SUMMARY Regulatory T cells (Treg) are critical in controlling alloreactive T cell responses in organ transplant recipients, but differences in suppressive capacity exist within phenotypically similar cells. Despite transcriptional heterogeneity within the Treg compartment, lack of available technical approaches has prevented any assessment of their functional heterogeneity. The Treg suppressive capacity is associated with a distinct metabolic signature defined by increased oxidative phosphorylation (OXPHOS) and decreased glycolysis. Cellular reduction-oxidation (redox) homeostasis mechanisms are fully intertwined with metabolic state; T cells engage specific metabolic pathways and also rearrange their redox system, to maintain T cell metabolic rewiring and cell fate. Regulation of de novo synthesis of the antioxidant glutathione (GSH) is a crucial redox homeostasis mechanism in Treg. These cells respond to activation-induced oxidative stress by increasing glutamate cysteine ligase (GCL) activity to form gamma-glutathione cysteine (-GC) and maintain high GC and low GSH intracellular levels (while Teff then shuttle -GC to increase intracellular GSH). This particular balance of high -GC and low GSH (GSH homeostasis) needs to be preserved for Treg to exert a suppressive function. The current techniques for capturing GSH homeostasis in Treg are inadequate and the only available assays measure -GC in cell lysates (not in live cells). In addition, most commercial GSH sensors bind irreversibly to GSH, eliminate it from the cells and thus completely disrupt GSH homeostasis and lose information on heterogeneity, which we contend is crucial. In this proposal, we will develop an approach to capture the heterogeneity in GSH homeostasis in Treg using a small-molecule dual -GC/GSH sensor that overcomes these limitations. We will use it to test the hypothesis that cell-to-cell differences in GSH homeostasis within Tregs underlies the heterogeneity in Treg suppressive capacity (Aim 1), and to provide proof-of-concept that this approach can capture clinically relevant heterogeneity in circulating Treg from kidney transplant recipients treated with anti-IL-6R antibody (tocilizumab) (Aim 2). At the end of the R21 we will have generated a small-molecule biosensor to obtain GSH-homeostasis profiles in Treg, mapped the relationship onto Treg suppressive capacity, and demonstrated the translational potential of this approach. This is a technically challenging approach (high-risk), but the results have the potential to provide a tool to identify previously undetected functional differences within the Treg compartment (high- reward), setting the basis for an R01 testing the mechanisms responsible for this heterogeneity.