PROJECT SUMMARY CD8 T cells specific for cancer cells are found within human tumors, but despite their presence, tumors progress, suggesting that T cells become unresponsive. To design predictably effective immunotherapies, we must elucidate the mechanisms controlling tumor-specific T cell dysfunction. We previously demonstrated that T cells in tumors enter an epigenetically encoded dysfunction state that becomes resistant to therapeutic reprogramming and found that TOX, a DNA-binding protein, is a key regulator enforcing the dysfunctional state. The factors that drive TOX expression, and how TOX precisely establishes the dysfunction program remain largely unknown. TCR signal strength impacts T cell differentiation, and while we know that antigen chronicity is a key driver of TOX-driven T cell dysfunction, we do not know how signal strength of chronic tumor antigen impacts TOX-driven dysfunction and amenability to immunotherapeutic reprogramming. In this application, we will determine how signal strength regulates TOX and TOX-driven dysfunction programs in mouse and human tumors, ask how TOX induces and maintains dysfunction, and target TOX and its downstream mediators to uncover the mechanisms underlying dysfunction imprinting. To achieve these goals, we will utilize clinically relevant genetic cancer mouse models and track T cells longitudinally within progressing tumors while encountering tumor antigens with varying signal strength. We will employ transcriptomic and epigenomic methods and innovative protein degradation strategies to determine what controls TOX, how TOX induces and/or maintains dysfunction, and how TOX downstream mediators regulate the epigenetic programs associated with plasticity and dysfunction imprinting. We will leverage human neoantigen- specific tumor-infiltrating T cell resources to understand how TCR signal strength determines TOX- dependent molecular signatures and functional states of T cells in human tumors. Importantly, we will test and design strategies to target TOX and TOX downstream mediators to improve cancer immunotherapy.