Costimulatory pathways are considered essential for T cell activation and differentiation. Manipulating these signals on antigen encountered T cells is a most attractive approach for inducing antigen specific tolerance to prevent and suppress autoimmunity. Co-inhibitors/repressor-receptors (negative regulators) that are upregulated significantly on activated T cells, and have been the molecules of attention as targets for therapy in last several years. Induction of antigen specific tolerance depends on concurrent engagement of the TCR and one or more of these repressor-receptors. Interestingly, our studies have shown that this T cell tolerance is mostly associated with an induction and/or expansion of antigen specific regulatory T cells (Tregs). Antigen specific Tregs can suppress effector T cell response and provide long-term sustained protection from autoimmunity. Our hypothesis is that dominant engagement of repressor-receptors along with TCR on antigen specific T cells using dendritic cells (DCs) engineered to stably overexpress negative regulatory ligands will induce effective long-lasting antigen specific T cell tolerance. Our studies have so far demonstrated that antigen specific engagement of repressor-receptors on T cells by DC directed approaches could induce significant suppression of antigen specific T cell response and generation of hypo- proliferative T cells with the ability to produce large amounts of suppressor cytokines such as IL-10 and TGF-21 both in vitro and in vivo. Therefore, the approach of stable exogenous over-expression of ligands, B7.1wa, PD-L1, and HVEM-CRD1 that are specific for T cell repressor-receptors, CTLA-4, PD-1 and BTLA is adopted for generating tolerogenic APCs. These DCs can present antigen to T cells and simultaneously engage their repressor-receptors with an enhanced strength. These tolerogenic DCs could induce profound suppression of antigen specific T cell and pro-inflammatory cytokine responses, but enhanced anti- inflammatory cytokine responses. The exceptional antigen presenting properties of DCs combined with the engineered DC's ability to predominantly engage repressor-receptors on activated T cells are exploited in this system. This negative regulatory ligand over-expressing DCs can also be a powerful tool to study the effect of enhanced engagement of T cell repressor-receptors during the antigen presentation. This study will be aimed at 1) understanding the potential of DCs that are over-expressing ligands for T cell repressor- receptors to induce and maintain antigen specific T cell hypo-responsiveness and tolerance, 2) understanding the mechanism of enhanced T cell repressor-receptor ligation induced signaling and the associated effects on effector T cell function and differentiation, and 3) characterizing the therapeutic potential of negative regulator over-expressing DCs using a T cell mediated autoimmune disease model. Public Health Relevance: This project will exploit the professional antigen presenting properties of dendritic cells (DCs) and the powerful inhibitory signals of T cell repressor-receptors (negative regulators) combined to achieve the goal of modulating T cell function and inducing antigen specific T cell hypo-responsiveness and tolerance. This will be achieved by exogenously over-expressing DCs with ligands specific for T cell repressor-receptors (CTLA-4, PD-1 and BTLA) and loading them with the antigen(s) of interest for actively suppressing T cells though delivering inhibitory signals during antigen presentation. Inducing antigen specific T cell tolerance by generating adaptive hypo-responsive and regulatory T cells will be the most effective way of treating autoimmunity and graft rejection and, we believe that, our proposed approach will be one of the most efficient ways of achieving that goal. Moreover, this approach would be one of the efficient ways to study the effect of enhanced engagement of T cell repressor-receptors during antigen presentation. Our study will have an immense impact on approaches to treat autoimmunity without risking opportunistic infections as well as by eliminating the need for a life-long treatment using general immunosuppressive agents.