PROJECT SUMMARY Response to immunotherapy remains less than 20% for Oral Cavity Squamous Cell Carcinoma (OSCC). The poor response is in part due to dysfunctional cytotoxic T cell activity resulting from terminal exhaustion as a response to chronic exposure to tumor antigen. New strategies are needed to sensitize OSCC to immunotherapy to improve response rates and overall survival. Neurotransmitters of the parasympathetic nervous system, such as Acetylcholine (Ach), can be secreted from non-neuronal tissues, including lymphocytes, which are abundant in microenvironment of OSCC. These lymphocytes express choline acetyltransferase (ChaT), the enzyme catalyzing the rate limiting step of Ach production. Ach binds to two classes of receptors, muscarinic and nicotinic receptors, to mediate downstream responses. Stimulation of muscarinic receptors by Ach restrains pancreatic and breast cancer but promotes gastric and late stage prostate cancer. The contribution of Ach signaling to the promotion of OSCC is poorly understood. The goal of this proposal is to elucidate the role of Ach signaling in maintaining homeostasis of T lymphocytes in the tumor immune microenvironment in OSCC. Preliminary studies have uncovered a putative autocrine pathway of signaling from Ach secreting CD8+T cells via the cholinergic muscarinic receptor-1 (CHRM1) supporting the central hypothesis that autocrine Ach-CHRM1 signaling by ChaT+ CD8+ T cells sustains the OSCC anti-tumor immune response by replenishing the memory T cell pool. The central hypothesis will be tested by pursuing two specific aims: 1. determine if loss of lymphocyte derived Ach and muscarinic signaling promotes OSCC growth and impairs T cell infiltration into the tumor immune microenvironment (TIME) and 2. determine if autocrine Ach-CHRM1 signaling on T lymphocytes maintains long-term activation potential and prevents exhaustion by maintaining the memory T cell pool. In the first aim the effect of targeted disruption of the Ach- CHRM1 pathway in CD8+T cells on orthotopic OSCC tumor growth kinetics will be evaluated. Topography of the TIME will be analyzed using quantitative multiplex immunofluorescence. For the second aim ex vivo activation and proliferation assays will be used to asses T cell dysfunction longitudinally over the course of tumor progression in OSCC models with targeted disruption of Ach-CHRM1 signaling in CD8+ T lymphocytes. The proposed research examines a role for non-neuronal, lymphocyte derived, Ach in maintaining anti-tumor immune responses by maintaining the memory T cell pool and preventing terminal exhaustion that can lead to failure of immunotherapy treatment.