The gastrointestinal mucosa is an important site of HIV acquisition, viral replication and pathogenesis. Immune cells in mucosal tissues frequently differ in phenotype and function from their non-mucosal counterparts. Although perforin-mediated cytotoxicity as measured in blood is a recognized correlate of HIV immune control, its role in gastrointestinal tissues is unknown. We sought to elucidate the cytotoxic features of rectal mucosal CD8+ T-cells in HIV infected and uninfected subjects. Perforin expression and lytic capacity were significantly reduced in rectal CD8+ T-cells compared to their blood counterparts, regardless of HIV clinical status; granzyme B (GrzB) was reduced to a lesser extent. Mucosal perforin and GrzB expression were higher in participants not on antiretroviral therapy compared to those on therapy and controls. Reduction in perforin and GrzB was not explained by differences in memory/effector subsets. Expression of T-bet and Eomesodermin was significantly lower in gut CD8+ T-cells compared to blood, and in vitro neutralization of TGF-β partially restored perforin expression in gut CD8+ T-cells. These findings suggest that rectal CD8+ T-cells are primarily non-cytotoxic, and phenotypically shaped by the tissue microenvironment. Further elucidation of rectal immune responses to HIV will inform the development of vaccines and immunotherapies targeted to mucosal tissues.

Tissue-resident memory (TRM) CD8+ T-cells are non-recirculating, long-lived cells housed in tissues that can confer protection against mucosal pathogens. HIV-1 is a mucosal pathogen and the gastrointestinal tract is an important site of viral pathogenesis and transmission. Thus, CD8+ TRM cells may be an important effector subset for controlling HIV-1 in mucosal tissues. This study sought to determine the abundance, phenotype, and functionality of CD8+ TRM cells in the context of chronic HIV-1 infection. We found that the majority of rectosigmoid CD8+ T-cells were CD69+CD103+S1PR1− and T-betLowEomesoderminNeg, indicative of a tissue-residency phenotype similar to that described in murine models. HIV-1-specific CD8+ TRM responses appeared strongest in individuals naturally controlling HIV-1 infection. Two CD8+ TRM subsets, distinguished by CD103 expression intensity, were identified. CD103Low CD8+ TRM primarily displayed a transitional memory phenotype and contained HIV-1-specific cells and cells expressing high levels of Eomesodermin, whereas CD103High CD8+ TRM primarily displayed an effector memory phenotype and were EomesoderminNeg. These findings suggest a large fraction of CD8+ T-cells housed in the human rectosigmoid mucosa are tissue-resident and that TRM contribute to the anti-HIV-1 immune response. Further exploration of CD8+ TRM will inform development of anti-HIV-1 immune-based therapies and vaccines targeted to the mucosa.

We previously reported that CD8 T cells in human gastrointestinal mucosa exhibit reduced perforin expression and weak or impaired cytotoxic capacity compared with their counterparts in blood. Nevertheless, these cells degranulate and express cytokines and chemokines in response to cognate Ag. In addition to weak expression of perforin, earlier studies suggested differential regulation of perforin and granzymes (Gzms), with GzmA and B expressed by significantly higher percentages of mucosal CD8 T cells than perforin. However, this topic has not been fully explored. The goal of this study was to elucidate the expression and coexpression patterns of GzmA, B, and K in conjunction with perforin in rectosigmoid CD8 T cells during HIV-1 infection. We found that expression of both perforin and GzmB, but not GzmA or GzmK, was reduced in mucosa compared with blood. A large fraction of rectosigmoid CD8 T cells either did not express Gzms or were single-positive for GzmA. Rectosigmoid CD8 T cells appeared skewed toward cytokine production rather than cytotoxic responses, with cells expressing multiple cytokines and chemokines generally lacking in perforin and Gzm expression. These data support the interpretation that perforin and Gzms are differentially regulated, and display distinct expression patterns in blood and rectosigmoid T cells. These studies may help inform the development of strategies to combat HIV-1 and other mucosal pathogens.

Purpose of review:This review summarizes our current understanding of HIV-1-specific T-cell responses in mucosal tissues, emphasizing recent work and specifically highlighting papers published over the past 18 months. Summary: HIV-1-specific T-cell responses have been extensively characterized; however, the vast majority of reports have focused on T-cells isolated from peripheral blood. Mucosal tissues of the genitourinary and gastrointestinal tracts serve as the primary sites of HIV-1 transmission, and provide "front line" barrier defenses against HIV-1 and other pathogens. In addition, the gastrointestinal tract remains a significant viral reservoir throughout the chronic phase of infection. Recent work on mucosal immunity has improved the standardization of tissue sampling approaches as well as provided new insights on the abundance, phenotype and distribution of HIV-1-specific T-cell populations in mucosal tissues.

Purpose of Review This review summarizes recent literature defining tissue-resident memory T cells (T RM) and discusses implications for HIV pathogenesis, vaccines, and eradication efforts. Recent Findings Investigations using animal models and human tissues have identified a T RM transcriptional profile and elucidated signals within the tissue microenvironment leading to T RM development and maintenance. T RM are major contributors to host response in infectious diseases and cancer; in addition, T RM contribute to pathogenic inflammation in a variety of settings. Although T RM are daunting to study in HIV infection, recent work has helped define their molecular signatures and effector functions and tested strategies for their mobilization. Summary Exclusive reliance on blood sampling to gain an understanding of host immunity overlooks the contribution of T RM , which differ in significant ways from their counterparts in circulation. It is hoped that greater understanding of these cells will lead to novel approaches to prevent and/or eradicate HIV infection.

CD8+ T-cells in human gastrointestinal mucosa exhibit reduced perforin expression and weak cytotoxicity compared to their counterparts in blood. We previously reported that these cells are predominantly T-bet-low and Eomesodermin-negative. Nevertheless, in response to antigenic stimulation these cells degranulate and express cytokines and chemokines. The goal of this study was to elucidate expression patterns of granzymes (Gzm) A, B and K in conjunction with perforin in rectosigmoid CD8+ T-cells during chronic HIV-1 infection and determine whether these patterns vary with disease status. We used flow cytometry to compare intracellular Gzm A, B, K and perforin in resting CD8+ T-cells from blood and rectosigmoid biopsies of HIV-1+ controllers; viremic individuals; virologically suppressed individuals on antiretroviral therapy; and seronegatives. Expression of perforin and GzmB, but not GzmA or GzmK, was reduced in mucosa compared to blood. A large fraction of rectosigmoid CD8+ T-cells did not express granzymes or were single-positive for GzmA. Rectosigmoid CD8+ T-cells appeared skewed towards cytokine production rather than cytotoxicity, with cells expressing multiple cytokines/chemokines lacking perforin and granzyme expression. Percentages of mucosal CD8+ T-cells expressing cytotoxic effectors were higher in HIV-1+ groups, compared to seronegatives. These data support the interpretation that perforin and granzymes are differentially regulated, and display distinct expression patterns in blood and rectosigmoid T-cells. These findings also reinforce our previous work, suggesting that rectosigmoid CD8+ T-cells are programmed for cytokine polyfunctionality rather than cytotoxicity.

Mucosal tissues including the gastrointestinal tract are important reservoirs for HIV replication. HIV-specific CD8 T cells are essential in controlling HIV infection. In order to gain insights into HIV-specific CD8 T cells in elite controllers (EC), we characterized this important cell population in rectal biopsies from EC using in situ tetramer staining combined with immunohistochemistry followed by quantitative analysis. Results showed tetramer+ HIV-specific CD8 T cells distributed unevenly throughout the rectal mucosa including within lymphoid aggregates. Frequencies of tetramer+ HIV-specific CD8 T cells ranged from 2 to 25 cells/mm2. A mean of 55% tetramer+ HIV-specific CD8 T cells expressed the effector protein perforin. Among the perforin+ HIV-specific T cells, the majority expressed low to medium levels of perforin and relatively few expressed high levels. In addition, we also observed a subset of perforin+ HIV-specific CD8 T cells in which perforin was located exclusively within the cell membrane, possibly representing a novel subset of armed resident or effector memory cells. Almost all tetramer+ HIV-specific CD8 T cells in the rectal mucosa were negative for Ki67, a marker for activation and proliferation (mean of 96%); many were PD-1+, a marker for recent T cell stimulation or exhaustion (mean of 59%), and some expressed CD103, a marker for a subset of resident memory T cells (mean of 6%). Taken together, these findings indicate that EC maintain diverse populations of memory HIV-specific CD8 T cells distributed throughout the rectal mucosa including within lymphoid aggregates and suggests that these cells are likely continually being exposed to antigen, but not dividing.

The gastrointestinal tract (GIT) is an important site of HIV transmission and pathogenesis. Preventing or curing HIV infection will likely entail maintenance of protective anti-HIV immune responses in the GIT. Tissue resident memory T-cells (TRM) are long-lived, non-recirculating memory T-cells localized in tissues including the GIT. Positioned near sites of infection, TRM have protective efficacy against several pathogens. The precise role CD8+ TRM in HIV infection is not known. Blood and GIT CD8+ TRM cells from HIV+ and HIV- adults were identified by flow cytrometry using CD45RO, CD69, CD103, S1PR1, T-bet, and Eomesodermin (Eomes). Functionality was assessed in a 6-hour ex vivo stimulation assay. Cells were stimulated with DMSO, Gag, or Staphylococcal Enterotoxin B, and stained for CD107a, IFNγ, Mip-1β, TNFα, and IL-2. The frequency of CD103+CD69+S1PR1− CD8+ T-cells was significantly greater (P=0.005) in gut compared to blood (medians 54.8% and 0.10% respectively), indicative of tissue residency. CD45RO+ cells in this subset were classified as TRM. Consistent with murine studies, CD8+TRM were mainly T-betLowEomesNeg; however a population of EomesHigh CD8+ TRM emerged in HIV+ individuals not on antiretroviral therapy. Although polyfunctional Gag-specific CD8+ TRM responses (CD107a+, IFNγ+, Mip-1β+) appeared strongest in participants who control HIV without therapy (“controllers”), they constituted a smaller proportion of the total rectal Gag-specific CD8+ T-cell response compared to viremic participants not on ART (approximately 15% and 29% respectively). In summary, a large subset of CD8+ T-cells in rectal mucosa are TRM and likely play an important role in HIV-1 containment.