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Role of the EHD Family of Endocytic Recycling Regulators for TCR Recycling and T Cell Function

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Role of the EHD Family of Endocytic Recycling Regulators for TCR Recycling and T Cell Function Role of the EHD Family of Endocytic Recycling Regulators for TCR Recycling and T Cell Function This information is current as Fany M. Iseka, Benjamin T. Goetz, Insha Mushtaq, Wei An, of September 27, 2021. Luke R. Cypher, Timothy A. Bielecki, Eric C. Tom, Priyanka Arya, Sohinee Bhattacharyya, Matthew D. Storck, Craig L. Semerad, James E. Talmadge, R. Lee Mosley, Vimla Band and Hamid Band J Immunol published online 6 December 2017 Downloaded from http://www.jimmunol.org/content/early/2017/12/05/jimmun ol.1601793 Supplementary http://www.jimmunol.org/content/suppl/2017/12/05/jimmunol.160179 http://www.jimmunol.org/ Material 3.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 6, 2017, doi:10.4049/jimmunol.1601793 The Journal of Immunology Role of the EHD Family of Endocytic Recycling Regulators for TCR Recycling and T Cell Function Fany M. Iseka,*,† Benjamin T. Goetz,* Insha Mushtaq,*,‡ Wei An,* Luke R. Cypher,* Timothy A. Bielecki,* Eric C. Tom,*,x,{ Priyanka Arya,*,†,1 Sohinee Bhattacharyya,*,‡,2 Matthew D. Storck,* Craig L. Semerad,‖ James E. Talmadge,‡ R. Lee Mosley,*,# Vimla Band,*,†,‡ and Hamid Band*,†,‡,x,{,# T cells use the endocytic pathway for key cell biological functions, including receptor turnover and maintenance of the immuno- logical synapse. Some of the established players include the Rab GTPases, the SNARE complex proteins, and others, which function together with EPS-15 homology domain–containing (EHD) proteins in non–T cell systems. To date, the role of the EHD protein family in T cell function remains unexplored. We generated conditional EHD1/3/4 knockout mice using CD4-Cre and crossed Downloaded from these with mice bearing a myelin oligodendrocyte glycoprotein–specific TCR transgene. We found that CD4+ T cells from these mice exhibited reduced Ag-driven proliferation and IL-2 secretion in vitro. In vivo, these mice exhibited reduced severity of experimental autoimmune encephalomyelitis. Further analyses showed that recycling of the TCR-CD3 complex was impaired, leading to increased lysosomal targeting and reduced surface levels on CD4+ T cells of EHD1/3/4 knockout mice. Our studies reveal a novel role of the EHD family of endocytic recycling regulatory proteins in TCR-mediated T cell functions. The Journal of Immunology, 2018, 200: 000–000. http://www.jimmunol.org/ nderstanding the mechanisms that underlie proper T cell CD28 and lymphocyte function–associated 1 (LFA-1), to form an function is of great interest in basic immunology and area of intimate contact with APCs, the immunological synapse U immunopathology. T cell activation requires recognition (IS) (2, 3). The endocytic pathways supply the IS with newly of an antigenic peptide bound to MHC proteins on APCs by the synthesized and recycled receptors to replenish those that are TCR and downstream signaling mediated by cytoplasmic regions targeted to the lysosome for degradation (4–6). of the TCR-CD3 complex. Effective generation of an immune Cells use clathrin-dependent as well as clathrin-independent response requires concurrent engagement of and signaling through pathways to internalize surface receptors into early endosome/ costimulatory proteins (e.g., CD28) on T cells by their cognate sorting endosome compartments, from where they traffic to late by guest on September 27, 2021 ligands on the APC surface (1). Therefore, the mechanisms that endosome/multivesicular bodies and lysosomes for degradation, or ensure optimal levels of the TCR and accessory receptors on the recycle back to the cell surface either through a Rab4+ fast T cell surface prior to Ag stimulation are fundamentally critical to recycling endosome (directly to the plasma membrane) or through generating an effective immune response. However, mechanisms a slow recycling route via the Rab11+ recycling endosome (7, 8). that regulate the surface pool of TCR and its accessory This sorting processes is known to be regulated by the Rab family receptors have been primarily investigated in the context of T cell of small GTPases and their interacting partners (9). TCRs such as activation. During T cell activation, TCR signaling elicited by the TCR-CD3 (1), CD28 (10), and LFA-1 (11, 12) are known to be APC-presented Ag, together with costimulatory signals, leads to internalized and recycled to the IS during T cell activa- spatial reorganization of the TCR and accessory receptors, such as tion. Internalized TCR-CD3 traverses the Rab5+ early endosome, *Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Department of Defense Grants W81XWH-07-1-0351 and W81XWH-11-1-0171 (to Medical Center, Omaha, NE 68198; †Department of Genetics, Cell Biology and V.B.), a University of Nebraska System Science Collaborative grant (to H.B.), and an Anatomy, University of Nebraska Medical Center, Omaha, NE 68198; Institutional Development Award from the National Institute of General Medical ‡Department of Pathology and Microbiology, University of Nebraska Medical Center, Sciences under National Institutes of Health Grant P30 GM1063. Support for the Omaha, NE 68198; xDepartment of Biochemistry and Molecular Biology, University University of Nebraska Medical Center Confocal, Flow Cytometry, and other core of Nebraska Medical Center, Omaha, NE 68198; {Department of Pharmacology and facilities was from National Cancer Institute Cancer Center Support Grant Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE P30CA036727 to the Fred and Pamela Buffett Cancer Center, as well as from funding 68198; ‖Flow Cytometry Research Facility, University of Nebraska Medical Center, by National Institute of General Medical Sciences Grant P30 GM1063 and the Omaha, NE 68198; and #Fred and Pamela Buffet Cancer Center, University of Nebraska Research Initiative. Nebraska Medical Center, Omaha, NE 68198 Address correspondence and reprint requests to Dr. Hamid Band, Eppley Institute for 1Current address: Department of Medical and Molecular Genetics, Indiana University Research in Cancer and Allied Diseases, BCC 10.12.390, 986805 Nebraska Medical School of Medicine, Indianapolis, IN. Center, Omaha, NE 68198-6805. E-mail address: [email protected] 2Current address: Department of Cell, Developmental and Cancer Biology, Oregon The online version of this article contains supplemental material. Health Science University, Portland, OR. Abbreviations used in this article: EAE, experimental autoimmune encephalomyeli- ORCIDs: 0000-0002-4029-8943 (M.D.S.); 0000-0002-6328-6056 (J.E.T.); 0000- tis; EHD, EPS-15 homology domain–containing protein; IS, immunological synapse; 0002-4241-6149 (R.L.M.); 0000-0002-4996-9002 (H.B.). KO, knockout; LFA-1, lymphocyte function–associated 1; LN, lymph node; MFI, mean fluorescence intensity; MOG, myelin oligodendrocyte glycoprotein; 4-OHT, Received for publication October 20, 2016. Accepted for publication November 1, 4-hydroxytamoxifen. 2017. This work was supported by National Institutes of Health Grants CA105489, Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 CA87986, CA99163, and CA116552 (to H.B.), as well as CA105489 (supplement; to F.M.I.), and CA96844 and CA144027 (to V.B.). This work was also supported by www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601793 2 EHD PROTEIN REGULATION OF TCR followed by transport through Rab4+ and Rab11+ endosomes, Ag-driven cell proliferation and IL-2 secretion. In vivo, these mice indicating the involvement of both fast and slow recycling path- exhibit a reduced severity of experimental autoimmune encepha- ways (13, 14). Naive and resting T cells constitutively internalize lomyelitis (EAE) elicited by immunization with the MOG peptide. and recycle their surface receptors, and the balance of these We show that surface levels of TCR, CD28, and LFA-1 are re- processes dictates their prestimulation surface levels and hence the duced and TCR recycling is impaired in CD4+ T cells of mice with levels of T cell activation (13). However, the mechanisms of a CD4-Cre–mediated EHD1/3/4 knockout. Therefore, our studies constitutive basal traffic of TCR-CD3 are not fully understood. reveal a novel role of the EHD family of endocytic recycling Recently, a new family of endocytic regulators, the EPS15 proteins in the surface display of TCR in unstimulated T cells that homology domain–containing proteins (EHD1–4), has been contributes to their positive functional role in Ag-induced T cell identified. These proteins exhibit a conserved domain architecture activation and immunopathology. with helical regions near the N terminus and the middle of the protein surrounding an ATP-binding G-domain that can hydrolyze Materials and Methods ATP slowly, and
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