SUMMARY Exploiting the immune system to eliminate cancer cells has been a goal for many years, but it has become apparent that tumors actively suppress immune cell functions. While inhibition of immunomodulatory receptors, such as through PD-1 checkpoint blockade therapy, holds tremendous promise, this treatment is effective in only a portion of patients. Factors that determine immune responsiveness against tumors remain largely uncertain. Our data show, however, that the metabolic demands of T cells may be a critical factor in the success of immunotherapy. We have shown that effector T cell (Teff) activation requires high rates of glucose and anabolic metabolism yet cancer cells and the tumor microenvironment can inhibit Teff metabolic pathways. This may represent a fundamental mechanism of tumor-mediated immune suppression. To better understand the influence of the tumor microenvironment on T cell metabolism and improve immunotherapies, we have examined tumor infiltrating lymphocytes (TIL) from surgically-excised human clear cell Renal Cell Carcinoma (ccRCC) tumor samples, a cancer responsive to PD-1 blockade and with a prognostic immune signature. ccRCC is highly associated with mutations and loss of the Von Hippel Lindau (VHL) tumor suppressor, which leads to stabilization of HIF1α and HIF2α and induction of a transcriptional pseudo-hypoxic response that alters the tumor to promote an immune suppressive microenvironment that can negatively impact ccRCC CD8 TIL function and anti-tumor immunity. We found CD8 TIL are abundant in ccRCC, yet these cells are uniformly PD-1high and functionally suppressed. In addition, CD8 TIL had multiple metabolic impairments and were unable to efficiently uptake glucose or perform glycolysis and had small, fragmented mitochondria that produced high levels of Reactive Oxygen Species (ROS). Importantly, neutralization of ROS or provision of the glycolytic end-product pyruvate could partially rescue ccRCC CD8 TIL function. Glutamine is also a key nutrient to support mitochondrial metabolism for T cells through glutaminolysis and we report here that inhibition or genetic deletion of the first enzyme in this pathway, Glutaminase 1 (GLS1), leads to a compensatory increase in glycolysis that can enhance cytotoxic CD8 function. This proposal will test the hypothesis that the ccRCC microenvironment impairs glycolysis and leads to accumulation of dysfunctional mitochondria in CD8 TIL and that rescue of TIL glycolysis will enhance T cell response to immunotherapy. We will study primary ccRCC tumors and mouse RCC models to: (1) Determine how mitochondria are dysregulated and impair activation and metabolism of ccRCC CD8 TIL; (2) Investigate if promoting glucose uptake or inhibiting GLS1 to enhance glucose metabolism can improve the metabolism and function of CD8 TIL; and (3) Test how PD-1 blockade therapy impacts T cell metabolism and functional populations in ccRCC. Together, these studies will establish the mechanism of m...