PROJECT SUMMARY/ABSTRACT The recent successes of checkpoint blockade therapies have highlighted the potential of immunotherapy for cancer treatment. Still, only about 30% of treatment recipients experience long term tumor regression. Thus, understanding the mechanisms that drive therapy resistance is essential for improving patient outcomes. We now understand that the efficacy of immunotherapy depends on the presence and persistence of functional immune cells within the tumor. Tumor-specific CD8 T cells are activated by tumor antigens and directly kill tumor cells. However, tumor infiltrating CD8 T cells encounter chronic antigen stimulation and a host of environmental stressors (e.g hypoxia, nutrient deprivation, etc) that suppress their function. This suppressive tumor microenvironment ultimately induces an altered CD8 T cell differentiation state called exhaustion, characterized by reduced effector function, immune inhibitory receptor expression, and a distinct epigenetic and transcriptional program. Our lab and others have demonstrated that the suppressive microenvironment drives mitochondrial stress and altered metabolism, which promotes CD8 T cell exhaustion. While we understand that mitochondrial insufficiency is a driver of CD8 T cell exhaustion, the mechanisms that underly this connection remain understudied. Interestingly, CD8 T cells accumulate lipids and repress lipolysis as they differentiate towards an exhausted state. In this study, we aim to understand the impact of increased lipid accumulation on CD8 T cell differentiation and function. Citrate transported from the mitochondria is converted to acetyl-CoA in the cytosol, where it acts as a substrate for de novo lipogenesis. Thus, citrate transport connects mitochondrial metabolism to cytosolic lipid synthesis and accumulation. Preliminary data show that in vitro inhibition of the mitochondrial citrate transporter (SLC25A1) preserves effector function in chronically stimulated T cells and reduces lipid content. These data suggest that as exhausted CD8 T cells may reprogram their metabolism to shuttle citrate from dysfunctional mitochondria, resulting in increased de novo lipogenesis and lipid accumulation in the cytosol. Thus, we hypothesize that chronically stimulated CD8 T cells inappropriately store mitochondrial citrate as lipids, which inhibits effector function and promotes differentiation to an exhausted state. We will test this hypothesis by (1) evaluating the impact of abolishing citrate transport on carbon utilization and effector function of CD8 T cells within the tumor microenvironment and (2) determining the role of stored lipids in CD8 T cell function. The insights gained by these studies will help us understand whether accumulated lipids represent ‘dead weight’ in exhausted CD8 T cells, or whether accumulated lipids represent an untapped source of fuel that may be the key to their reinvigoration.