Differential Expression of CD8+ T Cell Cytotoxic Effector Molecules in Blood and Gastrointestinal Mucosa in HIV-1 Infection

Abstract

We previously reported that CD8⁺ T-cells in human gastrointestinal mucosa exhibit reduced perforin expression and weak or impaired cytotoxic capacity compared to their counterparts in blood. Nevertheless, these cells degranulate and express cytokines and chemokines in response to cognate antigen. In addition to weak expression of perforin, earlier studies suggested differential regulation of perforin and granzymes, with granzymes A 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 co-expression patterns of granzymes (Gzm) A, 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 to blood. A large fraction of rectosigmoid CD8⁺ T-cells either did not express granzymes or were single-positive for GzmA. Rectosigmoid CD8⁺ T-cells appeared skewed towards cytokine production rather than cytotoxic responses, with cells expressing multiple cytokines/chemokines generally lacking in perforin and granzyme expression. These data support the interpretation that perforin and granzymes 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.

Introduction

Mucosal tissues are primary targets for human immunodeficiency virus (HIV-1) infection, and the gastrointestinal mucosa is a major site of virus transmission and pathogenesis. Accordingly, understanding mucosal immune responses to HIV-1 is important for the development of strategies to fight infection. CD8⁺ T-cells are thought to play a crucial role in containing HIV-1 infection, as depletion of these cells results in viral rebound (1, 2). However, the specific effector functions by which CD8⁺ T-cells mediate viral containment remain unclear. In blood, both perforin-mediated cytotoxicity and polyfunctionality, defined as the simultaneous production of multiple cytokines and chemokines, have been identified as possible correlates of immune-mediated protection from disease progression (3–9). CD8⁺ T-cell polyfunctionality, including robust production of MIP-1β, has also been identified as a potential correlate of protection in mucosa (10). However, gastrointestinal CD8⁺ T-cells exhibit strikingly low perforin expression and reduced cytotoxicity compared to blood T-cells, suggesting CD8⁺ T-cells housed in the gut are primed for cytokine release rather than cytotoxicity (11).

Despite significantly lower perforin levels in gut compared to blood, HIV-1⁺ individuals not on antiretroviral therapy (ART) display greater proportions of perforin and GzmB-positive CD8⁺ T-cells in gastrointestinal mucosa compared to those on ART and HIV-1 negative adults (10–12). Whether this indicates expression of cytotoxic effector proteins by polyfunctional HIV-1-specific CD8⁺ T-cells, the emergence of a cytotoxic HIV-1-specific effector T-cell subset, and/or bystander activation-induced perforin and granzyme B expression in non-HIV-1-specific CD8⁺ T-cells in the mucosa during chronic HIV-1 infection is unknown.

Interestingly, despite low perforin expression, gastrointestinal CD8⁺ T-cells express granzyme B, albeit at lower levels compared to blood, and display ample degranulation, measured by expression of CD107, suggesting that these mucosal CD8⁺ T-cells may produce and release “cytotoxic” granules containing little or no perforin (11, 13). Numerous in vitro and mouse in vivo studies have demonstrated non-cytolytic, extracellular functions of granzymes A, B, and K, including extracellular matrix degradation and the direct and indirect processing and release of pro-inflammatory cytokines (14–17). The concept of granzymes functioning in a non-cytolytic manner to elicit a pro-inflammatory state appears consistent with the strong propensity of gut CD8⁺ T-cells to produce chemokines and cytokines in response to antigenic stimulation. Accordingly, the major goal of this study was to more fully explore the expression and coexpression patterns of granzymes A, B and K in parallel with perforin in HIV-1-specific and non-HIV-1-specific CD8⁺ T-cells in rectosigmoid mucosa during chronic HIV-1 infection.

Materials and Methods

Participants and Sample Collection

HIV-1 positive and seronegative participants were enrolled through the SCOPE study based at San Francisco General Hospital. Written informed consent for phlebotomy and rectosigmoid biopsy was obtained through protocols approved by the Committee on Human Subjects Research, University of California, San Francisco [Protocols #10-01218, 10-00263 and 10-01330]. Participants were classified in four groups defined by plasma viral load (VL) and antiretroviral therapy (ART) as follows: Controllers (C, n=10) consistently maintained VL <2,000 copies/mL without ART; Viremic (V, n=15) subjects maintained VL ≥ 2,000 copies/mL without ART; ART treated (Tx, n=13) participants had VL < 40 copies/mL; and seronegative (SN, n=10) controls were negative for HIV-1. Seronegative participants were healthy volunteers from whom samples were collected for research purposes only. Exclusion criteria included active sexually transmitted infections other than HIV-1; inflammatory bowel disease or other known inflammatory conditions affecting the gastrointestinal tract; colorectal cancer or other malignancies; and severe anemia or blood clotting disorders. Additional demographic information (gender, race/ethnicity, age, viral load, CD4 count, and time since diagnosis) is supplied in Table I. Twenty to 40 ml of blood was collected by sterile venipuncture into tubes containing EDTA. Approximately 30 rectosigmoid biopsies were obtained by flexible sigmoidoscopy 10–30cm from the anal verge (18). Biopsies were collected in 50mL conical tubes containing 15mL of R-15 medium [RPMI-1640 supplemented with fetal calf serum (15%), penicillin (100U/mL), streptomycin (100 mg/mL), and glutamine (2 mM)]. Blood and biopsy samples were transported at room temperature to the laboratory and processed immediately upon arrival.

Table I.

Participant Characteristics

	Gender	Race/Ethnicity	Plasma viral load, RNA copies/mL* [Median] [Range]	CD4 count, Cells/mm3 [Median] [Range]	Time Post HIV+Diagnosis, years [Median] [Range]
Controller (n = 10)	M, 5 F, 3 T, 2	AA, 7 C, 2 Mx, 1	844 <40 – 1,727	716 353 – 2,542	20.5 4 – 29.8
Viremic Untreated (n = 15)	M, 15	AA, 7 C, 4 HL, 3 Mx, 1	20,544 393 – 186,307	506 236 – 1075	2.75 1 – 22.67
ART-Treated (n = 13)	M, 11 F, 1 T, 1	AA, 3 C, 7 HL, 3	<40 NA	603 193 – 811	18 2.7 – 27.1
Seronegative ** (n = 10)	M, 10	AA, 1 C, 6 HL, 2 Mx, 1	NA NA	950 600 – 1,211	NA NA1

¹M, Male; F, Female; T, Transgender (M to F)

AA, African American; C, Caucasian; HL, Hispanic/Latino; Mx, Mixed; NA, Not Applicable.

^*Median calculated using only detectable viral load data points.

^**CD4 count unavailable for one subject.

Blood and Tissue Processing

Ficoll-Paque^TM (Pfizer-Pharmacia, New York, NY) was used to isolate peripheral blood mononuclear cells (PBMCs) from blood. Rectosigmoid mucosal mononuclear cells (RMMCs) were liberated from biopsies using enzymatic and mechanical disruption as previously described (19, 20). Briefly, biopsies underwent shaking incubation at 37°C for 30 minutes in 25mg/mL Liberase DL (Roche, Indianapolis, IN), followed by passage through a 16-gauge blunt end needle to disrupt tissue. Free cells were collected through a sterile 70μm cell strainer. The disruption process was repeated until all biopsy tissue was digested. Free RMMCs were washed three times in 20mL of R-15. All isolated cells were rested overnight at 37°C, 5% CO₂ in R-15 supplemented with 200x Zozyn (Pfizer-Pharmacia).

Antibodies and Peptide Pools

The following fluorochrome-labeled monoclonal antibodies were used in flow cytometry: CD107a (H4A3: PE-Cy5, PE-Cy7), CD8 (SK1: APC-H7, FITC), CCR7 (3D12: PE-Cy7), Granzyme B (GB11: V450, PE-CF594), MIP-1β (D21-1351: APC-H7, PerCP-Cy5.5), IFNγ (B27: PE-Cy7), TNFα (Mab11: Ax700), CD69 (L78: PE) from BD Biosciences (San Jose, CA); Granzyme K (GM6C3: FITC) from Santa Cruz Biotechnology, Inc. (Dallas, Tx); CD4 (T4D11: ECD) from Beckman Coulter (Brea, CA); CD4 (RPA-TA: BV785), CD45RO (UCHL1: BV785), CD27 (O323: BV650), CD103 (Ber-ACT8: Ax488), Granzyme A (CB9: Ax647, PacBlue), and CD3 (UCHT-1: Ax700) from Biolegend (San Diego, CA). CD3 (S4.1: Qdot655) was purchased from Invitrogen (Carlsbad, CA). S1PR1 (SW4GYPP: eFluor660) was purchased from eBioscience (San Diego, CA); Unlabeled CD28 (L293), and CD49d (L25) were from BD Pharmingen (San Diego, CA). Perforin (B-D48: PE) was from Cell Sciences (Canton, MA). This perforin antibody detects de novo as well as pre-formed perforin and is suitable for monitoring perforin expression following TCR stimulation (21). The HIV-1 Gag peptide pool (Consensus Clade B) consisted of 123 15-mers overlapping by 11 residues (JPT Innovative Peptide Solutions, Berlin, Germany). Staphylococcal enterotoxin B (SEB) was from Sigma Aldrich.

Antigen stimulation and intracellular cytokine staining

Staining was performed as previously described with slight modifications (12) on ex vivo blood and mucosal mononuclear cells rested overnight at 37°C, 5% CO₂. For stimulation assays, cells were incubated at 2x10⁶ cells per 200μL R-15 for 5.5 hours in the presence of anti-CD28 (1μg/mL) and anti-CD49d (1μg/mL), anti-CD107a, 1μM GolgiStop^TM (BD Biosciences), brefeldin A (5μg/mL, Sigma Aldrich) and the appropriate antigenic stimulation: Gag-peptide pool (final concentration: 3.5μg/mL of each peptide), or medium containing an equivalent amount of DMSO (peptide solvent) as the negative control. Staphylococcal enterotoxin B (5μg/mL) was used as positive control. Following incubation, cells were stained for surface markers and viability (Aqua Dead cell stain kit, Invitrogen) for 20 minutes at room temperature. Cells were then fixed in 4% paraformaldehyde and permeabilized using FACS Perm 2 (BD Biosciences) prior to intracellular staining for CD3, cytotoxic effectors, chemokines, and cytokines. Staining for cytotoxic effectors following antigenic stimulation (Figs. 3, 4 and Supplemental Figs. 2, 3) utilized staining protocols and antibody clones previously demonstrated to reveal perforin and Granzyme B expression following TCR stimulation (21). Cells were re-suspended in 1% paraformaldehyde and stored at 4°C in the dark no longer than 24 hours until analysis.

FIGURE 3. Expression of cytokines, chemokines, perforin and GzmB in blood and rectosigmoid CD8⁺ T-cells responding to ex vivo stimulation.

(A) Representative flow cytometry plot displaying intracellular expression of perforin and GzmB in polyfunctional HIV-1 Gag-responding CD8⁺ T-cells (4 functions: CD107a⁺MIP-1β⁺IFNγ⁺TNFα⁺ and 3 functions: CD107a⁺MIP-1β⁺IFNγ⁺) in blood and rectosigmoid mucosa. (B) Expression of perforin (Perf) and/or GzmB in HIV-1 Gag or SEB-responding CD8⁺ T-cells showing differences related to stimulus and anatomical compartment. n=27. (C) Differences in intracellular expression of perforin and GzmB within mono- and polyfunctional HIV-1 Gag-responding CD8⁺ T-cells in blood and rectosigmoid mucosa visualized with a SPICE pie chart. Responsiveness was defined as the expression of one or more of the following effectors: CD107a, MIP-1β, IFNγ, and TNFα. Percentages indicate the median total HIV-1 Gag CD8⁺ T-cell response. Wedge colors correspond to the number of simultaneous functions, as indicated in the legend (perforin, GzmB, CD107a, MIP-1β, IFNγ, and TNFα). n=27. (D) Differences in expression of perforin and/or GzmB between mono- and polyfunctional HIV-1 Gag-specific CD8⁺ T-cells in blood and mucosa. Numbers on the x-axis correspond to the number of simultaneous functions (CD107a, MIP-1β, IFNγ, and TNFα). n=27. In B and D, wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; asterisks show level of significance as follows: * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

FIGURE 4. Expression/co-expression patterns of cytotoxic granule proteins in ex vivo stimulated CD8⁺ T-cells from blood and rectosigmoid mucosa.

(A) Representative flow cytometry plot displaying expression of GzmA, B, K, and perforin in CD8⁺ T-cells responding to ex vivo Gag-peptide stimulation as indicated by degranulation (CD107a) and MIP-1β expression. (B) Differences in expression of cytotoxic effectors between blood and rectosigmoid mucosal CD8⁺ T-cells responding to an HIV-1 Gag-peptide pool or SEB, summarized with SPICE pie charts. Responsiveness was defined as the expression of one or more of the following: CD107a and Mip-1β. n=18. (C) Differences in the expression of cytotoxic effectors between blood and rectosigmoid mucosal CD8⁺ T-cells responding to an HIV-1 Gag-peptide pool or SEB, detailing individual response combinations. n=18. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; asterisks show level of significance as follows: ** P <0.01, *** P <0.001, **** P <0.0001.

Data Acquisition and Analysis

An LSR II flow cytometer and FACSDiva^TM software (Becton Dickinson Immuno-cytometry Systems) were used to collect flow cytometry data. Flowjo software (Flowjo LLC, Ashland, OR) was used to perform initial flow cytometric analysis and SPICE was used to visualize multifunctional T-cell populations (22). CD8⁺ T-cells were identified using the following gating strategy: lymphocytes (SSC-A versus FSC-A) → singlets (FSC-H versus FSC-A) → live, CD3⁺ (CD3 versus viability) → CD8⁺, CD4^Neg (CD8 versus CD4). Memory subsets within the CD8⁺ T-cell population were identified by first establishing positive gates for each memory marker within the live, CD3⁺, CD4^Neg, CD8⁺ T-cell population followed by Boolean gating to define memory subsets as follows: naïve (T_naive CD45RO⁻CD27⁺CCR7⁺), central memory (T_CM CD45RO⁺CD27⁺CCR7⁺), transitional memory (T_TM CD45RO⁺CD27⁺CCR7⁻), effector memory (T_EM CD45RO⁺CD27⁻CCR7⁻), and effector (T_EFF CD45RO⁻CD27⁻CCR7⁻) (23). Polyfunctional cells and cells co-expressing cytotoxic effector molecules were identified by first establishing positive gates for each response analyte and/or cytotoxic effector molecule within live, CD3⁺, CD4^Neg, CD8⁺ T-cell populations, followed by Boolean gating to define subsets co-expressing these effector molecules. We then used Boolean gating to define memory marker expression within each cytotoxic effector subset. An in-house statistical algorithm was used to determine whether antigen-specific responses differed significantly from the negative control (12). For responses found to be statistically significant, net responses were then calculated by subtracting the negative control values from antigen-specific responses. Statistical analyses were performed with GraphPad Prism V.6 (GraphPad Software, San Diego, CA) or SPICE using non-parametric statistical test as follows: Wilcoxon matched-pairs signed rank test for paired samples, Mann-Whitney test for unpaired samples, and Spearman correlation and linear regression analysis for correlative analysis.

Results

Ex vivo expression of cytotoxic effector molecules differs between blood and gut and across HIV-1 disease status

Expression of perforin, and to a lesser extent GzmB, is reduced in resting and stimulated CD8⁺ T-cells in gastrointestinal mucosa compared to blood, but whether this trend extends to other granzymes, or to their coexpression, has not been fully explored (13). It is also unclear whether coexpression of these proteins varies with HIV-1-disease status. To address these questions, we compared intracellular expression of Granzymes A, B, K and perforin in resting CD8⁺ T-cells freshly isolated from blood and rectosigmoid mucosal biopsies of participants in the following groups: HIV-1⁺ controllers (C); HIV-1⁺ viremic individuals not on antiretroviral therapy (V); HIV-1⁺ individuals on antiretroviral therapy (Tx); and seronegative controls (SN) (Table I).

GzmB, GzmK, and perforin all displayed bi-modal expression patterns with distinctive positive and negative populations (Fig. 1A). GzmA displayed a tri-modal expression pattern, with a small fraction of CD8⁺ T-cells identified as GzmA^Bright, expressing GzmA at high fluorescence intensity, and a large subset displaying intermediate intensity, designated as GzmA^Int (Fig. 1A). The GzmA^Bright population was particularly prominent in mucosa. Irrespective of HIV-1 disease status, GzmA was the most abundantly expressed cytotoxic effector protein in both blood and mucosa, while perforin was the rarest (Fig. 1A, 1B). In agreement with our previous work, the proportion of CD8⁺ T-cells expressing GzmB and perforin was greater in blood compared to mucosa in both seronegative and HIV-1⁺ participants (11, 13). In contrast, the relative abundance of GzmA^Int cells in blood and gut differed between HIV-1⁺ and seronegative participants. In HIV-1⁺ individuals, the proportion of GzmA^Int CD8⁺ T-cells was similar in blood and mucosa; in seronegatives, it was significantly greater in mucosa. No significant differences were detected between blood and gut in the proportion of GzmK⁺ CD8⁺ T-cells in either HIV-1⁺ or seronegatives (Fig. 1B).

FIGURE 1. Expression of cytotoxic effector molecules in unstimulated blood and rectosigmoid CD8⁺ T-cells across HIV-1-disease status.

(A) Representative flow cytometry plot displaying intracellular protein staining for GzmA^Int/Bright, GzmB, GzmK, and perforin in unstimulated blood and rectosigmoid CD8⁺ T-cells CD8⁺ T-cells. (B) Differences in the intracellular expression of GzmA^Int/Bright, GzmB, GzmK, and perforin between unstimulated blood and rectosigmoid CD8⁺ T-cells in HIV-1-infected (HIV-1⁺) and seronegative participant groups (n=25 and 10, respectively). (C) Differences in the intracellular expression of GzmA^Int/Bright, GzmB, GzmK, and perforin in unstimulated rectosigmoid CD8⁺ T-cells across HIV-1-disease status as follows: C (Controllers) n=8, V (viremic untreated) n=7, Tx (ART-suppressed) n=10, SN (seronegatives) n=10. In B and C, wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; asterisks show level of significance as follows: * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Expression of cytotoxic effectors varied across HIV-1 disease status. With the exception of GzmA^Bright cells, which were most abundant in seronegatives, percentages of CD8⁺ T-cells expressing cytotoxic effectors were typically higher in HIV-1⁺ groups compared to seronegatives in both tissues, with the highest percentages observed in HIV-1⁺ individuals not on ART (Fig. 1C, Supplemental Fig. 1A). Among HIV-1⁺ groups, controllers and viremic individuals not on ART had higher percentages of GzmA^Int and GzmK-positive rectosigmoid CD8⁺ T-cells compared to ART-treated participants. Viremic individuals not on ART also had greater GzmB expression in gut compared to ART-treated subjects (Fig. 1C). Similar trends were observed in blood although not as pronounced (Supplemental Fig. 1A).

Taken together, these data demonstrate elevated expression of GzmA^Int, B, K and perforin in rectosigmoid CD8⁺ T-cells from HIV-1⁺ individuals, particularly those not on ART, compared to seronegatives, and highlight for the first time a novel subset of CD8⁺ T-cells expressing GzmA at high fluorescence intensity in the mucosa.

Co-expression of cytotoxic effectors is reduced in rectosigmoid CD8⁺ T-cells compared to PBMC but elevated in HIV-1-infection

Differences in the abundance of individual cytotoxic effectors led us to investigate the co-expression patterns of these molecules in mucosa. In blood, HIV-1-specific CD8⁺ T-cells have been previously characterized as Perforin^−/+ GzmB⁺ GzmA⁺ GzmK⁺ (24). As expression patterns of cytotoxic effectors were elevated in blood CD8⁺ T-cells of HIV-1⁺ individuals compared to seronegatives, we predicted that their co-expression would likewise be elevated in the mucosa of HIV-1 infected compared to healthy individuals. To test this prediction, we used flow cytometry and SPICE software to analyze co-expression of cytotoxic effectors in unstimulated CD8⁺ T-cells from rectosigmoid mucosa and blood of chronically HIV-1 infected and seronegative adults (Figure 2). This dataset captured information from all CD8⁺ T-cells, regardless of specificity.

FIGURE 2. Co-expression patterns of cytotoxic effectors vary with anatomical compartment and HIV-1 disease status.

(A) Representative flow cytometry plot displaying the intracellular co-expression patterns of GzmA^Int/Bright, GzmB, GzmK, and perforin in unstimulated blood and rectosigmoid CD8⁺ T-cells. (B) Differences in the proportions of CD8⁺ T-cells co-expressing GzmA^Int/Bright, GzmB, GzmK, and perforin between blood and rectosigmoid mucosa in HIV-1 infected (HIV-1⁺) and seronegative participants (n=25 and 10, respectively). Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; asterisks show level of significance as follows: * P <0.05, ** P <0.01, *** P <0.001. (C) Differences in the percentages of rectosigmoid CD8⁺ T-cells co-expressing cytotoxic effectors across HIV-1-disease status as follows: Viremic Untreated (V) n=7, Controllers (C) n=8, ART-suppressed (Tx) n=10, and seronegatives (SN) n=10. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; horizontal bars between participant subgroups indicate significance as follows: * P <0.05, ** P <0.01.

Irrespective of HIV-1 disease status, we observed significant differences in the percentages of CD8⁺ T-cells co-expressing certain cytotoxic effectors between blood and mucosa, most notably in the proportions of Perforin⁺GzmA^IntGzmB⁺ and GzmA^Bright CD8⁺ T-cells. Although in both tissues, perforin was typically co-expressed with GzmA^Int and GzmB, the percentage of Perforin⁺GzmA^IntGzmB⁺ CD8⁺ T-cells was significantly higher in blood compared to mucosa (Fig. 2A, 2B). This putative cytotoxic subset was one of the most abundant in blood (Blood_HIV+ median 17.1%, Blood_SN median 6.17%) but one of the rarest in mucosa (Rectosigmoid_HIV+ median 1.18%, Rectosigmoid_SN median 0.508%). In contrast, the mucosa contained a significantly higher percentage of GzmA^Int single positive CD8⁺ T-cells compared to blood (Rectosigmoid_HIV+ median 17.6%, Rectosigmoid_SN median 28.9%; Blood_HIV+ median 2.69%, Blood_SN median 1.43%). A similar trend was observed with GzmA^Bright CD8⁺ T-cells. Interestingly, in addition to differences in abundance, the co-expression pattern of GzmA^Bright also differed between blood and mucosa. In mucosa, GzmA^Bright CD8⁺ T-cells typically lacked expression of any other cytotoxic effectors; in contrast, blood GzmA^Bright CD8⁺ T-cells typically co-expressed perforin and GzmB (Fig. 2A, 2B).

Perhaps not surprisingly, HIV-1⁺ participants also had higher median frequencies of CD8⁺ T-cells co-expressing cytotoxic effectors compared to seronegatives in both blood and gut, including a large proportion of CD8⁺ T-cells co-expressing GzmA^Int, B, and K (Rectosigmoid_HIV+ median 21.7%, Rectosigmoid_SN median 6.89%; Blood_HIV+ median 15.9%, Blood_SN median 5.89%) (Fig. 2B). To elaborate on this observation, we compared the abundance of co-expressing subsets across HIV-1-disease status (Fig. 2C, Supplemental Fig. 1B). In mucosa, the proportions of GzmA^IntGzmB⁺GzmK⁺ and GzmA^IntGzmB⁺ CD8⁺ T-cells were significantly greater in all HIV-1⁺ subgroups compared to seronegatives. The proportion of GzmA^IntGzmB⁺GzmK⁺ CD8⁺ T-cells was also greater in viremic individuals not on ART compared to those on ART. Both controllers and viremic individuals not on ART had significantly higher percentages of ‘phenotypically cytotoxic’ CD8⁺ T-cells, defined as those co-expressing perforin and granzymes, compared to seronegatives. Specifically, controllers and viremic individuals not on ART had higher percentages of Perforin⁺GzmA^IntGzmB⁺GzmK⁺ CD8⁺ T-cells compared to seronegatives; controllers had higher percentages of Perforin⁺GzmA^IntGzmB⁺ CD8⁺ T-cells compared to seronegatives. In contrast, seronegatives displayed greater frequencies of GzmA^Int and GzmA^Bright single positive CD8⁺ T-cells compared to all HIV-1⁺ subgroups (Fig. 2C). Similar trends were observed in the blood (Supplemental Fig. 1B).

Taken together, these data reveal that in general, the rectosigmoid mucosa contains a lower percentage of ‘phenotypically cytotoxic’ CD8⁺ T-cells compared to blood, but an abundance of GzmA single-positive CD8⁺ T-cells. Furthermore, individuals with chronic HIV-1 infection, particularly those not on ART, have greater co-expression of cytotoxic effector proteins in mucosal CD8⁺ T-cells compared to uninfected individuals.

Expression of perforin and GzmB in antigen-specific CD8⁺ T-cells differs with anatomical compartment, antigenic stimulation, and cytokine polyfunctionality

We next turned our attention to antigen-specific CD8⁺ T-cells. To explore the range of functionality exhibited by antigen-specific rectosigmoid CD8⁺ T-cells, lymphocytes isolated from blood and mucosa of HIV-1⁺ individuals during early and chronic infection or seronegative controls (Table I) were stimulated with an HIV-1 Gag peptide pool or Staphylococcal enterotoxin B (SEB) in a 5.5-hour ex vivo stimulation assay followed by intracellular staining for MIP-1β, IFNγ, and TNFα along with CD107a, GzmB and perforin as surrogates of cytotoxicity. For these experiments we utilized reagents and protocols previously demonstrated to reveal perforin newly produced in response to TCR stimulation (21). Expression of perforin and GzmB was measured within Gag and SEB-responding CD8⁺ T-cells as defined by one or more of the following responses: degranulation (CD107a), MIP-1β, IFNγ, or TNFα production (Fig. 3A).

In both HIV-1-infected and seronegative participant groups, a large fraction of rectosigmoid CD8⁺ T-cells responding to antigenic stimulation did not express detectable perforin or GzmB (median_Gag 49.6%, median_SEB 74.5%) (Fig. 3B). For both antigens, the proportion of total responding CD8⁺ T-cells co-expressing perforin and GzmB was significantly greater in blood compared to mucosa (Fig. 3B); in contrast, the proportion of total responding CD8⁺ T-cells expressing neither perforin nor GzmB was greater in mucosa compared to blood, reaching statistical significance for SEB (Fig. 3, Supplemental Fig. 2). As no statistically significant differences were observed between HIV-1⁺ subgroups, HIV-1⁺ participant data were consolidated into a single group. Interestingly, in mucosa but not blood, the proportion of responding CD8⁺ T-cells expressing GzmB in the absence of perforin (Perforin^NegGzmB⁺) was significantly greater for Gag compared to SEB stimulation (medians 38.7% and 19.9% respectively) (Fig. 3B). The Gag-specific Perforin^NegGzmB⁺ CD8⁺ T-cell response was also greater in mucosa compared to blood (Fig. 3B).

We next assessed the expression of perforin and GzmB within CD8⁺ T-cell subsets producing multiple cytokines/chemokines and/or CD107, and again found differences between Gag and SEB-responding CD8⁺ T-cells. Similar to previous observations in blood (7), the proportion of Gag-responding CD8⁺ T-cells expressing perforin and/or GzmB tended to increase as cytokine polyfunctionality decreased in both blood and gut (Fig. 3D). This trend was especially pronounced in mucosa, where the majority of CD8⁺ T-cells responding to Gag with 3 or 4 soluble markers and/or CD107, did not express perforin or GzmB (medians 60.18% and 67.33% for 3- and 4-function cells, respectively) (Fig. 3D). In contrast, single-function mucosal Gag-responding CD8⁺ T-cells displayed the greatest GzmB expression (Fig. 3D). This was not observed for blood SEB-responding CD8⁺ T-cells, in which the proportion of perforin and/or GzmB-expressing cells appeared greater in polyfunctional compared to single-function CD8⁺ T-cells (Supplemental Fig. 2C, 2D). No obvious trend emerged for SEB in the mucosa, as the majority of rectosigmoid SEB-responding CD8⁺ T-cells did not express perforin or GzmB, irrespective of cytokine expression (Supplemental Fig. 2).

Taken together, these data demonstrate an apparent inverse relationship between polyfunctionality (defined as coexpression of multiple cytokines/chemokines and CD107a) and cytotoxic potential (defined as coexpression of perforin and GzmB) in rectosigmoid HIV-1 Gag-specific CD8⁺ T-cells, and highlight discordance in the expression of perforin and GzmB between Gag and SEB-responding CD8⁺ T-cells in the mucosa.

Expression of cytotoxic effectors in antigen-responding CD8⁺ T-cells differs by anatomic location and by antigenic stimulation

The data presented in Figure 3 suggest that most cytokine/chemokine-producing HIV-1-specific rectosigmoid CD8⁺ T-cells do not express perforin and granzyme B. The abundance of mucosal CD8⁺ T-cells expressing granzymes independently of perforin led us to re-examine this result using a broader range of cytotoxic effectors. As before, lymphocytes isolated from blood and rectosigmoid mucosa of chronically HIV-1 infected and seronegative adults were stimulated with an HIV-1 Gag-peptide pool or Staphylococcal enterotoxin B (SEB) in a 5.5 hour ex vivo stimulation assay. However, in this series of experiments, stimulation was followed by intracellular staining for Perforin, GzmA, GzmB and GzmK, along with MIP-1β and CD107 (Fig. 4). As CD107a and MIP-1β expression were the two strongest responses previously detected in rectosigmoid mucosa in response to HIV-1 Gag and SEB stimulation, they were used here as indicators of antigen responsiveness (10, 12).

For both HIV-1 Gag and SEB stimulation, the mucosa displayed a greater proportion of responding CD8⁺ T-cells not expressing any cytotoxic effectors compared to blood. These Perforin⁻GzmA⁻GzmB⁻GzmK⁻ responses dominated in the mucosa and were particularly striking for SEB-responding CD8⁺ T-cells (Fig. 4C). Rectosigmoid mucosa also exhibited greater proportions of GzmA single positive antigen-responding CD8⁺ T-cells compared to blood. In contrast, blood had greater proportions of Perforin^+/−GzmA⁺GzmB⁺ responding CD8⁺ T-cells compared to mucosa (Fig. 4C). Differences between antigenic stimulations were also observed. In mucosa but not blood, Gag-responding CD8⁺ T-cells exhibited a larger proportion of GzmA⁺GzmB⁺GzmK⁺ CD8⁺ T-cells compared to SEB-responding CD8⁺ T-cells (Fig. 4C and data not shown). In contrast, rectosigmoid SEB-responding CD8⁺ T-cells included a greater proportion of cells not expressing any cytotoxic effectors compared to Gag-responding CD8⁺ T-cells (Fig. 4C and data not shown).

These findings expand upon the data presented in Figures 1 and 3, and further support the interpretation that rectosigmoid CD8⁺ T-cells are typically primed for cytokine expression rather than cytotoxic effector production. However, this observation may be partly antigen-dependent, as rectosigmoid Gag-specific CD8⁺ T-cells displayed proportionately greater expression of granzymes A, B, and K compared to SEB-responding cells.

Memory phenotype varies with cytotoxic effector expression and anatomic location

Based on observations in blood, it is thought that CD8⁺ T-cells acquire expression of cytotoxic effectors as they mature into effector subsets. For example, Perforin⁻GzmA⁻GzmB⁻, GzmA⁺GzmB⁺GzmK⁺, and Perforin⁺GzmA⁺GzmB⁺ subsets are associated with central memory, transitional and effector maturation stages, respectively (24–26). However, despite housing a high proportion of effector-memory cells (27), the mucosa displays an abundance of CD8⁺ T-cells expressing either only GzmA or not expressing any cytotoxic effectors (Fig. 1). To address this apparent discrepancy, we assessed the intracellular expression of cytotoxic effectors in conjunction with T-cell memory markers CD45RO, CCR7, and CD27 on unstimulated blood and rectosigmoid CD8⁺ T-cells from chronically HIV-1 infected and seronegative adults.

Not surprisingly, blood CD8⁺ T-cells not expressing any cytotoxic effectors primarily had a naïve phenotype (Supplemental Fig. 3), whereas in rectosigmoid mucosa most “quadruple negative” Perforin⁻GzmA⁻GzmB⁻GzmK⁻ CD8⁺ T-cells were memory cells, with a large fraction displaying a transitional memory phenotype (Fig. 5). Because the proportion of GzmA^IntGzmB⁺GzmK⁺ was elevated in the mucosa of HIV-1⁺ individuals not on ART (Fig. 2C), we were interested in the memory phenotype of these cells. Rectosigmoid subsets expressing GzmK, such as GzmA^IntGzmB⁺GzmK⁺ and GzmA^IntGzmK⁺ CD8⁺ T-cells, exhibited a more pronounced transitional memory phenotype than their counterparts in blood (Fig. 5, Supplemental Fig. 3). Mucosal single-positive GzmA^Int/Bright CD8⁺ T-cells were predominantly effector-memory cells (Fig. 5). In blood, GzmA^Bright cells were absent, and GzmA^Int cells were distributed amongst T_TM, T_EM and T_EFF categories. The relatively small subset of rectosigmoid CD8⁺ T-cells expressing perforin, primarily Perforin⁺GzmA^IntGzmB⁺ CD8⁺ T-cells with or without GzmK, was comprised of effector T-cells with some T_TM and T_EM (Fig. 5B), a distribution similar to that of Perforin⁺GzmA^IntGzmB⁺ CD8⁺ T-cells in blood (Supplemental Fig. 3). Taken together, these data reveal differences between blood and gut regarding the memory phenotype of CD8⁺ T-cells lacking expression of cytotoxic effectors, and identify the GzmA^Bright CD8⁺ T-cell subset, limited to mucosa, as effector-memory cells.

FIGURE 5. Expression of memory/effector markers by prominent CD8⁺ T-cell subsets in rectosigmoid mucosa.

(A) Representative flow cytometry plot displaying expression of memory markers CD45RO and CD27 on the following rectosigmoid CD8⁺ T-cells subsets: GzmA^IntGzmB⁺GzmK⁺ (purple), GzmA^IntGzmK⁺ (orange), GzmA^Bright (green), GzmA^Int (brown), Perforin⁺GzmA^IntGzmB⁺ (red), and cells lacking expression of cytotoxic effectors (Perforin⁻GzmB⁻GzmA⁻GzmK⁻)(blue). The proportions of transitional memory (T_TM), effector-memory (T_EM), effector (T_EFF), and CD45RO⁻CD27⁺CCR7⁻ cells within each subset are displayed within the quadrants. (B) Differences in the percentages of naïve (T_naive), central-memory (T_CM), transitional memory (T_TM), effector-memory (T_EM), effector (T_EFF), and CD45RO⁻CD27⁺CCR7⁻ cells within GzmA^IntGzmB⁺GzmK⁺, GzmA^IntGzmK⁺, GzmA^Int/Bright single-positive, Perforin⁺GzmA^IntGzmB⁺, and Perforin⁻GzmB⁻GzmA⁻GzmK⁻ rectosigmoid CD8⁺ T-cells. Large asterisk identifies memory phenotypes with a significantly greater median frequency compared to all other memory subsets. n=11. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; small asterisks show level of significance as follows: * P <0.05, ** P <0.01, *** P <0.001.

GzmA^Bright CD8⁺ T-cells resemble tissue-resident T-cells

The human rectosigmoid mucosa houses a large fraction of CD8⁺ T-cells with a tissue resident phenotype, including in chronic HIV-1 infection (these authors, in press). As GzmA^Bright CD8⁺ T-cells were abundant in mucosa but not blood, and primarily displayed an effector-memory phenotype, we hypothesized that these cells might also express markers of tissue residency. To test this, we analyzed intracellular expression of GzmA and GzmB in conjunction with the canonical tissue-resident markers CD69, integrin αE (CD103), and sphingosine-1-phosphate receptor (S1PR1) on unstimulated blood and rectosigmoid CD8⁺ T-cells from chronically HIV-1 infected and seronegative adults.

Irrespective of HIV-1-disease status, the overwhelming majority of rectosigmoid GzmA^Bright CD8⁺ T-cells were CD69⁺CD103⁺S1PR1⁻, indicative of tissue-residency (Fig. 6). In contrast, the expression of tissue-resident markers on GzmB⁺ and GzmA^Int CD8⁺ T-cells varied across HIV-1-disease status. In ART-treated and healthy adults, GzmB⁺ and GzmA^Int CD8⁺ T-cells were primarily CD69⁺CD103⁺S1PR1⁻. In contrast, in HIV-1⁺ individuals not on ART, GzmB⁺ and GzmA^Int CD8⁺ T-cells were primarily single-positive for CD69 or lacked expression of all three markers (Fig. 6). Together, these data support the interpretation that GzmA^Bright CD8⁺ T-cells are likely a subset of tissue-resident CD8⁺ T-cells unique to the mucosa, whose functions and antigenic specificity remain to be determined.

FIGURE 6. Expression of tissue-resident markers on GrzA^Bright, GrzA^Int, and GrzB-expressing CD8⁺ T-cells in rectal mucosa.

(A) Representative flow cytometry plot showing expression of the tissue-resident markers early leukocyte activation marker CD69 and integrin αE (CD103) on GrzA^Bright, GrzA^Int, and GrzB-expressing CD8⁺ T-cells in rectal mucosa. (B) Proportion of rectal GrzA^Bright, GrzA^Int, and GrzB-positive CD8⁺ T-cells expressing the tissue residency markers CD69, CD103, and sphingosine-1-phosphate receptor (S1PR1) in HIV-1 Controllers n=4, Viremic Untreated n=5, ART-treated n=7, and Seronegatives n=4. Gray box outlines the canonical tissue residency phenotype. Co-expression analysis was generated using SPICE software. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; symbols indicate level of significance as follows: * P <0.05, ** P <0.01.

Discussion

The gastrointestinal mucosa is an important site of HIV-1 transmission and viral replication, and is generally considered as one of the “front lines” of defense against mucosal pathogens. Understanding how mucosal CD8⁺ T-cells respond to microbial challenge is important for developing therapeutic and preventative strategies targeted to mucosal tissues. Previous reports have demonstrated that rectosigmoid CD8⁺ T-cells are robust producers of chemokines and cytokines but have limited perforin expression, supporting the interpretation that gut CD8⁺ T-cells are primed for cytokine/chemokine secretion rather than cytotoxicity (11–13, 28). However, missing from earlier studies of mucosal T-cells were detailed analyses of cytolytic effector proteins granzymes A, B and K and their patterns of expression/coexpression with cytokines and chemokines. In the present study we provide a more comprehensive view of the effector molecules produced by rectosigmoid CD8⁺ T-cells, which may be of particular interest in light of recent literature demonstrating novel extracellular, pro-inflammatory functions of granzymes (14, 15, 17, 29, 30).

As previously reported, the percentages of CD8⁺ T-cells expressing perforin and GzmB were significantly reduced in rectosigmoid mucosa compared to blood (11–13, 28). In contrast, the percentages of GzmK and GzmA-positive CD8⁺ T-cells were similar between the two tissues. These observed differences in expression levels of cytotoxic effectors suggest differential regulation at the transcriptional and/or post-transcriptional level. Interestingly, the gene for human GzmB is located on chromosome 14 whereas genes for GzmA and GzmK, whose expression was not deficient in the gut, are located in close proximity to one another on chromosome 5 (29, 31). Little is known about the transcriptional regulation of GzmA and GzmK but their spatial separation from GzmB might suggest differing transcriptional regulation (32). Differences in post-transcriptional regulatory mechanisms may also account for the variations in expression levels. For example, although resting murine NK cells express mRNA for perforin, GzmB and GzmA, they must be activated to express perforin and GzmB protein; in contrast, both resting and activated NK cells express high levels of GzmA protein (33).

Although GzmA was the most abundantly expressed cytotoxic effector in both blood and gut, we observed discordance in its expression pattern between tissues. In mucosa, a large percentage of CD8⁺ T-cells were single-positive for GzmA; a subset of these expressed granzyme A at high levels (GzmA^Bright) and displayed an effector/memory, tissue-resident phenotype (CD103⁺CD69⁺S1PR1⁻) (34, 35). In blood, however, GzmA^Bright CD8⁺ T-cells were scarce and GzmA was nearly always co-expressed with other cytotoxic effectors. These data suggest that similar to perforin and GzmB, GzmA expression may be regulated differently in rectosigmoid CD8⁺ T-cells compared to blood.

In contrast to the abundance of GzmA, the percentage of CD8⁺ T-cells co-expressing perforin and granzymes was sharply reduced in gut compared to blood, further evidence of the diminished cytotoxic potential of rectosigmoid CD8⁺ T-cells (11). We previously speculated that perforin and GzmB are tightly regulated in mucosa to avoid ‘bystander’ damage to the fragile mucosal epithelium (11). As granzymes are believed to be unable to induce target cell apoptosis without perforin (36), one interpretation of these data is that limited perforin expression enables rectosigmoid CD8⁺ T-cells to utilize granzymes for non-cytolytic functions. Consistent with this interpretation, while the cytotoxicity of GzmB and perforin are well established, controversy remains as to whether other granzymes contribute significantly to target cell apoptosis in vivo (15, 37). Rather, GzmA and GzmK may function in extracellular, non-cytotoxic capacities in the mucosa. For example, GzmA and GzmK have been shown to facilitate cytokine responses to the microbial cell wall component lipopolysaccharide (LPS) (16, 38). Additionally, GzmA, B, and K have demonstrated extracellular activities that promote cellular migration and inflammation (14, 15, 17, 29, 30). Pro-inflammatory functions of granzymes appear consistent with the polyfunctional nature of rectosigmoid CD8⁺ T-cells, whose expression of cytokines and beta-chemokines likely have similar pro-inflammatory effects (39, 40).

Disparities in the expression levels of cytotoxic effectors varied with HIV-1-disease status. The percentages of CD8⁺ T-cells expressing GzmA^Int, GzmB, GzmK, and perforin were higher in HIV-1 infected compared to seronegative adults, with the highest percentages observed in HIV-1⁺ individuals not on ART. Interestingly, the opposite trend was observed for single-positive GzmA^Bright cells. One interpretation is that HIV-1 infection results in up-regulation of other cytotoxic effectors in GzmA^Bright CD8⁺ T-cells. The overwhelming majority of GzmA^Bright CD8⁺ T-cells expressed αE integrin (CD103), which is associated with proximity to epithelial cells expressing the CD103 ligand, E-cadherin (41–43). A characteristic of HIV-1 infection is loss of epithelial barrier integrity (44, 45). Accordingly, an alternative explanation is that lower proportions of GzmA^Bright CD8⁺ T-cells in HIV-1 infection reflects a loss of CD103⁺ CD8⁺ T-cells, due in part to reduced availability of epithelial-derived E-cadherin.

Notably, the proportion of CD8⁺ T-cells co-expressing GzmA, B, and K was significantly greater in HIV-1 infected compared to seronegative participants in both blood and gut. In blood, co-expression of GzmA, B, and K is associated both with an intermediate memory maturation status and HIV-1-specificity (24–26). Similarly, rectosigmoid GzmA⁺GzmB⁺GzmK⁺ CD8⁺ T-cells primarily displayed a transitional memory phenotype and a substantial fraction of rectosigmoid Gag-responding CD8⁺ T-cells co-expressed GzmA, B, and K. Whether co-expression of GzmA, B, and K in Gag-specific CD8⁺ T-cells is a reflection of maturation status or a consequence of HIV-1-specific priming of the immune response will require further exploration. However previous work has demonstrated that blood HIV-1-specific CD8⁺ T-cells are skewed towards an intermediate maturation stage, suggesting the former (46).

Consistent with the hypothesis that rectosigmoid CD8⁺ T-cells are skewed towards cytokine production rather than cytotoxicity, the proportions of Gag and SEB-responding CD8⁺ T-cells expressing cytotoxic effectors were significantly lower in rectosigmoid mucosa compared to blood. Furthermore, we observed that perforin and GzmB expression increased in Gag-specific CD8⁺ T-cells as polyfunctionality decreased, suggesting that cytotoxic and cytokine/chemokine-producing Gag-specific CD8⁺ T-cells are to some extent separate subsets. In the mucosa, responding CD8⁺ T-cells often lacked expression of perforin and granzymes. As postulated elsewhere (11), degranulation in the absence of cytotoxic effectors may reflect the release of other granule components like MIP-1α and MIP-1β (47). Alternatively, it may reflect differences in the kinetics of cytotoxic effector up-regulation between blood and gut CD8⁺ T-cells. In addition to differences between tissues, we also observed differences in expression of cytotoxic effectors between HIV-1 Gag and SEB-responding CD8⁺ T-cells in the mucosa. The proportion of rectosigmoid Gag-responding CD8⁺ T-cells expressing cytotoxic effectors was greater compared to SEB-responding CD8⁺ T-cells, indicating that expression of cytotoxic effectors is, to a certain degree, influenced by antigen-specificity. It is currently unclear whether elevated cytotoxic effector expression in Gag-responding rectosigmoid CD8⁺ T-cells reflects the up-regulation of cytotoxic effectors by tissue resident CD8⁺ T-cells or, alternatively, an influx of cytotoxic effector-expressing CD8⁺ T-cells from circulation. Addressing this question will require further work, likely involving experimental infection studies in nonhuman primate models.

Interestingly, unlike GzmA^Bright CD8⁺ T-cells that were overwhelmingly tissue-resident, a large fraction of GzmB⁺ and GzmA^Int rectosigmoid CD8⁺ T-cells did not express the phenotypic markers of tissue-residency, suggesting they might be recent immigrants. Notably, Gag-specific polyfunctional CD8⁺ T-cells were not completely devoid of perforin and granzyme B, suggesting a small fraction of Gag-specific CD8⁺ T-cells may be capable of both cytotoxic and polyfunctional responses. Determining whether the co-expression of cytotoxic effectors with cytokines/chemokines confers a protective advantage against HIV-1 will require further analysis. No differences were observed between HIV-1 Controllers and Non-controllers in the proportion of Gag-specific CD8⁺ T-cells expressing perforin and/or granzymes, although this analysis was limited by small sample size.

Taken together, these data demonstrate that rectosigmoid CD8⁺ T-cells produce granzymes independently of perforin, likely complementing dominant cytokine and chemokine responses to promote an inflammatory response. The work described here supports a model in which rectosigmoid CD8⁺ T-cells are skewed towards pro-inflammatory responses rather than direct cytotoxicity, suggesting new insights into the mucosal adaptive immune response that may inform the development of therapeutics to treat mucosal infections such as HIV-1.

Abbreviations used in this article

Gzm

granzyme

ART

antiretroviral therapy

SEB

Staphylococcal Enterotoxin B

MIP

macrophage inflammatory protein

CD

cluster of differentiation

C

controllers

V

viremic untreated

Tx

treated

SN

seronegative

S1PR1

sphingosine-1-phosphate receptor

VL

viral load

Perf

perforin