Project Summary Rejection remains a major hurdle to long lasting transplant survival and we aspire to uncover the fundamental mechanisms that hinder stable allograft survival. In the last funding period, we studied the mechanisms of Treg dysfunctions and identified BATF and BATF3 as potent repressors of Foxp3, suppressing Foxp3 expression and Treg induction. These studies also led to the discovery of the DHX15 helicase as a critical regulator of Foxp3+Tregs, as conditional deletion of Dhx15 in Tregs resulted in a profound depletion of Tregs in the periphery and lethal autoimmune diseases. In essence, DHX15 is an RNA helicase (an RNA binding motor protein) and traditionally thought to be involved in RNA processing. Its roles in the control of Tregs are unexpected and clearly reveal novel previously unknown aspects of Tregs that are different from currently known mechanisms, and uncovering those mechanisms is the central goal of this proposal. Our working hypothesis is that DHX15 controls Treg identity and/or survival at peripheral sites and that its deficiency results in either their reversion to Teff or die of apoptosis. We surmise that DHX15 may also control Tregs at the graft site, where they must overcome the inflammatory milieu to exert suppressive functions. We proposed 3 Aims to test this hypothesis in this application: the first Aim is to test whether DHX15 controls Treg identity by acting as an RNA splicing factor, processing mRNAs that encode the Foxp3 or Foxp3 controlled signature molecules in Tregs, the second Aim is to address whether DHX15 controls Tregs survival by regulating the IL-2 signaling complex, such that Tregs die of apoptosis in its absence, and the third Aim is to examine whether DHX15 is especially important for Tregs at graft sites where they are needed the most in promoting transplant tolerance. Overall, DHX15 is the first RNA helicase identified thus far that controls Tregs, and the genetically modified models as well as cutting-edge approaches we have developed put us in a unique position in dissecting mechanistically how the DHX15 helicase acts in regulating Tregs, an area of considerable importance in both basic Treg biology and Treg- based therapies.