SUMMARY Exploiting immunity to eliminate cancer cells offers tremendous new therapeutic opportunities, including the widely employed immune checkpoint blockade (ICB) agents. These approaches are challenged, however, by the multitude of mechanisms through which tumors can suppress anti-tumor immunity and render them effective in only a portion of patients. We have shown that effector T cells require high rates of glucose uptake for anabolic metabolism, and it is now apparent that cancer cells and the tumor microenvironment (TME) disrupt anti-tumor immunity in part through metabolic immune suppression. To address this barrier to immunotherapy, we examined tumor infiltrating lymphocytes (TIL) from surgically excised samples of human clear cell Renal Cell Carcinoma (ccRCC), a cancer with moderate rates of ICB response that is characterized by loss of the Von Hippel-Lindau (VHL) tumor suppressor. These tumors allowed us to show that both glucose and glutamine are available in the TME and while glucose metabolism promotes effector T cells, metabolism of glutamine restrains T cell effector function. It is unclear which glutamine-dependent enzymes or metabolites suppress T cells, but we found that Glutaminase-deficiency altered histone methylation and reduced expression of Pik3ip3, a PI3K- inhibitory protein that suppresses PI3K/mTORC1 signaling and production of inflammatory effector cytokines. To further explore mechanisms of T cell suppression by glutamine, we performed an in vivo CRISPR screen in primary TIL and found loss of Glutamine Synthetase among all glutamine-metabolizing enzymes to most effectively increase TIL accumulation. We also directly defined cell type-specific glucose and glutamine usage in the TME using radiolabeled Positron Emission Tomography tracers. In contrast to classic Warburg metabolism, tumor associated macrophages (TAM) were the dominant consumers of glucose, followed by TIL, and cancer cells, which instead preferentially consumed glutamine. Interestingly, loss of Vhl did not increase glucose uptake of RCC cells in vivo but instead increased glucose uptake in TIL and TAM. To explore these pathways in patients undergoing ICB therapy, we next performed high dimensional CyTOF analyses of peripheral blood from a longitudinal cohort of patients before and 3 weeks after start of therapy. This approach specifically identified rare but highly proliferative ICB-responsive CD8 and CD4 T cells with elevated mitochondrial potential. Based on these findings, we hypothesize that RCC genetics drive a metabolically immunosuppressive TME with abundant glutamine that suppresses PI3K signaling to impair T cell effector differentiation and function. We will study primary human ccRCC tumors and mouse RCC models to: (1) Test how nutrients in the ccRCC TME and tumor genetics influence TIL function and metabolism; and (2) Determine how glucose and glutamine metabolism in the TME promote or suppress anti-tumor immunity. Together, these studies wil...