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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.

Memory CD4 T cells in tissues fulfill numerous functions that are critical for local immune homeostasis and protection against pathogens. Previous studies have highlighted the phenotypic and functional heterogeneity of circulating and tissue-resident memory CD4 T cells across different human tissues such as skin, lung, liver and colon. Comparatively little is known in regard to memory CD4 T cells across tissues of the female reproductive tract (FRT). We examined CD4 T cells in donor-matched vaginal, ecto- and endo-cervical tissues, which differ in mucosal structure and exposure to external environmental stimuli. We hypothesized that this could be reflected by tissue-specific differences in the memory CD4 T cell compartment. We found differences in CD4 subset distribution across these tissues. Specifically, CD69+CD103+ CD4 T cells were significantly more abundant in vaginal than cervical tissues. In contrast, the transcriptional profiles of CD4 subsets were fairly conserved across FRT tissues. CD69+CD103+ CD4 T cells showed a TH17 bias independent of tissue niche. Our data suggest that FRT tissues affect T cell subset distribution but have limited effects on the transcriptome of each subset. We discuss the implications for barrier immunity in the FRT.

The immune system of the cervicovaginal tract (CVT) must balance immunosurveillance and active immunity against pathogens with maintenance of tolerance to resident microbiota and to fetal and partner antigens for reproductive purposes. Thus, we predicted that CVT immunity is characterized by distinctive features compared to blood and other tissue compartments. Indeed, we found that CVT CD8+ T-cells had unique transcriptional profiles, particularly in their cytokine signature, compared to that reported for CD8+ T-cells in other tissue sites. Among these CVT CD8+ T-cells, we identified a CD69- CD103- subset that was characterized by reduced migration in response to tissue-exit signals and higher pro-inflammatory potential as compared to their blood counterpart. These inflammatory mucosal CD8+ T-cells (Tim) were increased in frequency in the CVT of individuals with chronic infection, pointing to a potential role in perpetuating inflammation. Our findings highlight the specialized nature of immunity within the CVT and identify Tim cells as potential therapeutic targets to tame tissue inflammation upon chronic infection.

Tissue-resident memory CD8 T cells (CD8 TRM) are critical for maintaining barrier immunity. CD8 TRM have been mainly studied in the skin, lung and gut, with recent studies suggesting that the signals that control tissue residence and phenotype are highly tissue dependent. We examined the T cell compartment in healthy human cervicovaginal tissue (CVT) and found that most CD8 T cells were granzyme B+ and TCF-1–. To address if this phenotype is driven by CVT tissue residence, we used a mouse model to control for environmental factors. Using localized and systemic infection models, we found that CD8 TRM in the mouse CVT gradually acquired a granzyme B+, TCF-1– phenotype as seen in human CVT. In contrast to CD8 TRM in the gut, these CD8 TRM were not stably maintained regardless of the initial infection route, which led to reductions in local immunity. Our data show that residence in the CVT is sufficient to progressively shape the size and function of its CD8 TRM compartment.

Mucosal-associated invariant T (MAIT) cells acquire effector function in response to proinflammatory signals, which synergize with TCR-mediated signals. We asked if cell-intrinsic regulatory mechanisms exist to curtail MAIT cell effector function akin to the activation-induced expression of inhibitory receptors by conventional T cells. We examined human MAIT cells from blood and oral mucosal tissues by RNA sequencing and found differential expression of immunoregulatory genes, including CTLA-4, by MAIT cells isolated from tissue. Using an ex vivo experimental setup, we demonstrate that inflammatory cytokines were sufficient to induce CTLA-4 expression on the MAIT cell surface in the absence of TCR signals. Even brief exposure to the cytokines IL-12, IL-15, and IL-18 was sufficient for sustained CTLA-4 expression by MAIT cells. These data suggest that control of CTLA-4 expression is fundamentally different between MAIT cells and conventional T cells. We propose that this mechanism serves to limit MAIT cell–mediated tissue damage.

C - C chemokine receptor type 5 (CCR5) is thought to play a central role in orchestrating migration of cells in response to inflammation. CCR5 antagonists can reduce inflammatory disease processes which has led to an increased interest in using CCR5 antagonists in a wide range of inflammation-driven diseases. Paradoxically, these antagonists appear to function without negatively affecting host immunity at barrier sites. We reasoned that the resolution to this paradox may lie in the CCR5(+) T cell populations that permanently reside in tissues. We used a single-cell analysis approach to examine the human CCR5(+) T cell compartment in blood, healthy, and inflamed mucosal tissues to resolve these seemingly contradictory observations. We found that 65% of the tissue-resident (T(RM)) CD4 T cell compartment expressed CCR5. These CCR5(+) T(RM) cells were enriched in and near the epithelial layer and not limited to T(H)1 type cells but also contained a large T(H)17-producing and a stable regulatory T (Treg) cell population. Importantly, the CCR5(+) T(RM) compartment was stably maintained even in inflamed tissues including the preservation of T(H)17 and regulatory T cell populations. Further, using tissues from the CHARM-03 clinical trial we found that CCR5(+) T(RM) are preserved in human mucosal tissue during treatment with the CCR5 antagonist Maraviroc. Our data suggest that the human CCR5(+) T(RM) compartment is functionally and spatially equipped to maintain barrier immunity even in the absence of CCR5-mediated, de novo T cell recruitment from the periphery.

Tissue resident memory (Trm) CD8 T cells represent a newly described memory T cell population. We have previously characterized a population of Trm cells that persists within the brain after acute virus infection. Although capable of providing marked protection against a subsequent local challenge, brain Trm cells do not undergo recall expansion after dissociation from the tissue. Furthermore, these Trm cells do not depend on the same survival factors as the circulating memory T cell pool as assessed either in vivo or in vitro. To gain greater insight into this population of cells, we compared the gene expression profiles of Trm cells isolated from the brain with those of circulating memory T cells isolated from the spleen after an acute virus infection. Trm cells displayed altered expression of genes involved in chemotaxis, expressed a distinct set of transcription factors, and overexpressed several inhibitory receptors. Cumulatively, these data indicate that Trm cells are a distinct memory T cell population disconnected from the circulating memory T cell pool and display a unique molecular signature that likely results in optimal survival and function within their local environment.