CRACR2A-Mediated TCR Signaling Promotes Local Effector Th1 and Th17 Responses This information is current as Jin Seok Woo, Sonal Srikanth, Kyun-Do Kim, Heidi of September 24, 2021. Elsaesser, Jing Lu, Matteo Pellegrini, David G. Brooks, Zuoming Sun and Yousang Gwack J Immunol 2018; 201:1174-1185; Prepublished online 9 July 2018; doi: 10.4049/jimmunol.1800659 Downloaded from http://www.jimmunol.org/content/201/4/1174 Supplementary http://www.jimmunol.org/content/suppl/2018/07/06/jimmunol.180065 Material 9.DCSupplemental http://www.jimmunol.org/ References This article cites 52 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/201/4/1174.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 24, 2021 • No Triage! 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The Journal of Immunology CRACR2A-Mediated TCR Signaling Promotes Local Effector Th1 and Th17 Responses Jin Seok Woo,*,1 Sonal Srikanth,*,1 Kyun-Do Kim,*,1 Heidi Elsaesser,†,‡ Jing Lu,x Matteo Pellegrini,x David G. Brooks,†,‡ Zuoming Sun,{ and Yousang Gwack* Ca2+ release–activated Ca2+ channel regulator 2A (CRACR2A) is expressed abundantly in T cells and acts as a signal transmitter between TCR stimulation and activation of the Ca2+/NFATand JNK/AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies and was shown to be one of the most sensitive targets of the widely used statin drugs. However, the physiological role of CRACR2A in T cell functions remains unknown. In this study, using transgenic mice for tissue- specific deletion, we show that CRACR2A promotes Th1 responses and effector function of Th17 cells. CRACR2A was abundantly expressed in Th1 and Th17 cells. In vitro, deficiency of CRACR2A decreased Th1 differentiation under nonpolarizing conditions, whereas the presence of polarizing cytokines compensated this defect. Transcript analysis showed that weakened TCR signaling Downloaded from by deficiency of CRACR2A failed to promote Th1 transcriptional program. In vivo, conditional deletion of CRACR2A in T cells alleviated Th1 responses to acute lymphocytic choriomeningitis virus infection and imparted resistance to experimental autoimmune encephalomyelitis. Analysis of CNS from experimental autoimmune encephalomyelitis–induced mice showed im- paired effector functions of both Th1 and Th17 cell types, which correlated with decreased pathogenicity. Collectively, our findings demonstrate the requirement of CRACR2A-mediated TCR signaling in Th1 responses as well as pathogenic conversion of Th17 cells, which occurs at the site of inflammation. The Journal of Immunology, 2018, 201: 1174–1185. http://www.jimmunol.org/ 2+ 2+ 2+ uman Ca release–activated Ca (CRAC) channel (InsP3) that depletes endoplasmic reticulum Ca stores and trig- regulator 2A (CRACR2A, or alternatively EFCAB4B) gers activation of extracellular Ca2+ entry via CRAC channels in a gene is located on chromosome 12. Links between process termed as store-operated Ca2+ entry. Elevated cytoplasmic H 2+ polymorphism in CRACR2A and human diseases have been Ca concentration activates the downstream calcineurin/NFAT identified from numerous genome-wide association studies pathway. Vav1 is a guanine nucleotide exchange factor that re- (GWAS) of Parkinson disease, nonalcoholic fatty liver disease, cruits small G proteins to activate the JNK and p38 MAPK atrial fibrillation, and chronic infection of HIV type 1 (1–4). pathways that eventually turn on gene transcription by the AP-1 However, the mechanisms underlying this link are largely un- transcription factors (9). Previously, we reported a function of by guest on September 24, 2021 known because of the lack of information on the physiological CRACR2A in regulation of the Ca2+/NFAT and JNK MAPK role of CRACR2A. Recent studies have shed some light on the signaling pathways (10, 11). The short, cytoplasmic isoform of potential role of CRACR2A in T cell–mediated immunity. CRACR2A, CRACR2A-c, stabilizes CRAC channels by interac- Engagement of TCRs with cognate Ags induces clustering and tion with its key components, Orai1, the plasma membrane pore activation of enzymes and signaling adaptors including phospho- subunit, and STIM1, the endoplasmic reticulum Ca2+ sensor lipase C-g1 (PLCg1) and Vav1 at the immunological synapse, necessary for activation of Orai1 channels. Differing from which are responsible for activation of downstream signaling CRACR2A-c, the long isoform, CRACR2A-a is a component of cascades such as the Ca2+/NFAT and MAPK pathways (5–8). vesicles. It is a member of the unique, large Rab GTPase family PLCg1 produces a second messenger inositol 1,4,5-trisphosphate that also includes Rab44 and Rab45 (11). CRACR2A-a contains *Department of Physiology, David Geffen School of Medicine, University of maintained Cracr2afl/fl animals with help from J.L. and performed in vitro T cell California, Los Angeles, Los Angeles, CA 90095; †Princess Margaret Cancer Center, experiments and RNA sequencing analysis (in collaboration with J.L. and M.P.). University Health Network, Toronto, Ontario M5G 2M9, Canada; ‡Department of K.-D.K. analyzed development, homeostasis, and in vitro T cell phenotypes from Immunology, University of Toronto, Toronto, Ontario M5G 2M9, Canada; xDepart- Cracr2afl/fl animals. Lymphocytic choriomeningitis virus infection experiments ment of Molecular, Cell and Developmental Biology, University of California, were done by H.E., S.S., and D.G.B. Y.G., S.S., and J.S.W. wrote the manuscript Los Angeles, Los Angeles, CA 90095; and {Division of Molecular Immunology, with input from other authors. Y.G. supervised the project. Beckman Research Institute of the City of Hope, Duarte, CA 91010 Address correspondence and reprint requests to Dr. Yousang Gwack, Department of 1J.S.W., S.S., and K.-D.K. contributed equally to this work. Physiology, David Geffen School of Medicine, University of California, Los Angeles, 53-266 Center for the Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA ORCIDs: 0000-0001-8921-2832 (J.S.W.); 0000-0002-3034-095X (S.S.); 0000-0001- 90095. E-mail address: [email protected] 6872-5583 (K.-D.K.); 0000-0003-1896-6666 (Z.S.). The online version of this article contains supplemental material. Received for publication May 9, 2018. Accepted for publication June 12, 2018. Abbreviations used in this article: CRAC, Ca2+ release–activated Ca2+;CRACR2A, This work was supported by National Institutes of Health Grants AI083432 (to Y.G.) CRAC channel regulator 2A; C , threshold cycle; DAVID, Database for Annotation, and AI130653 (to S.S.). Flow cytometry was performed in the University of T Visualization and Integrated Discovery; EAE, experimental autoimmune encephalomy- California, Los Angeles (UCLA) Jonsson Comprehensive Cancer Center (JCCC) and elitis; GWAS, genome-wide association study; LCMV, lymphocytic choriomeningitis Center for AIDS Research (CFAR) Flow Cytometry Core Facility that is supported virus; MOG, myelin oligodendrocyte glycoprotein; shRNA, short hairpin RNA. by the JCCC Grants P30 CA016042 and 5P30 AI028697. Peripheral blood mononu- clear cells were obtained from the CFAR Virology Core Laboratory at UCLA that is Ó supported by Grant 5P30 AI028697. Copyright 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 S.S. and Y.G. designed research. J.S.W. performed in vitro T cell analysis and active/ passive experimental allergic encephalomyelitis experiments. S.S. generated and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800659 The Journal of Immunology 1175 multiple functional domains including the N-terminal domain that showed that Cracr2afl/fl/Cd4-Cre Th17 cells had weakened path- is identical with CRACR2A-c, a proline-rich protein-interacting ogenicity as judged by reduced clinical symptoms in recipients of domain, and a C-terminal Rab GTPase domain. GTP binding and Cracr2afl/fl/Cd4-Cre Th17 cells because of impaired transition into prenylation are essential for localization of CRACR2A in vesicles, Th1-like Th17 cells that produce IFN-g and GM-CSF cytokines. whereas its interaction with Vav1 is necessary for activation of the Therefore, our findings demonstrate that TCR signaling mediated JNK signaling pathway. Another interesting aspect of CRACR2A- by CRACR2A is crucial for Th1 differentiation as well as local a is its high sensitivity to statin drugs, which inhibit 3-hydroxyl-3- effector function of Th17 cells. methyl-glutaryl-CoA (HMG-CoA) reductase, a key rate-liming enzyme in cholesterol biosynthesis pathway. Statin treatment–in- Materials and Methods duced deprenylation causes dissociation of CRACR2A-a from Chemicals vesicles, leading to its degradation,