Project Summary CD8 T cells are a critical component of an effective anti-tumor response. Therefore, a major goal in cancer immunology, and more broadly in immunobiology, has been to understand how specific disease contexts govern T cell fate and function. A growing and largely descriptive atlas of chromatin accessibility has begun providing rich insight into the epigenetic landscape of CD8 T cells, yet it remains a challenge to understand cause-and-effect relationships from such data. Furthermore, T cell fate specification, like many cellular differentiation processes, develops with a continuum of phenotypic and functional intermediate states. This prompts the question, which epigenetic mechanisms are causal in driving state transitions within CD8 T cells? What are the earliest signaling pathways that drive commitment to particular T cell phenotype? How does tissue context shape anti-tumor T cell immunity? Our proposal addresses these questions by leveraging loss- of-function (LOF) and gain-of-function (GOF) approaches for enhancer editing to identify how, when and where CD8 T cell fate commitment happens in vivo. As an initial application, we will use this framework to understand the context-specific epigenetic mechanisms governing exhausted CD8 T cell differentiation in the tumor microenvironment. We focus in particular on comparing progenitor exhausted T cells in melanoma vs. hepatocellular carcinoma since these two diseases have markedly different tissue-specific signaling, have differential responsivity to immune checkpoint blockade, and most importantly, induce both shared and tumor- specific patterns of epigenetic changes. Although this project will yield fundamental insights into T cell regulation, we also aim to extend epigenetic reprogramming to preclinical models of adoptive T cell therapy with potential therapeutic application. The novel integration of technologies to achieve these goals promises to be useful for diverse disease contexts where CD8 T cells play a role, in cancer and beyond.