PROJECT SUMMARY/ABSTRACT Multiple sclerosis (MS) and other autoimmune diseases constitute a major healthcare burden at a cost of >$125 billion per year. Autoimmune disorders arise from a failure of immunoregulatory networks. Regulatory T (Treg) cells expressing transcription factor forkhead box protein 3 (Foxp3) are indispensable components of these networks. Moreover, recent studies from several groups suggest that Treg cells also facilitate tissue repair, in addition to exerting immunosuppression. During autoimmune diseases, Treg cells are activated in lymphoid organs and home to non-lymphoid target tissues where they persist in specialized niches to limit inflammation and facilitate tissue repair. Our overall goal is to determine how these Treg cell niches operate in the central nervous system (CNS) to ameliorate autoimmune neuroinflammation at cellular and molecular levels. Direct visualization of cell behavior often leads to surprises, new hypotheses, and follow-up experiments contributing to a better understanding of the mammalian immune system, and how autoimmunity and infectious diseases can be effectively treated. Building on our expertise in two-photon (2-P) imaging at the cellular level, and Ca2+ signaling at the molecular level, we will use the experimental autoimmune encephalomyelitis (EAE) mouse model of MS to: 1) elucidate the local cues that drive survival, functional organization in niches, and motility behaviors of Treg cells in the spinal cord; 2) investigate how Treg cells selectively target processes that incite neuroinflammation. Our experimental approach includes evaluation of the Piezo1 channels as promising therapeutic targets to selectively expand Treg cells, an ideal strategy to curb ongoing autoinflammatory responses while preserving the immune system’s ability to fight new infections. Although this proposal is targeted specifically to MS, in a broader context our project will provide fundamental insights into how Treg cells fine-tune tissue inflammation so that better Treg-modifying therapies can be developed for autoimmune disorders, organ transplantation, cancer, and infectious diseases.