STING Is Required in Conventional Dendritic Cells for DNA Vaccine Induction of Type I T Helper Cell- Dependent Antibody Responses

Abstract

DNA vaccines elicit antibody, T helper cell responses and CD8⁺ T cell responses. Currently, little is known about the mechanism that DNA vaccines employ to induce adaptive immune responses. Prior studies have demonstrated that stimulator of interferon genes (STING) and conventional dendritic cells (cDCs) play critical roles in DNA vaccine induced antibody and T cell responses. STING activation by double stranded (dsDNA) sensing proteins initiate the production of type I interferon (IFN),but the DC-intrinsic effect of STING signaling is still unclear. Here, we investigated the role of STING within cDCs on DNA vaccine induction of antibody and T cell responses. STING knockout (STING^-/- ) and conditional knockout mice that lack STING in cDCs (cDC STING cKO), were immunized intramuscularly with a DNA vaccine that expressed influenza A nucleoprotein (pNP). Both STING^-/- and cDC STING cKO mice had significantly lower type I T helper (Th1) type antibody (anti-NP IgG_2C) responses and lower frequencies of Th1 associated T cells (NP-specific IFN-γ⁺CD4⁺ T cells) post-immunization than wild type (WT) and cDC STING littermate control mice. In contrast, all mice had similar Th2-type NP-specific (IgG₁) antibody titers. STING^-/- mice developed significantly lower polyfunctional CD8⁺ T cells than WT, cDC STING cKO and cDC STING littermate control mice. These findings suggest that STING within cDCs mediates DNA vaccine induction of type I T helper responses including IFN-γ⁺CD4⁺ T cells, and Th1-type IgG_2C antibody responses. The induction of CD8⁺ effector cell responses also require STING, but not within cDCs. These findings are the first to show that STING is required within cDCs to mediate DNA vaccine induced Th1 immune responses and provide new insight into the mechanism whereby DNA vaccines induce Th1 responses.

Keywords: DNA vaccine; STING; cGAS; dendritic cells; type I interferon.

Figure 1

STING is required for DNA vaccine induction of antigen-specific Th1 associated IgG_2C antibody responses. Wild type (WT), cGAS^-/- and STING ^-/- mice were IM/EP vaccinated with a DNA vaccine (pNP) expressing influenza nucleoprotein (pNP). Sera were collected prior to vaccination (D0), and at 14 (D14), 21 (D21), and 28 days post-vaccination (D28). The concentration of anti-NP (A) IgG, (B) IgG_2C and (C) IgG₁ antibody responses was measured by ELISA. Each ELISA plate used goat anti-mouse IgG and known concentrations of mouse IgG, IgG_2C, or IgG₁ to create a standard curve to back calculate antibody titers (μg/ml). (D) The Th1:Th2 ratio was calculated as IgG_2C:IgG₁, a higher ratio is indicative of a Th1 response. Three independent experiments consisting of 3-9 mice were performed consisting of 3-9; representative data are the average ±SD 3-9 mice/genotype. At each timepoint a one-way ANOVA was used. ****p < 0.0001 and NS, not significant.

Figure 2

STING is required for DNA vaccine induction of antigen-specific CD8⁺ T cell polyfunctionality. WT, cGAS^-/- and STING^-/- mice were IM/EP vaccinated with pNP. Mice were sacrificed 21-days post-vaccination and splenocytes were isolated and made into single cell suspensions. (A) The frequency of tetramer positive cells within CD8⁺ T cells were measured for each genotype using a MHC-I tetramer that presents the NP immunodominant peptide (NP_366-374) for C57bl/6. CD8⁺ T cells and cells were gated as outlined in Supplementary Figure 3B . (B) Polyfunctional scores were determined by stimulating splenocytes overnight with 1μg/ml of NP_366-375 followed by ICS staining for IL-2, TNF-α, IFN-γ and CD107a/GranzymeB and analyzed by flow cytometry as shown in Supplementary Figure 4 . Polyfunctionality scores were calculated as described in the methods and Larsen et al (17). (C) Pie charts show the relative average proportion of CD8⁺ T cells producing 1-4 immune factors (IL-2, TNF-α, IFN-γ and/or CD107a/GranzymeB) after NP_366-374 stimulation. Three independent experiments consisting of 3-9 mice were performed consisting of 3-9 mice; representative data are the average± SD of 3-9 mice/genotype. Groups were compared using a one-way ANOVA, **p<0.01 and NS, not significant.

Figure 3

STING is not required within conventional dendritic cells to mediate DNA vaccine induction of CD8⁺ T cell responses. WT, STING^-/-, zbtb46^Cre xSTING^fl/fl (cDC STING cKO) and cDC STING littermate control (cDC STING LitC) mice were IM/EP vaccinated with pNP. Mice were sacrificed 21-days post-vacccination and splenocytes collected. (A) Tetramer positive cells within CD8⁺ T cells were measured for each genotype using a NP immunodominant peptide (NP_366-374) containing tetramer as described above and shown in Supplementary Figure 3C . (B) Polyfunctional scores were determined by stimulating splenocytes overnight with 1μg/ml of NP_366-374, staining for IL-2, TNF-α, IFN-γ and CD107a/GranzymeB and analysis via flow cytometry and Boolean gating as shown in Supplementary Figure 4 . Shown are CD8⁺ T cells’ polyfunctionality index scores. (C) Pie charts show the relative average proportion of responding CD8⁺ T cells producing at least one immune function (IL-2, TNF-α, IFN-γ and/or CD107a/GranzymeB) after NP_366-374 stimulation. Three independent experiments were performed consisting of 3-9 mice; representative data are the average ±SSD 3-9 mice/genotype. Groups were compared using a one-way ANOVA, ****p < 0.0001, and NS, not significant.

Figure 4

STING is required for DNA vaccine induction of innate pro-inflammatory cytokines, but not within cDCs. WT, STING^-/-, cDC STING cKO, and cDC STING LitC mice IM/EP vaccinated with pNP. Sera was collected prior to vaccination (baseline), 6 hours (6hrs) and/or 24 hours (24hrs) post-vaccinations, and sera concentrations of (A) TNF-α, (B) IL-6, (C) IFN-β and(D) IL-2 were calculated. Three independent experiments were carried out consisting of 3-6 mice; representative data are the averages± SD of 3-6 mice/genotype. At each timepoint a one-way ANOVA was performed, *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001 and NS, not significant.

Figure 5

STING is required within conventional dendritic cells for DNA vaccine induction of antigen-specific IgG_2C antibody responses. WT, STING^-/-, cDC STING cKO and cDC STING LitC mice IM/EP vaccinated with pNP. Sera were collected prior to vaccination (D0), 14 days (D14), 21 (D21) and 28 days post-vaccination (D28). Anti-NP (A) IgG, (B) IgG_2C and (C) IgG₁ antibody responses was measured by ELISA as described above. (D) The Th1:Th2 ratio was calculated as the ratio of IgG_2C:IgG₁. A Th1:Th2 score >1 indicates a predominantly Th1 responses with a higher score indicating a strong Th1 response. Antibody secreting cells (ASCs) in WT, STING^-/-, cDC STING cKO and cDC STING LitC mice vaccinated with pNP were measure 21 days post-vaccination by B cell ELIspot. Shown are (E) IgG, (F) IgG_2C and (G) IgG₁ ASCs. Three independent experiments were performed consisting of 3-7 mice; representative data shown are the average± SD of 3-7 mice/genotype. A one-way ANOVA was employed to compare groups, *p < 0.05, **p < 0.01 and NS, not significant.

Figure 6

DNA vaccines require STING within cDCs to induce vaccine generated IFN-γ⁺CD4⁺ T cells and IFN-γ production. WT, STING^-/- , cDC STING cKO and cDC STING LitC mice were IM/EP with pNP. Mice were sacrificed 21 days post-vaccination and splenocytes were isolated. (A) The frequency of tetramer positive cells within CD4⁺ T cells were measured for each genotype using a MHC-II tetramer that presents the NP immunodominant peptide (NP_311-325) for C57bl/6. Addtionally, splenocytes were stimulated with NP_311-325 to evaluate the cytokines produced by splenocytes. (B) IFN-γ production by CD4⁺ T cells was measured by stimulating splenocytes with NP_311-325 peptide in vitro and analyzing the supernatants. Production of IL-2, IL-4, IL-6, IL-10, and TNF-α were also measured ( Supplementary Figure 6 ). (C, D) frequencies of IFN-γ⁺CD4⁺ T cells measured by flow cytometry. (C) Gating scheme for detection of IFN-γ⁺CD4⁺ T cells by flow cytometry. (D) Frequencies of IFN-γ⁺CD4⁺ T cells. Three independent experiments were performed consisting of 3-6 mice; representative data shown are the average± SD of 3-6 mice/genotype. A one-way ANOVA was employed to compare groups, *p < 0.05, **p < 0.01 and NS, not significant.