Tissue-Resident-Memory CD8+ T Cells Bridge Innate Immune Responses in Neighboring Epithelial Cells to Control Human Genital Herpes.

Tissue-resident-memory T cells (TRM) populate the body's barrier surfaces, functioning as frontline responders against reencountered pathogens. Understanding of the mechanisms by which CD8TRM achieve effective immune protection remains incomplete in a naturally recurring human disease. Using laser capture microdissection and transcriptional profiling, we investigate the impact of CD8TRM on the tissue microenvironment in skin biopsies sequentially obtained from a clinical cohort of diverse disease expression during herpes simplex virus 2 (HSV-2) reactivation. Epithelial cells neighboring CD8TRM display elevated and widespread innate and cell-intrinsic antiviral signature expression, largely related to IFNG expression. Detailed evaluation via T-cell receptor reconstruction confirms that CD8TRM recognize viral-infected cells at the specific HSV-2 peptide/HLA level. The hierarchical pattern of core IFN-γ signature expression is well-conserved in normal human skin across various anatomic sites, while elevation of IFI16, TRIM 22, IFITM2, IFITM3, MX1, MX2, STAT1, IRF7, ISG15, IFI44, CXCL10 and CCL5 expression is associated with HSV-2-affected asymptomatic tissue. In primary human cells, IFN-γ pretreatment reduces gene transcription at the immediate-early stage of virus lifecycle, enhances IFI16 restriction of wild-type HSV-2 replication and renders favorable kinetics for host protection. Thus, the adaptive immune response through antigen-specific recognition instructs innate and cell-intrinsic antiviral machinery to control herpes reactivation, a reversal of the canonical thinking of innate activating adaptive immunity in primary infection. Communication from CD8TRM to surrounding epithelial cells to activate broad innate resistance might be critical in restraining various viral diseases.

Distinct populations of antigen-specific tissue-resident CD8+ T cells in human cervix mucosa.

The ectocervix is part of the lower female reproductive tract (FRT), which is susceptible to sexually transmitted infections (STIs). Comprehensive knowledge of the phenotypes and T cell receptor (TCR) repertoire of tissue-resident memory T cells (TRMs) in the human FRT is lacking. We took single-cell RNA-Seq approaches to simultaneously define gene expression and TCR clonotypes of the human ectocervix. There were significantly more CD8+ than CD4+ T cells. Unsupervised clustering and trajectory analysis identified distinct populations of CD8+ T cells with IFNGhiGZMBloCD69hiCD103lo or IFNGloGZMBhiCD69medCD103hi phenotypes. Little overlap was seen between their TCR repertoires. Immunofluorescence staining showed that CD103+CD8+ TRMs were preferentially localized in the epithelium, whereas CD69+CD8+ TRMs were distributed evenly in the epithelium and stroma. Ex vivo assays indicated that up to 14% of cervical CD8+ TRM clonotypes were HSV-2 reactive in HSV-2-seropositive persons, reflecting physiologically relevant localization. Our studies identified subgroups of CD8+ TRMs in the human ectocervix that exhibited distinct expression of antiviral defense and tissue residency markers, anatomic locations, and TCR repertoires that target anatomically relevant viral antigens. Optimization of the location, number, and function of FRT TRMs is an important approach for improving host defenses to STIs.

B cells, antibody-secreting cells, and virus-specific antibodies respond to herpes simplex virus 2 reactivation in skin.

Tissue-based T cells are important effectors in the prevention and control of mucosal viral infections; less is known about tissue-based B cells. We demonstrate that B cells and antibody-secreting cells (ASCs) are present in inflammatory infiltrates in skin biopsy specimens from study participants during symptomatic herpes simplex virus 2 (HSV-2) reactivation and early healing. Both CD20+ B cells, most of which are antigen inexperienced based on their coexpression of IgD, and ASCs - characterized by dense IgG RNA expression in combination with CD138, IRF4, and Blimp-1 RNA - were found to colocalize with T cells. ASCs clustered with CD4+ T cells, suggesting the potential for crosstalk. HSV-2-specific antibodies to virus surface antigens were also present in tissue and increased in concentration during HSV-2 reactivation and healing, unlike in serum, where concentrations remained static over time. B cells, ASCs, and HSV-specific antibody were rarely detected in biopsies of unaffected skin. Evaluation of samples from serial biopsies demonstrated that B cells and ASCs followed a more migratory than resident pattern of infiltration in HSV-affected genital skin, in contrast to T cells. Together, these observations suggest the presence of distinct phenotypes of B cells in HSV-affected tissue; dissecting their role in reactivation may reveal new therapeutic avenues to control these infections.

High Community SARS-CoV-2 Antibody Seroprevalence in a Ski Resort Community, Blaine County, Idaho, US. Preliminary Results.

Community-level seroprevalence surveys are needed to determine the proportion of the population with previous SARS-CoV-2 infection, a necessary component of COVID-19 disease surveillance. In May, 2020, we conducted a cross-sectional seroprevalence study of IgG antibodies for nucleocapsid of SARS-CoV-2 among the residents of Blaine County, Idaho, a ski resort community with high COVID-19 attack rates in late March and Early April (2.9% for ages 18 and older). Participants were selected from volunteers who registered via a secure web link, using prestratification weighting to the population distribution by age and gender within each ZIP Code. Participants completed a survey reporting their demographics and symptoms; 88% of volunteers who were invited to participate completed data collection survey and had 10 ml of blood drawn. Serology was completed via the Abbott Architect SARS-CoV-2 IgG immunoassay. Primary analyses estimated seroprevalence and 95% credible intervals (CI) using a hierarchical Bayesian framework to account for diagnostic uncertainty. Stratified models were run by age, sex, ZIP Code, ethnicity, employment status, and a priori participant-reported COVID-19 status. Sensitivity analyses to estimate seroprevalence included base models with post-stratification for ethnicity, age, and sex, with or without adjustment for multi-participant households. IgG antibodies to the virus that causes COVID-19 were found among 22.7% (95% CI: 20.1%, 25.5%) of residents of Blaine County. Higher levels of antibodies were found among residents of the City of Ketchum 34.8% (95% CI 29.3%, 40.5%), compared to Hailey 16.8% (95%CI 13.7%, 20.3%) and Sun Valley 19.4% (95% 11.8%, 28.4%). People who self-identified as not believing they had COVID-19 had the lowest prevalence 4.8% (95% CI 2.3%, 8.2%). The range of seroprevalence after correction for potential selection bias was 21.9% to 24.2%. This study suggests more than 80% of SARS-CoV-2 infections were not reported. Although Blaine County had high levels of SARS-CoV-2 infection, the community is not yet near the herd immunity threshold.

The human tissue-resident CCR5+ T cell compartment maintains protective and functional properties during inflammation.

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.

In Situ Detection of Regulatory T Cells in Human Genital Herpes Simplex Virus Type 2 (HSV-2) Reactivation and Their Influence on Spontaneous HSV-2 Reactivation.

BACKGROUND: Herpes simplex virus type 2 (HSV-2) reactivation is accompanied by a sustained influx of CD4(+) and CD8(+) T cells that persist in genital tissue for extended periods. While CD4(+) T cells have long been recognized as being present in herpetic ulcerations, their role in subclinical reactivation and persistence is less well known, especially the role of CD4(+) regulatory T cells (Tregs). METHODS: We characterized the Treg (CD4(+)Foxp3(+)) population during human HSV-2 reactivation in situ in sequential genital skin biopsy specimens obtained from HSV-2-seropositive subjects at the time of lesion onset up to 8 weeks after healing. RESULTS: High numbers of Tregs infiltrated to the site of viral reactivation and persisted in proximity to conventional CD4(+) T cells (Tconvs) and CD8(+) T cells. Treg density peaked during the lesion stage of the reactivation. The number of Tregs from all time points (lesion, healed, 2 weeks after healing, 4 weeks after healing, and 8 weeks after healing) was significantly higher than in control biopsy specimens from unaffected skin. There was a direct correlation between HSV-2 titer and Treg density. CONCLUSIONS: The association of a high Treg to Tconv ratio with high viral shedding suggests that the balance between regulatory and effector T cells influences human HSV-2 disease.

Image analysis for accurately counting CD4+ and CD8+ T cells in human tissue.

In situ detection of specific cells offers a unique perspective on the spatial interactions between host immune cells and specific viral pathogens or cancers. Most immunohistochemistry techniques require manual cell counting on biopsied and fixed tissue sections. The availability of sophisticated software packages for analyzing fluorescently labeled tissue has made it possible to quickly and accurately quantitate the number of positive cells on such slides. Manual cell counting was compared to automatic cell counting using the program CellProfiler. The two techniques were used to count CD4+ and CD8+ T cells in human genital skin biopsies from herpesvirus type 2 (HSV-2) infected subjects. Manual counting and CellProfiler demonstrated high correlation both in cell counting as well as detection of immune cell "clustering" in tissue, an important visceral component of localized inflammation and characteristic of most chronic infections. Overall, CellProfiler is an effective and accurate method in addition to or replacement of manual cell counting of fluorescently labeled biopsies.

Virologic and immunologic evidence of multifocal genital herpes simplex virus 2 infection.

UNLABELLED: Genital herpes simplex virus (HSV) reactivation is thought to be anatomically and temporally localized, coincident with limited ganglionic infection. Short, subclinical shedding episodes are the most common form of HSV-2 reactivation, with host clearance mechanisms leading to rapid containment. The anatomic distribution of shedding episodes has not been characterized. To precisely define patterns of anatomic reactivation, we divided the genital tract into a 22-region grid and obtained daily swabs for 20 days from each region in 28 immunocompetent, HSV-2-seropositive persons. HSV was detected via PCR, and sites of asymptomatic HSV shedding were subjected to a biopsy procedure within 24 h. CD4(+) and CD8(+) T cells were quantified by immunofluorescence, and HSV-specific CD4(+) T cells were identified by intracellular cytokine cytometry. HSV was detected in 868 (7%) of 11,603 genital swabs at a median of 12 sites per person (range, 0 to 22). Bilateral HSV detection occurred on 83 (67%) days with shedding, and the median quantity of virus detected/day was associated with the number of sites positive (P < 0.001). In biopsy specimens of asymptomatic shedding sites, we found increased numbers of CD8(+) T cells compared to control tissue (27 versus 13 cells/mm(2), P = 0.03) and identified HSV-specific CD4(+) T cells. HSV reactivations emanate from widely separated anatomic regions of the genital tract and are associated with a localized cellular infiltrate that was demonstrated to be HSV specific in 3 cases. These data provide evidence that asymptomatic HSV-2 shedding contributes to chronic inflammation throughout the genital tract. IMPORTANCE: This detailed report of the anatomic patterns of genital HSV-2 shedding demonstrates that HSV-2 reactivation can be detected at multiple bilateral sites in the genital tract, suggesting that HSV establishes latency throughout the sacral ganglia. In addition, genital biopsy specimens from sites of asymptomatic HSV shedding have increased numbers of CD8(+) T cells compared to control tissue, and HSV-specific CD4(+) T cells are found at sites of asymptomatic shedding. These findings suggest that widespread asymptomatic genital HSV-2 shedding is associated with a targeted host immune response and contributes to chronic inflammation throughout the genital tract.

Immune surveillance by CD8αα+ skin-resident T cells in human herpes virus infection.

Most herpes simplex virus 2 (HSV-2) reactivations in humans are subclinical and associated with rapid expansion and containment of virus. Previous studies have shown that CD8(+) T cells persist in genital skin and mucosa at the dermal-epidermal junction (DEJ)--the portal of neuronal release of reactivating virus--for prolonged time periods after herpes lesions are cleared. The phenotype and function of this persistent CD8(+) T-cell population remain unknown. Here, using cell-type-specific laser capture microdissection, transcriptional profiling and T-cell antigen receptor ß-chain (TCRß) genotyping on sequential genital skin biopsies, we show that CD8αα(+) T cells are the dominant resident population of DEJ CD8(+) T cells that persist at the site of previous HSV-2 reactivation. CD8αα(+) T cells located at the DEJ lack chemokine-receptor expression required for lymphocyte egress and recirculation, express gene signatures of T-cell activation and antiviral activity, and produce cytolytic granules during clinical and virological quiescent time periods. Sequencing of the TCR ß-chain repertoire reveals that the DEJ CD8αα(+) T cells are oligoclonal with diverse usage of TCR variable-ß genes, which differ from those commonly described for mucosa-associated invariant T cells and natural killer T cells. Dominant clonotypes are shown to overlap among multiple recurrences over a period of two-and-a-half years. Episodes of rapid asymptomatic HSV-2 containment were also associated with a high CD8 effector-to-target ratio and focal enrichment of CD8αα(+) T cells. These studies indicate that DEJ CD8αα(+) T cells are tissue-resident cells that seem to have a fundamental role in immune surveillance and in initial containment of HSV-2 reactivation in human peripheral tissue. Elicitation of CD8αα(+) T cells may be a critical component for developing effective vaccines against skin and mucosal infections.

Evasion of the mucosal innate immune system by herpes simplex virus type 2.

Understanding the mechanisms by which herpes simplex virus (HSV) evades host immune defenses is critical to defining new approaches for therapy and prevention. We performed transcriptional analyses and immunocytochemistry on sequential biopsy specimens of lesional tissue from the acute through the posthealing phases of recurrent mucocutaneous HSV-2 infection. Histological analysis of these biopsy specimens during the acute stage revealed a massive infiltration of T cells, as well as monocytes/macrophages, a large amount of myeloid, and a small number of plasmacytoid dendritic cells, in the dermis of these lesional biopsy specimens. Type I interferon (IFN-beta and IFN-alpha) was poorly expressed and gamma IFN (IFN-gamma) potently induced during time periods in which we detected abundant amounts of HSV-2 antigens and HSV-2 RNA. IFN-stimulated genes were also markedly upregulated, with expression patterns that more closely matched those in primary human fibroblasts treated by IFN-gamma than those in fibroblasts treated by IFN-beta. Transcriptional arrays of the same lesional biopsy sites during healing and at 2 and 4 weeks posthealing revealed no HSV nucleic acids or antigen; however, there was persistent expression of IFN-gamma, with very low levels of IFN-beta and IFN-alpha. The findings of extremely low levels of IFN-alpha and IFN-beta, despite the presence of a large number of cells capable of synthesizing these substances, suggest a potent alteration in host defense during HSV-2 infection in vivo. HSV-2's blockade of the innate immune system's production of type I IFN may be a major factor in allowing the virus to break through host mucosal defenses.

Persistence of HIV-1 receptor-positive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition.

To explore the mechanism by which herpes simplex virus (HSV)-2 infection is related to HIV-1 acquisition, we conducted in situ analysis of the cellular infiltrate from sequential biopsies of HSV-2 lesions from patients on and off antiviral therapy. CD4(+) and CD8(+) T cells and a mixed population of plasmacytoid and myeloid dendritic cells (DCs), including cells expressing the C-type lectin receptor DC-SIGN, persisted at sites of HSV-2 reactivation for months after healing, even with daily antiviral therapy. The CD4(+) T cells that persisted reacted to HSV-2 antigen, were enriched for expression of the chemokine receptor CCR5, and were contiguous to DCs expressing the interleukin-3 receptor CD123 or DC-SIGN. Ex vivo infection with a CCR5-tropic strain of HIV-1 revealed greater concentrations of integrated HIV-1 DNA in cells derived from healed genital lesion biopsies than in cells from control skin biopsies. The persistence and enrichment of HIV receptor-positive inflammatory cells in the genitalia help explain the inability of anti-HSV-2 therapy to reduce HIV acquisition.