Her Mucosa, Her Rules: Regulation of Memory T Cells in the Female Genital Tract

Abstract

The lower female genital tract (FGT) is a mucosal barrier and site of exposure to various pathogens, including fungi, parasites, bacteria, and viruses. The FGT faces the unique challenge of maintaining tolerance to a semi-allogenic fetus and the optimal vaginal microbiome, in addition to preventing infection through cell-mediated surveillance for pathogens and subsequent measured immune activation without excessive damage to host tissues. The lower FGT (vagina and ectocervix) is covered in multi-layered squamous epithelial cells, whereas the upper FGT (endocervix, uterus and fallopian tubes) is covered by a single layer of columnar epithelial cells. Beneath the epithelium is a layer of stromal fibroblasts providing structure to the tissue. Dynamic populations of immune cells are distributed throughout the stroma, with T lymphocytes being the most abundant immune cell subset within the lower FGT. Mechanisms involved in immune responses within the FGT are further complicated by changes in sex-hormones throughout an individual’s life. In addition to regulating changes in epithelial and stromal structural reorganization and repair, hormonal fluctuations throughout the menstrual cycle can also participate in modulation immune cell function to maintain tolerance to innocuous antigens and protection against pathogens. Tissue-resident memory T cells (Trm) mediate protection within tissue sites of prior or persisting infection. Upon pathogen re-exposure and antigen recognition, Trm elicit robust immunity locally within the tissue and orchestrate the recruitment of other T cells and innate immune cells. To avoid excessive tissue damage associated with this robust tissue immune reaction, we predicted that tissue recall T cell responses must be subject to regulation. We previously demonstrated that regulatory T cells (Treg) accumulate within infected tissue and coordinate early immune responses and T cell priming during primary infection, though their role in shaping tissue memory T cell responses remains unclear. Using transient Treg depletion in mice, we show a requirement for Treg in limiting tissue pathology following vaginal HSV-2 challenge, despite normal viral clearance. While Treg-depleted mice exhibit an elevated frequency and number of vaginal CD44+Ki67+ T cells and granzyme-B+CD8+ T cells by day 3 post-challenge, there is no difference in the magnitude of the antigen-specific CD8+ T cell response. Using adoptive transfer of TCR-transgenic OT-1 CD8 T cells, we show that Treg-depleted mice have a significantly stronger vaginal bystander-activated cytotoxic T cell (BA-CTL) response upon challenge. In vivo antibody blocking demonstrates that Treg limit cytotoxicity of both HSV-2-specific CD8 and BA-CTL responses via an IL-2-dependant mechanism that additionally restricts IL-15 trans-presentation by antigen presenting cells. Our findings highlight Treg’ role in selectively restraining cytotoxic function during a tissue recall response, while preserving the pathogen-specific tissue T cell response, to balance viral clearance with restriction of immunopathology. In addition to regulation by Treg, immunity can also be modulated by fluctuations in sex hormones. Characterization local FGT and systemic T cell responses throughout the menstrual cycle is paramount to better understanding mechanisms underlying susceptibility to sexually transmitted infections and correlates of protection for improved design of vaccines and therapeutics. We sought to characterize the impact of menstrual phase on local cervicovaginal and systemic T cell responses in Kenyan women. Using high-parameter, high-throughput flow cytometry and a large panel we compared follicular vs luteal phase phenotypes in T cell populations isolated from vaginal tract (VT) and ectocervix (CX) mucosal tissue biopsies and PBMC samples. Given the abundance of T cells in the lower FGT, cervicovaginal tract (CVT) biopsies may better capture the mucosal immune environment than other minimally invasive sampling methods such as cervicovaginal lavage, vaginal/cervical swabs, or cytobrushes. Additionally, we evaluated 71 immunomodulatory molecules in serum and cervicovaginal secretions in follicular vs luteal phase. Overall, we did not find changes in the frequencies of T cell subsets (CD4+, CD8+, Treg) or increases in HIV susceptibility markers on CD4+ T cells (CCR5, HLA-DR, CD38) between follicular vs luteal phase in any of the tissue sample types. Interestingly, all phenotypic differences were observed in CD8+ T cells within CX and VT. Specifically, CCR7+ CD45RA- central memory CD8 T cells were significantly elevated during the luteal phase in both the CX and PBMCs, whereas CCR7- CD45RA+ terminally differentiated effector memory T cells CD8 T cells were more prevalent in the CX during the follicular phase. Additionally, CD8 T cells in the CX during the luteal phase were more activated, exhibiting higher frequencies of HLA-DR, CD38, and the exhaustion marker CD39. We also found altered expression of soluble factors in follicular phase, with increased concentrations of CCL15 and IL-2 in cervicovaginal secretions and increased CXCL12 in serum samples. These findings suggest that menstrual phase modulates CD8 T cell memory subsets and intrinsic regulation of activation, providing new insights into how hormonal fluctuations influence FGT immunity to infection.