The STING ligand cGAMP potentiates the efficacy of vaccine-induced CD8+ T cells

Abstract

Pathogen recognition receptor (PRR) agonists are currently being developed and tested as adjuvants in various formulations to optimize the immunogenicity and efficacy of vaccines. Using an original in vitro approach to prime naive precursors from unfractionated human peripheral blood mononuclear cells, we assessed the influence of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a ligand for the stimulator of interferon genes (STING), on the induction of antigen-specific CD8+ T cells. We found that 2'3'-cGAMP and 3'3'-cGAMP were especially potent adjuvants in this system, driving the expansion and maturation of functionally replete antigen-specific CD8+ T cells via the induction of type I IFNs. The biological relevance of these findings was confirmed in vivo using two mouse models, in which 2'3'-cGAMP-adjuvanted vaccination elicited protective antitumor or antiviral CD8+ T cell responses. These results identify particular isoforms of cGAMP as effective adjuvants that may find utility in the development of novel immunotherapies and vaccines.

Keywords: T cells; Vaccines.

Figure 1. STING ligands enhance the induction of effector CD8⁺ T cells.

(A) Left: Representative flow cytometry data showing EV10-specific CD8⁺ T cells expanded in the presence of Flt3 ligand and various adjuvants. Cytokines: TNF, IL-1β, IL-7, and PGE2. Plots are gated on viable CD3⁺ events. Percentage values represent HLA-A2-EV10 tetramer⁺ cells among total CD8⁺ T cells. Right: Data summary. Each dot represents 1 HLA-A2⁺ donor per condition. The dotted line shows the mean percentage of HLA-A2–EV10 tetramer⁺ cells among total CD8⁺ T cells incubated with cytokines in the absence of peptide (n = 20). (B) Intracellular expression of T-bet among EV10-specific CD8⁺ T cells primed as in A. Left: Representative flow cytometry plots gated on viable HLA-A2–EV10 tetramer⁺ CD8⁺ T cells. The dotted line shows the limit for positive expression. Right: data summary. Each dot represents 1 HLA-A2⁺ donor per condition. (C) Intracellular expression of perforin and granzyme B among EV10-specific CD8⁺ T cells primed as in A. Details as in B. Horizontal bars indicate median values. P values are represented as circles with variable size and color intensity (Mann-Whitney U test).

Figure 2. STING ligands enhance the functionality of effector CD8⁺ T cells.

(A) Intracellular expression of perforin and granzyme B among EV10-specific CD8⁺ T cells primed in the presence of 2′3′-cGAMP or TLR4 and expanded for a further 2 weeks in culture after restimulation with EV10. (B) Intracellular expression of IFN-γ among EV10-specific CD8⁺ T cells generated as in A after exposure to HLA-A2⁺ or HLA-A2^– melanoma cells (effector/target ratio 1:10). (C) Lysis of HLA-A2⁺ or HLA-A2^– melanoma cells by EV10-specific CD8⁺ T cells generated as in A. Error bars indicate mean ± SEM (n = 3 replicates). **P < 0.01, ***P < 0.001 (unpaired t test).

Figure 3. STING ligands drive the maturation of DCs.

(A) Expression profile of PRR pathway genes in CD1a⁺CD14^– moDCs 24 hours after stimulation with 2′3′-cGAMP or 3′3′-cGAMP at concentrations of 10 μg/ml. Data are shown relative to unstimulated moDCs from the same donor. stim, stimulation. (B) Expression of maturation markers on CD1a⁺CD14^– moDCs stimulated with 2′3′-cGAMP or 3′3′-cGAMP at concentrations of 10 μg/ml. Error bars indicate mean ± SEM (n = 3 replicates). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test).

Figure 4. STING ligands elicit a type I IFN response.

(A) Extracellular concentrations of various cytokines secreted by PBMCs in response to overnight stimulation with various PRR ligands. Each dot represents 1 donor per condition. Horizontal bars indicate median values. P values are represented as circles with variable size and color intensity (Mann-Whitney U test). (B) Frequency of EV10-specific CD8⁺ T cells and intracellular expression of T-bet, perforin, and granzyme B among EV10-specific CD8⁺ T cells primed in the presence of IFN-α (0.1 μg/ml), IFN-γ (0.1 μg/ml), or IL-12 (0.1 μg/ml). Each dot represents 1 HLA-A2⁺ donor per condition. Horizontal bars indicate median values. **P < 0.01, ***P < 0.001 (Kruskal-Wallis test). (C and D) Frequency of EV10-specific CD8⁺ T cells and intracellular expression of T-bet and granzyme B among EV10-specific CD8⁺ T cells primed in the presence of 2′3′-cGAMP, either alone or together with type I IFN blocking antibodies (C) or IL-10 (0.1 μg/ml) (D). *P < 0.05 (paired t test).

Figure 5. cGAMP enhances the induction of protective antitumor CD8⁺ T cells in vivo.

C57BL/6J mice were vaccinated intramuscularly with PBS (negative control) or OVA, either alone or together with various adjuvants, on days 0 and 14. (A) Top: Representative flow cytometry data showing SL8-specific CD8⁺ T cells 1 week after the last vaccination. Plots are gated on viable CD3⁺ events. Percentage values refer to H-2K^b-SL8 tetramer⁺ cells among total CD8⁺ T cells. Bottom: Data summary (n = 4 mice per group). (B) Left: Representative flow cytometry data showing IFN-γ production among CD8⁺ T cells restimulated with SL8 1 week after the last vaccination. Plots are gated on viable CD3⁺ events. Percentage values refer to IFN-γ⁺ cells among total CD8⁺ T cells. Right: Data summary. (C) Tumor size after subcutaneous injection of EG7 cells on day 21 (n = 10 mice per group). Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01 (unpaired t test).

Figure 6. cGAMP enhances the induction of protective antiviral CD8⁺ T cells in vivo.

CB6F1 mice were vaccinated intranasally with PBS (negative control) or NP-p24, either alone or together with 2′3′-cGAMP, on days 0, 7, 14, and 21. (A) Frequency of H-2K^d-AI9 tetramer⁺ CD8⁺ T cells 1 week after the last vaccination. (B) Frequency of IFN-γ–producing cells among splenocytes restimulated with AI9 1 week after the last vaccination. Results are expressed as the number of spot-forming units (SFU) per 3 × 10⁵ splenocytes. (C) Weight loss after challenge with VV-Gag. Unchallenged naive mice were included as a negative control. (D) Viral titers 1 week after challenge with VV-Gag. Error bars indicate mean ± SEM ( n = 5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test or Mann-Whitney U test).