SUMMARY T cell exhaustion (TEX) is a dysfunctional state marked by reduced cytokine production resulting from persistent antigen stimulation in chronic viral infections and cancer. The differences between TEX cells and functional effector T cells (TEFF) is marked by significant change in their epigenetic landscapes and a global decline in histone acetylation as TEX cells form during chronic viral infection. The major goal of this project is to elucidate mechanisms that induce these epigenetic changes that drive CD8+ T cell exhaustion, with a focus on the role of nutrient metabolism as a major regulator of histone acetylation and gene expression in TEX cells. Histone acetylation requires a nuclear acetyl-CoA pool, which is sourced from extracellular nutrients including acetate, citrate, and pyruvate and relies on the nuclear location of acetyl-CoA synthetases. Thus, how cells metabolize particular nutrients in the surrounding environment can dictate specific epigenetic changes to promote or suppress T cell function. We propose that changes in how T cells metabolize specific nutrients underlies key epigenetic changes that lead to CD8+ T cell exhaustion. In particular, we hypothesize that expression of acetyl-CoA synthetase short-chain family member-2 (ACSS2), which synthesizes acetyl-CoA from acetate, is critical for maintaining histone acetylation and activation of TEFF genes because ACSS2 expression and H3K27acetylation plummets as TEX cells form. In contrast, the expression of another acetyl-CoA synthetase ATP citrate synthase (ACLY), which converts citrateàacetyl-CoA, is sustained in TEX cells. This result suggests that either ACLY activity alone is insufficient to maintain TEFF gene expression or that it actually promotes expression of TEX signature genes. This proposal will focus on the role of ACSS2 and ACLY on the epigenetic landscape of TEFF and TEX cells and their effector functions. For both ACSS2 and ACLY, we will knock out or overexpress these enzymes and study their effects on T cells in acute and chronic LCMV infections that induce TEFF and TEX cells, respectively. We will use a combination of flow cytometry and ChIP-Seq to determine how these enzymes affect the epigenetic landscape, histone acetylation and effector functions in TEFF and TEX cells. We will also examine how TEFF and TEX cells differentially utilize relevant nutrients to modulate their acetylated histone pool using carbon isotope tracing. Lastly, we will examine if it is possible to metabolically reprogram TEX cells in order to rejuvenate them back to TEFF cells by improving nutrient availability and drugs that modify the epigenome. Understanding the link between nutrient metabolism and epigenetic change in T cells would reveal how a ‘metabolic code’ influences the histone code that governs T cell differentiation, and open the possibility of metabolically rejuvenating TEX cells to fight chronic viral infections and cancer.