ABSTRACT Many inflammatory diseases and cancer are driven by dysregulation of CD4 T helper cells (Th cells). Our group has shown that different T cell subsets utilize distinct metabolic programs. Proinflammatory Th1 and Th17 T cells utilize aerobic glycolysis and increase lipid synthesis, while anti-inflammatory T regulatory cells (Tregs) utilize fatty acid oxidation and oxidative phosphorylation (OXPHOS). Importantly, the lab has shown that manipulation of metabolic pathways can affect Th cell differentiation, thus offering new approaches to modify immune-related diseases. Fatty acid and mitochondrial metabolism are critical processes that may be targeted to alter CD4 T cell fate. Utilizing established in vivo CRISPR screens in models of inflammatory bowel disease and lung inflammation, I identified several metabolic genes involved in lipid metabolism for this potential purpose. These screens found mitochondrial fatty acid synthesis (mtFAS) and the mtFAS enzyme Mitochondrial trans-2-enoyl- coenzyme A reductase (Mecr) to be important in T cell-mediated inflammation. mtFAS is a pathway parallel to cytosolic fatty acid synthesis that creates acyl-ACP and lipoic acid or longer fatty acid chains crucial for electron transport chain assembly and OXPHOS. Mecr is the final mtFAS enzyme and humans with MECR loss-of- function mutations develop a rare neurometabolic disorder. Mechanistically, Mecr-deficient skeletal myoblasts have reduced OXPHOS and Mecr-knockout in patient fibroblasts and drosophila cause increased levels of iron and impaired iron-sulfur (Fe-S) cluster biogenesis. Despite cytosolic fatty acid synthesis being well-characterized in T cells, it is currently unclear what effect Mecr and mtFAS play in immune cells. Therefore, I proposed studying the effects of Mecr and mitochondrial fatty acid synthesis on T cell function and metabolism. In a Th17 model of transfer inflammatory bowel disease, Mecr-knockout cells were depleted compared to non-targeting control cells in the spleens, mesenteric lymph nodes, lamina propria, and intra-epithelial lymphocytes. Mecr-knockout cells had lower Tbet expression and reduced IFNγ+ T cells, showing reduced Th1 function. In addition, Mecr-knockout cells had reduced proliferation, increased rates of cell death by apoptosis, and an increase in intracellular iron. These preliminary data demonstrate that Mecr plays a key role in inflammatory T cells as a rate limiting step in mtFAS. I hypothesize that mtFAS and Mecr activity supports TCA cycle flux and are required for the mitochondrial metabolism of Th1 CD4+ T cells. I will utilize control and Mecrfl/fl; Cd4cre conditional knockout mice that I generated and validated for my experiments in which Mecr will be knocked out specifically in T cells. I have also developed and utilized single-guide CRISPR/Cas9 knockouts for Mecr and have validated their functionality. I will: (1) Test the requirement of Mecr expression in the differentiation and function of CD4+ T cells ...