Immune Checkpoint Blockade Restores HIV-Specific CD4 T Cell Help for NK Cells

Abstract

Immune exhaustion is an important feature of chronic infections, such as HIV, and a barrier to effective immunity against cancer. This dysfunction is in part controlled by inhibitory immune checkpoints. Blockade of the PD-1 or IL-10 pathways can reinvigorate HIV-specific CD4 T cell function in vitro, as measured by cytokine secretion and proliferative responses upon Ag stimulation. However, whether this restoration of HIV-specific CD4 T cells can improve help to other cell subsets impaired in HIV infection remains to be determined. In this study, we examine a cohort of chronically infected subjects prior to initiation of antiretroviral therapy (ART) and individuals with suppressed viral load on ART. We show that IFN-γ induction in NK cells upon PBMC stimulation by HIV Ag varies inversely with viremia and depends on HIV-specific CD4 T cell help. We demonstrate in both untreated and ART-suppressed individuals that dual PD-1 and IL-10 blockade enhances cytokine secretion of NK cells via restored HIV-specific CD4 T cell function, that soluble factors contribute to these immunotherapeutic effects, and that they depend on IL-2 and IL-12 signaling. Importantly, we show that inhibition of the PD-1 and IL-10 pathways also increases NK degranulation and killing of target cells. This study demonstrates a previously underappreciated relationship between CD4 T cell impairment and NK cell exhaustion in HIV infection, provides a proof of principle that reversal of adaptive immunity exhaustion can improve the innate immune response, and suggests that immune checkpoint modulation that improves CD4/NK cell cooperation can be used as adjuvant therapy in HIV infection.

Figure 1.. Stimulation of PBMCs by HIV Gag induces a CD4 T cell-dependent production of cytokines by NK cells.

PBMCs were stimulated in vitro with an HIV Gag peptide pool or left unstimulated (negative control) and assessed by a delayed ICS assay, in which cells were incubated with Ag for 12–36 before addition of brefeldin and monensin for 12h and collection after 24–48h. The flow cytometry plots show IFNγ production by CD4 T cells (CD3+CD4+), B cells (CD19+), NK cells (CD56+) and monocytes (CD14+) after doublet exclusion on FSC-A/FSC-H and dead cell exclusion. (A) Example plots for one representative donor showing IFN-γ production by CD4 T cells (CD3+CD4+), B cells (CD3-CD19+), NK cells (CD3-CD56+) and CD14+ monocytes, after doublet exclusion on FSC-A/FSC-H and dead cell exclusion after 24 or 48h of stimulation. (B) ICS frequencies of IFN-γ-producing NK cells 48h after Gag stimulation of PBMCs (n=21 untreated subjects) correlated (C) with HIV viral loads; and (D) with HIV Gag-specific CD4 T cell responses. (E) Representative example and (F) summary data on 7 subjects of the impact of CD3+ or CD8+ cell depletion on IFN-γ production by NK cells. Statistical tests: Wilcoxon paired test (BF) and Spearman correlation (CD).

Figure 2.. CD4 T cell-dependent enhancement of NK cell function after combined blockade of the PD-1 and IL-10 pathways.

(ABC) PBMCs from untreated donors were stimulated for 48h with an HIV Gag peptide pool or left unstimulated and subsequently stained for (A) PD-L1 and (B) IL-10. (C) shows representative dot plots illustrating PD-L1 and IL-10 co-expression. (D-K) PBMCs were stimulated in vitro with an HIV Gag peptide pool or left unstimulated, this in the presence of blocking antibodies against PD-L1 and IL-10Rα or corresponding isotype controls. A delayed ICS assays performed after 48h. (D) Net IFNγ induction after Gag stimulation combined with single or dual immune checkpoint blockade are shown. (EF) Example plots for one representative donor showing IFN-γ production by NK (E) cells and CD4 (F) T cells after doublet exclusion on FSC-A/FSC-H and dead cell exclusion in the presence of combined blockade with anti-PD-L1 and anti-IL-10Rα or isotype controls. (GHI) Summary data for the frequencies of (G) IFN-γ-producing; (H) IFN-γ/CD107a co-expressing and (I) TNF-α-producing NK cells observed in the presence of combined blockade with anti-PD-L1 and anti-IL-10Rα compared with isotype controls. (J) Summary data for the frequencies of IFN-γ-producing CD4 T cells observed in the presence of combined blockade with anti-PD-L1 and anti-IL-10Rα compared with isotype controls. (K) Correlation between the HIV Gag-specific IFN-γ+ CD4 T cell response to dual PD-1/IL-10 blockade and the increase in the fraction of IFN-γ+ NK cells with this intervention. Number of subjects: (D) n=15; (GJK) n=21; (HI) n = 14. Statistical tests: Non-parametric Friedman test for multiple paired comparisons with Dunn’s post tests (p˂0.01) (A,D). Wilcoxon paired test (G-J) and Spearman correlation (K).

Figure 3.. NK cells from ART-suppressed individuals respond to combined blockade of the PD-1 and IL-10 pathways

(A-E) PBMCs from ART donors were stimulated in vitro with an HIV Gag peptide pool or left unstimulated, this in the presence of blocking antibodies against PD-L1 and IL-10Rα or corresponding isotype controls. A delayed ICS assays performed after 48h. (A-D) Summary data of the frequencies of (A) IFN-γ-producing NK cells, (B) IFN-γ/CD107a co-expressing NK cells and (C) TNF-α-producing NK cells and (D) IFN-γ-CD4 T cells. (E) Fold increase in net cytokine expression upon PD-L1/IL-10 dual blockade in ART donors compared to untreated individuals presented in Figure 2. Number of ART subjects: (A-E) n=6. Wilcoxon paired test (a A-D). Non-parametric Mann-Whitney U test (p˂0.05) (E).

Figure 4.. NK cell activation after HIV antigen stimulation is mainly dependent on soluble IL-2 and IL-12 secretion.

(A-D) CD8-depleted PBMCs were stimulated for 48h with an HIV Gag peptide pool or left unstimulated, this in the presence of combinations of blocking antibodies against PD-L1, IL10Rα, IL-2 and/or IL-12. The last 12 hours were performed in the presence of Brefeldin A and monensin and anti-CD107a antibody before staining and flow cytometric acquisition. (A) Representative example of IFN-γ and CD107a expression by NK cells in the different Gag stimulated conditions. (BCD) Summary plots of the impact of IL-12 or IL-2 blockade on NK cell functions observed in the presence of combined blockade of the PD-1 and IL-10 pathways (n=14): (B) IFN-γ expression; (C) Degranulation as measured by CD107a and IFN-γ co-expression; baseline expression of CD107a precludes reliable analysis of the single positive CD107a+ cells; (D) TNF-α expression. Statistical tests: Non-parametric Friedman test for multiple paired comparisons with Dunn’s post tests. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. (E) Schematic representation of the transwell experiment. (FG) CD8-depleted PBMCs and purified NK cells were placed in the bottom and top compartment, respectively. PBMCs were then stimulated for 48h with an HIV Gag peptide pool or left unstimulated, this in the presence of a combination of blocking antibodies against PD-L1 and IL10Rα. BFA was added 12h before harvesting the cells for staining. (F) Summary plots of the impact of blockade on IFNγ+ CD4 and NK cells are shown. (G) Net frequencies upon blockade for matched top and bottom NK cell populations are also shown to illustrate the effect of the transwell on crosstalk.

Figure 5.. Combined blockade of the PD-1 and IL-10 pathways restores NK cell killing.

CD8-depleted PBMCS were incubated overnight with an HIV Gag peptide pool, SEB or left unstimulated in the presence of isotype control antibodies or blocking antibodies against PD-L1 or IL-10Rα. On the next day, the PBMCs were mixed with target K562 cells, with effector and target cells identified by cell tracker dyes. Killing was measured by a live/dead dye. (AB) Representative examples of (A) the gating strategy used and (B) results obtained in the killing assay. (C) Summary of the pooled data of the specific killing of K562 cells observed in the Unstimulated, Gag-stimulated and SEB-stimulated conditions. (D) Increase in specific killing elicited by combined PD-1/IL-10 blockade in the Gag-stimulated conditions. Statistical tests: (C) Non-parametric Friedman test for multiple paired comparisons with Dunn’s post tests; (D) Wilcoxon matched-pairs test. * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.