T lymphocyte-mediated protection of the reproductive mucosa - PROJECT SUMMARY The female reproductive tract (FRT) mucosa is routinely invaded by pathogenic viruses. Resident memory CD8 T cells (TRM) have proven antiviral roles in this barrier tissue. Recent studies in mice and non-human primates have demonstrated that when present in sufficient numbers, TRM can mediate rapid antiviral protection. Responding CD8 TRM elicit immediate effector functions, recruit a network of antiviral immune cells and limit pathogen spread beyond the initial replication site. However, efforts to establish high density CD8 TRM that are durably maintained have yet to be realized. This is largely due to an incomplete understanding of the cellular and molecular signals that regulate CD8 TRM differentiation, enables their poised effector state and maintain them long term. Heterogeneity among TRM populations, which has recently become appreciated, further compounds this lack of understanding. This proposal seeks to understand the CD8 TRM diversity and define the processes that control CD8 TRM differentiation in the reproductive mucosa. Our phenotypic and single cell RNA sequencing studies have identified two major subsets of FRT TRM that are marked by mutually exclusive expression of CD103 and Ly6C. While both subsets were largely resident as determined by parabiosis, CD103+ cells resembled classical epithelial TRM cells but Ly6C+ cells displayed properties of circulating effector cells. Further exploration into their biogenesis showed that CD103+ cells were dependent on TGF-β signaling for their formation as well as maintenance. Interestingly, exposure to inflammation drove the formation of Ly6C+ TRM. We will utilize both in vivo infection models and a novel in vitro vaginal organoid system to understand the cellular and molecular drivers of these two distinct TRM subsets. We will pursue two aims. In aim-1, we will determine the role of type-1 interferon in promoting Ly6C+ TRM generation. We will also test if CD103+ and Ly6C+ subsets represent terminal TRM states or are capable of further differentiation. Importantly we will assess the relative protective roles of both TRM subsets against pathogen challenges. Finally, we will examine if vaginal microbial dysbiosis modulates the differentiation of each subset. In aim-2, we will investigate the cellular and molecular circuitry responsible for regulating TGF-β signaling in FRT TRM. Specifically, we will assess the timing and source of TGF-β signals necessary for establishing and maintaining FRT TRM. We will identify cells that support integrin-mediated activation of TGF-β in the FRT. These studies will be achieved by a combination of inducible genetic mouse models and antibody-based approaches. The overall purpose of this project is to understand the mechanisms driving the generation and maintenance of FRT TRM. The findings from this work will inform targeted therapies to improve TRM lodgment, functional tuning, and survival to achieve effective immune surveillance in the reproductive mucosa.
