PROJECT SUMMARY/ABSTRACT Recent research has shown that metabolic byproducts of tumors prevent effector T-cells from destroying malignant tissue. The mechanisms by which this occurs is a currently an area of intense investigation. However, CD4+Foxp3+ Regulatory T-cells (Tregs), central regulators of anti-tumor immunity that accumulate within tumors, thrive under the same metabolic constraints that hinder effector T-cell responses. Our data shows that in conditions of extracellular acidification that mimic the tumor microenvironment (TME), Tregs preferentially survive while effector T-cells are potently inhibited. Extracellular acidification that is produced by tumors inhibits glycolysis, which is critical to effector T-cell metabolism, and suggests that a separate bioenergetic pathway is being utilized by Tregs to thrive within the tumor. Our preliminary studies suggest Tregs preferentially utilize fatty-acid (FA) uptake within the tumor, and may be an alternative energy source for Tregs in the TME. The objectives of this proposal are to: establish how the Treg program is stabilized under conditions of acidification, if they rely on exogenous FA to thrive in the TME, and if inhibition of FA production and extracellular acidification can alleviate glioma immunosuppression. The overall goal of Aim 1 is to determine why Tregs preferentially thrive under conditions of extracellular acidification. Using a previously established lineage tracking model of Foxp3 expression, we can verify if acidic conditions of the tumor are potentiating Treg survival or maintaining their expression of Foxp3. We will also compare the metabolic phenotype of Tregs compared to other T-cells subsets under acidic culture or ex-vivo from the TME. The two major epigenetic modifications required for Foxp3 expression are demethylation of its promoter regions, and acetylation of the histone binding its DNA. Using a series of biochemical and molecular biology assays, we will determine how acidity contributes to the epigenetic regulation of Foxp3. As Tregs are thought to preferentially obtain exogenous FA for the purposes of fatty-acid oxidation, Aim 2 will seek to determine if this metabolic pathway allows for Foxp3 survival and stability under conditions of acidity. Using established metabolic assays, we can determine how important FA uptake/oxidation is by Tregs under acidic culture and within the tumor. In Aim 3, we will determine how inhibition of extracellular acidification and fatty acid oxidation influence tumor induced immunosuppression. Inhibition of the enzyme Fatty Acid Synthase (FASN) effectively prevents de-novo FA generation, and in this aim we will inhibit glioma generated FASN using both shRNA expression and pharmacologic inhibition to understand the role de-novo fatty acid generation has on Treg function in glioma. We will also use the same approaches to understand how inhibition of a major contributor of tumor acidification, Carbonic Anhydrase IX, influences tumor induced immunosuppression.