STING-dependent type I IFN production inhibits cell-mediated immunity to Listeria monocytogenes

Abstract

Infection with Listeria monocytogenes strains that enter the host cell cytosol leads to a robust cytotoxic T cell response resulting in long-lived cell-mediated immunity (CMI). Upon entry into the cytosol, L. monocytogenes secretes cyclic diadenosine monophosphate (c-di-AMP) which activates the innate immune sensor STING leading to the expression of IFN-β and co-regulated genes. In this study, we examined the role of STING in the development of protective CMI to L. monocytogenes. Mice deficient for STING or its downstream effector IRF3 restricted a secondary lethal challenge with L. monocytogenes and exhibited enhanced immunity that was MyD88-independent. Conversely, enhancing STING activation during immunization by co-administration of c-di-AMP or by infection with a L. monocytogenes mutant that secretes elevated levels of c-di-AMP resulted in decreased protective immunity that was largely dependent on the type I interferon receptor. These data suggest that L. monocytogenes activation of STING downregulates CMI by induction of type I interferon.

Figure 1. Mice lacking the STING signaling pathway generate a protective adaptive immune response following L. monocytogenes reinfection.

B6, Gt, IRF3^−/− and IRF3/7^−/− mice were immunized intravenously with 10³ CFU ActA⁻Lm-OVA (ΔactA) (open circles) or administered PBS (closed circles) and 30–38 days later challenged with 2×10⁵ CFU WT Lm-OVA. Three days post challenge, A. bacterial CFU in the spleens and livers were enumerated. The dashed line represents the limit of detection. B. Splenocytes were stained with anti-mouse CD8, CD44 and K^b/OVA_257–264 tetramer and analyzed by flow cytometry. Quantitative analysis shows the total number of K^b/OVA_257–264 tetramer⁺ CD44⁺ CD8⁺ T cells/spleen. C. Splenocytes were either unstimulated (un) or stimulated with OVA_257–264 (OVA) or LLO_190–201 (LLO) peptides followed by intracellular staining with anti-mouse IFN-γ, TNF-α and IL-2. Quantitative analysis shows the percentage of IFN-γ⁺/TNF-α⁺/IL-2⁻ (solid bars) or IFN-γ⁺/TNF-α⁺/IL-2⁺ (lined bars) within the CD8⁺ or CD4⁺ population. Data are presented as the cumulative results from 5–12 (A), 3–6 (B) or 2–4 (C, mean ± SEM) independent experiments using at least five mice per group. Asterisks represent significance as compared to ΔactA-immunized B6 mice (*p<0.05, **p<0.005, ***p<0.0005).

Figure 2. MyD88 and STING contribute to dendritic cell and T cell activation in vivo.

B6 (closed bars), Gt (open bars), MyD88^−/− (lined bars) and MyD88^−/−Gt (dotted bars) mice were intravenously injected with either PBS (shaded histogram) or 10⁵ CFU ActA⁻Lm-OVA (ΔactA) (black line). One day post injection, A. splenocytes were isolated and stained with anti-mouse CD86, CD40, CD11b, CD11c or B. anti-mouse CD8, CD4 and CD69 and analyzed by flow cytometry. Quantitative analysis shows the fold increase of median fluorescence intensity over uninfected mice. C. Serum was measured for IL-6 and TNF-α, MCP-1 and IL-12p70 by Cytometric Bead Array. Data represent the mean ± SEM from 3 independent experiments with 3 mice per group (ns = not significant, *p<0.05, **p<0.005, ***p<0.0005).

Figure 3. Mice lacking MyD88 and STING are protected from secondary challenge.

B6, MyD88^−/− and MyD88^−/−Gt mice were immunized intravenously with 10³ CFU ActA⁻Lm-OVA (ΔactA) (open circles) or administered PBS (closed circles) and 30–38 days following immunization, challenged with 2×10⁵ CFU WT Lm-OVA. Three days post challenge, A. bacterial numbers in the spleens and livers were enumerated. An X marks each mouse that succumbed to infection prior to the conclusion of experiment. The dashed line represents the limit of detection. B. Splenocytes were stained with anti-mouse CD8, CD44 and K^b/OVA_257–264 tetramer and analyzed by flow cytometry. Quantitative analysis shows the total number of K^b/OVA_257–264 tetramer⁺ CD44⁺ CD8⁺ T cells/spleen. Data are presented as the cumulative results from 4–6 (A) or 3 (B) independent experiments (ns = not significant, *p<0.05, **p<0.005, ***p<0.0005).

Figure 4. Administration of c-di-AMP during immunization inhibits CD8⁺ T cell expansion and protective immunity upon L. monocytogenes reinfection.

A and B. B6 or Gt mice were intravenously injected with either 50 µg c-di-AMP (black line) or PBS (shaded histogram) and splenocytes were isolated 24 hours were stained with either A. anti-mouse CD86 and CD40 or B. anti-mouse CD8, CD4 and CD69 and analyzed by flow cytometry. Data are quantified as the fold increase of median fluorescence intensity over uninfected mice and presented as the mean ± SEM from 3 independent experiments using 3 mice per group (ND = not detectable). C. B6 mice were immunized intravenously with either 10³ ActA⁻Lm-OVA (ΔactA) or 10⁴ LLO⁻Lm-OVA (Δhly) in the presence (open triangles) or absence (open circles) of 50 or 100 µg c-di-AMP, or D. B6, Gt or IRF3/7^−/− mice were immunized with either 10³ CFU ΔactA in the presence (open triangles) or absence (open circles) of 100 µg c-di-AMP. Naive controls were administered sterile PBS (closed circles). Mice were challenged 30–38 days post immunization with 2×10⁵ CFU WT Lm-OVA and 3 days later CFU were enumerated in spleens. The dashed line represents limit of detection. E. Splenocytes isolated from D were stained with anti-mouse CD8, CD44 and K^b/OVA_257–264 tetramer and analyzed by flow cytometry. Quantitative analysis shows the total number of K^b/OVA_257–264 tetramer⁺ CD44⁺ CD8⁺ T cells/spleen. Data are presented as the cumulative results from 3 (C and D) or 2–3 (E) independent experiments (ns = not significant, *p<0.05, **p<0.005, ***p<0.0005).

Figure 5. Immunization with a c-di-AMP hyper-secreting strain of L. monocytogenes reduces CD8⁺ T cell expansion and protective immunity following challenge.

B6, Gt or IRF3/7^−/− mice were immunized with either 10³ CFU ΔactA (open circles) or tetRActA⁻Lm-OVA (tetRΔactA) (open triangles). Naive controls were administered sterile PBS (closed circles). Mice were challenged 30–38 days post immunization with 2×10⁵ CFU WT Lm-OVA and 3 days later A. CFU were enumerated in spleens. The dashed line represents limit of detection. B. Splenocytes were stained with anti-mouse CD8, CD44 and K^b/OVA_257–264 tetramer and analyzed by flow cytometry. Quantitative analysis shows the total number of K^b/OVA_257–264 tetramer⁺ CD44⁺ CD8⁺ T cells/spleen. Data are presented as the cumulative results from 3–4 independent experiments (ns = not significant, *p<0.05, **p<0.005, ***p<0.0005).

Figure 6. T cell priming is inhibited in the presence of enhanced STING activation.

B6 mice were immunized with either 10³ CFU ActA⁻Lm-OVA (ΔactA) (open circles), ΔactA in the presence of 50 µg c-di-AMP or tetRActA⁻Lm-OVA (tetRΔactA) (open triangles). Naive controls were administered sterile PBS (closed circles). Splenocytes were isolated 7 days post immunization and stained with anti-mouse CD8, CD44, CD62L and K^b/OVA_257–264 tetramer and analyzed by flow cytometry. A. Quantitative analysis shows the total number of K^b/OVA_257–264 tetramer⁺ CD44⁺ CD8⁺ T cells/spleen. B. Histograms represent mice administered PBS (shaded), ΔactA (solid line) or ΔactA in the presence of c-di-AMP or tetRΔactA (dotted line). Quantitative analysis shows the percentage of CD62L high-expressing cells of the CD8⁺ CD44⁺ K^b/OVA_257–264 tetramer⁺ population. C. Splenocytes were either unstimulated (un) or stimulated with OVA_257–264 (OVA) or LLO_190–201 (LLO) peptides followed by intracellular staining for anti-mouse IFN-γ, TNF-α and IL-2. Quantitative analysis shows the percentage of IFN-γ⁺/TNF-α⁺/IL-2⁻ (solid bars) or IFN-γ⁺/TNF-α⁺/IL-2⁺ (lined bars) within the CD8⁺ or CD4⁺ population. Data are presented as the cumulative results from 2–3 independent experiments (ns = not significant, *p<0.05, **p<0.005, ***p<0.0005).

Figure 7. Mice lacking type I IFNs are partially rescued from c-di-AMP-mediated immune inhibition.

A. B6 or IFNAR1^−/− mice were immunized with 10³ CFU ActA⁻Lm-OVA (ΔactA) in the presence (open triangles) or absence (open circles) of 100 µg c-di-AMP or B. tetRActA⁻Lm-OVA (tetRΔactA) (open triangles). Naïve mice were administered PBS (closed circles). Mice were challenged 30–38 days later with 2×10⁵ CFU WT Lm-OVA and 3 days post challenge, CFU were enumerated from spleens. The dashed line represents limit of detection. Data are presented as the cumulative results from 3 independent experiments (ns = not significant, *p<0.05, **p<0.005, ***p<0.0005).

Figure 8. Type I IFN-mediated immune suppression is T cell extrinsic.

A. B6, IFNAR1^−/− and Gt mice were injected with 1∶1 ratio WT GFP⁺ Ly5.2⁺ and IFNAR1^−/− Ly5.1⁺ OT-I cells 1 day prior to immunization with ActA⁻Lm-OVA (ΔactA) in the presence (open triangles) or absence (open circles) of 50 µg c-di-AMP or administered PBS (closed circles). Splenocytes were isolated 7 days later and stained with anti-mouse CD8 and CD45.1 and analyzed by flow cytometry. Values in representative FACs plots show the median percentage of WT or IFNAR1^−/− OT-I cells ± SEM within the CD8⁺ T cell population. B. Quantitative analysis shows the percentage (left panels) within the CD8⁺ T cell population or the total number of CD8⁺ T cells/spleen (right panels) of WT OT-I T cells (top panels) and IFNAR1^−/− OT-I T cells (bottom panels). Data are presented as the cumulative results from 2 of 3 independent experiments (ns = not significant, *p<0.05, **p<0.005, ***p<0.0005).