Summary. Transplantation is a curative treatment for end-stage organ failure, but rates of significant morbidity and graft loss due to immunosuppression-induced toxicities remain unacceptably high. Belatacept, a CTLA-4Ig fusion protein and the first new therapy for immunosuppression in transplantation in over 20 years, offers a significant benefit to renal transplant recipients in that it carries a 43% reduced risk of death or graft loss after 7 years as compared to calcineurin inhibitor-based regimens. However, belatacept confers a significantly increased risk of acute rejection as compared to calcineurin inhibitors. The two main cellular subsets that have been implicated in belatacept-resistant rejection are 1) Foxp3+ Treg and 2) CD8+ memory T cells. First, numerous studies have shown that because belatacept/CTLA-4Ig binds to the CD80/86 ligands for CTLA-4, the CTLA-4-mediated suppressive function of Treg is compromised under these conditions. Second, a separate body of work has shown that distinct CD8+ memory T cell populations exhibit reduced requirements for CD28 costimulation. Studies in mouse, NHP, and humans have identified CD8+ alloreactive memory T cells as forming a barrier to graft acceptance during transplantation. As such, identifying alternate pathways that 1) augment Treg suppressive function in the context of CTLA-4 blockade and 2) control memory CD8+ T cell populations during rejection or tolerance are clinically relevant questions in transplantation. Our preliminary data show that that while agonism of TIGIT alone had no effect on graft survival, agonism of TIGIT mitigated the costimulation blockade-resistant rejection observed in the setting of treatment with CTLA-4Ig, resulting in prolonged allograft survival. However, the mechanisms underlying these observations are not understood. What is the impact of TIGIT agonism on Tregs in the context of CTLA-4Ig? What are the cellular and molecular pathways downstream of TIGIT agonism on Tregs? Is there a cell-intrinsic role for TIGIT agonism on memory CD8+ T cells in the context of CTLA-4Ig? How does TIGIT agonism of Foxp3+ Treg impact memory CD8+ T cells? What is the impact of belatacept treatment of human T cells isolated from transplant recipients on these pathways? In this proposal, we will parse apart the effect of TIGIT agonism on Foxp3+ Treg (which could secondarily impact graft-reactive CD8+ T cell responses) and a cell-intrinsic effect on CD8+ memory T cells using conditional knockouts of TIGIT on either Foxp3+ Treg or memory CD8+ T cells. This proposal will answer these fundamental questions, thereby filling a gap in our current understanding of the role of TIGIT coinhibition in T cell alloimmunity during transplantation. Finally, we propose to directly test the clinical relevance of these findings and hypotheses by interrogating the impact of TIGIT agonism on both Foxp3+ Treg and CD8+ effector/memory T cells isolated from belatacept-treated human renal transplant recipient...