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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.

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 the blood, healthy, and inflamed mucosal tissues to resolve these seemingly contradictory observations. We found that 65% of the CD4 tissue-resident memory T (TRM) cell compartment expressed CCR5. These CCR5+ TRM cells were enriched in and near the epithelial layer and not only limited to TH1-type cells but also contained a large TH17-producing and a stable regulatory T cell population. The CCR5+ TRM compartment was stably maintained even in inflamed tissues including the preservation of TH17 and regulatory T cell populations. Further, using tissues from the CHARM-03 clinical trial, we found that CCR5+ TRM are preserved in human mucosal tissue during treatment with the CCR5 antagonist Maraviroc. Our data suggest that the human CCR5+ TRM 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.

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.

The memory CD8 T cell compartment in human cervicovaginal tissue (CVT) is phenotypically and functionally distinct compared to other non-lymphoid tissues. To determine if these distinctive features are driven by residence in the tissue itself, rather than a consequence of local antigenic insults, we used a mouse model to assess memory CD8 T cell differentiation in the CVT. Following systemic immunization to elicit a CD8 T cell response to a herpes simplex virus (HSV) epitope, HSV-specific memory CD8 T cells were stably maintained in other tissues but gradually declined in the CVT over 5 months post-immunization, correlating with a delay in viral control upon HSV challenge. While memory cells in the periphery and small intestine maintained a fairly stable phenotype after 30 days, memory CD8 T cells in the CVT gradually acquired a CD103 + , TCF-1 − , granzyme B + phenotype over the 5 month time span. Notably, a sizable and phenotypically similar population of CD8 T cells was also found in human CVT. Our data indicate that the CVT environment itself considerably affects memory CD8 T cell maintenance and differentiation, and call for a reassessment of the prevalent model of memory T cell maintenance which does not currently account for tissue-specific divergence.

Herpes simplex virus 2 (HSV-2) is a chronic sexually transmitted infection that causes recurrent genital ulcers and increases HIV susceptibility. Efforts to design a vaccine have assumed regulatory T cells (Tregs) restrain immune activation and impede viral clearance. However, we have previously shown that in a mouse model of vaginal primary HSV-2 infection, Treg removal leads to impaired T cell priming and migration. This suggests an alternative model where Treg function is location- and context-dependent. Additionally, research suggests that tissue-resident memory T cells (Trm) localized in the vaginal tract (VT) are crucial for efficient viral control, and Trm are likely regulated by local Tregs. These findings underscore the importance of understanding the specific contributions of VT Tregs during viral infection and vaccination. We performed RNA sequencing of VT and lymph node (LN) Tregs after HSV-2 infection to reveal a highly activated transcriptional profile in VT Tregs compared to LN Tregs. Using flow cytometry, we confirmed an elevated activation and tissue residency phenotype in VT Tregs, exacerbated after HSV-2 infection. Notably, we found that VT Tregs express Granzyme B (GzmB) by both RNA-sequencing and flow cytometry. These Tregs persist in the VT up to 90 days, suggesting that they remain poised to respond to HSV-2 reencounter by restraining memory Trm to prevent tissue damage, possibly through GzmB-mediated toxicity. Tregs from healthy human VT corroborated mouse VT Treg phenotypes. These results suggest that VT Tregs are distinct and uniquely poised to respond to viral infection. Studies are ongoing to interrogate the signals that drive Treg migration into the VT and that lead to the tissue-specific Treg phenotype.

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.

The study of T cell immunity at barrier surfaces has largely focused on T cells bearing the αβ TCR. However, T cells that express the γδ TCR are disproportionately represented in peripheral tissues of mice and humans, suggesting they too may play an important role responding to external stimuli. In this article, we report that, in a murine model of cutaneous infection with vaccinia virus, dermal γδ T cell numbers increased 10-fold in the infected ear and resulted in a novel γδ T cell population not found in naive skin. Circulating γδ T cells were specifically recruited to the site of inflammation and differentially contributed to dermal populations based on their CD27 expression. Recruited γδ T cells, the majority of which were CD27+, were granzyme B+ and made up about half of the dermal population at the peak of the response. In contrast, recruited and resident γδ T cell populations that made IL-17 were CD27−. Using a double-chimera model that can discriminate between the resident dermal and recruited γδ T cell populations, we demonstrated their divergent functions and contributions to early stages of tissue inflammation. Specifically, the loss of the perinatal thymus-derived resident dermal population resulted in decreased cellularity and collateral damage in the tissue during viral infection. These findings have important implications for our understanding of immune coordination at barrier surfaces and the contribution of innate-like lymphocytes on the front lines of immune defense.

During an infection the antigen non-specific memory CD8 T cell compartment is not simply an inert pool of cells, but becomes activated and cytotoxic. It is unknown how these cells contribute to the clearance of an infection. We measured the strength of T-cell receptor (TCR) signals that bystander activated, cytotoxic CD8 T-cells (BA-CTLs) receive in vivo and found evidence of limited TCR signaling. Given this marginal contribution of the TCR, we asked how BA-CTLs identify infected target cells. We show that target cells express NKG2D ligands following bacterial infection and demonstrate that BA-CTLs directly eliminate these target cells in an innate-like, NKG2D-dependent manner. Selective inhibition of BA-CTL mediated killing led to a significant defect in pathogen clearance. Together these data suggest a previously unappreciated, innate role for memory CD8 T-cells in the early immune response before the onset of a de-novo generated, antigen-specific CD8 T-cell response.

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.

The brain is not routinely surveyed by lymphocytes and is defined as an immuno-privileged site. However, viral infection of the brain results in the infiltration and long-term persistence of pathogen-specific CD8 + T cells. These cells survive without replenishment from the circulation and are referred to as resident memory T cells (Trm). Brain Trm selectively express the integrin CD103, the expression of which is dependent on antigen recognition within the tissue. After clearance of virus, CD8 + T cells persist in tight clusters, presumably at prior infection hot spots. Antigen persistence is not a prerequisite for T-cell retention, as suggested by the failure to detect viral genomes in the T-cell clusters. Furthermore, we show that an intracranial dendritic cell immunization regimen, which allows the transient introduction of antigen, also results in the generation of memory T cells that persist long term in the brain. Brain Trm die rapidly on isolation from the tissue and fail to undergo recall expansion after adoptive transfer into the bloodstream of antigen-challenged recipients. These ex vivo defects imply a dependency on the local milieu for function and survival. Cumulatively, this work shows that Trm are a specialized population of memory T cells that can be deposited in tissues previously thought to be beyond routine immune surveillance.