Project Summary/Abstract Allograft rejection is characterized by effector CD4+ T cell activation in response to donor antigen and an intense cellular and humoral attack on the graft. However, multiple intracellular signals within CD4+ T cells operate co-incidentally to enhance the expansion and function of CD4+Foxp3+ T regulatory cells that collectively serve to control the alloimmune response. Furthermore, the potency of this process of immunoregulation prevents and restrains alloimmune T effector cell activation and rejection. Importantly, recent advances indicate that CD4+Foxp3+ T cell differentiation and function is negatively regulated by the cell intrinsic activity of mTOR and specifically mTORC1. However, little is known about the regulation of intracellular mTOR signaling within alloreactive CD4+ T cell effectors, or how its relative activity may be modulated in Foxp3+ subsets, or whether it is possible to exploit modulatory signals to augment physiological Treg activity in pathological states to prevent disease, including the development of chronic allograft rejection. DEPTOR is a recently discovered cell intrinsic factor that modulates mTOR-induced signaling responses in highly proliferative cancer cells, and it has more recently been observed to function in normal cell types including vascular endothelial cells. In preliminary studies, we find that DEPTOR is expressed at high levels in unactivated CD4+ T cells, and further, that its expression is reduced upon cellular activation. In addition, we find that forced overexpression of DEPTOR modulates CD4+ T cell activation responses in vitro, promotes immunoregulation and prolongs graft survival following fully MHC mismatched transplantation in vivo. We suggest that these observations identify DEPTOR as a critical upstream intracellular modulator of CD4+ T cell activation as well as the phenotypic and functional outcome of the alloimmune response. Our objectives in this R01 are to further evaluate these observations using novel transgenic mice, and 1), define the select function of DEPTOR in CD4+ T effector and regulatory subsets in vivo, and 2), evaluate the consequences of CD4+ T cell DEPTOR expression in models of transplant rejection. We will test the hypothesis that DEPTOR is a cell intrinsic molecule that modulates CD4+ T effector cell activation and augments CD4+ T regulatory cell function to enhance immunoregulation and promote long-term graft survival. We propose two specific aims in which we will: 1), determine the consequences and mechanism of function of cell intrinsic DEPTOR in CD4+ T cell subsets, and 2), determine the function of CD4+ T cell DEPTOR in long-term allograft survival. Collectively, these innovative studies will have broad scientific and biological implications of great significance and relevance to transplantation immunobiology.