Project Summary Commensalism in the gut has been suggested to influence many aspects of human health and diseases. It is now widely accepted that gut commensal microbes have a crucial impact on immune homeostasis in the gut. Emerging evidence suggest that the gut microbiota shapes systemic immunity, but the underlying mechanisms are poorly understood. Recent reports from other groups and our preliminary findings indicate that the gut microbiome and its composition are important factors that determine responsiveness to immune checkpoint blockade 1-3. Immune checkpoint inhibitors are novel therapeutic interventions which reinvigorate tumor- specific T cells to attack tumor cells by targeting co-inhibitory pathways in T cells, such as PD-1:PD-L1 and CTLA-4. Despite the clinical success of these checkpoint blockade immunotherapies, a significant fraction of patients do not respond to the checkpoint inhibitors 4. Recent studies showed that one of the critical determinants of responsiveness to checkpoint blockade may be the composition of the gut microbiome. However, how the gut microbiota modulates systemic immune responses to tumor is not known. The goals of this project are to determine mechanisms by which the gut microbiome regulates anti-tumor immunity and response to PD-L1 blockade. To achieve these goals, we will 1) Use gnotobiotic techniques and antibiotics to identify human gut microbes that promote tumor clearance upon PD-L1 blocking antibody treatment; 2) elucidate cellular and molecular mechanisms of gut microbiota dependent anti-tumor immunity, focusing on how changes in the gut microbiota controls co-inhibitory pathways that regulate anti-tumor immunity; and 3) investigate how microbial products from the gut contribute to anti-tumor immunity. The proposed study will provide critical insights into mechanisms by which the gut microbiota regulates anti-tumor immunity, and new therapeutic strategies for patients who do not respond to PD-1 pathway blockade.