PROJECT SUMMARY/ABSTRACT Activating a patient's immune system to target cancer cells has emerged as an effective therapeutic strategy. However, many cancer subtypes, including epithelial malignancies such as pancreatic, breast, and colorectal cancers still respond poorly to existing T-cell therapies. Why immune-based strategies have shown such poor success in treating these common cancers is not known. In addition to characterizing the inhibitory checkpoints that restrain T-cell activation, our laboratory previously demonstrated that activated T-cells depend on extracellular glucose and amino acids to sustain their viability and effector function. Over the last several years, we have been studying the metabolic properties of the multiple cell types present in the tumor microenvironment. In addition to tumor cell consumption of glucose and free amino acids, we have found that tumor-associated fibroblasts can deplete glutamine and other nonessential amino acids from the extracellular space. In particular, the tumor and fibroblast-dependent depletion of glutamine and cystine (cysteine) makes it difficult for cells in the tumor microenvironment to maintain protein translation and redox homeostasis. We have uncovered multiple novel mechanisms by which tumor and stromal cells adapt to amino acid depletion, including utilizing extracellular proteins as a source of amino acids through macropinocytosis, adaptive alterations in translation, and activation of the transsulfuration pathway to maintain endogenous cysteine levels and glutathione levels. Whether tumor-infiltrating immune cells can use any or all of these adaptations is unknown. Our preliminary evidence suggest that T-cells are unable to maintain their viability and effector function when depleted of either glutamine and/or cysteine. Therefore, we hypothesize that the tumor microenvironment drives immunosuppression by depleting these non-essential amino acids. To address this hypothesis, we propose three Specific Aims: 1) Study whether cancer-associated fibroblasts contribute to immunosuppression by depleting extracellular free amino acids, 2) Determine the mechanism by which glutamine depletion impairs anti-tumor immune function, and 3) Examine whether cysteine depletion in tumor- infiltrating immune cells results in impaired mitochondrial redox homeostasis and/or reduced effector function. Through these studies, we hope to demonstrate how depletion of glutamine and cysteine drives immunosuppression within the tumor microenvironment and identify mechanisms by which these amino acids can be restored to levels that will sustain an anti-tumor immune response.