Primary role of type I interferons for the induction of functionally optimal antigen-specific CD8+ T cells in HIV infection

Abstract

Background: CD8⁺ T cells equipped with a full arsenal of antiviral effector functions are critical for effective immune control of HIV-1. It has nonetheless remained unclear how best to elicit such potent cellular immune responses in the context of immunotherapy or vaccination. HIV-2 has been associated with milder disease manifestations and more commonly elicits functionally replete virus-specific CD8⁺ T cell responses compared with HIV-1. We aimed to learn from this immunological dichotomy and to develop informed strategies that could enhance the induction of robust CD8⁺ T cell responses against HIV-1.

Methods: We developed an unbiased in vitro system to compare the de novo induction of antigen-specific CD8⁺ T cell responses after exposure to HIV-1 or HIV-2. The functional properties of primed CD8⁺ T cells were assessed using flow cytometry and molecular analyses of gene transcription.

Findings: HIV-2 primed functionally optimal antigen-specific CD8⁺ T cells with enhanced survival properties more effectively than HIV-1. This superior induction process was dependent on type I interferons (IFNs) and could be mimicked via the adjuvant delivery of cyclic GMP-AMP (cGAMP), a known agonist of the stimulator of interferon genes (STING). CD8⁺ T cells elicited in the presence of cGAMP were polyfunctional and highly sensitive to antigen stimulation, even after priming from people living with HIV-1.

Interpretation: HIV-2 primes CD8⁺ T cells with potent antiviral functionality by activating the cyclic GMP-AMP synthase (cGAS)/STING pathway, which results in the production of type I IFNs. This process may be amenable to therapeutic development via the use of cGAMP or other STING agonists to bolster CD8⁺ T cell-mediated immunity against HIV-1.

Funding: This work was funded by INSERM, the Institut Curie, and the University of Bordeaux (Senior IdEx Chair) and by grants from Sidaction (17-1-AAE-11097, 17-1-FJC-11199, VIH2016126002, 20-2-AEQ-12822-2, and 22-2-AEQ-13411), the Agence Nationale de la Recherche sur le SIDA (ECTZ36691, ECTZ25472, ECTZ71745, and ECTZ118797), and the Fondation pour la Recherche Médicale (EQ U202103012774). D.A.P. was supported by a Wellcome Trust Senior Investigator Award (100326/Z/12/Z).

Keywords: CD8(+) T cells; HIV-1; HIV-2; STING; Type I IFN.

Fig. 1

Virus production and experimental validation. (A) Graphical representation of Δenv HIV-1 and Δenv HIV-2 expressing ELA. (B) SAMHD1 expression in MDDCs 24 h after infection with the indicated pNLAD8 (HIV-1, n = 3) or pJK7312As (HIV-2, n = 4) viruses. NI, no infection. Left: representative flow cytometry plots. Right: data summary. Each dot represents one donor. Bars indicate mean values. ∗∗∗∗p < 0.0001 (unpaired repeated measures ANOVA). (C) Expression of CD86 and Siglec 1 on MDDCs and production of IFN-λ1 by MDDCs 48 h after infection with the indicated pNLAD8 (HIV-1, n = 4) or pJK7312As (HIV-2, n = 4) viruses. SIVmac VLPs were added to provide Vpx. NI, no infection. Each dot represents one donor. ∗p < 0.05, ∗∗p < 0.01, ns = not significant (paired repeated measures ANOVA). (D) Schematic representation of the in vitro priming approach using ELA20 Δenv HIV-1 and ELA20 Δenv HIV-2. (E) Left: representative flow cytometry plots showing ELA-specific CD8⁺ T cells expanded in the presence of 10 ng of ELA20 Δenv HIV-1 or ELA20 Δenv HIV-2. Right: HLA-A∗02:01/ELA tetramer⁺ CD8⁺ T cell frequencies after priming with 1 or 10 ng of ELA20 Δenv HIV-1 or ELA20 Δenv HIV-2. Each connected dot represents one donor. ∗p < 0.05 (Wilcoxon signed rank test).

Fig. 2

Qualitative and quantitative analyses of CD8⁺T cells primed by HIV-1 or HIV-2. (A) Frequencies of ELA-specific CD8⁺ T cells after priming with 10 ng of ELA20 Δenv HIV-1 or ELA20 Δenv HIV-2. Each connected dot represents one donor. ∗∗p < 0.01 (Wilcoxon signed rank test). (B) PCA map showing the spatial distribution of ELA-specific CD8⁺ T cells primed with ELA20 Δenv HIV-1 (red) or ELA20 Δenv HIV-2 (blue), grouped according to gene expression. Each symbol represents one donor. (C) Heatmap showing genes that were differentially expressed between ELA-specific CD8⁺ T cells primed with ELA20 Δenv HIV-1 (n = 5) or ELA20 Δenv HIV-2 (n = 5). (D) ELA-specific CD8⁺ T cell frequencies and intracellular expression of granzyme B, perforin, and T-bet among tetramer⁺ CD8⁺ T cells after priming with ELA20 Δenv HIV-1 or ELA20 Δenv HIV-2. Each connected dot represents one donor. ∗p < 0.05 (Wilcoxon signed rank test). Representative flow cytometry plots are shown in the top left panel. Numbers indicate percentages in the drawn gates. (E) Viability of ELA-specific CD8⁺ T cells after priming with ELA20 Δenv HIV-1 or ELA20 Δenv HIV-2 (n = 5). Top: representative flow cytometry histograms. Numbers indicate percentages in the drawn gates. Bottom: data summary. Data are shown as median ± IQR. ∗p < 0.05 (Wilcoxon signed rank test).

Fig. 3

CD8⁺T cell priming efficacy as a function of type I IFNs. (A–C) Concentrations of various chemokines/cytokines in culture supernatants 3 days after exposure of PBMCs to ELA20 Δenv HIV-1 or ELA20 Δenv HIV-2. Each dot represents one donor. Bars indicate median values. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns = not significant (Mann–Whitney U test). (D) ELA-specific CD8⁺ T cell frequencies after priming with ELA20 in the presence of proinflammatory cytokines (IL-1β, TNF, and PGE2) or IFN-α. Each connected dot represents one donor. Bars indicate median values. ns = not significant (Wilcoxon signed rank test). (E) Intracellular expression of granzyme B, perforin, and T-bet among tetramer⁺ CD8⁺ T cells after priming with ELA20 in the presence of proinflammatory cytokines (IL-1β, TNF, and PGE2) or IFN-α. Each connected dot represents one donor. Bars indicate median values. ∗p < 0.05, ∗∗∗p < 0.001 (Wilcoxon signed rank test).

Fig. 4

Comparative transcriptomic analyses of CD8⁺T cells primed under different conditions. (A) Heatmap showing genes that were differentially expressed among ELA-specific CD8⁺ T cells primed in the presence of proinflammatory cytokines (IL-1β, TNF, and PGE2, n = 7), IFN-α (n = 7), or cGAMP (n = 7). (B) PCA map showing the spatial distribution of ELA-specific CD8⁺ T cells primed with ELA20 in the presence of proinflammatory cytokines (IL-1β, TNF, and PGE2, grey), IFN-α (orange), or cGAMP (green), grouped according to gene expression. Each symbol represents one donor. (C) PCA map showing the spatial distribution of ELA-specific CD8⁺ T cells primed with ELA20 Δenv HIV-1 (red), ELA20 Δenv HIV-2 (blue), or ELA20 in the presence of cGAMP (green), grouped according to gene expression. Each symbol represents one donor. (D) ELA-specific CD8⁺ T cell frequencies and intracellular expression of granzyme B and perforin after priming with ELA20 Δenv HIV-1, ELA20 Δenv HIV-2, or ELA20 in the presence of cGAMP ± anti-IFN-α and anti-IFN-β. Each connected dot represents one donor. ∗p < 0.05, ∗∗p < 0.01 (Wilcoxon signed rank test).

Fig. 5

Qualitative and quantitative analyses of antigen-specific CD8⁺T cells primed from people living with HIV-1. (A, B) Radar plots showing the mean extracellular concentrations (pg/mL) of various chemokines/cytokines in culture supernatants 3 days after stimulation of PBMCs from HUDs (n = 9) (A) or PLWH on ART (n = 10) (B) with proinflammatory cytokines (IL-1β, TNF, and PGE2) or cGAMP. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (Mann–Whitney U test). (C) ELA-specific CD8⁺ T cell frequencies primed from HUDs or PLWH on ART with ELA20 in the presence of proinflammatory cytokines (IL-1β, TNF, and PGE2) or cGAMP. Each connected dot represents one donor. ∗∗p < 0.01 (Wilcoxon signed rank test). (D) PCA map showing the spatial distribution of ELA-specific CD8⁺ T cells primed from HUDs or PLWH on ART with ELA20 in the presence of proinflammatory cytokines (IL-1β, TNF, and PGE2) or cGAMP, grouped according to gene expression. Each symbol represents one donor. (E) Intracellular expression of granzyme B, perforin, and T-bet among tetramer⁺ CD8⁺ T cells primed from HUDs or PLWH with ELA20 in the presence of proinflammatory cytokines (IL-1β, TNF, and PGE2) or cGAMP. Each connected dot represents one donor. ∗p < 0.05, ∗∗p < 0.01 (Wilcoxon signed rank test).

Fig. 6

Antigen sensitivity and functionality of CD8⁺T cells primed from people living with HIV-1. (A) Representative flow cytometry plots showing the mobilization of CD107a and the production of IFNγ, MIP-1β, TNF, and IL-2 in response to antigen stimulation. ELA-specific CD8⁺ T cells primed with ELA20 in the presence of proinflammatory cytokines (IL-1β, TNF, and PGE2) or cGAMP were incubated with medium alone (top) or HLA-A2⁺ B-LCL target cells pulsed with ELA at concentration of 10⁻⁷ M (middle and bottom). Function plots were gated on viable tetramer⁺ CD3⁺ CD8⁺ lymphocytes (center and right). Numbers indicate percentages in the drawn gates. (B) Combinatorial analysis of effector functions among ELA-specific CD8⁺ T cells primed from HUDs (n = 2) or PLWH (n = 2). Data are shown as mean ± SEM. Details as in (A). (C) Normalized IFN-γ and MIP-1β production curves for ELA-specific CD8⁺ T cells primed from HUDs or PLWH as in (A). HLA-A2⁺ B-LCL target cells were pulsed with the indicated concentrations of ELA. Data are representative of two independent experiments. (D) Functional profiles of ELA-specific CD8⁺ T cells primed from HUDs or PLWH as in (A). HLA-A2⁺ B-LCL target cells were pulsed with the indicated concentrations of ELA. Pie chart segments and colors represent the proportions of ELA-specific CD8⁺ T cells expressing the indicated number of functions, respectively. Data are representative of two independent experiments.