PROJECT SUMMARY/ABSTRACT T helper (Th) cell differentiation into specialized lineages is critical for effective immune responses against pathogens, but dysregulated Th differentiation can contribute to autoimmune diseases. Emerging evidence suggests that metabolism can influence Th cell development and function, but which metabolites and metabolic pathways are involved remains poorly understood. Somatic mutations in the isocitrate dehydrogenase (IDH) enzymes contribute to the pathogenesis of acute myeloid leukemia (AML) and other malignancies via production of the ‘oncometabolite’ D-2-hydroxyglutarate (D-2HG). D-2HG blocks differentiation of malignant cells by inhibiting alpha-ketoglutarate (⍺KG)-dependent enzymes that regulate chromatin structure and gene expression. Recent reports suggest that D-2HG can influence the balance of Th17 and Treg differentiation, but the findings are contradictory and inconclusive. Whether CD4+ cells lacking IDH mutations produce physiologically relevant levels of D-2HG remains unclear. This raises the question as to whether analogous metabolic pathways might influence physiologic Th cell differentiation. Intriguingly, 2HG is a chiral molecule that can exist in either the D- or L- enantiomer. Although cancer-associated IDH mutants exclusively produce D-2HG, our lab and others have shown that virtually all normal and malignant cells produce L-2HG in response to hypoxia. Hypoxia-induced L- 2HG potently inhibits ⍺KG-dependent enzymes, including chromatin modifiers and regulators of hypoxia- inducible factor (HIF) stability. Nonetheless, physiologic sources and functions of L-2HG remain poorly understood. I discovered that activated Th cells produce L-2HG and that L-2HG levels are enhanced by hypoxia and hydrogen sulfide (H2S), microenvironmental factors that are prominent within the intestine. Furthermore, I found that genetic manipulations to elevate endogenous L-2HG in Th cells can promote T helper 17 (Th17) and impair regulatory T cell (Treg) differentiation. Thus, I hypothesize that CD4+ helper T cells produce L-2HG in response to specific microenvironmental cues to fine-tune the balance of Th17 and Treg cells and enhance inflammatory responses. I will rigorously test this hypothesis in two Specific Aims. Aim 1 will dissect the molecular mechanisms by which L-2HG regulates the balance of Th17 and Treg cells in vitro. In this aim, I will use novel genetically engineered mouse models (GEMM) that allow for tissue-specific manipulation of endogenous L-2HG levels to determine the effects of L-2HG on metabolism, chromatin structure, and gene expression. Aim 2 will elucidate how both T cell-intrinsic and microenvironmental L-2HG regulate CD4+ T cell- mediated inflammatory immune responses in vivo. Here, I will use novel GEMM to elevate and deplete L-2HG in Th cells or intestinal epithelial cells in models of intestinal bacterial infection and autoimmune colitis. The proposed studies will provide important insights in the ...