Project Summary The cGAS/STING pathway is an essential mediator of many anti-viral and anti-tumor immune responses because it senses cytosolic double stranded DNA (dsDNA), which can accumulate as a result of infection, tumor cell death, or cell stress-induced DNA leakage from the nucleus or mitochondria. STING interacts with metabolic regulators of T cell activation, differentiation, and plasticity, and was recently shown to promote CD4+ T cell differentiation into inflammatory Th1 and Th9 cells with enhanced functions. There are almost no reports of STING activation in regulatory T cells (Tregs), which have an opposing, but crucial, role in suppressing excessive inflammation. Inflammation can also coincide with elevated temperatures (i.e. fever-range temperature, FRT, 39°C), which can cause self-DNA leakage, yet the effects of FRT on STING in Tregs are unknown. Tregs have a unique capacity to be reprogrammed into inflammatory effector-like cells by an increase in glycolysis, which suppresses the lineage-driving transcription factor FoxP3, and in turn, inhibits their suppressive function. The goal of this research is to dissect the mechanisms intertwining STING activation and FRT and their effect on Treg function and metabolism. We propose to accomplish this through two Specific Aims: 1) We will establish the effects of STING activation at physiological temperatures on Treg function and metabolism. We will first profile transcriptional and epigenetic changes in STING-activated Tregs to unbiasedly identify mechanisms underlying the phenotype observed in our preliminary data. Effector functions of STING-activated Tregs will be evaluated in vitro and in vivo. Metabolic reprogramming will be assessed in vitro by performing metabolic flux analyses of STING-activated Tregs and comparing the effect of metabolic inhibitors on STING-activated Treg function. 2) We will investigate the effect of FRT on STING activation in Tregs via functional and metabolic analyses. We will first determine if FRT alone activates the cGAS/STING pathway in Tregs. We will then test if the effects of FRT in Tregs are dependent on cGAS/STING by evaluating Treg phenotype, function, and metabolism at FRT in cGAS and STING deficient cells. The impact of FRT on STING activation in Tregs will be investigated by treating Tregs with a STING agonist at 37°C and 39°C and comparing their gene expression, suppressive versus inflammatory function, and metabolism in vitro and in vivo. Given that STING can increase in glycolysis in innate cells, we hypothesize that STING activation induces a glycolytic switch in Tregs that inhibits their suppressive function and reprograms them into inflammatory Th1/Th9-like cells, and that this is potentiated at FRT. This research will define the role of STING in Tregs at physiological and fever temperatures, motivate further investigation of immune cell function in the context of inflammation, and has the potential to identify Treg- intrinsic STING activati...