PROJECT SUMMARY CD8+ T cells are a fundamental component of the anti-tumor response; however, tumor-infiltrating CD8+ T cells (TIL) are rendered dysfunctional by the tumor microenvironment. CD8+ TIL display an exhausted phenotype with decreased cytokine expression and increased expression of co-inhibitory receptors (IRs), such as PD-1 and Tim-3. The acquisition of IRs marks the progression of dysfunctional TIL from progenitors (PD1lo) to terminally exhausted (PD1hiTim3+). We have described a distinct increase in bivalent genes in terminally exhausted CD8+ TIL, which are characterized by presence of both the active mark H3K4me3 and the repressive mark H3K27me3, as well as low gene expression. While this state has traditionally been found in pluripotent cells and described as a “poised” state, new research suggests that it is not specific to stem-like cells. The increase in bivalent chromatin in terminally exhausted cells suggests increased methylation in response to the tumor microenvironment. We suspect that hypoxia-mediated regulation of histone modifiers is responsible for this increase in bivalent genes and plays a critical role in mediating dysfunction. Understanding the role of bivalent genes and their regulation will provide crucial information about the regulation of exhaustion in the tumor microenvironment. This proposal seeks to use a combination of traditional immunology techniques and sequencing-based experiments to better understand the regulation of these chromatin features. We hypothesize that dysregulated chromatin modifiers of H3K27me3 drive bivalency and exhaustion in terminally exhausted T cells and can be targeted to improve anti-tumor immunity. We will test this hypothesis in two aims; we will 1) determine whether inhibition of H3K27 demethylation in hypoxia is sufficient to drive bivalency and terminal exhaustion using demethylase inhibitors and innovative in vitro assays of T cell exhaustion. We will 2) determine whether Ezh2 can be targeted to prevent bivalency in exhaustion using both inhibitors of Ezh2 activity and inducible deletion of Ezh2 in in vitro and in vivo systems, respectively. The presence of bivalent genes specific to terminally exhausted TIL supports the notion that while exhausted cells are epigenetically resistant to immunotherapy, such as anti-PD1 treatment, chromatin and transcriptional regulators can be targeted to reinvigorate exhausted TIL by increasing expression of key functional genes to improve the anti-tumor response.