Regulatory T cells (Treg) selectively permit or prevent immune responses based on recognition of foreign vs self-antigen. Tregs are dependent on the transcription factor FOXP3 for formation and fitness, and loss of FOXP3 is lethal in both mice and humans. Treg defects contribute to many autoimmune diseases but changes in regulation of FOXP3 or its target genes are most commonly implicated in this loss of function, rather than loss of FOXP3 itself. Mouse models have been instrumental in identifying a population of Treg that are induced in the periphery in response to cytokine stimulation (pTreg). The contribution of pTregs to human tolerance remains unclear due to lack of biomarkers to distinguish between thymic and peripheral CD25+ cells. In vitro induced Tregs (iTreg) can be generated from naïve CD4+ CD25- cells are thought to be equivalent to mouse pTregs. It is unclear whether human pTregs are stable as some evidence suggests plasticity and conversion to other T cell subsets. MicroRNAs are also essential for Treg generation and function, as demonstrated by autoimmune defects in microRNA deficient mouse models. MicroRNAs play a critical role in fine tuning key gene networks to reinforce the Treg phenotype and we have identified microRNAs that are differentially regulated by FOXP3 in human tTregs. Further, we investigated the function of microRNAs and their downstream target genes in human Treg subsets (tTreg/iTreg) in order to understand whether or not microRNAs contribute to stable Treg function. Once a profile of the miR regulatory network in healthy donor tTreg and iTreg is established it will be compared with tTregs and iTregs generated from IBD/Type 1 diabetes patients. Our preliminary data suggests that human iTregs express a unique microRNA signature comprised of both Treg and Tconv specific miRs. iTregs significantly up-regulate miR-31, a miR known for its role in suppressing FOXP3 that is commonly over-expressed in Tconv cells and under-expressed in tTregs. This may explain functional plasticity of iTregs.