We have focused our studies in a number of different areas in FY2014: 1. For several years we have been interested in the role of cell surface and secreted TGF-beta in Treg Function. TGF-beta can have pleotropic effects on different cell types ranging from immune suppression, the promotion of fibrosis, to promotion/suppression of tumor growth. Activated regulatory T-cells (Tregs), but not activated T conventional cells, express the leucine rich repeat protein, GARP, which is responsible for surface localization of latent TGF-beta1. Although TGF-beta1 has been implicated in the suppressor function of Tregs, Treg conditional knock outs of TGF-beta1 or GARP display normal suppressor function in vitro. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, but are unable to produce biologically active TGF-beta1, as measured by their inability to drive Th17 differentiation in vitro in the presence of IL-6 or to induce Foxp3+ Treg in the presence of IL-2. Although integrins have been implicated in mediating the release of active TGF-beta1 from the complex of latent TGF-beta1 and latent TGF-beta1 binding protein, their role in processing latent TGF-beta1 from the latent TGF-beta1/GARP complex is unclear. Mouse CD4+Foxp3+ Treg, but not CD4+Foxp3- T cells, expressed integrin beta8 (Itgb8) as detected by qRT-PCR. Itgb8 expression was a marker of thymically-derived (t)Treg, as it could not be detected on Foxp3+Helios- Tregs or on Foxp3+ T cells induced in vitro. Tregs from Itgb8 conditional knockouts exhibited normal suppressor function in vitro and in vivo in a model of colitis, but failed to provide TGF-beta1 to drive Th17 or iTreg differentiation in vitro. In addition, Itgb8 knockout Tregs expressed higher levels of latent TGF-beta1 on their cell surface consistent with defective processing. Thus, integrin alphaVbeta8 is a marker of tTregs and functions in a cell intrinsic manner in mediating the processing of latent TGF-beta1 from the latent TGF-beta1/GARP complex on the surface of tTregs. Tregs may primarily exert their effects via recognition of self-antigens during the process of priming and differentiation of naive T cells on the surface of antigen-presenting dendritic cells. It is likely that not all dendritic cells express Itgb8 and that Treg cell intrinsic expression of Itgb8 may be required to facilitate activation of Treg-expressed latent TGF-beta1 during the priming/differentiation of nave T cells. 2. One of the critical issues regarding the potential therapeutic use of Tregs in vivo is the specificity of their inhibitory effects. To address this question, we generated allo-specific iTregs in vitro by stimulation of nave polyclonal Foxp3- T cells from Foxp3-GFP reporter mice with allogeneic DCs, TGFbeta1, and retinoic acid. Foxp3+ T cells were isolated by cell sorting and expanded with anti-CD3 and IL-2 and then resorted. The iTregs specifically suppressed responses to the inducing alloantigen in vitro, but had no effect on 3rd party stimulators. Similarly, when the allo-specific iTregs were transferred to immunocompetent recipients, they specifically suppressed responses induced by the inducing alloantigen, but not by 3rd party DC. To determine whether the allo-iTregs would suppress responses to foreign antigens driven by the target alloantigen presenting DC, we transferred allo-iTregs with antigen-specific T effector (Teff) cells specific for a foreign antigen presented by DCs exoressubg the target alloantigen. When the alloantigen and the foreign antigen were present on the same DC, marked suppression of both the alloantigen and antigen responses were observed. However, when the alloantigen and foreign antigen were present on separate populations of DCs, only the responses to alloantigen were inhibited. Although it remains possible that under certain conditions, alloantigen-specific Tregs can inhibit responses to foreign antigen, this is likely to be a rare event. We conclude that allo-specific iTreg represent a safe, promising strategy for the treatment of both GVHD and organ transplant rejection. 3. Foxp3+ regulatory T cells (Treg) are critical for the maintenance of immunological tolerance, and a mutation of the Foxp3 gene leads to the absence of Treg and a fatal systemic autoimmune disease in Scurfy mice. Transfer of Scurfy lymphocytes to RAG-/- recipients reproduces the inflammatory phenotype of the scurfy donor including hepatitis and pneumonitis. Here, we show that TCRalpha/beta-/- recipients, that lack TCR alpha/beta+ T cells, but have TCR gamma/delta+ T cells and B cells, are significantly protected from autoimmunity induced by adoptive transfer of Scurfy lymphocytes. Co-transfer of Scurfy lymphocytes and B cells from TCRalpha-/- mice into RAG-/- recipients failed to prevent inflammation. Furthermore, transfer of Scurfy lymphocytes to TCRbeta/delta-/- mice, which lack both TCR alpha/beta+ and TCR gamma/delta+ T cells, but have B cells, resulted in more severe inflammation in the liver and lungs than that seen in TCRalpha-/- recipients. Taken together, these studies demonstrate that TCR gamma/delta+ T cells, but not B cells, in the TCRalpha/beta-/- recipients mediate protection from disease induced by Scurfy T cells. The TCR gamma/delta+ T cells in the TCR alpha -/- recipients of Scurfy cells markedly expanded and expressed a highly activated (CD44hi CD62Llo) phenotype, but did not produce IL-10. Surprisingly, the Scurfy T cells in these recipients produced more IL-10 and less IL-4 than Scurfy T cells transferred to RAG-/- or TCRbeta/delta-/- recipients, suggesting that gamma/delta+ T cells may protect by modulating cytokine production by Scurfy T cells.

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Akkaya, Billur; Holstein, Amanda H; Isaac, Christopher et al. (2017) Ex-vivo iTreg differentiation revisited: Convenient alternatives to existing strategies. J Immunol Methods 441:67-71
Shevach, Ethan M (2017) Garp as a therapeutic target for modulation of T regulatory cell function. Expert Opin Ther Targets 21:191-200
Vermeersch, Elien; Denorme, Frederik; Maes, Wim et al. (2017) The role of platelet and endothelial GARP in thrombosis and hemostasis. PLoS One 12:e0173329
Akkaya, Billur; Miozzo, Pietro; Holstein, Amanda H et al. (2016) A Simple, Versatile Antibody-Based Barcoding Method for Flow Cytometry. J Immunol 197:2027-38
Edwards, Justin P; Hand, Timothy W; Morais da Fonseca, Denise et al. (2016) The GARP/Latent TGF-?1 complex on Treg cells modulates the induction of peripherally derived Treg cells during oral tolerance. Eur J Immunol 46:1480-9
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
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
Chattopadhyay, Gouri; Shevach, Ethan M (2013) Antigen-specific induced T regulatory cells impair dendritic cell function via an IL-10/MARCH1-dependent mechanism. J Immunol 191:5875-84
Edwards, Justin P; Fujii, Hodaka; Zhou, Angela X et al. (2013) Regulation of the expression of GARP/latent TGF-?1 complexes on mouse T cells and their role in regulatory T cell and Th17 differentiation. J Immunol 190:5506-15
Davidson, Todd S; Shevach, Ethan M (2011) Polyclonal Treg cells modulate T effector cell trafficking. Eur J Immunol 41:2862-70

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