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CD8 T Cell-Intrinsic GITR Is Required for T Cell Clonal Expansion and Mouse Survival following Severe Influenza Infection This information is current as Laura M. Snell, Ann J. McPherson, Gloria H. Y. Lin, of October 2, 2021. Shimon Sakaguchi, Pier Paolo Pandolfi, Carlo Riccardi and Tania H. Watts J Immunol 2010; 185:7223-7234; Prepublished online 12 November 2010; doi: 10.4049/jimmunol.1001912 http://www.jimmunol.org/content/185/12/7223 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/11/12/jimmunol.100191 Material 2.DC1 http://www.jimmunol.org/ References This article cites 47 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/185/12/7223.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 2, 2021 • No Triage! Every submission reviewed by practicing scientists • 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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology CD8 T Cell-Intrinsic GITR Is Required for T Cell Clonal Expansion and Mouse Survival following Severe Influenza Infection Laura M. Snell,* Ann J. McPherson,* Gloria H. Y. Lin,* Shimon Sakaguchi,† Pier Paolo Pandolfi,‡,x,{ Carlo Riccardi,‖ and Tania H. Watts* The regulation of T cell expansion by TNFR family members plays an important role in determining the magnitude of the immune response to pathogens. As several members of the TNFR family, including glucocorticoid-induced TNFR-related protein (GITR), are found on both regulatory and effector T cells, there is much interest in understanding how their effects on these opposing arms of the immune system affect disease outcome. Whereas much work has focused on the role of GITR on regulatory T cells, little is known about its intrinsic role on effector T cells in an infectious disease context. In this study, we demonstrate that GITR signaling on CD8 T cells leads to TNFR-associated factor (TRAF) 2/5-dependent, TRAF1-independent NF-kB induction, resulting in increased Bcl- Downloaded from xL. In vivo, GITR on CD8 T cells has a profound effect on CD8 T cell expansion, via effects on T cell survival. Moreover, GITR is required on CD8 T cells for enhancement of influenza-specific CD8 T cell expansion upon administration of agonistic anti-GITR Ab, DTA-1. Remarkably, CD8 T cell-intrinsic GITR is essential for mouse survival during severe, but dispensable during mild respiratory influenza infection. These studies highlight the importance of GITR as a CD8 T cell costimulator during acute viral infection, and argue that despite the similarity among several TNFR family members in inducing T lymphoctye survival, they clearly have nonredundant functions in protection from severe infection. The Journal of Immunology, 2010, 185: 7223–7234. http://www.jimmunol.org/ lucocorticoid-induced TNFR-related protein (GITR) is studied in the context of Tregs and generated much excitement a member of the TNFR superfamily that modulates im- when it was observed that agonistic anti-GITR Ab in cocultures of mune responses. Perhaps most extensively studied for its CD4+CD252 effector T cells and CD4+CD25+ Tregs caused the G + + + high basal expression on CD4 CD25 Foxp3 regulatory T cells abrogation of suppression by Tregs (1, 2). Interestingly, it was (Tregs) (1, 2), GITR is also expressed on many other immune cell shown that GITR signaling on the effector T cells was necessary types, including CD4 and CD8 T cells, NK cells, B cells, and mac- to render the cells less refractory to Treg-mediated suppression rophages (3). The ligand for GITR (GITRL) can be expressed on (7). A recent report showed that this phenomenon is not unique to by guest on October 2, 2021 dendritic cells, macrophages, B cells, endothelial cells, and acti- CD4 T cells, and GITR signaling can induce resistance to CD4+ vated T cells (3). CD25+ Treg suppression in CD8 T cells as well (8). The role of GITR in immune responses is complex and may In addition to enhancing T cell responses by raising the threshold be cell-type specific. Stimulation of macrophages with an anti- for Treg suppression, GITR has also been shown to costimulate GITR Ab induces a proinflammatory response (4); however, re- CD4 and CD8 T cells in vitro by enhancing proliferation, survival, cent reports have shown that GITR can play an inhibitory role for and cytokine secretion (9, 10). GITR acts early in the immune human NK cell activation (5, 6). GITR has also been extensively response to lower the threshold for CD28 signaling in both CD4 and CD8 T cells (11). Whereas GITR’s costimulatory role for T cells is well documented in vitro, much remains to be elucidated *Department of Immunology, University of Toronto, Toronto, Ontario, Canada; as to its function in vivo, particularly on CD8 T cells. Notably †Department of Experimental Pathology, Institute for Frontier Medical Sciences, lacking from the literature is any thorough assessment of GITR’s Kyoto University, Kyoto, Japan; ‡Cancer Genetics Program, Beth Israel Deaconess Cancer Center, xDepartment of Medicine, and {Department of Pathology, Beth Israel physiological role on CD8 T cells in an infectious disease context. Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and ‖De- Although a few studies have shown that agonistic anti-GITR partment of Clinical and Experimental Medicine, University of Perugia, Perugia, Ab can enhance antiviral CD8 T cell responses in vivo (12–14), Italy the mechanism whereby this occurs remains to be determined, as Received for publication June 9, 2010. Accepted for publication October 8, 2010. GITR is broadly expressed and its signaling on other immune cells This work was supported by Grant #84419 from the Canadian Institutes of Health may influence the CD8 T cell response indirectly. Clearly, more Research (CIHR) to T.H.W. L.M.S. is funded by a fellowship from the Fonds de la Recherche en Sante´ du Que´bec. G.H.Y.L.is funded by a CIHR Doctoral Award. T.H.W. defined models are needed to study the intrinsic role of GITR on holds the Sanofi Pasteur Chair in Human Immunology at the University of Toronto. CD8 T cells in response to pathogens in vivo. Address correspondence and reprint requests to Dr. Tania H. Watts, Department of In this study, we sought to address the role of GITR on CD8 Immunology, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S T cells during viral infection. By using an adoptive transfer model 1A8, Canada. E-mail address: [email protected] in which GITR is only absent on the responding CD8 T cells, we The online version of this article contains supplemental material. show that GITR is intrinsically required on CD8 T cells for Abbreviations used in this paper: DC, dendritic cell; GITR, glucocorticoid-induced TNFR-related protein; GITRL, ligand for GITR; HAU, hemagglutinin units; i.n., maximal primary and secondary CD8 T cell responses to influenza. intranasal; NP, nucleoprotein; PDTC, 1-pyrrolidinecarbodithioic acid; siRNA, small Moreover, we demonstrate that GITR-specific agonistic Abs en- interfering RNA; TCID50, tissue culture infectious dose 50; TRAF, TNFR-associated hance the response to influenza virus through direct effects of GITR factor; Treg, regulatory T cell; WT, wild-type. on CD8 T cells. Although GITR does not influence cell division, it Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 augments CD8 T cell survival by upregulating the prosurvival www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001912 7224 GITR AS A CD8 T CELL-INTRINSIC COSTIMULATOR molecule Bcl-xL in a NF-kB–dependent manner. We found that (StemCell Technologies, Vancouver, Canada). For memory experiments, 2/2 GITR-induced NF-kB activation is positively regulated by both memory-like WT and GITR OT-I cells were generated, as previously TNFR-associated factor (TRAF) 2 and 5, whereas TRAF1 is described (19). In certain experiments, T cells were stained with 1 mM CFSE for 10 min at 37˚C. T cells were injected i.v. at 104 cells/mouse (for dispensable for this survival signaling. Although the absence of ratio experiments 5 3 103 of each cell type/mouse) with the exception of GITR on T cells did not affect disease outcome during mild in- CFSE experiments, which used 106 cells/mouse, and PR8-OVA experi- fluenza virus infection, during a severe and potentially lethal ments, which used 103 cells/mouse. A day later, the mice were infected model of influenza infection we show that GITR on CD8 T cells with X31-OVA or PR8-OVA, as described above. For experiments using agonistic Abs, X31-OVA was administered i.p. at a dose of 100 HAU/ augmented viral clearance and protected mice from death. These mouse with either 200 mg anti-GITR (DTA-1) hybridoma (2) or purified results highlight the importance of GITR as a CD8 T cell-intrinsic rat IgG (Sigma-Aldrich, St. Louis, MO). costimulatory receptor. Cell viability assay Materials and Methods WT and GITR2/2 OT-I CD8 T cells were isolated from naive mice and Mice cultured in vitro with irradiated splenocytes from C57BL/6 mice at a ratio of 1:4. Cultures were stimulated with a suboptimal dose of SIINFEKL 2 C57BL/6 mice were obtained from Charles River Laboratories (Wil- (10 10 M), and in some cases T cells were CFSE labeled, as described mington, MA).

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