PROJECT SUMMARY-ABSTRACT In new data presented in this proposal, we show that nerve driven, adrenergic stress signaling via the β-adrenergic receptors (β-ARs) inhibits responsiveness to checkpoint inhibitor therapy in two different transplantable tumor models. By reducing β-AR signaling using three separate approaches, (including through the use of a warmer room temperature, pharmacological antagonists of β-ARs, and β-AR knock out mice), we observed a significant remodeling of the immune microenvironment of tumors. From a tumor microenvironment (TME) largely devoid of CD8+ T cells, the TME became T cell rich, with an enhanced ratio of activated CD8+ cells to Treg cells, a change we associated with the improved sensitivity to anti-PD-1 checkpoint therapy. Other new, mechanistic data reveals that β-AR signaling suppresses metabolic reprogramming required for optimal T cell activation. These, and other novel data, raise the provocative hypothesis that reducing adrenergic signaling in the TME (by repurposing safe and well- studied β-blockers) could significantly increase the overall response rate of patients to immunotherapy. To rigorously expand these data to additional tumor models and to identify the mechanistic pathways involved, this revised proposal outlines a comprehensive set of experiments that will provide an in depth understanding of how adrenergic stress signaling significantly and broadly affects baseline immunity, and ultimately, the response to immunotherapy. Three interactive aims are proposed: Aim 1 will test the hypothesis that adrenergic stress signaling influences tumor formation or progression in autochthonous tumor models induced by either carcinogen or genetic manipulation wherein the host and tumor evolve together over a prolonged period of time. These experiments offer the unique opportunity to investigate the equilibrium phase of the interaction between host immune cells and tumor cells. Using these models, we will also test whether β-blockers can improve the efficacy of immune checkpoint inhibitors. In Aim 2, we will define how adrenergic stress influences the immune contexture of the TME. Aim 3 arises from our new mechanistic data showing that adrenergic signaling inhibits the bioenergetic changes required for both CD8+ T cell activation and effector function. Since metabolic reprogramming is required for an optimal T cell-mediated anti-tumor immune response, reducing adrenergic stress could provide a novel mechanism underlying the broad effects of adrenergic stress signaling seen in our studies. Impact: These data highlight the potential of adrenergic stress signaling to regulate the immune status of the tumor microenvironment and support the strategic repurposing use of clinically available β-blockers in patients to improve responses to immunotherapy.