PROJECT SUMMARY Cancer and chronic virus infections are significant causes of morbidity and mortality. While cytotoxic CD8 T cells are the main killers of tumors or virus-infected cells, persistent stimulation of CD8 T cells during chronic infections or cancer results in a gradual loss of their cytotoxic function as T cells progress towards a fully- exhausted state. While immune checkpoint blockade (ICB) therapy allows partially-exhausted CD8 T cells to functionally recover by blocking inhibitory signals, terminally-exhausted T cells remain nonresponsive to this therapy. The inability of terminally-exhausted T cells to recover after ICB may explain why many cancer patients fail to mount durable responses to ICB. We recently showed that de novo DNA methylation programming is causally linked to the progression of T cells toward terminal exhaustion and poor response to ICB. Importantly, we discovered that targeting T cell-intrinsic epigenetic programs synergized the efficacy of ICB during chronic infection or cancer. Yet, the following questions represent major gaps in our current understanding of T cell exhaustion: (1) How are these epigenetic changes acquired in exhausted T cells? (2) What are the upstream signals that regulate the specificity of de novo DNA methylation programs in exhausted versus functional T cells? (3) Can we reverse the epigenetic programming in exhausted T cells to the functional state while avoiding transformation? Bridging these gaps will allow us to identify and target factors that apply “epigenetic brakes” to CD8 T cell function. To address these questions, we have developed a novel in-vitro model of stable human T cell dysfunction as a tractable tool that can guide our in-vivo experiments by providing first-line mechanistic studies. First, we will employ cutting-edge approaches, such as CRISPR-Cas9 gene editing and retroviral transduction, to test the hypothesis that specific tumor microenvironmental signals regulate epigenetic programming in persistently stimulated CD8 T cells, which promotes their resistance to ICB therapy. We aim to block and/or revert the progression toward the terminally-exhausted state by targeting components of this signaling pathway while promoting counteracting pathways. Second, we aim to rebalance specific microenvironmental signals to restore functionality and response in terminally-exhausted T cells. Using novel in- vitro model systems of T cell dysfunction and complementary in-vivo models of chronic viral infection and cancer, as well as cutting-edge technologies to profile DNA methylation, open chromatin landscape, and transcriptome in CD8 T cells, our proposed studies can determine if targeting specific factors can remodel chromatin back into an accessible state at effector and/or stemness-associated genes, leading to functionally-rejuvenated T cells. These proposed studies will provide insights into how epigenetic programming can be reversed during progression to T cell exhaustion that ca...