Multiple sclerosis (MS) is a debilitating demyelinating disease of the central nervous system (CNS) that affects approximately 2.5 million people worldwide. US military personnel are at special risk to develop MS: the incidence rate in the US military population (12.9 per 100,000 person-years) is 1.7x higher than the civilian population, and 3x higher than the global population. Experimental autoimmune encephalomyelitis (EAE) is a widely studied animal model that shares many features of human MS. Tissue injury in EAE and MS is caused by inflammatory leukocytes that enter the CNS and destroy myelin. CNS-infiltrating, myelin-reactive CD4+ T cells play key roles in the pathology of MS. Although a number of MS treatments are available, due to the heterogeneity of the MS disease process, individual patient responses, and medication toxicities, there is a substantial unmet clinical need for improved therapeutics. Leukocyte differentiation and function is profoundly affected by the engagement of metabolic pathways for lipid or retinoid processing. Diacylglycerol O-acyltransferase-1 (DGAT1) is a metabolic enzyme that can catalyze the synthesis of triglycerides (via DGAT activity), and retinyl esters (via acyl CoA:retinol acyltransferase (ARAT) activity). Little is known regarding the role of DGAT1 in leukocyte biology. We published that DGAT1 is selectively upregulated in activated mouse CD4+ T cells both in vitro and in vivo during EAE. DGAT1 is selectively expressed in brain lesions and CD4+ blood T cells obtained from MS patients. Both conventional DGAT1 KO mice and mice we generated with CD4+ T cell-selective deficiency in DGAT1 (tamoxifen-treated CD4CreERT2+/-DGAT1fl/fl) were protected against EAE and histological CNS inflammation and demyelination. Furthermore, DGAT1 pharmaco-inhibition can be used therapeutically to improve clinical EAE. Our preliminary studies indicate that genetic deficiency in DGAT1 leads to increased regulatory T cell (Treg) numbers and frequency; that polyfunctional Treg proliferation is enhanced in vitro in DGAT1 KO cells; and that suppressor activity is increased in DGAT1 KO Treg from normal and EAE mice. We therefore hypothesize that DGAT1 serves as a novel immune checkpoint for Treg that can be targeted to enhance Treg proliferation and activity to combat MS. In Aim 1, we propose to investigate if DGAT1 small molecule antagonists enhance human and mouse regulatory T cell proliferation/suppressor activity and extend remission in the relapsing-remitting PLP-SJL EAE model. Notably, DGAT1 inhibitors are already being tested in clinical trials for treatment of obesity-associated diseases. Thus, the studies outlined in Aim 1 have the potential to prompt swift repurposing of existing, safe, well-characterized DGAT1 inhibitors and offer real translational opportunities to improve the lives of Veterans and other patients with MS. In Aim 2, we will define the role of DGAT1 in regulatory T cell trafficking to the CNS during demyel...