PROJECT SUMMARY The long-term goal of our research is to determine how the immune system interfaces with cells in the dystrophic niche to impact the pathogenesis of Duchenne muscular dystrophy (DMD), to provide a foundation for developing novel treatments for this devastating disease. The objective of the current study is to define how regulatory T cells (Treg) regulate macrophage-stromal interactions in muscle, and how these interactions can be pharmacologically manipulated to alter disease progression. We provide strong scientific premise for proposing the central hypothesis that Tregs regulate the severity of DMD by suppressing a distinct galectin-3+ muscle macrophage population that promotes fibrosis. This hypothesis is supported by our preliminary data and prior research, showing that 1) dystrophic muscle is populated by a putative fibrogenic galectin-3+/Spp1+ macrophage that we identified by single-cell RNA sequencing (scRNAseq); 2) prior research showing that spp1 promotes fibrosis in mdx mice; 3) macrophage-specific deletion of Spp1 in mdx mice causes remarkable changes in the transcriptional profile of PGFRa+ stromal cells, as assessed by scRNAseq; 4) galectin-3+ macrophages are expanded when Tregs are depleted in mdx mice, suggesting that Tregs suppress fibrogenic macrophages and 5) depletion of Tregs in mdx mice causes an increased expression of pro-fibrotic genes. We will test our central hypothesis by addressing two specific aims. Specific aim 1 will define how galectin-3+ muscle macrophages promote fibrosis. Two subaims will be addressed under specific aim 2, identify the molecular basis for Treg suppression of galectin-3+ muscle macrophages. In subaim 2a we will determine whether the Treg-mediated suppression of IFNg-producing cells is IL-10 dependent; and in subaim 2b we will examine whether the therapeutic expansion of Tregs suppresses fibrosis. Although a role for Spp1 in promoting fibrosis in mdx mice has been documented, a mechanism for this pathological process is largely unknown. Thus, our proposed study is significant as it will define the cellular basis by which Spp1 promotes muscle fibrosis, and advance our understanding of how Tregs ameliorate the severity of muscular dystrophy. The proposed research is innovative because it will define mechanisms of cellular cross talk that promote fibrosis in dystrophic muscles. The proposed studies use innovative technologies (e.g. scRNAseq) and experimental therapeutics (e.g. IL-2c) that will provide the framework for developing novel approaches to inhibit the development of fibrosis. These potential discoveries will advance the field by establishing interactions between the lymphoid and myeloid compartment of the immune system that operate in DMD to suppress or limit fibrogenic responses. Further, findings from the proposed studies are expected to have significant clinical implications for DMD, as the immunosuppressive function of Tregs can be therapeutically augmented to ameliorate...