Foxp3+CD4+ regulatory T cells (Tregs) control most types of immune responses. Recently, distinct compartments of tissue-localized Tregs that help maintain local homeostasis were also discovered. These so- called “tissue-Tregs” have transcriptomes, TCR repertoires and growth/survival factor dependencies that differ from those of lymphoid-organ Tregs and of Tregs in other tissues. Tissue-Tregs regulate non-immunological processes such as metabolism and regeneration via influences on innate and adaptive immunocytes as well as on local stromal and parenchymal cells. A paradigmatic tissue-Treg compartment, first reported by the PI's lab in 2009, is that found in visceral adipose tissue (VAT), especially the epidydimal VAT (eVAT) depot of lean mice. eVAT Tregs, which depend on the supreme regulator of adipogenesis, PPARγ, for effective accrual and function, help assure local and systemic metabolism, notably insulin sensitivity. This important function is known to reflect direct eVAT-Treg influences on local immunocytes and on eVAT-resident, immunocyte-promoting mesenchymal stromal cells (VmSCs). The focus of this proposed project is on a newly discovered function of eVAT Tregs. Our preliminary data, primarily derived from transcriptomic analyses and in vitro adipogenesis assays, indicate that they control the differentiation of stroma-resident adipocyte precursor cells into mature adipocytes (and potentially adipocyte turnover and fibrosis). Moreover, these data point to Oncostatin M (OSM) as a potential mediator of these influences. THE MAJOR GOAL OF THIS PROPOSED PROJECT IS TO ELUCIDATE THE AVENUES OF COMMUNICATION BETWEEN eVAT Tregs AND ADIPOCYTE PRECURSORS. We propose: (1) To determine the extent to which OSM production by eVAT-Tregs is responsible for their constraint of stroma- resident adipocyte precursors. This Aim addresses the need to validate our transcriptomic and in vitro adipogenesis data with in vivo examinations. (2) To evaluate the effects of aging on eVAT-Treg control of local inflammation and adipogenesis. Discrepancies in the literature imply that the impact of eVAT Tregs may be very different in aging vs old mice. We propose to evaluate this notion in a head-to-head comparison of mice specifically lacking eVAT-Tregs or not. (3) To determine whether and how eVAT-Treg control of adipocyte differentiation shows sexual dimorphism. Male and female rodents and humans differ in the deposition and function of their adipose tissue. A recent report (on which the PI's lab collaborated) highlights a male:female dichotomy in IL-33-producing, immunocyte- promoting VmSCs, leading to a dimorphism in gonadal VAT accrual. Our preliminary data uncovered a difference in adipocyte dynamics as well, which is the focus of this Aim. Upon successful completion of this proposed project, we should know in what contexts eVAT-Tregs regulate adipocyte dynamics and how they do so. This represents a novel function of eVAT Tregs, one that fits within the emerging ...