ABSTRACT Regulatory T cells (Tregs) are critical for the maintenance of immunological tolerance but more recently their importance in regulating other aspects of tissue homeostasis has been an area of intense interest. Most relevant to this application are populations of tissue-resident Tregs that accumulate upon injury and facilitate tissue repair by producing factors such as the EGF family member amphiregulin (Areg). These repair functions are distinct from the suppressive function classically attributed to Tregs, but the signals that instruct Tregs to adopt these distinct functional modalities have not been well defined. The IL-1 family cytokines IL-18 and IL-33 are involved at some level, at least in certain tissues, but it remains unclear whether these cytokines act as the key initial determinants of Treg function. This proposal will test the hypothesis that Toll-like receptor 7 (TLR7) signaling in Tregs is a key determinant of Treg tissue repair function. We propose that TLR7 enables Tregs to sense pathogen-derived nucleic acids as well as self RNA released from damaged host tissues. Our hypothesis is based on our analysis of a panel of TLR reporter mice, which revealed that only TLR7 is expressed on Tregs, as well as strong preliminary data demonstrating that TLR7 can induce expression of the signature tissue repair gene, Areg, in both murine and human Tregs. Using newly generated mice with Treg-specific deletion of TLR7, we will examine the importance of TLR7 signaling in Tregs during lung damage (Aim 1). Single-cell RNA sequencing will identify which subsets of lung Tregs are controlled by TLR7 and will define TLR7-dependent genes in Tregs. We will also investigate the importance of IL18R and IL33R signaling in Tregs, using mice with Treg-specific deletion of these receptors, and will determine the extent to which TLR7, IL18R, and IL33R regulate distinct aspects of Treg expansion and/or differentiation in response to diverse lung damaging agents (Aim 2). Finally, we will build on our recent work that identified a mechanism by which the TLR chaperone Unc93b1 specifically dampens TLR7 signaling. Using Tregs from mice with mutant Unc93b1 that have enhanced TLR7 signaling, we will test whether adoptive therapy of Tregs with enhanced TLR7 responses to viral and self RNA can mediate more effective repair of lung damage (Aim 3). Altogether, these studies will define the signals that control Treg tissue repair functions and test the therapeutic potential of amplifying these signals in the context of lung damage, a key first step toward therapeutic manipulation of Treg function for clinical benefit.