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

Abstract

In people with HIV (PWH) on antiretroviral therapy (ART), immune dysfunction persists, including elevated expression of immune checkpoint (IC) proteins on total and HIV-specific T cells. Reversing immune exhaustion is one strategy to enhance the elimination of HIV-infected cells that persist in PWH on ART. We aimed to evaluate whether blocking 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) alone or in combination would enhance HIV-specific CD4⁺ and CD8⁺ T cell function ex vivo. Intracellular cytokine staining was performed using human PBMCs from PWH on ART (n = 11) and expression of CD107a, IFN-γ, TNF-α, and IL-2 was quantified with HIV peptides and Abs to IC. We found the following: 1) IC blockade enhanced the induction of CD107a and IL-2 but not IFN-γ and TNF-α in response to Gag and Nef peptides; 2) the induction of CD107a and IL-2 was greatest with multiple combinations of two Abs; and 3) Abs to LAG-3, CTLA-4, and TIGIT in combinations showed synergistic induction of IL-2 in HIV-specific CD8⁺ and CD107a and IL-2 production in HIV-specific CD4⁺ and CD8⁺ T cells. These results demonstrate that the combination of Abs to LAG-3, CTLA-4, or TIGIT can increase the frequency of cells expressing CD107a and IL-2 that associated with cytotoxicity and survival of HIV-specific CD4⁺ and CD8⁺ T cells in PWH on ART. These combinations should be further explored for an HIV cure.

Figure 1:. Fold change in the frequency of cytokine+ T cells in response to HIV peptides in the presence of antibodies to immune checkpoints (ICs) relative to isotype control.

Total CD4⁺ and CD8⁺ T-cells collected from PWH on ART were incubated with antibodies to ICs either alone (red), as dual combinations (blue) or a cocktail of six antibodies (green) following incubation with overlapping peptides to either (A) gag or (B) nef and the frequency of cells expressing CD107a, IFNγ, TNFα and IL-2 quantified by flow cytometry. The fold change increase in the presence of IC antibodies relative to isotype control is shown. Data are summarised with box plots indicating the median and inter-quartile range for the 9 participants. Asterisks indicate the significant differences between the specific antibody combination and the respective IgG isotype control(s). Statistical significance was determined by Wilcoxon Signed-Rank tests. * p < 0.05, ** p < 0.01, *** p < 0.005

Figure 2:. The fold change in the frequency of cytokine+ T cell subsets in response to HIV peptides in the presence of antibodies to immune checkpoints (ICs) relative to isotype control.

CD4⁺ and CD8⁺ T-cell subsets collected from PWH on ART were incubated with antibodies to ICs either alone (red), as dual combinations (blue) or a cocktail of six antibodies (green) following incubation with overlapping peptides to either gag or nef and the frequency of cells expressing CD107a, IFNγ, TNFα and IL-2 quantified. The fold change relative to isotype control for some combinations of IC antibodies is shown for (A) Gag-stimulated CD4⁺, (B) Gag-stimulated CD8⁺, (C) Nef-stimulated CD4⁺ and (D) Nef-stimulated CD8⁺ T-cell subsets. Only IC antibodies alone or in combination, that have a statistically significant effect on the fold change production of cytokine relative to isotype control are shown. Data is summarised with box plots indicating the median and inter-quartile range for the 9 participants. Asterisks indicate the significant differences between the specific antibody combination and the respective IgG isotype control (s). Statistical significance was determined by Wilcoxon Signed-Rank tests. * p < 0.05, ** p < 0.01, *** p < 0.005

Figure 3:. Synergistic effects of antibodies to immune checkpoints. Intracellular cytokine staining for the frequency of T cells that produced CD107a, IFNγ, TNFα and IL-2 in response to Gag and Nef peptides in the presence of antibodies to immune checkpoints (ICs) either alone or in combination was compared to staphylococcal enterotoxin B (SEB).

The Bliss independence model was used to calculate the difference between the predicted (response to each antibody alone) and observed fractional responses of combinations of antibodies to ICs relative to SEB. A calculated number of predicted – observed response > 0 demonstrated synergism for (A) the expression of CD107a or (B) the production of IL-2 in CD4⁺ (upper) and CD8⁺ (lower) T cells for the various combinations shown. Data are summarised with box plots indicating the median and inter-quartile range for the 9 participants. Wilcoxon Signed-Rank tests were used to determine the statistical differences between the predicted and the experimentally observed effect for a given combination. * p < 0.05, ** p < 0.005

Figure 4:. The magnitude and synergism of antibodies to immune checkpoints (IC) used in combination to enhance the frequency of HIV-specific T-cells producing either CD107a or IL-2.

Heat maps showing the magnitude of the fold change of number of T cells expressing (A) CD107a and (B) IL-2 following stimulation with either Gag (left panels) or Nef (right panels) peptides in CD4⁺ (upper) and CD8⁺ (lower) T cells following stimulation with one or two IC antibodies relative to isotype controls. Numbers indicate the magnitude of the fold change with one antibody (top row) or two antibodies to ICs compared to IgG isotype control. The bold numbers indicate the fold changes that were significantly higher than isotype control. The asterisks represent the statistical significance for the Bliss independence tests for the specific antibody combinations. * p < 0.05, ** p < 0.005

Figure 5:. Heat maps showing the correlations between clinical parameters and the fold change increase in the frequency of cytokine positive cells in response to Gag and Nef peptides in the presence of antibodies to immune checkpoints.

Heatmap illustrating the strength and significance of correlation coefficients between the immune checkpoint blockade response and (A) CD8 count and (B) CD4:CD8 ratio. Statistical significance was determined by Spearman’s rank correlation. Positive and negative correlations are indicated as blue and red, respectively. * p < 0.05, ** p < 0.01