Combination Immune Checkpoint Blockade Enhances IL-2 and CD107a Production from HIV-Specific T Cells Ex Vivo in People Living with HIV on Antiretroviral Therapy

Antiretroviral therapy (ART) efficiently controls HIV replication, allowing for immune recovery and a normal life expectancy in people with HIV (PWH); however, virus persists indefinitely in a latent form, meaning that treatment is required lifelong (1, 2). Immune dysfunctions can also persist in PWH on ART, with evidence of increased microbial translocation, innate immune and T cell activation as well as T cell exhaustion (reviewed in Refs. 1, 2) characterized by the upregulation of inhibitory immune checkpoint (IC) markers (3–5). To eliminate long-lived latently infected cells in PWH on ART, multiple approaches are being evaluated, including strategies to enhance HIV-specific T cell function to increase clearance of infected cells, reduce the size of the reservoir, as well as maintain long-term control of virus replication (1). In this study, we evaluated the ability of Abs to multiple IC, used either alone or in combination, to reinvigorate HIV-specific CD4⁺ and CD8⁺ T cell function.

Multiple IC markers, including CTL-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), T cell Ig domain and mucin domain 3 (TIM-3), T cell Ig and ITIM domain (TIGIT), and lymphocyte activation gene-3 (LAG-3), remain elevated on the surface of total and HIV-specific CD4⁺ and CD8⁺ T cells in PWH on ART compared with uninfected individuals (4–11). Blockade of each of these IC with Abs has been previously investigated using different ways to quantify recovery of immune function. For example, in HIV-specific CD4⁺ T cells, there was improved production of IFN-γ and IL-2 as well as T cell proliferation following administration of Abs to either PD-1 or CTLA-4 ex vivo (6, 12). HIV-specific CD8⁺ T cells also showed increased proliferation and higher expression of IFN-γ and TNF-α ex vivo following administration of Abs to PD-1 or PD-L1 ex vivo (13, 14). Similarly, recombinant LAG-3–Fc, which competes with the binding of native membrane-bound LAG-3 to MHC class II, has been shown to enhance the production of TNF-α and IL-2 in HIV-specific CD4⁺ and CD8⁺ T cells ex vivo (11). The relationship of these ex vivo findings to effects in vivo remains to be determined.

There is far more limited information available on the effects of IC blockade in PWH on ART in vivo. One small prospective dose finding clinical trial of anti–PD-L1 in PWH on ART without cancer used an ELISPOT assay to demonstrate enhancement of HIV-specific T cell function in two of six anti–PD-L1 recipients (15). Other small case series or case reports in PWH on ART with concomitant malignancy have similarly shown an increase in HIV-specific CD4⁺ and CD8⁺ T cells in some but not all participants following anti–PD-1 treatment (16, 17). There are currently seven clinical trials of anti–PD-1 enrolling PWH on ART, including three studies enrolling people without cancer (https://clinicaltrials.gov) and one study evaluating the impact of anti–PD-1 and anti–CTLA-4 (18).

Beyond the blockade of single IC markers, there is increasing evidence that the clinical impact of IC blockade can be enhanced when used in combination. The best evidence has been in individuals with metastatic malignant melanoma, in which there was enhanced efficacy and long-term survival with the use of anti–PD-1 together with anti–CTLA-4 compared with either Ab alone; however, this has also been associated with an increased risk of toxicity (19, 20). Combinations of other Abs to IC are in preclinical and clinical development (21), with clinical trials investigating anti–PD-1 in combination with anti-TIGIT or anti–LAG-3 currently underway.

In HIV infection, combination IC blockade could also potentially have an enhanced effect on restoring HIV-specific T cell function, as multiple inhibitory IC are expressed on HIV-specific CD4⁺ (6, 11, 22, 23) and CD8⁺ (6, 10, 11, 13, 22, 23) T cells. Ex vivo, some combinations of IC Abs compared with either Ab alone have shown enhanced proliferation and cytokine production of HIV-specific CD8⁺ T cells, including the combination of anti–PD-L1 with anti-TIGIT (13), or anti–PD-1 with anti–TIM-3, anti-BTLA, or anti-CD160 (14). Whether there are additive or synergistic effects on HIV-specific T cell function following the inhibition of multiple other IC in blood from PWH on ART has not been systematically explored.

We hypothesized that combination blockade of multiple ICs compared with blockade with a single Ab would lead to an increase in cytokine-producing in HIV-specific T cells in both CD4⁺ and CD8⁺ T cells obtained from PWH on ART. To address this, we quantified the effects of monoclonal Abs to six ICs (CTLA-4, PD-1, PD-L1, TIM-3, TIGIT, and LAG-3) alone or in combination to determine the effect on the frequency of HIV-specific T cells that express CD107a, IFN-γ, TNF-α, and IL-2 in response to HIV peptide stimulation. Similar to previous reports (24, 25), we found that in PWH on ART there was a robust expression of IFN-γ and TNF-α in HIV-specific CD4⁺ and CD8⁺ T cells in the absence of Abs to IC. However, we only observed modestly enhanced expression of IFN-γ and TNF-α following anti–PD-1 blockade alone, with no additive effects when multiple Abs were used in combination. In contrast, the production of IL-2 and the expression of surface CD107a in both CD4⁺ and CD8⁺ HIV-specific T cells was infrequent in the absence of IC blockade upon antigenic stimulation but was markedly enhanced with specific combinations of Abs. These data demonstrate that significant functional impairment in IL-2 production and CD107a expression in PWH persists on ART and that this can be overcome with combination IC blockade.

Eleven PWH on combination ART were enrolled at the Alfred Hospital, Melbourne, Australia with the inclusion criteria of plasma HIV RNA fewer than 50 copies/ml for at least 3 y. Clinical details are summarized in Table I. The participants were all male with suppressed viral load for at least 3 y, and at the time of sample collection they had plasma HIV RNA <20 copies/ml. Leukapheresis samples were collected from HIV-1–infected individuals at the Alfred Hospital, with informed consent and under institutional guidelines. The study was approved by Human Research Ethics Committees at the Alfred and Avenue Hospitals in Melbourne and the University of Melbourne Ethics Committee.

Overlapping 15-mer peptides for HIV-clade B Gag (8117) and Nef (5189) as well as 8- to 11-mer peptides to CMV, EBV, and influenza (cumulative distribution function) (CEF) (9808) were obtained from the National Institutes of Health AIDS Reagent Program. DMSO was used to reconstitute peptides to 500 µg/ml/peptide as the working concentration. The final concentration for peptide stimulation was 2 µg/ml/peptide.

Abs to ICs, including IgG1 Abs (BMS-734016, CTLA-4, ipilimumab; BMS-986207, TIGIT; BMS-986258, TIM-3) and IgG4 (BMS-936558, PD-1, nivolumab; BMS-936559, PD-L1; or BMS-986016 LAG-3) and the relevant isotype controls (IgG1: DT1D12-g1f-N297Q; clone 1182_RAS_Ab; or IgG4 g4P-DT1D12; clone 4A09_RAS_Ab) were a kind gift from Bristol-Myers Squibb. Other isotype Abs evaluated included anti–β-Gal (catalog nos. bgal-mab1 and bgal-mab114; InvivoGen), tumor Ag (a kind gift from Prof. Andrew Scott, Olivia Newton-John Cancer Research Institute, Heidelberg, Australia), and an unknown Ag from BioLegend (clones QA16A12 and QA16A15; BioLegend). Abs to each IC were used at 10 µg/ml, consistent with studies using therapeutic Abs to PD-1/PD-L1 (26, 27) and CTLA-4 (28).

PBMCs were isolated by leukapheresis and cryopreserved. Upon thawing, the cryopreserved PBMC in warm RF10 (RPMI 1640 with 10% FBS) PBMC were adjusted in RF10 to 2 × 10⁶ cells/ml in tissue culture flasks and incubated at 37°C and 5% CO₂ overnight. Rested PBMC were washed and adjusted to 1 × 10⁷ viable cells/ml.

Stimulation of PBMC was performed in 200-µl reactions in a 96-well plate for 6 h at 37°C with 5% CO₂. Each well contained 1 × 10⁶ PBMC, a cytokine secretion inhibitor mixture (5 µg/ml brefeldin A [B7651; Sigma-Aldrich] and 5 µg/ml monensin [M5273; Sigma-Aldrich]), anti-CD107a (clone H4A3) and antiretrovirals (18 µM azidothymidine, 10 µM efavirenz, and 20 µM raltegravir) to inhibit further rounds of viral replication following stimulation ex vivo. The cells were stimulated with either 0.4% DMSO, 2 µg/ml Gag, Nef, or a mix of CEF peptides (catalog no.8117, 5189, and 9808; National Institutes of Health AIDS Reagent Program) or 1 µg/ml staphylococcal enterotoxin B (SEB;S4881; Sigma-Aldrich]). Blocking Abs to CTLA-4, TIGIT, TIM-3, PD-1, PD-L1, and LAG-3 were added at 10 µg/ml each in various combinations, and the equivalent total concentration of isotype Abs were used as a control.

After stimulation, cells were washed in wash buffer (1% FBS and 1 mM EDTA in PBS) and stained for the live/dead marker (catalog no. L34957; Invitrogen) and with Abs to the following surface markers: CD4 (clone RPA-T4), CD14 (clone M5E2), CD19 (clone HIB19), CD45RA (clone HI100), and CCR7 (clone 3D12) at ambient temperature in the dark for 30 min. Separately, anti–PD-1 PE (clone EH12.1) was added to the same surface staining mix in a separate well to assess PD-1 expression for the given donor. After cell fixation and permeabilization, cells were washed in Perm/Wash buffer (catalog no. 554714; BD Biosciences). Staining with Abs to CD3 (clone UCHT1), CD8 (clone RPA-T8), IFN-γ (clone B27), TNF-α (clone MAb11), and IL-2 (clone MQ1-17H12) was performed at ambient temperature in the dark for 30 min. For wells with anti–PD-1, IL-2 was excluded from the postpermeabilization staining mix, as both were conjugated with the same fluorochrome.

After washing cells with Perm/Wash buffer twice, cells were fixed in 100 µl of 1% formaldehyde at ambient temperature in the dark for 15 min. All staining Abs were obtained from BD Biosciences, unless indicated otherwise. Within 2 h, the LSRFortessa cytometer (BD Biosciences) was used to acquire between 210,000 and 300,000 lymphocyte events. Anti-Mouse and Anti-Rat Compensation Beads (catalog nos. 552843 and 552844; BD Biosciences) were used for compensation. The sequential gating strategy (Supplemental Fig. 1) on the cytometric data was performed and quantified in FlowJo 9.9.6.

The fold change in percentage of cells expressing a specific cytokine following incubation with one (single), two (duo), or all six (mixture) Abs to ICs was assessed relative to isotype control. Normal distribution of the fold change in cytokine-positive cells was evaluated by the Shapiro–Wilk test. The Wilcoxon signed-rank test was used to compare the percentage of cells expressing a specific cytokine following incubation with IC Abs (alone or in combination) or appropriate IgG isotype controls. The effect size for the fold-change increase of experimentally observed effects of a given Ab (ICB) relative to isotype control was calculated by post hoc analyses using a sample size of 11, 80% power, and a significance level at 0.05 for two-tailed Wilcoxon signed-ranked tests in G*Power 3.1 (29). Synergism of two Abs was evaluated by Bliss independent models as previously described (30). Briefly, the ICB relative to maximal stimulation with SEB was calculated as (ICB − IgG isotype)/(SEB − DMSO), where all parameters denote the frequency of cytokine-producing cells in response to HIV peptide stimulation. This was repeated for all examined cellular subsets and cytokines. To determine synergism, we calculated the predicted effect of multiple Abs (e.g., blocking Abs to IC1 [ICB1] and IC2 [ICB2], using the following formula: ICB1 [observed] + ICB2 [observed] − ICB1 [observed] × ICB2 [observed]). A Wilcoxon signed-rank test was used to formally compare the difference between the observed and predicted fractions. If the observed minus predicted effect was >0 with statistical significance, this indicated synergism. Correlations between the frequency of cytokine-producing cells following ICB and the T cell expression of PD-1 or the CD4/CD8 ratio were assessed using Spearman correlation. RStudio (version 1.3.1073) and R package ggplot2 (version 3.3.1) were used for statistical analyses and graphs.

We initially observed that two commercial IgG isotype controls (clone QA16A12 and QA16A15 from BioLegend) induced cytokine production above DMSO-treated controls (Supplemental Fig. 2). To determine whether the higher readouts by the commercial IgG isotype were specific, we titrated the commercial IgG isotype Abs without any additional stimulus and observed dose-dependent increases in cytokine production in both CD4⁺ and CD8⁺ T cells (Supplemental Fig. 2).

To identify a suitable IgG isotype with minimal background, we then quantified the frequency of CD4⁺ and CD8⁺ T cells that produced CD107a, IFN-γ, TNF-α, and IL-2 following incubation with three IgGs targeting irrelevant Ags (bacterial β-galactosidase, tumor-induced and diphtheria). All three IgGs showed lower background than the commercial IgG isotype, but the IgG isotype control that targeted bacterial β-galactosidase and tumor showed dose-dependent stimulation at a concentration of 15 µg/ml or higher in CD8⁺ T cells in two of 10 donors (D49 and D81). In contrast, the diphtheria-specific IgG did not show dose-dependent stimulation in any of the 10 donors (Supplemental Fig. 2), and was therefore selected as the IgG isotype control for subsequent experiments.

We first evaluated cytokine production in response to HIV peptides, CEF peptides, and a maximal stimulus of SEB which were each added to PBMC isolated from blood collected from PWH on ART (Table I). Following stimulation with peptides to either Gag or Nef, compared with DMSO, we observed a significant increase in the production of IFN-γ and TNF-α but no increase in IL-2 in both CD4⁺ and CD8⁺ T cells, with CD45RA⁻CCR7⁻ effector CD4⁺ and CD8⁺ T cells as the main subset showing a response (Supplemental Fig. 3). Both Gag and Nef peptides led to an increase in expression of CD107a in CD8⁺ effector T cells but not in CD4⁺ T cells (Supplemental Fig. 3). All responses to HIV peptides were significantly lower than responses to either SEB on both CD4⁺ and CD8⁺ T cells or CEF peptides on CD4⁺ T cells (Supplemental Fig. 3). Together, these findings demonstrated a low frequency of HIV-specific CD4⁺ and CD8⁺ T cells on ART with a far lower frequency of HIV-specific CD4⁺ and CD8⁺ T cells that produce IL-2 than IFN-γ or TNF-α compared with the expression of surface CD107a,.

We next compared the effects of IC Abs, either alone or in combination, on the function of T cells in response to either Gag or Nef peptide stimulation, looking at both the absolute frequency of cytokine-expressing cells (Supplemental Fig. 4) and the fold change relative to isotype control (Fig. 1). Surprisingly, most Abs, when administered alone, had minimal effect on the frequency of cytokine-positive cells (Fig. 1), with the exception of anti–PD-1, which induced a modest but statistically significant increase in IFN-γ and TNF-α production in CD4⁺ T cells but had no effect in CD8⁺ T cells (Fig. 1). The mixture of six Abs showed increased frequency of CD107a, IFN-γ, and TNF-α in Gag-specific CD4⁺ T cells and CD8⁺ T cells, with the exception of IFN-γ in CD4⁺ T cells (green bars, Fig. 1A). Interestingly, the mixture of Abs did not induce a greater magnitude fold change than the use of two Abs (green versus red-single/blue-dual bars, Fig. 1). The most dramatic effect of IC Abs was seen in the frequency of IL-2⁺ HIV-specific T cells in response to multiple combinations of Abs, predominantly those that included either anti–CTLA-4, anti-TIGIT, or anti–LAG-3. These effects were observed in total CD4⁺ and CD8⁺ T cells (Fig. 1) as well as multiple T cell subsets (Fig. 2). Similar patterns of IL-2 expression were seen following incubation with either Gag (Fig. 1A) or Nef (Fig. 1B) peptides. Combinations of two IC Abs also increased expression of CD107a in CD4⁺ T cells. We observed that combinations that included anti–LAG-3 and anti–CTLA-4 showed the greatest fold change in the frequency of CD4⁺and CD8⁺ T cells expressing CD107a and IL-2 (blue bars, Fig. 1). Interestingly, apart from CD107a production in CD4⁺ T cells, we found no increased activity with combinations that included anti–PD-1 (Fig. 1).

We observed a significant increase in the frequency of HIV-specific CD4⁺ and CD8⁺ T cells that produced either IL-2 or CD107a following incubation with various combinations of two IC Abs. To determine whether the effects observed were additive or synergistic, we next used Bliss independence modeling, as previously described (30), to obtain the predicted readouts for every donor for each cytokine. Paired analyses were used to determine the significant differences between the experimentally observed and predicted readouts.

For expression of CD107a, we observed that Abs to LAG-3, when combined with Abs to either CTLA-4, TIGIT, or TIM-3, resulted in a significant synergistic response in CD4⁺ T cells (Fig. 3A). For production of IL-2, the combination of Abs to CTLA-4 with all other Abs (except anti–PD-1 but including anti–PD-L1) led to a significant synergistic response in both CD4⁺ and CD8⁺ T cells (Fig. 3B). Interestingly, none of the synergistic combinations of Abs to ICs that enhanced CD107a or IL-2 responses included anti–PD-1.

Next, we calculated the magnitude of effect of IC Abs relative to isotype controls with regard to CD107a and IL-2 production and the presence of synergism as determined by the Bliss independence model (Fig. 4). This revealed that all three synergistic combinations for CD107a response in Gag- or Nef-stimulated CD4⁺ T cells included anti–LAG-3, of which anti–LAG-3 with anti-TIGIT showed the greatest fold-change increase above IgG isotype controls (1.76-fold) (Fig. 3A). For IL-2 responses, combinations that included anti–CTLA-4 generally resulted in the greatest fold change relative to isotype controls, especially in Gag-stimulated CD8⁺ T cells (Fig. 3B).

Although the combination of Abs to CTLA-4 and either TIGIT or TIM-3 resulted in the largest fold-change response, anti–CTLA-4 and anti–PD-L1 showed synergism with more breath, as indicated by the response to both Gag or Nef peptides, and in CD4⁺ and CD8⁺ T cells but at a lower magnitude fold change than anti–CTLA-4 with either anti-TIGIT or anti–TIM-3 (Fig. 3B).

We next hypothesized that IC Ab combinations that induced a greater fold increase in the frequency of cytokine-positive cells following stimulation with either Gag or Nef peptides (Fig. 1A, 1B) may be due to certain clinical or cellular factors. Hence, we determined the correlation between the fold change in CD107a and IL-2 responses to combination IC blockade that were significantly higher than isotype (Fig. 1) and five clinical parameters (CD8 counts, CD4/CD8 ratio, expression of surface PD-1, and nadir and current counts of CD4).

The CD8 count was the only clinical parameter that positively correlated with the frequency of IL-2⁺ CD8⁺ T cells in response to Gag (r = 0.76, p = 0.016) or Nef (r = 0.66, p = 0.04) peptide alone (Fig. 5A). Gag-stimulated CD8⁺ T cell subsets showed significant positive correlations between CD8 count and IL-2 production with blockade to CTLA-4 and TIM-3 (total: r = 0.53, p = 0.148; central memory T cells (Tcm): r = 0.81, p = 0.022; effector memory T cells reexpressing CD45RA (Temra): r = 0.70, p = 0.043), CTLA-4 and TIGIT (total: r = 0.63, p = 0.076; Tcm: r = 0.90, p = 0.005; Temra: r = 0.77, p = 0.021), or TIM-3 and TIGIT (total: r = 0.70, p = 0.043; Temra: r = 0.72, p = 0.037). Interestingly, these three combinations were identified as synergistic in previous analyses (Fig. 3B). IL-2 production in Nef-stimulated total CD8⁺ T cells in response to blockade to PD-L1 and TIGIT (total: r = −0.73, p = 0.031; Temra: r = −0.73, p = 0.031) was the only combination that negatively correlated with CD8 count.

The CD4/CD8 ratio also showed a positive correlation with the frequency of CD107a⁺ in Gag-stimulated CD4⁺ and CD8⁺ T cells, with the fold-change increase in cells following blockade to six IC (total CD4⁺: r = 0.65, p = 0.042; Tcm CD4⁺: r = 0.83, p = 0.003) and CTLA-4 and TIM-3 (total CD8⁺: r = 0.68, p = 0.045), respectively (Fig. 5B).

Finally, we observed no significant correlations between the responses to HIV peptides alone or with IC blockade and 1) the expression of surface PD-1 for IC blockade to PD-1/PD-L1 or 2) nadir/current CD4 count in CD4 cells. Interestingly, PD-1 frequency correlated with responses to ICB that did not target PD-1 or PD-L1. Whereas lower frequency of PD-1 showed a tendency of higher CD107a response in Gag-stimulated CD8 with blockade to CTLA-4 and TIM-3 (r = −0.72, p = 0.037), positive correlation between PD-1 frequency and the IL-2 response in Nef-stimulated CD4 with blockade to TIGIT and LAG-3 was observed (r = 0.84, p = 0.01).

Abs that block ICs, alone or in combination, can enhance HIV-specific T cell responses ex vivo (13, 14), but synergistic and additive effects, including which combinations provide a superior response, remain unclear. In this study, we determined the effect on HIV-specific T cell function of single and combination IC blockade in PBMCs obtained from PWH on ART. Whereas anti–PD-1 showed a modest increase in the frequency of CD4⁺ T cells expressing IFN-γ and TNF-α, Abs to LAG-3, CTLA-4, and TIGIT in different combinations showed synergistic induction of CD107a and IL-2 production in HIV-specific T cells. Collectively, these results suggest that combination blockade involving LAG-3, CTLA-4, or TIGIT can enhance the cytokine production of HIV-specific T cells in PWH on suppressive ART.

IC proteins are coexpressed on different CD4⁺ and CD8⁺ T cells subsets, as we have previously shown using PBMC from healthy donors (31). Although the blockade of multiple IC, specifically anti–PD-1 and anti–CTLA-4, for the management of melanoma has shown significant clinical benefit (32), few studies have evaluated combinations of IC blockade on HIV-specific T cell function (13, 14). To systematically assess the effect of combination IC blockade, we first demonstrated that the choice of IgG isotype control could alter the interpretation of whether a given IC Ab was effective in reversing T cell function (Supplemental Fig. 2). High background levels of activation and expression of cytokines in HIV-specific cells could potentially underestimate the effects of a specific Ab to IC. We overcame this issue using isotype controls that targeted diphtheria. Given our findings, it is important to confirm and use the isotype Abs with minimal background for studies on the effect of IC blocking Abs.

Prior reports have shown that, in the presence of persistent Ag, as seen in chronic viral infections, exhausted Ag-specific T cells initially lose the capacity to produce IL-2, followed by TNF-α and IFN-γ as well as, in some cases, degranulation (3, 33–35). Given that we detected IFN-γ– and TNF-α– (but not IL-2 and CD107a) producing cells following stimulation with either Gag or Nef HIV peptides (Fig. 1), our findings are consistent with PWH on ART having partially (not fully) exhausted T cells.

The use of anti–PD-1 and the mixture of six Abs to a range of IC markers showed a modest but significant fold increase of HIV-specific T cells expressing IFN-γ and TNF-α. It is possible that the use of cytokine secretion inhibitors for the intracellular cytokine assay that we used in this study limited the availability of proinflammatory cytokines, such as IL-12 (36), in the supernatant that are a prerequisite for further induction of IFN-γ and TNF-α in CD4⁺ and CD8⁺ T cells (37–40). However, we observed clear increases in production of both IFN-γ and TNF-α following stimulation with HIV peptides, CEF peptides, and SEB using this same method (Supplemental Fig. 3). Our findings suggest that the production of IFN-γ and TNF-α in HIV-specific T cells from PWH on ART might already be maximal and unable to be further enhanced. In contrast, we saw minimal production of IL-2 or CD107a in response to HIV peptides in both CD4⁺ and CD8⁺ T cells (Supplemental Fig. 3), but production could be significantly enhanced with combinations of Abs (Fig. 1). Interestingly, the frequency of cytokine-positive HIV-specific CD4⁺ and CD8⁺ T cells was less following incubation with six compared with two IC Abs, suggesting that there is a limit to the number of IC blocking Abs that can be used together. Multiple Abs used together could inhibit or potentially compete for Fc receptor binding sites to induce an effective Ag-specific response.

To our surprise, the largest fold increase in cytokine production (specifically IL-2 and to a lesser extent CD107a) were not seen with combinations that included anti–PD-1 but, instead, various combinations of Abs to CTLA-4, LAG-3, and TIGIT. We saw remarkably similar responses for Nef- and Gag-specific IL-2–producing CD4⁺ and CD8⁺ T cells following combination IC blockade. These data suggest that the effects of combination IC blockade extend to T cells that target either early or late viral gene products, although the frequency of Nef-specific T cells is highly stable on ART, whereas Gag-specific cells decay over time (41). There may be several explanations for the additive and synergistic effects with these Abs. First, there are distinct and different signaling pathways activated following blockade of ICs; For example, Abs to CTLA-4 and LAG-3 inhibit calcium-independent and calcium-dependent signaling pathways, respectively (42–44). Calcium influx, as a result of LAG-3 blockade (42), and protein kinase C (PKC) activation, through TCR ligation, are required for degranulation in T cells, which is indicated by expression of CD107a (45, 46). Therefore, the specific combination of anti–CTLA-4 and anti–LAG-3 can also potentially enhance CD107a expression. Second, induction of IL-2 by combination IC blockade might be explained by enabling multiple transcription factors, such as AP-1, NF-κB, and NFAT, all of which are required to simultaneously bind to the IL-2 promoter for IL-2 induction (47). One or more of these IL-2–specific transcription factors are inhibited following engagement of CTLA-4 (43), PD-1/PD-L1 (48), TIM-3 (49, 50), TIGIT (51), and LAG-3 (52). Consistent with our observations, treatment of PBMC from PWH on ART with Abs to PD-L1 and TIGIT in combination but not alone increased IL-2 production in Gag-specific CD4⁺ and CD8⁺ T cells (13). Last, additive or synergistic effects of using two Abs to ICs could also be a result of the distribution of expression of each of the IC markers on Ag-specific T cells. Coexpression of the ICs that we evaluated in this study on CD4⁺ and CD8⁺ T cells have been reported in PWH off ART (10, 22, 23, 53), on ART (54, 55), or both off and on ART (6, 9, 11, 13, 53).

The most striking observation was the enhanced production of IL-2 in HIV-specific CD4⁺ and CD8⁺ T cells as a result of combining Abs to CTLA-4 with either TIGIT or TIM-3. This strategy could have several beneficial effects on the clearance of latently infected cells in PWH on ART. First, a previous study showed that the production of IL-2 from HIV-specific CD4⁺ T cells can enhance NK-mediated cytotoxicity (56), potentially leading to enhanced clearance of infected cells. Second, IL-2 production by HIV-specific CD8⁺ T cells could lead to CD4-independent proliferation and differentiation of HIV-specific CD8⁺ T cells (57) as seen in HIV long-term nonprogressors (58) as well as in PWH with a lower viral set point following primary infection (59). Third, any enhanced degranulation in CD4⁺ T cells from combination IC blockade might enhance the cytotoxicity of CD4⁺ T cells, although this is considered controversial, which in turn might assist the elimination of MHC class II–restricted HIV-infected macrophages (60). In contrast, there could be potential counterproductive effects from increased production of IL-2 in HIV-specific CD4⁺ and CD8⁺ T cells, such as the expansion of regulatory T cells due to their expression of CD25, a high-affinity IL-2R (61). Any induction of proliferation of CD4⁺ T cells could potentially increase the number of infected CD4⁺ T cells on ART, although this remains to be determined. Further work investigating the effects of the combinations of IC Abs that enhance IL-2 production on target cell killing and number of latently infected cells is ongoing in our laboratories.

To our knowledge, this is the first comprehensive analysis of the effects of combinations of Abs to IC on HIV-specific T cell function; however, there are several limitations in this study. First, we did not use anti-CD28 and anti-CD49d as costimulation during the peptide stimulation and IC blockade for the intracellular cytokine assay. Previous work has shown that the effects of IC blockers, including proliferation or the production of IFN-γ and TNF-α in HIV-specific cells can vary, depending on whether these costimulatory Abs were included (14). We chose to not include these costimulatory models because anti-CD28 might enhance CTLA-4 inhibitory signaling due to less competition for CD80 (62). Second, we intentionally limited the stimulation period to 6 h, with the secretion inhibitor added at the beginning. Because other immune cells, such as dendritic cells and macrophages, express IC (63–66) and respond to blocking Abs to IC, the addition of secretion inhibitors at the beginning minimized the effect of the secreted cytokines on T cells. This approach allowed us to observe the direct and immediate effects of the tested IC blockade on T cells. A longer duration of stimulation with HIV peptides and IC blockade with anti–PD-L1 (12) has previously been shown to induce a higher frequency of cells producing IFN-γ; however, the observed effect of IC blockade on T cells might be indirect (56, 67). Third, we only measured protein expression of cytokines using intracellular cytokine assay. It is possible that IC blockade induced changes in mRNA but there were downstream blocks to expression of the particular cytokine. Finally, the effect size we could detect differed for each measured cytokine. Effect sizes of 0.2, 0.5, and 0.8 are considered as small, medium, and large, respectively. With 11 participants, the effect size (expressed as median interquartile range [IQR]) for the fold-change increase of cytokine-positive cells for CD107a was 0.418 (0.295–0.608), IFN-γ was 0.236 (0.095– 0.353), TNF-α was 0.186 (0.125–0.295), and IL-2 was 0.644 (0.295–0.922). With statistical significance at 0.05 for two-tailed Wilcoxon signed-rank tests, ICB combinations with no or small biological effect would both shown as p > 0.05. This limitation, however, does not change the conclusion of the study that identified specific dual ICB combinations that significantly increased CD107a and IL-2 responses. It is important to highlight that toxicity remains a major obstacle to the clinical development of using IC blockade for PWH on ART as a cure strategy. Both anti–CTLA-4 and, to a lesser extent, anti–PD-1 have been associated with grade 3 or 4 immune-related adverse events (19, 20) that would be unacceptable for PWH on ART without cancer as part of a cure strategy, as recently highlighted in work on developing a target product profile for an HIV cure (68). One strategy to reduce toxicity could be using a low dose of Ab. For example anti–PD-1 administered at one tenth the licensed dose in people with chronic hepatitis B was recently shown to be safe and have prolonged and high receptor occupancy (69). Multiple early-phase studies of anti-TIGIT, anti–LAG-3, and other Abs are now underway alone and in combination, and these Abs may well be associated with lower toxicity (reviewed in Ref. 70). Either way, significant advances will still be needed to understand, predict, and ultimately reduce these toxicities to enable the use of these Abs in PWH.

In conclusion, we identified multiple additive as well as synergistic combinations of two Abs that blocked ICs and enhanced the frequency of cytokine-producing HIV-specific T cells. The most dramatic increases were observed in the production of IL-2 in HIV-specific CD4⁺ and CD8⁺ T cells and to a lesser extent the expression of CD107a in HIV-specific CD4⁺ T cells. The largest fold increases in the frequency of CD107a or IL-2–positive cells were with the use of two Abs targeting either CTLA-4, LAG-3, or TIGIT. Surprisingly, none of the Ab combinations that induced enhanced production of cytokines targeted PD-1. Further mechanistic studies are required to determine the effects of Abs to CTLA-4 with LAG-3 or TIGIT on functional effects of HIV-specific T cells, including proliferation and killing of latently infected cells, as well as potential effects of increased IL-2 on the number of regulatory T cells and expansion of latently infected cells. Given the emerging improved safety profiles of Abs to LAG-3 and TIGIT compared with anti–PD-1 or anti–CTLA-4 (71), these Abs could potentially be attractive for further clinical development for use in HIV cure strategies.

We thank Alan Korman for the intellectual discussion and helpful suggestions throughout the design and analysis phases of the study and Andrew Scott, from Olivia Cancer Research Institute, for providing the tumor-specific IgG. We acknowledge the generous provision of IC Abs from Bristol Myers Squibb. We thank the Infectious Diseases Unit at the Alfred Hospital and the Lewin/Cameron Clinical Research Group at the Doherty Institute for providing the leukapheresis samples.

S.R.L. has received honoraria for participation in advisory boards and/or educational seminars from Gilead Sciences, Merck, ViiV Healthcare, AbbVie, Immunocore, AELIX Therapeutics, and COVAXX. S.R.L. has received investigator-initiated research grant support from Gilead Sciences, Merck, and ViiV Healthcare and has received funds for contract research with Leidos. T.A.R has received speaker's fees from ViiV and Gilead. T.A.R. has received funding from Gilead for research outside the submitted work. The other authors have no financial conflicts of interest.