Advances and challenges in studying the tissue-resident T cell compartment in the human female reproductive tract.

Tissue-resident memory T cells (TRM ) are considered to be central to maintaining mucosal barrier immunity and tissue homeostasis. Most of this knowledge stems from murine studies, which provide access to all organs. These studies also allow for a thorough assessment of the TRM compartment for each tissue and across tissues with well-defined experimental and environmental variables. Assessing the functional characteristics of the human TRM compartment is substantially more difficult; thus, notably, there is a paucity of studies profiling the TRM compartment in the human female reproductive tract (FRT). The FRT is a mucosal barrier tissue that is naturally exposed to a wide range of commensal and pathogenic microbes, including several sexually transmitted infections of global health significance. We provide an overview of studies describing T cells within the lower FRT tissues and highlight the challenges of studying TRM cells in the FRT: different sampling methods of the FRT greatly affect immune cell recovery, especially of TRM cells. Furthermore, menstrual cycle, menopause, and pregnancy affect FRT immunity, but little is known about changes in the TRM compartment. Finally, we discuss the potential functional plasticity of the TRM compartment during inflammatory episodes in the human FRT to maintain protection and tissue homeostasis, which are required to ensure reproductive fitness.

Baseline T cell immune phenotypes predict virologic and disease control upon SARS-CoV infection in Collaborative Cross mice.

The COVID-19 pandemic has revealed that infection with SARS-CoV-2 can result in a wide range of clinical outcomes in humans. An incomplete understanding of immune correlates of protection represents a major barrier to the design of vaccines and therapeutic approaches to prevent infection or limit disease. This deficit is largely due to the lack of prospectively collected, pre-infection samples from individuals that go on to become infected with SARS-CoV-2. Here, we utilized data from genetically diverse Collaborative Cross (CC) mice infected with SARS-CoV to determine whether baseline T cell signatures are associated with a lack of viral control and severe disease upon infection. SARS-CoV infection of CC mice results in a variety of viral load trajectories and disease outcomes. Overall, a dysregulated, pro-inflammatory signature of circulating T cells at baseline was associated with severe disease upon infection. Our study serves as proof of concept that circulating T cell signatures at baseline can predict clinical and virologic outcomes upon SARS-CoV infection. Identification of basal immune predictors in humans could allow for identification of individuals at highest risk of severe clinical and virologic outcomes upon infection, who may thus most benefit from available clinical interventions to restrict infection and disease.

Cervicovaginal Tissue Residence Confers a Distinct Differentiation Program upon Memory CD8 T Cells.

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.

Regulatory T cells limit unconventional memory to preserve the capacity to mount protective CD8 memory responses to pathogens.

Immunological memory exists so that following infection an expanded population of pathogen-specific lymphocytes can rapidly and efficiently control infection in the case of reexposure. However, in the case of CD8+ T lymphocytes, a population of unconventional CD44+CD122+ virtual memory T cells (TVM) has been described that possesses many, though not all, features of "true memory" T cells, without the requirement of first encountering cognate antigen. Here, we demonstrate a role for regulatory T cell-mediated restraint of TVM at least in part through limiting IL-15 trans-presentation by CD11b+ dendritic cells. Further, we show that keeping TVM in check ensures development of functional, antigen-specific "true" memory phenotype CD8+ T cells that can assist in pathogen control upon reexposure.

Herpes simplex virus-2 dynamics as a probe to measure the extremely rapid and spatially localized tissue-resident T-cell response.

Herpes simplex virus-2 infection is characterized by frequent episodic shedding in the genital tract. Expansion in HSV-2 viral load early during episodes is extremely rapid. However, the virus invariably peaks within 18 hours and is eliminated nearly as quickly. A critical feature of HSV-2 shedding episodes is their heterogeneity. Some episodes peak at 108 HSV DNA copies, last for weeks due to frequent viral re-expansion, and lead to painful ulcers, while others only reach 103 HSV DNA copies and are eliminated within hours and without symptoms. Within single micro-environments of infection, tissue-resident CD8+ T cells (TRM ) appear to contain infection within a few days. Here, we review components of TRM biology relevant to immune surveillance between HSV-2 shedding episodes and containment of infection upon detection of HSV-2 cognate antigen. We then describe the use of mathematical models to correlate large spatial gradients in TRM density with the heterogeneity of observed shedding within a single person. We describe how models have been leveraged for clinical trial simulation, as well as future plans to model the interactions of multiple cellular subtypes within mucosa, predict the mechanism of action of therapeutic vaccines, and describe the dynamics of 3-dimensional infection environment during the natural evolution of an HSV-2 lesion.

Immune Predictors of Mortality After Ribonucleic Acid Virus Infection.

BACKGROUND: Virus infections result in a range of clinical outcomes for the host, from asymptomatic to severe or even lethal disease. Despite global efforts to prevent and treat virus infections to limit morbidity and mortality, the continued emergence and re-emergence of new outbreaks as well as common infections such as influenza persist as a health threat. Challenges to the prevention of severe disease after virus infection include both a paucity of protective vaccines as well as the early identification of individuals with the highest risk that may require supportive treatment. METHODS: We completed a screen of mice from the Collaborative Cross (CC) that we infected with influenza, severe acute respiratory syndrome-coronavirus, and West Nile virus. RESULTS: The CC mice exhibited a range of disease manifestations upon infections, and we used this natural variation to identify strains with mortality after infection and strains exhibiting no mortality. We then used comprehensive preinfection immunophenotyping to identify global baseline immune correlates of protection from mortality to virus infection. CONCLUSIONS: These data suggest that immune phenotypes might be leveraged to identify humans at highest risk of adverse clinical outcomes upon infection, who may most benefit from intensive clinical interventions, in addition to providing insight for rational vaccine design.

A Fixed Spatial Structure of CD8+ T Cells in Tissue during Chronic HSV-2 Infection.

Tissue-resident CD8+ T cells (Trm) can rapidly eliminate virally infected cells, but their heterogeneous spatial distribution may leave gaps in protection within tissues. Although Trm patrol prior sites of viral replication, murine studies suggest they do not redistribute to adjacent uninfected sites to provide wider protection. We perform mathematical modeling of HSV-2 shedding in Homo sapiens and predict that infection does not induce enough Trm in many genital tract regions to eliminate shedding; a strict spatial distribution pattern of mucosal CD8+ T cell density is maintained throughout chronic infection, and trafficking of Trm across wide genital tract areas is unlikely. These predictions are confirmed with spatial analysis of CD8+ T cell distribution in histopathologic specimens from human genital biopsies. Further simulations predict that the key mechanistic correlate of protection following therapeutic HSV-2 vaccination would be an increase in total Trm rather than spatial reassortment of these cells. The fixed spatial structure of Trm induced by HSV-2 is sufficient for rapid elimination of infected cells but only in a portion of genital tract microregions.

Immune Correlates of Protection From West Nile Virus Neuroinvasion and Disease.

BACKGROUND: A challenge to the design of improved therapeutic agents and prevention strategies for neuroinvasive infection and associated disease is the lack of known natural immune correlates of protection. A relevant model to study such correlates is offered by the Collaborative Cross (CC), a panel of recombinant inbred mouse strains that exhibit a range of disease manifestations upon infection. METHODS: We performed an extensive screen of CC-F1 lines infected with West Nile virus (WNV), including comprehensive immunophenotyping, to identify groups of lines that exhibited viral neuroinvasion or neuroinvasion with disease and lines that remained free of WNV neuroinvasion and disease. RESULTS: Our data reveal that protection from neuroinvasion and disease is multifactorial and that several immune outcomes can contribute. Immune correlates identified include decreased suppressive activity of regulatory T cells at steady state, which correlates with peripheral restriction of the virus. Further, a rapid contraction of WNV-specific CD8+ T cells in the brain correlated with protection from disease. CONCLUSIONS: These immune correlates of protection illustrate additional networks and pathways of the WNV immune response that cannot be observed in the C57BL/6 mouse model. Additionally, correlates of protection exhibited before infection, at baseline, provide insight into phenotypic differences in the human population that may predict clinical outcomes upon infection.

A Mouse Model of Chronic West Nile Virus Disease.

Infection with West Nile virus (WNV) leads to a range of disease outcomes, including chronic infection, though lack of a robust mouse model of chronic WNV infection has precluded identification of the immune events contributing to persistent infection. Using the Collaborative Cross, a population of recombinant inbred mouse strains with high levels of standing genetic variation, we have identified a mouse model of persistent WNV disease, with persistence of viral loads within the brain. Compared to lines exhibiting no disease or marked disease, the F1 cross CC(032x013)F1 displays a strong immunoregulatory signature upon infection that correlates with restraint of the WNV-directed cytolytic response. We hypothesize that this regulatory T cell response sufficiently restrains the immune response such that a chronic infection can be maintained in the CNS. Use of this new mouse model of chronic neuroinvasive virus will be critical in developing improved strategies to prevent prolonged disease in humans.

HIV-1-Neutralizing IgA Detected in Genital Secretions of Highly HIV-1-Exposed Seronegative Women on Oral Preexposure Prophylaxis.

Although nonhuman primate studies have shown that simian immunodeficiency virus/simian-human immunodeficiency virus (SIV/SHIV) exposure during preexposure prophylaxis (PrEP) with oral tenofovir can induce SIV immunity without productive infection, this has not been documented in humans. We evaluated cervicovaginal IgA in Partners PrEP Study participants using a subtype C primary isolate and found that women on PrEP had IgA with higher average human immunodeficiency virus type 1 (HIV-1)-neutralizing magnitude than women on placebo (33% versus 7%; P = 0.008). Using a cutoff of ≥90% HIV-1 neutralization, 19% of women on-PrEP had HIV-1-neutralizing IgA compared to 0% of women on placebo (P = 0.09). We also estimated HIV-1 exposure and found that the proportion of women with HIV-1-neutralizing IgA was associated with the level of HIV-1 exposure (P = 0.04). Taken together, our data suggest that PrEP and high levels of exposure to HIV may each enhance mucosal HIV-1-specific humoral immune responses in sexually exposed but HIV-1-uninfected individuals. IMPORTANCE: Although there is not yet an effective HIV-1 vaccine, PrEP for at-risk HIV-1-uninfected individuals is a highly efficacious intervention to prevent HIV-1 acquisition and is currently being recommended by the CDC and WHO for all individuals at high risk of HIV-1 acquisition. We previously demonstrated that PrEP use does not enhance peripheral blood HIV-1-specific T-cell responses in HIV-exposed individuals. Here, we evaluate for cervicovaginal HIV-neutralizing IgA responses in genital mucosal secretions of HIV-exposed women, which is likely a more relevant site than peripheral blood for observation of potentially protective immune events occurring in response to sexual HIV-1 exposure for various periods. Furthermore, we assess for host response in the context of longitudinal quantification of HIV-1 exposure. We report that HIV-neutralizing IgA is significantly correlated with higher HIV-1 exposure and, furthermore, that there are more women with HIV-1-neutralizing IgA in the on-PrEP group than in the placebo group.

Regulatory T cells shape the resident memory T cell response to virus infection in the tissues.

Regulatory T cells (Tregs) are well known for their role in dampening the immune responses to self-Ags and, thereby, limiting autoimmunity. However, they also must permit immune responses to occur against foreign infectious agents. Using a mouse model of West Nile virus infection, we examined the role of Tregs in the generation of effector and memory T cell responses in the secondary lymphoid organs, as well as the infected tissues. We found that Treg numbers and activation increased in both the secondary lymphoid organs and CNS postinfection. Using Foxp3(DTR) knock-in mice, we found that Treg-deficient mice had increased Ag-driven production of IFN-γ from both CD4(+) and CD8(+) T cells in the spleen and CNS during the effector phase. In mice lacking Tregs, there were greater numbers of short-lived effector CD8(+) T cells in the spleen during the peak of the immune response, but the memory CD8(+) T cell response was impaired. Specifically, we demonstrate that Treg-dependent production of TGF-ß results in increased expression of CD103 on CD8(+) T cells, thereby allowing for a large pool of resident memory T cells to be maintained in the brain postinfection.

Antiretroviral Pre-Exposure Prophylaxis Does Not Enhance Immune Responses to HIV in Exposed but Uninfected Persons.

BACKGROUND: Antiretroviral preexposure prophylaxis (PrEP), using daily oral combination tenofovir disoproxil fumarate plus emtricitabine, is an effective human immunodeficiency virus (HIV) prevention strategy for populations at high risk of HIV acquisition. Although the primary mode of action for the protective effect of PrEP is probably direct antiviral activity, nonhuman primate studies suggest that PrEP may also allow for development of HIV-specific immune responses, hypothesized to result from aborted HIV infections providing a source of immunologic priming. We sought to evaluate whether PrEP affects the development of HIV-specific immune response in humans. METHODS AND RESULTS: Within a PrEP clinical trial among high-risk heterosexual African men and women, we detected HIV-specific CD4(+) and CD8(+) peripheral blood T-cell responses in 10%-20% of 247 subjects evaluated. The response rate and magnitude of T-cell responses did not vary significantly between those assigned PrEP versus placebo, and no significant difference between those assigned PrEP and placebo was observed in measures of innate immune function. CONCLUSIONS: We found no evidence to support the hypothesis that PrEP alters either the frequency or magnitude of HIV-specific immune responses in HIV-1-exposed seronegative individuals. These results suggest that PrEP is unlikely to serve as an immunologic prime to aid protection by a putative HIV vaccine.

Regulatory T cells restrict immunity and pathology in distal tissue sites following a localized infection.

Regulatory T cells (Tregs) are well-known to mediate peripheral tolerance at homeostasis, and there is a growing appreciation for their role in modulating infectious disease immunity. Following acute and chronic infections, Tregs can restrict pathogen-specific T cell responses to limit immunopathology. However, it is unclear if Tregs mediate control of pathology and immunity in distal tissue sites during localized infections. We investigated the role of Tregs in immunity and disease in various tissue compartments in the context of "mild" vaginal Zika virus infection. We found that Tregs are critical to generating robust virus-specific CD8 T cell responses in the initial infection site. Further, Tregs limit inflammatory cytokines and immunopathology during localized infection; a dysregulated immune response in Treg-depleted mice leads to increased T cell infiltrates and immunopathology in both the vagina and the central nervous system (CNS). Importantly, these CNS infiltrates are not present at the same magnitude during infection of Treg-sufficient mice, in which there is no CNS immunopathology. Our data suggest that Tregs are necessary to generate a robust virus-specific response at the mucosal site of infection, while Treg-mediated restriction of bystander inflammation limits immunopathology both at the site of infection as well as distal tissue sites.

Unique immune profiles in collaborative cross mice linked to survival and viral clearance upon infection.

The response to infection is generally heterogeneous and diverse, with some individuals remaining asymptomatic while others present with severe disease or a diverse range of symptoms. Here, we address the role of host genetics on immune phenotypes and clinical outcomes following viral infection by studying genetically diverse mice from the Collaborative Cross (CC), allowing for use of a small animal model with controlled genetic diversity while maintaining genetic replicates. We demonstrate variation by deeply profiling a broad range of innate and adaptive immune cell phenotypes at steady-state in 63 genetically distinct CC mouse strains and link baseline immune signatures with virologic and clinical disease outcomes following infection of mice with herpes simplex virus 2 (HSV-2) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This work serves as a resource for CC strain selection based on steady-state immune phenotypes or disease presentation upon viral infection, and further, points to possible pre-infection immune correlates of survival and early viral clearance upon infection.

Bacterial vaginosis-driven changes in vaginal T cell phenotypes and their implications for HIV susceptibility.

Bacterial vaginosis (BV) is a dysbiosis of the vaginal microbiome that is prevalent in reproductive-age women worldwide. Adverse outcomes associated with BV include an increased risk of sexually acquired Human Immunodeficiency Virus (HIV), yet the immunological mechanisms underlying this association are not well understood. To investigate BV driven changes to cervicovaginal tract (CVT) and circulating T cell phenotypes, participants with or without BV provided vaginal tract (VT) and ectocervical (CX) tissue biopsies and peripheral blood mononuclear cells (PBMC). Immunofluorescence analysis of genital mucosal tissues revealed a reduced density of CD3+CD4+CCR5+ cells in the VT lamina propria of individuals with compared to those without BV (median 243.8 cells/mm2 BV- vs 106.9 cells/mm2 BV+, p=0.043). High-parameter flow cytometry of VT biopsies revealed an increased frequency in individuals with compared to those without BV of dysfunctional CD39+ conventional CD4+ T cells (Tconv) (median frequency 15% BV- vs 30% BV+, padj=0.0331) and tissue-resident CD69+CD103+ Tconv (median frequency 24% BV- vs 38% BV+, padj=0.0061), previously reported to be implicated in HIV acquisition and replication. Our data suggests that BV elicits diverse and complex VT T cell alterations and expands on potential immunological mechanisms that may promote adverse outcomes including HIV susceptibility.

A gH/gL-encoding replicon vaccine elicits neutralizing antibodies that protect humanized mice against EBV challenge.

Epstein-Barr virus (EBV) is associated with several malignancies, neurodegenerative disorders and is the causative agent of infectious mononucleosis. A vaccine that prevents EBV-driven morbidity and mortality remains an unmet need. EBV is orally transmitted, infecting both B cells and epithelial cells. Several virally encoded proteins are involved in entry. The gH/gL glycoprotein complex is essential for infectivity irrespective of cell type, while gp42 is essential for infection of B cells. gp350 promotes viral attachment by binding to CD21 or CD35 and is the most abundant glycoprotein on the virion. gH/gL, gp42 and gp350, are known targets of neutralizing antibodies and therefore relevant immunogens for vaccine development. Here, we developed and optimized the delivery of several alphavirus-derived replicon RNA (repRNA) vaccine candidates encoding gH/gL, gH/gL/gp42 or gp350 delivered by a cationic nanocarrier termed LION™. The lead candidate, encoding full-length gH/gL, elicited high titers of neutralizing antibodies that persisted for at least 8 months and a vaccine-specific CD8+ T cell response. Transfer of vaccine-elicited IgG protected humanized mice from EBV-driven tumor formation and death following high-dose viral challenge. These data demonstrate that LION/repRNA-gH/gL is an ideal candidate vaccine for preventing EBV infection and/or related malignancies in humans.

Recurrent infection transiently expands human tissue T cells while maintaining long-term homeostasis.

Chronic viral infections are known to lead to T cell exhaustion or dysfunction. However, it remains unclear if antigen exposure episodes from periodic viral reactivation, such as herpes simplex virus type-2 (HSV-2) recrudescence, are sufficient to induce T cell dysfunction, particularly in the context of a tissue-specific localized, rather than a systemic, infection. We designed and implemented a stringent clinical surveillance protocol to longitudinally track both viral shedding and in situ tissue immune responses in a cohort of HSV+ volunteers that agreed to avoid using anti-viral therapy for the course of this study. Comparing lesion to control skin biopsies, we found that tissue T cells expanded immediately after reactivation, and then returned numerically and phenotypically to steady state. T cell responses appeared to be driven at least in part by migration of circulating T cells to the infected tissue. Our data indicate that tissue T cells are stably maintained in response to HSV reactivation, resembling a series of acute recall responses.

Oral tolerance to systemic vaccination remains intact without RORγt expression in regulatory T cells.

Many promising vaccine candidates and licensed vaccines lead to variable immune responses within humans. Studies suggest that environmental exposures in the gastrointestinal tract could contribute to a reduction in vaccine efficacy via immune tolerance at this site; this is partly achieved by a high abundance of regulatory T cells (Tregs). It is unclear if Treg subsets regulate systemic vaccine responses following oral antigen pre-exposure. Here, we implemented a conditional knock-out mouse model of RORγt+ Tregs to examine the role of these cells in mediating this process. Following oral exposure to the model antigen ovalbumin (OVA) prior to immunization, we found similar induction of vaccine-induced antibody responses in mice lacking RORγt expression in Tregs compared to sufficient controls. Use of various adjuvants led to distinct findings. Our data suggest that expression of RORγt+ within Tregs is not required to regulate tolerance to systemic vaccination following oral antigen exposure.

Memory T cells possess an innate-like function in local protection from mucosal infection.

Mucosal infections pose a significant global health burden. Antigen-specific tissue-resident T cells are critical to maintaining barrier immunity. Previous studies in the context of systemic infection suggest that memory CD8+ T cells may also provide innate-like protection against antigenically unrelated pathogens independent of T cell receptor engagement. Whether bystander T cell activation is also an important defense mechanism in the mucosa is poorly understood. Here, we investigated whether innate-like memory CD8+ T cells could protect against a model mucosal virus infection, herpes simplex virus 2 (HSV-2). We found that immunization with an irrelevant antigen delayed disease progression from lethal HSV-2 challenge, suggesting that memory CD8+ T cells may mediate protection despite the lack of antigen specificity. Upon HSV-2 infection, we observed an early infiltration, rather than substantial local proliferation, of antigen-nonspecific CD8+ T cells, which became bystander-activated only within the infected mucosal tissue. Critically, we show that bystander-activated CD8+ T cells are sufficient to reduce early viral burden after HSV-2 infection. Finally, local cytokine cues within the tissue microenvironment after infection were sufficient for bystander activation of mucosal tissue memory CD8+ T cells from mice and humans. Altogether, our findings suggest that local bystander activation of CD8+ memory T cells contributes a fast and effective innate-like response to infection in mucosal tissue.