SUMMARY: CD8 T cells are a critical part of the immune system that protect against intracellular pathogens and cancer. This protection is achieved by the T cell’s ability to target and kill tumor cells or cells infected with a pathogen. Upon clearance of the diseased cells, pathogen-specific CD8 T cells can persist for the life of the host, ready to rapidly recall their killing functions if the source of the antigen returns. This poised state of memory T cells is the basis for long-lived immunity. However, if the source of the disease is not initially cleared, as occurs during chronic infections or cancers, the killing functions of pathogen-specific CD8 T cells are progressively reduced, commonly referred to as T cell exhaustion. This reduction in T cell mediated killing limits the ability of the immune system to control tumor progression. Recent breakthroughs in our understanding of T cell exhaustion have revealed that the non-functional state can be temporarily reversed by therapies that block receptor signaling (PD-1) on the T cell, enabling T cell mediated tumor control. In light of the tremendous therapeutic effect PD-1 blockade has on controlling tumor progression, the FDA has recently approved it for clinical use. While PD-1 blockade therapy clearly controls tumor progression, the temporarily reactivated CD8 T cells retain a memory of the non-functional state. Therefore, a current challenge for the field is to identify the cell-intrinsic properties that maintain T cell exhaustion after PD-1 treatment. We have recently demonstrated that epigenetic modifications (modifications to the genome that are maintained during cell division) acquired during prolonged antigen exposure reinforces T cell exhaustion by maintaining exhaustion-specific gene expression programs. We hypothesize that these epigenetic programs are a major barrier for therapeutic strategies that aim to reprogram exhausted tumor-specific T cells. Therefore, the aims of our proposal are 1) To identify de novo DNA methylation programs that reinforce commitment of T cell exhaustion in mouse and human tumor-specific CD8 T cells. 2) To erase de novo DNA methylation programs that constrain rejuvenation of exhausted CD8 T cells during immune checkpoint blockade (ICB). 3) To determine if CAR T cell exhaustion is regulated by de novo DNA methylation. The research proposed here will broadly identify gene expression programs in antigen-specific CD8 T cells that inhibit anti-tumor functions, and will provide new insight into the cell-intrinsic mechanisms for maintenance of exhaustion programs. These studies will provide a foundation for developing methods to reprogram exhausted CD8 T cells to sustain effector potential during and after immune checkpoint blockade and CAR T-cell therapies.