PROJECT SUMMARY/ABSTRACT Treg cells are enriched within the tissues, one of their main sites of action and possess a number of adaptations that allow them to thrive and maintain a stable lineage identity. One of these critical features is an altered metabolic profile. We have explored how the metabolism of various tissue environments, especially within tumors, stabilize regulatory T cell function. Tumors produce a local metabolic environment that is toxic to conventional, effector T cells, but regulatory T cells thrive there, being highly proliferative and maintaining stable function. Metabolic derangement of cancer cells and the potentiation of regulatory T cell function are linked: we have recently shown that Treg cells are supported by tumor-derived metabolites, most notably lactic acid. Treg cells eschew glucose metabolism, upregulating genes allowing them to withstand lactic acid-rich conditions and utilize this metabolite to fuel their function. Foxp3-restricted deletion of the lactate transporter monocarboxylate transporter 1 (MCT1, encoded by Slc16a1), hindered Treg function within tumors, resulting in a more immunologically active environment. Importantly, Treg cell-targeting immunotherapies like CTLA-4 blockade drives Treg cells to utilize glucose rather than lactate. Notably, this Treg utilization of glucose vs. lactate was not limited to tumors, but also found in the peripheral tissues of mice. While deletion of MCT1 resulted in no autoimmunity at the steady state, MCT1-deficient Treg cells were unable to sufficiently control intestinal inflammation in a T cell transfer model. Even in isolation, high glucose concentrations can hinder Treg cell function and stability, while lactate can protect against these differentiation events. Mechanistically, lactate broadly supports Treg cell proliferation and function, but how alternative pathways like lactate metabolism support and drive Treg cell identity remains unclear. Treg cells are not solely programmed by Foxp3, but rather rely on an established epigenetic landscape that supports their function, both in where Foxp3 can bind but also other key transcription factors. It now is clear that metabolic intermediates play critical roles in epigenetic remodeling, as histones can be either directly modified by metabolites (acetylation) or modified through metabolic processes (demethylation requiring αKG). Recently, lactate itself has been shown to directly modify histones, although the epigenetic consequences of histone lactylation remain incompletely described. Our preliminary data suggest that Treg cells harbor elevated lactylation of histones in an MCT1-dependent manner, and that Treg cells with increased lactylation harbor a more stable Treg cell signature. Here we will address the hypothesis that metabolites, most notably lactate, enriched in the tissues drive regulatory T cell functional identity, using in vivo systems in which Treg cells are either unfavorably stabilized (cancer) or struggl...