Peculiar Phenotypic and Cytotoxic Features of Pulmonary Mucosal CD8 T Cells in People Living with HIV Receiving Long-Term Antiretroviral Therapy

Oussama Meziane, Yulia Alexandrova, Ronald Olivenstein, Franck P. Dupuy, Syim Salahuddin, Elaine Thomson, Marianna Orlova, Erwin Schurr, Petronela Ancuta, Madeleine Durand, Nicolas Chomont, Jérôme Estaquier, Nicole F. Bernard, Cecilia T. Costiniuk and Mohammad-Ali Jenabian
J Immunol February 1, 2021, 206 (3) 641-651; DOI: https://doi.org/10.4049/jimmunol.2000916

Key Points

  • Pulmonary mucosal CD8 T cells are highly differentiated, activated, and exhausted.

  • BAL versus blood CD8 T cells showed lower perforin/granzyme B expression.

  • BAL CD8 T cells display reduced target killing ability compared to blood CD8 T cells.

Abstract

People living with HIV have high burdens of chronic lung disease, lung cancers, and pulmonary infections despite antiretroviral therapy (ART). The rates of tobacco smoking by people living with HIV vastly exceed that of the general population. Furthermore, we showed that HIV can persist within the lung mucosa despite long-term ART. As CD8 T cell cytotoxicity is pivotal for controlling viral infections and eliminating defective cells, we explored the phenotypic and functional features of pulmonary versus peripheral blood CD8 T cells in ART-treated HIV+ and uninfected controls. Bronchoalveolar lavage fluid and matched blood were obtained from asymptomatic ART-treated HIV+ smokers (n = 11) and nonsmokers (n = 15) and uninfected smokers (n = 7) and nonsmokers (n = 10). CD8 T cell subsets and phenotypes were assessed by flow cytometry. Perforin/granzyme B content, degranulation (CD107a expression), and cytotoxicity against autologous Gag peptide-pulsed CD4 T cells (Annexin V+) following in vitro stimulation were assessed. In all groups, pulmonary CD8 T cells were enriched in effector memory subsets compared with blood and displayed higher levels of activation (HLA-DR+) and exhaustion (PD1+) markers. Significant reductions in proportions of senescent pulmonary CD28−CD57+ CD8 T cells were observed only in HIV+ smokers. Pulmonary CD8 T cells showed lower perforin expression ex vivo compared with blood CD8 T cells, with reduced granzyme B expression only in HIV+ nonsmokers. Bronchoalveolar lavage CD8 T cells showed significantly less in vitro degranulation and CD4 killing capacity than blood CD8 T cells. Therefore, pulmonary mucosal CD8 T cells are more differentiated, activated, and exhausted, with reduced killing capacity in vitro than blood CD8 T cells, potentially contributing to a suboptimal anti-HIV immune response within the lungs.

Introduction

Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) suffer from a high burden of infectious and noninfectious pulmonary complications and chronic lung diseases, associated with high mortality rates (1, 2). As the lungs are constantly exposed to environmental Ags, pulmonary immune cells differ in phenotype and function from pulmonary immune cells in the blood (1, 3). HIV seeds the lungs during primary infection and is detectable in different intrapulmonary immune cells, notably alveolar macrophages and CD4 T cells, contributing to chronic pulmonary inflammation and mucosal tissue damage (1, 3, 4). Studies in SIV-infected rhesus macaques have shown that besides the gut, the lungs also contain high amounts of SIV during suppressive ART (5, 6). In humans, HIV DNA and HIV RNA were detectable in the lungs even after 6 mo of effective ART (7). We recently reported 13-fold-higher levels of HIV DNA in total bronchoalveolar lavage (BAL) cells and pulmonary mucosal CD4 T cells compared with cells from the peripheral blood of HIV+ adults despite long-term suppressive ART (median of 9 y) (3). Furthermore, despite ART, PLWH continue to suffer from a high burden of nonopportunistic infections, suggesting that pulmonary immunity is not fully restored. PLWH also have rates of tobacco smoking as high as 70% (8), which induces proinflammatory effects and perturbs pulmonary immune function (9). Thus, elucidating HIV-specific immune responses in the lungs versus the peripheral blood is an essential step along the path to develop immune-based therapies for HIV and for treatment strategies to slow or reverse pulmonary disease and lung cancer risk. Given the high prevalence of tobacco smoking among PLWH, decorticating how tobacco smoking affects the pulmonary immune environment is also a critical element along this path of discovery. However, data on the effect of tobacco smoking on pulmonary CD8 T cell function in PLWH are scarce.

Cytotoxic CD8 T cells are essential in controlling chronic viral infections and eliminating infected, defective, or precancerous cells (10, 11). Proliferation and maintenance of Ag-specific CD8 responses are critical for eradication of acute respiratory viral infections, such as influenza and respiratory syncytial virus (12–14). During chronic viral infections or lung cancers, there is a loss of CD8 cytotoxic activity during their late-stage of differentiation due to CD8 exhaustion (15–18). Importantly, the severity of inflammatory lung diseases such as chronic obstructive pulmonary disease (COPD), which is more prevalent in PLWH, is associated with increased CD8 exhaustion and dysfunction (2, 19, 20).

HIV elicits potent CD8 T cell virus-specific cytotoxic responses. However, initial suppression of viremia is insufficient for complete viral clearance, and there is a gradual decline in CD8 cytotoxic function (11). Cytotoxic CD8 T cells are needed to maintain the suppression of viral replication during ART, but their exhaustion and senescence lead to disrupted anti-HIV immune responses and HIV persistence (11, 17, 21–24). In the absence of opportunistic infections, asymptomatic PLWH frequently develop “CD8 T cell alveolitis” characterized by accumulation of functionally impaired HIV-specific pulmonary CD8 T cells, associated with increased respiratory symptoms and poor clinical outcomes (25). ART initiation has been shown to partially restore CD8 T cell immune functions (11) and resolve CD8 alveolitis (26). Nevertheless, in contrast to CD4 T cells, HIV- or CMV-specific pulmonary mucosal CD8 T cells remain dysfunctional despite ART (26, 27). However, it is unclear why pulmonary mucosal CD8 T cells fail to recover their Ag-specific functions despite ART.

In this study, we sought to assess the phenotypical and cytotoxic functional features of pulmonary CD8 T cells versus blood CD8 T cells from PLWH and uninfected controls. A secondary goal was to determine how tobacco smoking status, a habit highly prevalent in the HIV community, may influence pulmonary CD8 T cells.

Materials and Methods

Study population, sample collection, and cell isolation

ART-treated PLWH smokers (n = 11) and nonsmokers (n = 15) and uninfected smokers (n = 7) and nonsmokers (n = 10) were recruited without any respiratory symptoms or active infection at the McGill University Health Centre (Montreal, QC, Canada) (Table I, Supplemental Table I). Participants underwent spirometric testing several weeks prior to bronchoscopy to ensure the absence of any undiagnosed obstructive airflow disease. For each participant, data were captured on age, sex, ethnicity, cannabis use, and tobacco smoking at the time of bronchoscopy. Participants were labeled as smokers if they smoked at least one tobacco cigarette daily. Additionally, for participants living with HIV, data were captured on duration of HIV infection, time since viral load suppression, components of their antiretroviral regimens, and history of pulmonary infections. HIV-infected participants were all ART treated with suppressed plasma viral load and CD4 count higher than 350 cells/mm3 for at least 3 y. A total of 50 to 100 ml of BAL fluid was obtained via bronchoscopy, and 40 ml of blood was collected from each participant. BAL cells and PBMCs were isolated as we previously described (28). Of note, because of the limited numbers of BAL cells and interindividual variations between their CD8 T cell frequencies, we were not able to assess all study measures described below in all individuals. According to available cell numbers, we prioritized the measures to be assessed on each specimen as described in Supplemental Table II.

Ethical considerations

The study was approved by the Institutional Review Boards of the McGill University Health Centre (no. 15-031) and Université du Québec à Montréal (no. 602). All study participants signed a written informed consent.

CD8 T cell phenotyping and staining ex vivo

Live/Dead Aqua Stain Kit (Thermo Fisher Scientific, Carlsbad, CA) was used to exclude dead CD3+CD4−CD8+ T cells from the analysis. The frequencies of naive, central memory (CM), effector memory (EM), and terminally differentiated (TD) live CD8 T cell populations were measured by their differential CD28/CD45RA expression (Fig. 1A). Markers of CD8 T cell activation (HLA-DR), senescence (CD57+CD28−), and exhaustion (PD-1) as well as ectonucleotidases CD39/CD73 were also assessed. All Abs/fluorochromes used for phenotyping are listed in Supplemental Table III. All extracellular staining was performed in PBS, 2% FBS, and 2 mM EDTA at 4°C for 1 h. Intracellular staining was used to detect granzyme B and perforin. After extracellular staining, the cells were washed and permeabilized using the BD Cytofix/Cytoperm Kit (BD Biosciences, Mississauga, ON, Canada). Permeabilized cells were incubated with the appropriate Abs in a Perm/Wash buffer at 4°C for 1 h and stored in PBS until acquisition. A three-laser BD Fortessa-X20 was used for acquisition, and results were analyzed by FlowJo software V10.0.7 (BD Biosciences).

In vitro degranulation assay and expression of cytotoxic molecule

PBMCs or BAL cells (1 × 106 cells/ml) were either stimulated or not with soluble anti-human CD3 and CD28 Abs (1 μg/ml each) (BioLegend, San Diego, CA) for 6 h at 37°C in RPMI 1640 and 10% FBS. CD107a is located on the internal side of granules, and its expression on cell surface is usually low because of the endocytic pathway that reinternalizes the molecule after the granule contents have been released. Because of this reinternalization process, and based on protocols optimized previously (29), cells were stained with CD107a Ab during the activation period. After stimulation, cells were washed and stained extracellularly for anti-human CD3, CD4, and CD8 and a Live-Dead stain for 1 h at 4°C. Cells were then washed and stained intracellularly with anti-human granzyme B and anti-perforin Abs for 1 h at 4°C using the Cytofix/Cytoperm kit (BD Biosciences).

In vitro killing of autologous CD4 T cells

Our killing assay was adapted from previously published protocols (30–32). Briefly, autologous target CD4 T cells were isolated from PBMCs using a negative selection EasySep Human CD4+ T Cell Isolation Kit (Stemcell Technologies, Vancouver, BC, Canada) and stained with CFSE dye (Thermo Fisher Scientific) to distinguish them from CD4 T cells that are present in the effector cell pool. Purified CD4 T cells were pulsed or not with 10 μg/ml each of a pool of 15-mer peptides with 11 aa overlaps corresponding to the HIV-1 consensus B Gag sequence (National Institutes of Health AIDS Reagent Program; ref no. 12425) for 1 h at 37°C and then stained with a Live-Dead Aqua vivid to distinguish between CFSE+ live and dead CD4 T cells prior to coculture with effector cells. Effector cells were either total BAL or PBMCs that were stimulated with anti-CD3 and anti-CD28 for 6 h. Coculture was performed at a ratio of 1:10 target to effector cells (5 × 104 target cells/5 × 105 effector cells). Target and effector cells were cocultured in RPMI 1640 and 10% FBS for 6 h at 37°C. Following the coculture, cells were stained with Annexin V (BD Biosciences), followed by fluorochrome-conjugated Abs to CD3, CD4, and CD8 in 1× Annexin V buffer. Frequencies of CFSE+CD4+ Annexin V+ T cells were measured by flow cytometry. The cells were fixed in 1× Annexin V buffer and 2% paraformaldehyde and stored until acquisition.

Statistical analyses

GraphPad Prism V7 (San Diego, CA) and Stata (V 13.1; StataCorp, College Station, TX) were used for statistical analyses. Data were summarized using means, medians, and proportions. To compare distribution of covariates between HIV+ and HIV− study groups and between smokers and nonsmokers, we used Fisher exact tests and Mann–Whitney rank-sum tests. Wilcoxon matched-pairs signed rank test was used to compare paired study variables, and Mann–Whitney rank-sum test was used to compare unpaired study variables.

Results

Characteristics of included participants

Of the 43 participants included in our study, 26 (60%) were living with HIV, and 18 (42%) were tobacco smokers. There were no differences in the distribution of covariates (age, sex, ethnicity, cannabis smoking) across those groups. Baseline characteristics of participants according to the HIV status and smoking status are presented in Table I and Supplemental Table IV, respectively.

View this table:
Table I. Participant characteristics at time of bronchoscopy

Phenotypic characterization of pulmonary mucosal CD8 T cell subsets ex vivo

To assess the overall functionality of pulmonary mucosal CD8 T cells in PLWH and the impact of tobacco smoking on the profile of these cells, we first analyzed their differentiation status (Fig. 1A). In all studied groups, compared with PBMCs, pulmonary mucosal CD8 T cells had lower frequencies of naive (CD28+CD45RA+) and TD (CD28−CD45RA+) subsets and a higher frequency of the EM (CD28−CD45RA−) subset (Fig. 1B). Interestingly, in contrast to HIV− individuals, larger frequencies of CM subset were observed exclusively among the BAL cells of HIV+ smokers versus nonsmokers. Conversely, an enrichment in the EM subset was observed in BAL cells from HIV− smokers versus nonsmokers. Overall, the observed differential proportions of CD8 subsets suggest higher recruitment potential of CD8 T cells toward the lung mucosa of HIV+ smokers.

FIGURE 1.
FIGURE 1.

BAL CD8 T cells show an increased EM subset compared with CD8 T cells in blood in all study groups. (A) Gating strategy used to analyze the distribution of CD8 T cell subsets in PBMCs and BAL from HIV-infected and -uninfected study groups. CD8 T cell subsets are defined as follows: naive (CD28+CD45RA+), CM (CD28+CD45RA−), EM (CD28−CD45RA−), and TD (CD28−CD45RA+) cells. (B) Frequencies of naive, CM, EM, and TD CD8 subsets in HIV− nonsmokers, n = 9; HIV− smokers, n = 4; HIV+ nonsmokers, n = 12; HIV+ smokers, n = 9.

BAL CD8 T cells are highly activated and exhausted and differ in senescence levels from blood CD8 T cells

To investigate the functional profile of pulmonary mucosal CD8 T cells, we analyzed the expression of an activation marker (HLA-DR), an exhaustion marker (PD1), and the senescence profile (CD57+CD28−) (Fig. 2A). We observed both higher frequencies of HLA-DR+ and PD1+ CD8 T cells in BAL than in blood (Fig. 2B, 2D) in all study groups. The frequency of senescent CD8 T cells did not differ significantly in BAL and PBMC from uninfected individuals. In HIV+ individuals, however, we observed a trend toward a higher frequency of senescent CD8 T cells in BAL than in PBMC in nonsmokers (Fig. 2C, p = 0.07). Interestingly, the frequency of senescent CD8 T cells in BAL from HIV+ smokers was significantly lower than that of PBMCs (Fig. 2C, p = 0.004). As per differential proportions of CD8 subsets in BAL versus blood, we also compared levels of activation, exhaustion, and senescence of memory CD8 T cells in BAL versus blood, and similar greater activation/exhaustion status of CD8 T cells has been observed in BAL (data not shown). We then assessed the expression of the immunosuppressive ectonucleotidases CD39 and CD73 known to be involved in CD8 T cell Ag-specific and immunoregulatory functions (Fig. 3A) (28). CD73 was usually expressed at lower levels in CD8 T cells from BAL versus blood. No significant difference between the study groups was observed (Fig. 3B, 3C).

FIGURE 2.
FIGURE 2.

Frequency of CD8 T cell blood versus BAL exhibiting markers of immune activation, senescence, and exhaustion. (A) Representative flow cytometry plots show the frequencies of activated (HLA-DR+), senescent (CD57+CD28−), and exhausted (PD1+) CD8+ T cells. (B) Frequencies of HLA-DR+ cells among total CD8 T cells in PBMCs and BAL from HIV− nonsmokers (n = 8), HIV− smokers (n = 5), HIV+ nonsmokers (n = 12), and HIV+ smokers (n = 7). (C) Frequencies of senescent CD8 T cells in PBMCs and BAL from HIV− nonsmokers (n = 7), HIV− smokers (n = 5), HIV+ nonsmokers (n = 9), and HIV+ smokers (n = 9). (D) Frequencies of CD8+PD1+ T cells in PBMCs and BAL from HIV− nonsmokers (n = 3), HIV− smokers (n = 3), HIV+ nonsmokers (n = 5), and HIV+ smokers (n = 8).

FIGURE 3.
FIGURE 3.

BAL CD8 T cells have a lower frequency of the ectonucleotidase CD73 compared with those from blood. (A) Representative flow cytometry pseudo-color plots showing the frequencies of CD73 and CD39; frequency of (B) CD73+ and (C) CD39+ CD8 T cells in PBMCs and BAL from HIV− nonsmokers (n = 7). HIV− smokers, n = 4; HIV+ nonsmokers, n = 10; and HIV+ smokers, n = 8.

Lack of ex vivo perforin expression and lower expression of granzyme B by pulmonary mucosal CD8 T cells

CD8 T cell cytotoxicity primarily comes from their ability to release granules containing perforin and granzyme molecules. Thus, we assessed the expression of granzyme B (a serine protease that initiates apoptosis in target cells) together with perforin, which polymerizes to form a pore in the target cell’s membrane (Fig. 4A) (33). These measures revealed that perforin was rarely detected in pulmonary mucosal CD8 T cells in both HIV− and HIV+ individuals (Fig. 4B). Furthermore, although granzyme B expression was detected in both compartments, lower levels are observed in pulmonary mucosal CD8 T cells than in PBMC of HIV+ nonsmokers (Fig. 4C). In addition, despite higher frequencies of CD8 T cells expressing granzyme B detected in both BAL and PBMCs of PLWH compared with HIV− individuals, the amount of granzyme B+ CD8 T cells in PBMCs of HIV+ smokers was significantly lower compared with that in HIV+ nonsmokers (Fig. 4C).

FIGURE 4.
FIGURE 4.

Differential ex vivo expression of perforin and granzyme B in BAL and PBMC CD8 T cells from HIV-infected individuals. (A) Representative flow cytometry pseudo-color plots show the expression of perforin and granzyme B in total CD8+ T cells of HIV+ and HIV− study groups. Frequency of (B) perforin+ and (C) granzyme B+ CD8 T cells in PBMCs and BAL from HIV− nonsmokers (n = 4), HIV− smokers (n = 5), HIV+ nonsmokers (n = 6), and HIV+ smokers (n = 8).

Weaker degranulation capacity of pulmonary rather than circulating CD8 T cells following in vitro stimulation

To make the link between the significant phenotypical differences in pulmonary versus blood in both PLWH and uninfected individuals, we assessed the degranulation capacity upon stimulation of CD8 T cells with anti-CD3/anti-CD28 Abs as higher frequencies of CD8 T cells degranulate in response to anti-CD3/anti-CD28 stimulation versus antigenic peptides (34). Another reason to select anti-CD3/anti-CD28 instead of HIV-specific stimulation was to be able to compare CD8 degranulation capacities of PLWH versus uninfected individuals using a similar method. Of note, because of the very limited numbers of available BAL cells, we were capable to perform this set of in vitro experiments only on a subset of specimens. As per consistent functional degranulation of pulmonary CD8 T cells from PLWH and HIV− individuals, results of both groups are shown together in Fig. 5. BAL CD8 T cells in both HIV+ and HIV− individuals had significantly higher ex vivo baseline frequencies of CD107a+ cells, a marker of degranulation (35), than CD8 T cells in PBMCs (Fig. 5A, 5B). However, following stimulation, the frequency of CD107a PBMCs increases significantly, up to 80-fold (p = 0.03), reaching similar levels as seen in BAL. In contrast, the frequency of CD107a+ BAL CD8 T cells barely changed and was limited to 3-fold (p = 0.3) (Fig. 5C). We further assessed whether these degranulating CD8 T cells produced perforin and granzyme B upon stimulation. BAL CD8 T cells, whether CD107a+ or not, had lower frequencies of perforin- and granzyme B–coexpressing cells, which were lower than the PBMC counterparts following in vitro anti-CD3/CD28 costimulation (p = 0.0006). Indeed, blood CD8 T cells that were actively degranulating (CD107a+) had upregulated perforin expression up to 10-fold (p = 0.01) compared with nondegranulating (CD107a−) CD8 T cells, whereas this increase was only up to 3-fold (p = 0.04) in pulmonary CD8 T cells (Fig. 5A, 5D).

FIGURE 5.
FIGURE 5.

Lower frequency of degranulating and perforin secreting CD8 T cells in BAL and PBMCs upon in vitro TCR costimulation. (A) Representative flow cytometry pseudo-color plots showing the frequency of degranulating CD8 T cells assessed by CD107a expression as well as the frequency of perforin+ and granzyme B+ cells among CD107+ and CD107− CD8 T cells from PBMCs and BAL following CD3/CD28 costimulation. (B) Frequencies of CD107a+ CD8 T cells in PBMC and BAL before and after a 6-h CD3/CD28 costimulation. (C) Fold changes in the frequency of CD107a+ CD8 T cells following in vitro CD3/CD28 costimulation of PBMCs and BAL. (D) Frequencies of degranulating versus nondegranulating perforin+ CD8 T cells after a 6-h CD3/CD28 costimulation (HIV−: n = 3, including n = 2 smokers; HIV+: n = 4, including n = 2 smokers).

Lower in vitro killing capacity of BAL cells versus PBMC to Gag-pulsed autologous CD4 T cells

To further evaluate cytotoxic capacity in BAL versus PBMCs, specifically in the context of HIV infection, we designed an in vitro HIV-specific killing assay of pulmonary CD8 T cells from PLWH using Gag-pulsed autologous CD4 T cells as target cells (Fig. 6A, 6B). CFSE-labeled autologous CD4 T cells were pulsed or not with consensus B 15-mer HIV Gag peptide for 1 h and used as targets for the HIV-specific CD8 T cell killing assay (32). CFSE labeling of Gag-pulsed cells is used to distinguish CD4 T cell targets from other cell types. After subtracting the frequency of negative control (unpulsed) CFSE+CD4+Annexin V+ cells from the frequency of Gag-pulsed CFSE+CD4+ Annexin V+ cells (the experimental condition), we observed a significantly lower frequency of Annexin V+ target cells in BAL cells than when PBMCs were used as effector cells (p = 0.03) (Fig. 6C, 6D). Specimens from an HIV− donor used as a negative control did not induce the Gag-specific killing (Fig. 6C).

FIGURE 6.
FIGURE 6.

Lower killing capacity of BAL cells versus PBMC to Gag-pulsed autologous CD4 T cells. (A) Representative schema of the protocol used for the in vitro killing assay. (B) Representative flow cytometry pseudo-color plots showing the gating strategy and (C) different coculture conditions used to detect the frequency of Annexin V+ CFSE+ CD4+ T cell targets. Specimens from an HIV− donor have been used as a negative control for the Gag-specific killing. (D) Frequencies of apoptotic Annexin V+ CD4+ T cells were calculated using the following formula: (sum of % of Annexin V+ Gag-pulsed CD4 T cells) − (% of Annexin V+ CD4 T cells); HIV+: n = 6, including n = 3 smokers.

Discussion

The anatomical location of CD8 T cells at the time of viral infection impacts their ability to induce a quick Ag-specific immune response. Although various studies have provided evidence of differential phenotypes and function between CD8 T cells from the gastrointestinal mucosa and blood in PLWH on ART (36, 37), there is a paucity of data describing such differences in CD8 T cells from the lungs versus the peripheral blood in PLWH and between smoking versus nonsmoking PLWH. This information is important given the high burden of chronic lung disease, lung cancer, and pulmonary infection in PLWH on ART (2). In this article, we provide evidence that pulmonary mucosal CD8 T cells are characterized by enriched EM phenotypes with higher percentages of immune activation and exhaustion markers. Importantly, pulmonary mucosal CD8 T cells ex vivo express much lower frequencies of perforin-expressing cells than peripheral CD8 T cells, along with exhibiting decreased in vitro killing capacity.

Our results show that, regardless of HIV infection or tobacco smoking status, pulmonary mucosal CD8 T cells are enriched in EM cells. These results are not confounded by age, sex, or ethnicity because we observed no differences in the distribution of any of the covariates between smokers and nonsmokers, nor between HIV+ and HIV− study groups. EM CD8 T cells have the ability to migrate into nonlymphoid tissues in response to infections and remain present as long-lived memory cells (38, 39). CD8 EM T cells are highly specialized and actively participate in pathogen clearance in peripheral organs, including recall responses to secondary pulmonary infections (40). Thus, it is not surprising that lung CD8 T cells are composed of mostly the EM subset, which should be able to mediate antiviral immune responses. Furthermore, pulmonary mucosal CD8 T cells were characterized by higher expression of HLA-DR (immune activation) and PD-1 (exhaustion) compared with blood CD8 T cells, regardless of the HIV infection or tobacco smoking status. Excessive expression of exhaustion molecules, notably PD-1, has been proposed to explain the dysfunction of effector CD8 T cells from HIV/SIV-infected individuals (17, 23), and PD-1 expression by pulmonary CD8 T cells has also been shown to be involved in dysregulation of the antiviral functions of CD8 T cells in COPD (20). Another recent study has identified disease-relevant exhausted CD8 T cell populations in both HIV infection and lung cancer based on their epigenetic accessibility and single-cell proteomic profiling (41). Similar CD8 T cell activated/exhausted EM phenotype has also been observed in other tissues, such as female genital tract, spleen, and liver (42, 43). Furthermore, levels of HIV DNA were recently shown to be linked to CD8 T cell activation and memory expansion (44). Indeed, residual immune activation and/or inflammation despite ART may favor the generation of target cells, facilitate the migration of target cells to sites of HIV spread, and induce dysfunctional T cell responses. Thus, the dominant CD8 EM memory phenotype, along with higher frequencies of activation and PD-1 expression by pulmonary mucosal CD8 T cells, could contribute to HIV persistence within the lungs despite ART as we recently reported (3). Of note, we acknowledge that downregulation of CD45RA and CD28 and upregulation of PD-1 could also be indicative of recent cell activation rather than cell function (45–47). For instance, long-lived yellow fever virus-specific memory CD8 T cells have been shown to express CD45RA, CD28, and CCR7, which is characteristic of naive CD8 T cells. However, despite their naive-like phenotype, they retain their ability to express cytotoxic effector molecules, and their epigenetic landscape resembled that of effector CD8 T cells (48). Similarly, some studies have shown that PD-1 is not a definitive marker of loss of function but rather cell adaptation to an inflammatory milieu in context of chronic inflammation (49). Notably, virus-specific pulmonary CD8 T cells have been documented to show an activated phenotype in the absence of Ag for at least a year after initial infection (50), suggesting that their activated phenotype can be modulated by the tissue environment in an Ag-independent manner.

Chronic T cell immune activation and exhaustion also result in advanced T cell differentiation and immunosenescence. Indeed, senescent CD8 T cells, characterized as CD28−CD57+, are one of the major sources of proinflammatory cytokines in PLWH. HIV inhibits terminal differentiation and proliferation of EM CD8 T cells and promotes expansion of less-differentiated CD28− CD8 T cells (51). CD57+ HIV-specific CD8 T cells fail to proliferate in response to Ag-specific stimulation in vitro and undergo activation-induced cell death instead (52). We observed higher percentages of senescent CD8 T cells in the lung than in PBMCs only in PLWH. However, in contrast to HIV+ nonsmokers, tobacco smokers had a significantly lower frequency of senescent pulmonary CD8 T cells. Accordingly, the proportion of CM CD8 T cells, which maintain their proliferation capacity because of the expression of CD28, is higher in HIV+ smokers versus nonsmokers. It is well known that regular tobacco smoking induces an active inflammatory milieu within the small airway mucosa (53), which might explain such differences. Overall, these observations suggest higher recruitment of CD8 T cells within the lungs of smoker PLWH. Accordingly, smoking asthmatics show distinct airway pathology with bronchial infiltrate of CD8 T cells (54).

CD8 T cells expressing CD73, which is both a T cell coactivator molecule and an immunosuppressive ecto-enzyme via adenosine synthesis, are involved in HIV-specific CD8 T cell expansion and are present at high frequencies in HIV elite controllers (55). Decreased CD73+ CD8 T cells have been associated with immune activation and exhaustion in PLWH (55). Accordingly, we observed much lower frequencies of CD73-expressing cells among pulmonary mucosal than circulating CD8 T cells in all studied groups in line with their higher levels of activated and exhausted CD8 T cells. Lower frequencies of CD73+ pulmonary mucosal CD8 T cells could explain, partially, their dysfunctional anti-HIV–specific responses despite effective ART. CD39 is another ecto-enzyme, which in concert with CD73, hydrolyzes ATP into adenosine. CD39 expression is induced after exposure to proinflammatory cytokines, and its expression by CD8 T cells is upregulated during experimental respiratory tolerance induction in mice because of their ability to comprise CD8 T cell expansion (56). However, no significant difference was observed in CD39+ CD8 T cell percentages between BAL and PBMC or between study groups.

A well-described correlate of HIV immune control is perforin-mediated cytotoxicity (31, 57). Perforin is a protein that creates a pore to disrupt plasma and endosomal membrane. These pores provide a pathway for granzyme entry into the cell and ultimately lead to apoptosis of the cells (33). However, within tissue compartments, attenuated toxicity is thought to be protective against tissue damage. Our results demonstrate that pulmonary mucosal CD8 T cells ex vivo rarely express perforin, regardless of HIV status or smoking. This is of particular importance as ex vivo perforin expression by HIV-specific CD8 T cells correlates directly with HIV control in elite controllers (57). Moreover, CD8 T cells from the lymphoid tissues of PLWH on ART are less effective at killing target cells compared with CD8 T cells from the peripheral circulation because of lower perforin expression (37, 58), thus contributing to HIV persistence in lymphoid tissues. Furthermore, as expected for HIV+ smokers, we observed a significant decrease in the frequency of ex vivo granzyme B+ pulmonary mucosal CD8 T cells compared with CD8 T cells from blood, which could potentially be explained by the differential recruitment of CD8 T cells from blood into the lungs of smoker PLWH. Therefore, based on the important overall lack of perforin expression and diminished expression of granzyme B, mucosal CD8 T cells are likely unable to eliminate HIV-infected cells in lung mucosa, which in turn could contribute to the establishment of viral reservoirs in this tissue.

CD8 T cells begin to release cytotoxic granules in response to TCR ligation within 30 min to 6 h of activation (34, 59). CD107a is routinely used as a marker of degranulation in immune cells (35). We observed that the ex vivo baseline (nonactivated) expression of CD107a+ CD8 T cells was higher in BAL than in blood, whereas frequencies of CD107a+ BAL CD8 T cells did not increase significantly following 6 h of in vitro stimulation. In contrast, CD8 T cells in blood were much more potent in upregulating their degranulation levels in response to the stimulus. Moreover, these CD107+ CD8 T cells in BAL included much lower frequencies of perforin and granzyme B+ cells versus blood. In contrast to blood, the lung mucosa is not a sterile tissue because it is exposed to various airborne particles, resulting to much larger Ag load compared with the blood. Therefore, it is not surprising that lung CD8 T cells are highly activated (HLA-DR+) and have higher ex vivo frequency of CD107a+ cells. These findings suggest that lung CD8 T cells have been previously stimulated in vivo by pulmonary mucosal Ags prior to our in vitro assay and, unlike CD8 T cells from PBMCs, cannot degranulate more than they already have done. In addition, as we showed, BAL CD8 T cells express higher levels of PD-1 and lower CD28 compared with blood CD8 T cells, which could also explain their deficiencies in degranulation and perforin/granzyme B production. Although, because of the very limited numbers of cells recovered from BAL, we were unable to perform in vitro functional assays on smokers and nonsmokers separately; a previous study demonstrated greater CD107a expression by circulating blood CD8 T cells from nonsmokers following in vitro stimulation in both HIV+ and HIV− individuals (60).

The capacity of BAL cells versus PBMCs from HIV-infected subjects to kill Gag-pulsed autologous CD4 T cells confirmed that BAL cells had lower HIV Gag-specific in vitro killing capacity. Besides the lower cytotoxic capacity of BAL than PBMC CD8 T cells, such a difference could also be due to differential proportions of CD8 T cells as well as the Ag presentation in BAL cells and PBMCs as BAL is dominated by alveolar macrophages (28). However, we chose not to normalize these cell ratios because it would not reflect the real pool of pulmonary mucosal immune cells, and our aim was to mimic the natural environment the CD8 T cells to the best of our ability. Considering the natural composition of pulmonary mucosal immune cells in functional assays is of particular importance as antiviral CD8 cytokine secretion has been shown previously to be altered by the lung mucosal environment compared with that found in secondary lymphoid tissues (61). In line with our results, similar CD8 T cell deficiency in both perforin and granzyme B expressions as well as significantly reduced CD8 toxicity have been also reported in human rectal mucosa regardless of HIV status (36).

The majority of T cells within the lungs are known as tissue resident memory T cells (TRM), which are mainly characterized by the expression of CD103, CD69, and CD49a. Chronic pulmonary inflammatory conditions affect these cells’ dynamics (62). Tuberculosis, for instance, has been shown to be associated with an enrichment in TRM populations in BAL along with their increased activation (CD38/HLA-DR expression) and exhaustion (PD-1 expression) (63). Furthermore, the cytotoxic potential of lung CD8 T cells increases with progression of chronic lung mucosal inflammatory diseases, such as COPD, characterized by increased expression of perforin, granzyme B, and TNF-α (64–66). Importantly, HIV-specific TRM are more numerous in tissues where HIV persists, such as the gut and the lymph nodes, and their frequencies are inversely correlated with HIV viral replication (67). Thus, the assessment of TRM dynamics within the lungs of PLWH should be considered in our upcoming studies.

Taken together, our findings provide additional support for the notion that, independently of the HIV status, pulmonary CD8 T cells display increased exhaustion and advanced differentiation as well as reduced in vitro cytotoxicity. Smoking seems to result in higher infiltration of CM CD8 T cells into the lung mucosa of PLWH. Such a unique phenotypical and functional feature of pulmonary mucosal CD8 T cells could, in turn, contribute to a suboptimal anti-HIV immune response within the lungs. Furthermore, these findings have broad implications for understanding immunological susceptibility to pulmonary infectious, including coronaviruses, and noninfectious pulmonary disease in addition to mucosal vaccine strategies, which aim to boost CD8 responses.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank all the participants who underwent research procedures in this study as well as research nurse Josée Girouard for nursing care and the respiratory therapists for assistance with bronchoscopies throughout this study.

Footnotes

  • This work was supported by the Canadian Institutes of Health Research (CIHR) (Grant 153082) (to C.T.C., M.-A.J., N.C., and E.S.), the CIHR Canadian HIV Cure Enterprise 2.0 (Team Grant HIG-133050) (to P.A., N.C., J.E., C.T.C., and M.-A.J.), the Réseau SIDA et Maladies Infectieuses du Fonds de Recherche du Québec-Santé (FRQ-S) (to C.T.C. and M.-A.J.), and the McGill Department of Medicine (to C.T.C.). C.T.C., M.D., and N.C. hold FRQ-S Junior 1, Junior 2, and senior research salary awards, respectively. J.E. holds the CIHR Canada Research Chair Tier 1 in Pathophysiology of Cell Death in Host-Pathogen Interactions. M.-A.J. holds the CIHR Canada Research Chair Tier 2 in Immuno-Virology. E.T. is supported by an M.Sc. scholarship from FRQ-S. S.S. received a training scholarship from the Research Institute of the McGill University Health Centre. The funding institutions played no role in the design, collection, analysis, and interpretation of data.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ART
    antiretroviral therapy
    BAL
    bronchoalveolar lavage
    CM
    central memory
    COPD
    chronic obstructive pulmonary disease
    EM
    effector memory
    PLWH
    person living with HIV
    TD
    terminally differentiated
    TRM
    tissue resident memory T cell.

  • Received August 5, 2020.
  • Accepted November 13, 2020.

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