Beyond its primary function of repelling microbial challenges, the immune system plays important roles in safeguarding tissue homeostasis. Macrophages have long been recognized to exercise such secondary functions and, over the past several years, there has been growing interest in the implication of Foxp3+CD4+ regulatory T cells (or Tregs) in homeostatic processes. For example, unique Treg compartments in a number of parenchymal tissues promote local repair/regeneration after acute or chronic injury – even in zebrafish! Skeletal-muscle Tregs serve as a paradigmatic pro-regenerative regulatory T cell population. First reported by our lab in 2012, muscle Tregs increase rapidly after acute injury, differing from lymphoid-organ Tregs in their elevated representation, tissue-adapted transcriptome, distinct – clonally expanded – T cell receptor repertoire, and growth/survival factor dependencies. One intriguing axis, discovered during the last funding-cycle, entails nociceptive neuron production of the peptide, CGRP; which elicits IL-33 production from stromal cells; which, in turn, promotes local Treg accumulation. Muscle Tregs exert multiple influences along the course of tissue repair – on both lymphoid and non-lymphoid cells during both the early, pro-inflammatory, and late, pro-regenerative, phases. This broad range of activities highlights the need to go beyond the current static image of muscle-Treg phenotype and function to obtain a dynamic view spanning the entire process. Temporal single-cell RNAseq data on the muscle-Treg compartment from 1-14 days post-injury (generated during the last funding-cycle) revealed five distinct subtypes that waxed and waned over time: circulating, recently activated, RORγ+, T-bet+ and GATA3+ (or reparative). Our long-term goal is to understand how the five muscle-Treg subtypes integrate with each other and with neighboring lymphoid and non-lymphoid cells to promote muscle regeneration. Our overall hypothesis is that individual subtypes emerge and/or expand to deal with particular biological issues that arise during the repair/regeneration process. In particular, this proposed project aims to: 1. Identify the provenance of RORγ+ skeletal-muscle Tregs. 2. Determine what critical role(s) RORγ+ Tregs play in effective muscle repair/regeneration. 3. Determine whether endogenous CGRP orchestrates increased IL-33 production by muscle MSCs and consequent expansion of the reparative (GATA3+) muscle-Treg subtype. Completion of these studies will provide us with a more accurate and nuanced picture of Treg activities during muscle regeneration. Potential therapeutic applications are many: catastrophic wound healing, exercise- induced damage, age-related sarcopenias, muscular dystrophies, autoimmune myositides and chronic muscle infections.