Four distinct areas were studied in this project in FY2017: 1. The transcription factor Helios is expressed in a large subset of Foxp3+ Tregs. We previously proposed that Helios is a marker of thymic derived Treg (tTreg), however, other studies have suggested that Helios is primarily a marker of T cell activation. In order to examine and compare the two Treg subpopulations, we have generated Helios-GFP reporter mice and crossed them to Foxp3-RFP reporter mice. The Helios+ Treg population expressed a more activated phenotype and had a slightly higher suppressive capacity in vitro. Both populations expressed a highly demethylated TSDR and both subsets were equivalent in their ability to suppress inflammatory bowel disease in vivo. However, Helios+ Treg more effectively inhibited the proliferation of autoreactive splenocytes from scurfy mice. When Helios+ and Helios- Treg were transferred to lymphoreplete mice, both populations maintained comparable Foxp3 expression, but Foxp3 expression was less stable in Helios- Treg when transferred to lymphopenic mice. Gene expression profiling of the two populations demonstrated a large number of differentially expressed genes and TCR repertoire analysis indicated very little overlap between Helios+ and Helios- Treg. Thus, Helios expression can differentiate two phenotypically distinct populations of Treg with differing stability, function and origin. 2. The transcription factor Helios is required for the maintenance of the suppressive phenotype of Foxp3+ regulatory T cells (Tregs) as evidenced by the spontaneous autoimmune activation that develops in Treg-specific Helios deficient mice. Helios is also present in a small subset of CD4+ Foxp3- T conventional (Tconv) cells, and its expression is upregulated upon T cell activation, but its role in Tconv cells remains unknown. Mice with a CD4-Cre driven Helios deletion do not exhibit autoimmunity despite the defect in the suppressor function of their Treg population, pointing to a requisite function of Helios in Tconv cells. We examined the capacity of Helios-deficient, naive Tconv cells to respond to their cognate antigen in vivo and found that Helios was not required for proliferation, differentiation into helper subsets, or the production of effector cytokines. However, Helios appears to play a role in the reactivation of memory T cells as Helios-deficient T cells failed to proliferate upon secondary challenge with antigen. However, we did not find an intrinsic defect in the Helios deficient memory T cells, but rather, we observed that a small population of Helios-deficient naive T cells differentiated into antigen specific peripheral Tregs and that these pTregs suppressed the recall response in an antigen specific manner. In conjunction, these findings suggest that increased pTreg generation in the periphery in mice with a CD4-cre specific Helios deletion may prevent the development of autoimmunity normally associated with Helios deficiency in Tregs. 2. In addition to Helios, a second member of the Ikaros gene family, Eos, is preferentially expressed in Treg cells and activated T-conventional (T conv) cells. Our previous studies had shown that young mice with a global deletion of Eos did not appear to have defective Treg. To fully characterize the role of Eos in Treg function, we generated Eosfl/flx Foxp3Cre (Eos cKO) mice. Beginning at 34 months of age, both CD4+ and CD8+ T cells, as well as Treg in the Eos cKO mice expressed an activated/memory phenotype. The percentage of Tregs in the Eos cKO mice was higher than controls, suggesting that the Eos deficient Tregs were attempting to inhibit the activated immune response. H&E staining revealed severe lymphoid infiltration of lungs, kidneys, liver, small intestine, salivary glands of Eos cKO mice. The autoimmune state in the Eos cKO mice was characterized by enhanced levels of IFN-gamma production by CD4+ Foxp3 T cells, elevated percentages of T-follicular helper cells, and elevated levels of serum anti-nuclear antibodies. Tregs from Eos cKO mice failed to protect in the IBD transfer model and cKO mice also demonstrated enhanced susceptibility to EAE. Nevertheless, the in vitro suppressive function of Treg from cKO was normal. We determined the phenotype of Eos deficient Tregs under non-inflammatory conditions in heterozygous Eosfl/fl Foxp3cre/+ female mice. Although the thymic development of Eos deficient Tregs was not impaired, a marked reduction in Eos deficient Tregs was present in secondary lymphoid tissues indicating a defect in Treg survival in a competitive environment. We conclude that Eos is not required for the development of Tregs, but plays a critical role in controlling many, but not all, of the suppressive functions of Treg. 4. Foxp3+Tregs, CD4+Foxp3- effector T cells and CD8+ T cells are composed of naive (NP, CD44loCD62Lhi) and memory (MP, CD44hiCD62Llo) subsets. About 30% of MP T cells are cycling (Ki-67+) in-vivo. To determine the factors that drive T cell homeostatic proliferation in-vivo, we investigated the roles of cytokines (TNFalpha, IL-2, IL-7, and IL-33), TCR-MHC-II interactions, co-stimulatory (CD28, ICOS, CD40, GITR, OX40), and co-inhibitory (CTLA-4, PD1, BTLA, TIGIT) receptors. Blockade of CD28-CD80/86 signaling completely inhibited MP Treg and MP CD4+Foxp3 effector T cell proliferation, but did not affect MP CD8+ T cell proliferation. Inhibition of cytokine activity did not affect the proliferation of any of the T cell populations. Marked enhancement of homeostatic proliferation of MP Treg and MP CD4+Foxp3- cells was observed by blocking TCR-MHCII interactions, CTLA-4-CD80/CD86 interactions, or by agonistic mAbs to CD40, GITR, and OX40. Blocking PD1-PDL1 interactions modestly enhanced proliferation, while blocking BTLA, TIM3, ICOS and TIGIT did not affect MP T cell proliferation. Importantly, blocking of CD28-CD80/86 signaling completely abrogated the augmentation of proliferation observed after blocking TCR and CTLA-4, PD1, or by agonistic mAbs to GITR, OX40, and CD40. Our results support a complex pathway in which CD28-CD80/CD86 interactions play a critical role in Treg and MP CD4+Foxp3- T cell proliferation in-vivo. CTLA4-CD80/CD86 interactions act as a brake, while MHCII-TCR interactions negatively regulate the inhibitory functions of CTLA-4. These findings have important implications for the use of biologic agents to enhance Treg numbers/functions, as well as for the use of checkpoint inhibitors.

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13
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2018
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Shevach, Ethan M (2018) Foxp3+ T Regulatory Cells: Still Many Unanswered Questions-A Perspective After 20?Years of Study. Front Immunol 9:1048
Holt, Michael P; Punkosdy, George A; Glass, Deborah D et al. (2017) TCR Signaling and CD28/CTLA-4 Signaling Cooperatively Modulate T Regulatory Cell Homeostasis. J Immunol 198:1503-1511
Sebastian, Mathew; Lopez-Ocasio, Maria; Metidji, Amina et al. (2016) Helios Controls a Limited Subset of Regulatory T Cell Functions. J Immunol 196:144-55
Myers, Jennifer M; Cooper, Leslie T; Kem, David C et al. (2016) Cardiac myosin-Th17 responses promote heart failure in human myocarditis. JCI Insight 1:
Ujiie, Hideyuki; Shevach, Ethan M (2016) ?? T Cells Protect the Liver and Lungs of Mice from Autoimmunity Induced by Scurfy Lymphocytes. J Immunol 196:1517-28
Metidji, Amina; Rieder, Sadiye Amcaoglu; Glass, Deborah Dacek et al. (2015) IFN-?/? receptor signaling promotes regulatory T cell development and function under stress conditions. J Immunol 194:4265-76
Rieder, Sadiye Amcaoglu; Metidji, Amina; Glass, Deborah Dacek et al. (2015) Eos Is Redundant for Regulatory T Cell Function but Plays an Important Role in IL-2 and Th17 Production by CD4+ Conventional T Cells. J Immunol 195:553-63
Shevach, Ethan M; Thornton, Angela M (2014) tTregs, pTregs, and iTregs: similarities and differences. Immunol Rev 259:88-102
Edwards, Justin P; Thornton, Angela M; Shevach, Ethan M (2014) Release of active TGF-?1 from the latent TGF-?1/GARP complex on T regulatory cells is mediated by integrin ?8. J Immunol 193:2843-9
Zhu, Jinfang; Shevach, Ethan M (2014) TCR signaling fuels T(reg) cell suppressor function. Nat Immunol 15:1002-3

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