PROJECT SUMMARY/ABSTRACT There is ample evidence that patients with melanoma can develop immune responses directed against antigens expressed by their tumor. However, high tumor antigen (TA)-specific cytotoxic T cell (CTL) frequencies often fail to induce melanoma rejection. Understanding the failure of TA-specific T cells to promote tumor regression is, therefore, critical for the design of novel therapeutic interventions aimed at overcoming tumor-induced immune escape. Among the numerous mechanisms of tumor-induced immunosuppression that contribute to the resistance of tumors to CTLs, a number of studies in animals and humans have suggested the role of inhibitory pathways in impeding CTL effector functions. TA-specific CD8+T cells in peripheral blood lymphocytes (PBLs) or tumor-infiltrating lymphocytes (TILs) of patients with advanced melanoma upregulate the expression of multiple inhibitory receptors (IRs), including PD1, Tim3, and TIGIT. We have previously shown that these IRs interact with their ligands expressed in the tumor microenvironment to impede of TA- specific CD8+ T cell expansion and functions in the context of chronic antigen stimulation. In particular, we have shown for the first time that TIGIT blockade adds to PD-1 blockade to increase TA-specific CD8+T cell functions in vitro. We have also reported that CD8+TILs in metastatic melanoma downregulate the costimulatory receptor CD226, which competes with TIGIT for binding to the same ligands. Interestingly, TIGIT is also upregulated by highly suppressive CD4+ regulatory T cells (Tregs). The mechanisms supporting the role of TIGIT in regulating Tregs and CD8+T cells remains poorly understood. Based on novel findings, we propose to investigate the mechanisms supporting the role of TIGIT and TIGIT/CD226 imbalance in regulating Tregs and CD8+T cells, respectively, in melanoma. We will also investigate the role of CD226 in regulating anti-tumor immune responses and tumor rejection upon dual PD-1/TIGIT blockade in vivo in mouse-bearing melanoma. Finally, we will test the efficacy of novel Fc-engineered anti-mouse TIGIT antibodies to target Tregs or CD8+TILs in vivo, and promote tumor rejection in combination with PD1 blockade. Collectively, the information derived from the outlined studies will serve as a rationale for the development of novel therapeutic strategies to reverse tumor-induced T cell dysfunction in patients with advanced melanoma and increase the likelihood of clinical benefits.