses. He later developed the anti-IL6 operated by IFReC and RIMD. The Core Instrumentation Facil- Using these common facilities, IFReC researchers are able to changes at IFReC. The fellowships are open to international receptor-based therapy, tocilizumab, to treat immune disorders ity is equipped with various highly advanced instruments and effectively and smoothly carry out their experiments to pro- postdoctoral researchers who seek to collaborate with IFReC such as Castleman's disease or rheumatoid arthritis. skilled technicians provide in-house services to IFReC and mote their world-leading research at IFReC. researchers. The recipients are provided with a salary and an

IFReC–RIMD Research Complex at Suita Campus of Osaka University FY2017 Kishimoto Fellowship Recipients Position of Recipient Name (initials) Nationality Host researcher Period

Specially Appointed Researcher D. H. Australia Sakaguchi Oct. 16, 2015 - Oct 15, 2018

1 Specially Appointed Researcher J. P. Vietnam Kishimoto Apr. 1, 2016 - Mar. 31, 2019 6 2

Quantitative Specially Appointed Researcher N. T. Slovenia Immunology Oct. 1, 2016 - Jul. 15, 2017 3 Research Unit 5

Specially Appointed Researcher H. J. China Arase May 1, 2017 - Apr. 30, 2018

4 Visiting Researcher H. H. Jordan Kishimoto Jun. 26, 2017 - Aug. 18, 2017

Visiting Researcher P. K . Turkey Coban Mar. 1, 2018 - May 28, 2018

Photo : S. Higashiyama 1. IFReC Research Building Visiting Researcher E. E. Turkey Coban Mar. 1, 2018 - May 28, 2018 2. Integrated Life Science Building 3. Main Building, Research Institute for Microbial Diseases, RIMD Visiting Researcher N. N. Vietnam Kishimoto Mar. 10, 2018 - Mar. 22, 2018 4. South Building, Research Institute for Microbial Diseases, RIMD 5. Cutting-edge Research Building for Infectious Diseases Visiting Researcher D. L. Vietnam Kishimoto Mar. 10, 2018 - May 8, 2018 6. Animal Resource Center for Infectious Diseases

Animal Resource Center for Infectious Diseases Network Administration Office

・ Specific pathogen-free (SPF) animal facility ・ Provision and maintenance of network infrastructure:

・ Sperm/ embryo freezing and preservation LAN system and servers (web, mail, mailing lists, etc.)

・ In vitro fertilization and embryo transplantation ・ Intracytoplasmic sperm injection Core Instrumentation Facility ・ Transgenic and knock-out animals ・ Basic and advanced instruments ・ Genome editing in experimental animals ・ In-house service

・ DNA sequencing, cell sorting, electron microscopy, mass Live Immuno-Imaging Facility spectrometry and next-generation DNA sequencing analysis

・ SPF animal experiment facility with 11.7T MRI, in-vivo imager ・ Radio isotope facility & two-photon microscope.

82 83 Major Awards Data Shimon Sakaguchi Person of Cultural Merit of Japan Cevayir Coban JSI Women Immunologist Award

The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Ja- Cevayir Coban (PI, Malaria Immunology) was awarded the 4th Japanese Society for Immunology (JSI) Prize for Women Immunolo- pan announced that Prof. Shimon Sakaguchi, Deputy Director of IFReC was named gists. The awarded title is "Revealing the mechanism of host-Plasmodium parasite interactions". a Person of Cultural Merit. MEXT commented that Prof. Sakaguchi was awarded for his outstanding achievements in studies of Regulatory T cell (Treg); discovering and Taroh Kinoshita Takeda Prize for Medical Science, JSI Human Immunology Research Award clarifying the roles of Treg in autoimmune diseases and allergies. This is the third time for the scientist of IFReC to be honored as a Person of Cultural Merit, others include This prize is given to scientists who have made remarkable contributions in the field of medical science. Kinoshita was honored for Shizuo Akira (Director of IFReC) in 2009 and Toshio Yanagida in 2013. research on GPI-anchored proteins and mechanisms of its deficiencies.

Four IFReC researchers Highly Cited Researchers 2017

Highly Cited Researchers are researchers with papers that have a large number of citations from all over the world as selected by ©Clarivate Analytics. They are thought to be leading the way in solving the world’s biggest challenges. In 2017, some 3,300 research- ers from 21 fields were selected as Highly Cited Researchers. Eight researchers in three fields were selected from Osaka University, and four of them are from IFReC. Cevayir Coban (L) and Taroh Kinoshita (R) at the venue of the commemorative lectures

Michelle Sue Jann Lee JSPS Ikushi Prize

IFReC Assistant Professor Michelle Sue Jann Lee was awarded the 8th JSPS • Shizuo Akira • Shimon Sakaguchi • Kiyoshi Takeda • Masahiro Yamamoto Ikushi Prize. The 18 prize winners including Dr. Lee were unequivocally recog- Director of IFReC Deputy Director of IFReC Deputy Director of IFReC Immunoparasitology, IFReC nized for their excellent results in each research area. The award ceremony was held at the headquarters of Japan Academy (Gakushi-in) in the presence of their Imperial Highnesses Prince and Princess Akishino.

Satoh and Segawa The Young Scientists' Prize by MEXT

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

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

84 85 Composition Finance Data

Breakdown of total expenditure at IFReC in FY2017 Number of IFReC Staff

350

Administrative Sta 300 Research Support Sta 28 Researchers Individual Research 28 24 Project 250 Personnel 29 28 30.1% 25 74 89 30.3% 82 28 200 63 28 45 62 Total Expenditure 21 77 3,072,273,597 150 31 74 (JPY) 15 173 183 186 100 23 171 Project 135 168 179 Activities 13 127 115 Equipment 3 50 89 23.3% 16.0% 52 0 Mar.2008 Mar.2009 Mar.2010 Mar.2011 Mar.2012 Mar.2013 Mar.2014Mar.2015 Mar.2016 Mar.2017 Mar.2018 Travel 0.2%

Number of Researchers

200 26% Travel From the 30% 23% Equipment Grant-in-Aid for University From Overseas 31% 0.4% Scientific Research 32% 26% Japanese 18.8% 13.7% 48 20.4% 55 42 150 31% 54 54 44 Comprehensive 31% Other Sources collaboration Personnel 29% agreement Grant Management 2,102,806,447 Expenses Grant 42 969,467,150 49.0% Contract 40 Research (JPY) 26.7% 100 33 (JPY) 27% Project 22.3% Activities 24 138 137 128 124 31.7% 117 119 Donation 15% 50 8 93 87 82 16.8% 65 44

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

86 87 Selected Articles Data

BATF2 inhibits immunopathological Th17 responses by suppressing Il23a expression during Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host Trypanosoma cruzi infection. defense. J. Exp Med. 214(5):1313-1331, 2017. doi: 10.1084/jem.20161076. Nature Immunology. 18(8):899-910, 2017. doi: 10.1038/ni.3767.

Kitada S, Kayama H, Okuzaki D. et al. Sasai M, Sakaguchi N, Ma JS. et al.

Kiyoshi Takeda's group and others have revealed the regula- Masahiro Yamamoto's group and others revealed evealed a tion of immune response after Chagas disease causing Try- unique role of GABARAPs, in particular Gate-16, in interferon-γ panosoma cruzi (T. cruzi) infection. (IFN-γ)-mediated antimicrobial response. The group showed T. cruzi infection induces IL-23 production by host immune that Gate-16 specifically associates with Arf1 to mediate uniform cells, and antigen-specific Th17 responses are then promoted. distribution of IFN-inducible GTPases, and that lack of Gate-16 However, whether the IL-23-Th17 axis is controlled by IFN- reduces Arf1 activation, leading to formation of IFN-inducible γ-dependent mechanisms during T. cruzi infection remains GTPase-containing aggregates, hampering their function. Fur- unclear. In this study, they have demonstrated that IFN-γ- thermore, mice lacking Gate-16 alone are susceptible to Toxo- inducible BATF2 exerts its immunoregulatory function through plasma. Thus, GABARAPs are uniquely required for antimicrobial the suppression of Il23a by interacting directly with c-JUN in host defense through cytosolic distribution of IFN-inducible innate immune cells, thereby preventing immunopathological GTPases. Th17 responses during T. cruzi infection. These results will help Gate-16 targeted strategy for the treat- ment of toxoplasmosis and alimentary intoxication caused by Toxoplasma and Salmonella.

Plasmodium products persist in the bone marrow and promote chronic bone loss The activated conformation of integrin β7 is a novel multiple myeloma-specific target for CAR T Science Immunology. 2(12) eaam8093, 2017. doi: 10.1126/sciimmunol. aam8093. cell therapy. Nature Medicine. 23(12):1436-1443, 2017. doi: 10.1038/nm.4431. Lee MSJ, Maruyama K, Fujita Y. et al. Hosen N, Matsunaga Y, Hasegawa K. et al.

Cevayir Coban's group revealed that malaria infection in- malaria-mediated bone loss was found to be more severe in duces robust immune activation and invasion of parasite by- those of younger age, leading to growth retardation. Naoki Hosen, Athushi Kumanogoh and their research group Thus, MMG49 CAR T cell therapy is promising for MM, and a products into the bone marrow leading to harmful outcomes showed that the active conformer of an integrin can serve as receptor protein with a rare but physiologically relevant confor- on bone homeostasis. a specific therapeutic target for multiple myeloma (MM). They mation can serve as a cancer immunotherapy target. The Coban Team has used mouse malaria models to show screened >10,000 anti-MM mAb clones and identified MMG49 that even after one time malaria infection, Plasmodium prod- as an MM-specific mAb specifically recognizing a subset of ucts gradually accumulate in the bone marrow niche and integrin β7 molecules. The MMG49 epitope, in the N-terminal are "eaten-up" by bone resorbing cells osteoclasts. These ac- region of the β7 chain, is predicted to be inaccessible in the cumulated malarial products in bone marrow niche induce resting integrin conformer but exposed in the active conforma- MyD88-dependent inflammatory responses in osteoclast and tion. Elevated expression and constitutive activation of integrin osteoblast precursors, leading to increased RANKL expression β7 conferred high MMG49 reactivity on MM cells, whereas and over-stimulation of osteoclastogenesis favoring bone MMG49 binding was scarcely detectable in other cell types resorption. Infection with a mutant parasite with impaired including normal integrin β7+ lymphocytes. T cells transduced hemoglobin digestion that produces little hemozoin, a major with MMG49-derived chimeric antigen receptor (CAR) exerted Plasmodium by-product, did not cause bone loss. Importantly, anti-MM effects without damaging normal hematopoietic cells.

88 89 Data

Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors. Regulation of inflammatory responses by dynamic subcellular localization of RNA-binding Nature. 552(7683):101-105, 2017. doi: 10.1038/nature24994. protein Arid5a. Proc Natl Acad Sci USA. 115(6):E1214-E1220, 2018. doi: 10.1073/pnas.1719921115. Saito F, Hirayasu K, Satoh T. et al. Higa M, Oka M, Fujihara Y. et al.

Malaria, one of the world's three major infectious diseases interact with LILRB1 than erythrocytes from patients with non- alongside tuberculosis and AIDS, has been reported to affect severe malaria, although an extended study with larger sample Tadamitsu Kishimoto and his research group revealed the approximately 300 million people every year, accounting for sizes is required to confirm this finding. Our results suggest that regulatory mechanism of subcellular localization of Arid5a in about 500,000 deaths, and there is still no effective vaccine. P. falciparum has acquired multiple RIFINs to evade the host im- response to inflammation. It has been known that an inflam- Hisashi Arase and others showed that P. falciparum uses im- mune system by targeting immune inhibitory receptors. matory accelerator, Arid5a, is localized in the nucleus, and an mune inhibitory receptors to achieve immune evasion. RIFIN inflammatory brake, Regnase-1, is localized in the cytoplasm. In proteins are products of a polymorphic multigene family com- this study, they showed that 1) Arid5a translocates to the cyto- prising approximately 150–200 genes per parasite genome3 plasm from the nucleus in response to inflammation, 2) bimax, that are expressed on the surface of infected erythrocytes. We which inhibit cNLS-dependent nuclear import via high-affinity found that a subset of RIFINs binds to either leucocyte immu- interactions with NLS-binding sites of importin-α, inhibits the noglobulin-like receptor B1 (LILRB1) or leucocyte-associated nuclear import of Arid5a, 3) CRM1 inhibitor, Leptomycin B, in- immunoglobulin-like receptor 1 (LAIR1). LILRB1-binding RIFINs hibits the nuclear export of Arid5a after LPS stimulation. inhibit activation of LILRB1-expressing B cells and natural killer (NK) cells. Furthermore, P. falciparum-infected erythrocytes isolated from patients with severe malaria were more likely to

Direct cell–cell contact between mature osteoblasts and osteoclasts dynamically controls their Combination of multiple optical measurements can assess macrophage activation at single-cell functions in vivo. level without any contrast agent. Nat Commun. 9(1):300, 2018. doi: 10.1038/s41467-017-02541-w. Proc Natl Acad Sci USA. 115(12):E2676-E2685, 2018. doi: 10.1073/pnas.1711872115.

Furuya M, Kikuta J, Fujimori S. et al. Pavillon N, Hobro AJ, Akira S, and Smith N.

Masaru Ishii and his group showed, by using an intravital Nicholas I. Smith and the collaborators developed a label- imaging technique, that mOB and mOC functions are regulated free multimodal microscopy platform that allows the non-inva- via direct cell–cell contact between these cell types. The mOBs sive study of cellular preparations without the need of any ad- and mOCs mainly occupy discrete territories in the steady state, ditional chemicals or contrast agent. The parameters extracted although direct cell–cell contact is detected in spatiotempo- from these measurements, coupled with machine algorithms, rally limited areas. In addition, a pH-sensing fluorescence probe enable the study of fine cellular processes such as macrophage reveals that mOCs secrete protons for bone resorption when cells activation upon exposure to lipopolysaccharide (LPS). The they are not in contact with mOBs, whereas mOCs contacting authors demonstrate that activation, as well as partial activa- mOBs are non-resorptive, suggesting that mOBs can inhibit tion inhibition, can be observed at single-cell level through bone resorption by direct contact. Intermittent administration phenotypic and molecular characterization purely through of parathyroid hormone causes bone anabolic effects, which non-invasive optical means. lead to a mixed distribution of mOBs and mOCs, and increase cell–cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeo- stasis in vivo.

90 91 Publications

Akiyama, M; Okada, Y; Kanai, M; Takahashi, A; Momozawa, Y; Eid, MMA; Shimoda, M; Singh, SK; Almofty, SA; Pham, P; Goodman, Hemmi, M; Tachibana, M; Fujimoto, N; Shoji, M; Sakurai, F; Ikeda, K; Kinoshita, M; Kayama, H; Nagamori, S; Kongpracha, P; Data Ikeda, M; Iwata, N; Ikegawa, S; Hirata, M; Matsuda, K; Iwasaki, M; MF; Maeda, K; Sakaguchi, N. Integrity of immunoglobulin vari- Kobiyama, K; Ishii, KJ; Akira, S; Mizuguchi, H. T helper 17 Pro- Umemoto, E; Okumura, R; Kurakawa, T; Murakami, M; Mikami, N; Yamaji, T; Sawada, N; Hachiya, T; Tanno, K; Shimizu, A; Hozawa, 13 able regions is supported by GANP during AID-induced somatic 26 motes induction of antigen-specific gut-Mucosal cytotoxic 39 Shintani, Y; Ueno, S; Andou, A; Ito, M; Tsumura, H; Yasutomo, K; 1 A; Minegishi, N; Tsugane, S; Yamamoto, M; Kubo, M; Kamatani, hypermutation in germinal center B cells. International Immu- T lymphocytes following adenovirus Vector Vaccination. Fron- Ozono, K; Takashima, S; Sakaguchi, S; Kanai, Y; Takeda, K. Slc3a2 Y. Genome-wide association study identifies 112 new loci for nology 29, 211-220 (2017). tiers in Immunology 8, 1456 (2017). Mediates Branched-Chain Amino-Acid-Dependent Mainte- body mass index in the Japanese population. Nature Genetics nance of Regulatory T Cells. Cell Reports 21, 1824-1838 (2017). 49, 1458-1467 (2017). Endo, T; Freinkman, E; de Rooij, DG; Page, DC. Periodic produc- Higa, M; Oka, M; Fujihara, Y; Masuda, K; Yoneda, Y; Kishimoto, T. Ikeda, T; Shimoda, M; Ebrahimi, D; VandeBerg, JL; Harris, RS; 14 tion of retinoic acid by meiotic and somatic cells coordinates 27 Regulation of inflammatory responses by dynamic subcellular Ando, T; Kashiwakura, J; Itoh-Nagato, N; Yamashita, H; Baba, M; four transitions in mouse spermatogenesis. Proceedings of the localization of RNA-binding protein Arid5a. Proceedings of the 40 Koito, A; Maeda, K. Opossum APOBEC1 is a DNA mutator with Kawakami, Y; Tsai, SH; Inagaki, N; Takeda, K; Iwata, T; Shimojo, N; National Academy of Sciences USA 114, E10132-E10141 (2017). National Academy of Sciences USA 115, E1214-E1220 (2018). retrovirus and retroelement restriction activity. Scientific Re- 2 Fujisawa, T; Nagao, M; Matsumoto, K; Kawakami, Y; Kawakami, ports 7, 46719 (2017). T. Histamine-releasing factor enhances food allergy. Journal of Foster, AJ; Nagata, M; Lu, XY; Lynch, AT; Omandi, Z; Ishikawa, E; Hill, A; DeZern, AE; Kinoshita, T; Brodsky, RA. Paroxysmal noctur- Clinical Investigation 127, 4541-4553 (2017). Yamasaki, S; Timmer, MSM; Stocker, BL. Lipidated Brartemicin 28 nal haemoglobinuria. Nature Reviews Disease Primers 3, 17028 Ilkovicova, L; Trost, N; Szentpeteriova, E; Solar, P; Komel, R; 15 Analogues Are Potent Th1-Stimulating Vaccine Adjuvants. Jour- (2017). Debeljak, N. Overexpression of the erythropoietin receptor in Andrabi, M; Hutchins, AP; Miranda-Saavedra, D; Kono, H; Nussi- nal of Medicinal Chemistry 61, 1045-1060 (2018). 41 RAMA 37 breast cancer cells alters cell growth and sensitivity to tamoxifen. International Journal of Oncology 51, 737-746 3 nov, R; Mizuguchi, K; Ahmad, S. Predicting conformational Hirata, J; Hirota, T; Ozeki, T; Kanai, M; Sudo, T; Tanaka, T; Hizawa, ensembles and genome-wide transcription factor binding sites Fuchs, A; Gliwinski, M; Grageda, N; Spiering, R; Abbas, AK; Appel, N; Nakagawa, H; Sato, S; Mushiroda, T; Saeki, H; Tamari, M; (2017). from DNA sequences. Scientific Reports 7, 4071 (2017). Okada, Y. Variants at HLA-A, HLA-C, and HLA-DQB1 Confer Risk S; Bacchetta, R; Battaglia, M; Berglund, D; Blazar, B; Bluestone, 29 Inoue, N; Ogura, S; Kasai, A; Nakazawa, T; Ikeda, K; Higashi, S; of Psoriasis Vulgaris in Japanese. Journal of Investigative Der- 16 JA; Bornhauser, M, et al. Minimum Information about T Regula- Isotani, A; Baba, K; Mochizuki, H; Fujimura, H; Ago, Y; Hayata- Arase, N; Tanimura, K; Jin, H; Yamaoka, T; Kishibe, M; Nishioka, tory Cells: A Step toward Reproducibility and Standardization. matology 138, 542-548 (2018). M; Kiyohara, E; Tani, M; Matsuoka, S; Ohmura, K; Takasugi, Takano, A; Seiriki, K; Shintani, Y; Shintani, N; Hashimoto, H. Frontiers in Immunology 8, 1844 (2018). 42 Knockdown of the mitochondria-localized protein p13 protects K; Yamamoto, T; Murota, H; Arase, H; Katayama, I. Novel auto- Hirata, T; Mishra, SK; Nakamura, S; Saito, K; Motooka, D; Takada, 4 antibody against the beta 2-glycoprotein I/human leucocyte against experimental parkinsonism. Embo Reports 19, e44860 Fujitani, M; Sato, R; Yamashita, T. Loss of p73 in ependymal cells Y; Kanzawa, N; Murakami, Y; Maeda, Y; Fujita, M; Yamaguchi, Y; (2018). antigen-DR complex in patients with refractory cutaneous ul- 17 during the perinatal period leads to aqueductal stenosis. Scien- 30 Kinoshita, T. Identification of a Golgi GPI-N-acetylgalactosamine cers. British Journal of Dermatology 178, 272-275 (2018). tific Reports 7, 12007 (2017). transferase with tandem transmembrane regions in the cata- Inoue, T; Shinnakasu, R; Ise, W; Kawai, C; Egawa, T; Kurosaki, T. lytic domain. Nature Communications 9, 405 (2018). The transcription factor Foxo1 controls germinal center B cell Arima, Y; Ohki, T; Nishikawa, N; Higuchi, K; Ota, M; Tanaka, Furukawa, A; Kakita, K; Yamada, T; Ishizuka, M; Sakamoto, J; 43 Y; Nio-Kobayashi, J; Elfeky, M; Sakai, R; Mori, Y; Kawamoto, T; proliferation in response to T cell help. Journal of Experimental Hatori, N; Maeda, N; Ohsaka, F; Saitoh, T; Nomura, T; Kuroki, K; Hobro, AJ; Smith, NI. An evaluation of fixation methods: Spatial Medicine 214, 1181-1198 (2017). 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96 97 Access Map SUITA CAMPUS Osaka University ADDRESS 2Fl. IFReC Research Building, Osaka University

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Graduate School of Medicine Handaiguchi Ibaraki Mihogaoka

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Graduate School of Engineering

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

Handai- guchi Handai- By Osaka Monorail Honbu-Mae 25 min. walk from Handai Byoin Mae Station on the Osaka Monorail. Yamada

By Hankyu Bus Graduate School of Frontier Biosciences Route1: Expo ’70 Kita Fire Commemorative Get on the bus bound for "Handai Honbu Mae" or Park Station "Ibaraki Mihogaoka" from Senri-Chuo Station. Get Hankyu Railway o at "Handai Honbu Mae", walk 15 min. JR-Ibaraki Route 2: Get on the bus bound for "Onohara Higashi, Fuji Kasai " from Senri-Chuo Station. Get o at "Handai- guchi", walk 5 min. Umeda Osaka Awaji Minami-Ibaraki Ibaraki-shi By Kintetsu Bus Get on the bus bound for "Handai Honbu Mae" or "Ibaraki Mihogaoka" from Hankyu Ibarakishi Station (via JR Ibaraki Station). Get o at "Handai Honbu Mae", walk 15 min.

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