Vaginal regulatory T cells in health and infection

Abstract

Regulatory T cells (Tregs) mediate immune homeostasis, yet also facilitate nuanced immune responses during infection, balancing pathogen control while limiting host inflammation. Recent studies have identified Treg populations in non-lymphoid tissues that are phenotypically distinct from Tregs in lymphoid tissues (LT), including performance of location-dependent roles. Mucosal tissues serve as critical barriers to microbes while performing unique physiologic functions, so we sought to identify distinct phenotypical and functional aspects of mucosal Tregs in the female reproductive tract. In healthy human and mouse vaginal mucosa, we found that Tregs are highly activated compared to blood or LT Tregs. To determine if this phenotype reflects acute activation or a general signature of vaginal tract (VT)-residency, we infected mice with HSV-2 to discover that VT Tregs express granzyme-B (GzmB) and acquire a VT Treg signature distinct from baseline. To determine the mechanisms that drive GzmB expression, we performed ex vivo assays to reveal that a combination of type-I interferons and interleukin-2 is sufficient for GzmB expression. Together, we highlight that VT Tregs are activated at steady state and become further activated in response to infection; thus, they may exert robust control of local immune responses, which could have implications for mucosal vaccine design. Additionally, little is known about the role of Tregs in recurrent HSV-2 outbreaks or secondary exposure to HSV-2. Tissue-resident memory T cells (Trm), which reside in tissue sites without recirculation, have been shown to be crucial for protection from HSV-2 in mouse models and HSV-2 control. However, HSV-2 symptoms and viral load are highly variable between infected individuals, despite the presence of vaginal Trm. Therefore, we predicted that Tregs prevent immune-mediated vaginal tissue damage during HSV-2 challenge by restraining CD4+ and CD8+ Trm responses, possibly at the expense of impaired viral clearance. Additionally, we hypothesized that Tregs may directly contribute to the repair of vaginal HSV-2 lesions through production of amphiregulin (Areg), a growth factor known to facilitate tissue repair in other tissue Tregs. We used immunohistochemistry to assess vaginal pathology in mice lacking Tregs at the time of HSV-2 challenge and found that Treg-depleted mice experienced delayed tissue healing by day 7 post-challenge, compared to Treg-replete controls. However, PCR revealed that vaginal HSV-2 viral titers were comparable in both groups, suggesting that tissue damage was immune-mediated. We also found that vaginal Tregs in mice express Areg transcript and Areg protein by day 7 post-HSV-2 primary infection, suggesting that Tregs may directly contribute to vaginal tissue repair of HSV-2 lesions, but further studies are necessary to functionally link Treg-produced Areg to vaginal mucosa healing and confirm Treg production of Areg in human tissues. Experiments are ongoing to assess the role of Tregs on Trm abundance, activation, and cytotoxic activity after HSV-2 challenge, as well as to define the role of Treg-produced Areg in tissue pathology using conditional knockout mice. Together, these findings suggest that vaginal Tregs may have vagina-specific roles in tissue protection and immune control during viral infection, further evidencing the need to consider Tregs in mucosal vaccine design. Likewise, Treg-based therapeutics could be an important tool for reversing pathology in mucosal infection or autoimmunity.