CCR2- and Flt3-Dependent Inflammatory Conventional Type 2 Dendritic Cells Are Necessary for the Induction of Adaptive Immunity by the Human Vaccine Adjuvant System AS01

Abstract

The Adjuvant System AS01 contains monophosphoryl lipid A (MPL) and the saponin QS-21 in a liposomal formulation. AS01 is included in recently developed vaccines against malaria and varicella zoster virus. Like for many other adjuvants, induction of adaptive immunity by AS01 is highly dependent on the ability to recruit and activate dendritic cells (DCs) that migrate to the draining lymph node for T and B cell stimulation. The objective of this study was to more precisely address the contribution of the different conventional (cDC) and monocyte-derived DC (MC) subsets in the orchestration of the adaptive immune response after immunization with AS01 adjuvanted vaccine. The combination of MPL and QS-21 in AS01 induced strong recruitment of CD26⁺XCR1⁺ cDC1s, CD26⁺CD172⁺ cDC2s and a recently defined CCR2-dependent CD64-expressing inflammatory cDC2 (inf-cDC2) subset to the draining lymph node compared to antigen alone, while CD26^-CD64⁺CD88⁺ MCs were barely detectable. At 24 h post-vaccination, cDC2s and inf-cDC2s were superior amongst the different subsets in priming antigen-specific CD4⁺ T cells, while simultaneously presenting antigen to CD8⁺ T cells. Diphtheria toxin (DT) mediated depletion of all DCs prior to vaccination completely abolished adaptive immune responses, while depletion 24 h after vaccination mainly affected CD8⁺ T cell responses. Vaccinated mice lacking Flt3 or the chemokine receptor CCR2 showed a marked deficit in inf-cDC2 recruitment and failed to raise proper antibody and T cell responses. Thus, the adjuvant activity of AS01 is associated with the potent activation of subsets of cDC2s, including the newly described inf-cDC2s.

Keywords: AS01; CD64; Fc receptor; MAR-1; adjuvant; dendritic cell; inf-cDC2; vaccine.

Figure 1

cDC subsets are recruited to the draining lymph node after i.m. AS01 immunization. (A–D) Gating strategy of migratory MHCII^hi DC subsets pre-gated on live CD3^-CD19^- cells (left panel) and pie charts depicting relative distribution of DC subset (right panel) in the dLN 24 h after i.m. (M. gastrocnemius) immunization with 2 µg VZV gE antigen formulated in 1 µg AS01 (A), MPL (B), QS-21 (C), or buffer (D) per injection site. (E) Histograms showing surface expression of cDC, monocyte and macrophage markers on different DC subsets in the dLN 24 h after i.m. immunization with gE/AS01 as in Figure 1A. (F) Absolute number of DC subsets in the dLN 24 h after i.m. immunization with the different compounds as in Figures 1A–D. (G) Absolute number of total cells, cDCs, monocytes and neutrophils in the dLN 24 h after i.m. immunization with OVA/gE (0.5/2.5 µg) formulated in 2.5 µg AS01 or buffer per injection site. AS01, MPL, and QS-21 are all in liposome. Data are representative of at least 2 independent experiments (n = 4–6 mice per group). Size of the pie chart is proportional to the absolute number of DCs in the dLN (A–D). Error bars indicate mean ± SEM (G). Data analyzed with a Mann-Whitney test **p < 0.01.

Figure 2

AS01 activated cDCs effectively prime antigen-specific T cells. (A, B) Expression of CD40 (A) and CD86 (B) shown as MFIs by cDC subsets in the dLN 24 h after i.m. (M. gastrocnemius) immunization with OVA/gE antigen (0.5/2.5 µg) formulated in 2.5 µg AS01, MPL, QS-21, or buffer per injection site. AS01, MPL, and QS-21 are all in liposome. Data analyzed with Two-way ANOVA and Tukey’s multiple comparisons test. Data are representative of at least two independent experiments (n = 5 mice per group). **p < 0.01, ns, non-significant. (C) Expansion index of CD8⁺ OVA-specific TCR transgenic T cells (OTI) cocultured for 3 days with the different migratory cDC subsets sorted from pooled dLNs (n = 80 mice) 24 h after i.m. immunization with OVA/AS01 (5/1 µg) per injection site. (D) Proliferation prolife of CTV-labeled OTI T cells cocultured in 1:4 DC:T cell ratio with the different migratory cDC subsets for 3 days sorted from pooled dLNs (n = 80 mice) 24 h after i.m. immunization with OVA/AS01 (5/1 µg) per injection site. (E) Expansion index of CD4⁺ OVA-specific TCR transgenic T cells (OTII) cocultured for 4 days with the different migratory cDC subsets sorted from pooled dLNs (n = 80 mice) 24 h after i.m. immunization with OVA/AS01. (5/1 µg) per injection site (F) Proliferation prolife of CTV-labeled OTII T cells cocultured in 1:1 DC:T cell ratio with the different migratory cDC subsets for 4 days sorted from pooled dLNs (n = 80 mice) 24 h after i.m. immunization with OVA/AS01 (5/1 µg) per injection site.

Figure 3

Cellular and humoral adaptive immune responses depend on CD11c⁺ cells. (A) Schematic representation of Itgax-DTR BM chimeras. (B–D) Absolute number of distinct cDC subsets (B), monocytes (C), and neutrophils (D) in the dLN 24 h after i.m. immunization with OVA/gE/AS01 (0.5/2.5/2.5 µg) per injection site with or without prior DT-mediated depletion of CD11c⁺ cells. (E) Schematic representation of Itgax-DTR BM chimeras treated 12 h prior to or 24 h after OVA/gE/AS01 (0.5/2.5/2.5 µg) per injection site for both sensitization and challenge. (F, G) Percentage OVA_257–264 MHCI-Tetramer⁺ CD8⁺ T cells in the blood (F) and spleen (G) of Itgax-DTR BM chimeric mice 21 days after i.m. immunization at d0 and challenge at d14 with OVA/gE/AS01 (0.5/2.5/2.5 µg) per injection site and DT treatment. (H) Percentage IFN-γ and/or IL2 producing CD8⁺ T cells upon OVA restimulation in the spleen of Itgax-DTR BM chimeric mice 21 days after i.m. immunization at d0 and challenge at d14 with OVA/gE/AS01 (0.5/2.5/2.5 µg) per injection site and DT treatment. (I) Percentage IFN-γ and/or IL2 producing CD4⁺ T cells upon VZV gE restimulation in the spleen of Itgax-DTR BM chimeric mice 21 days after i.m. immunization at d0 and challenge at d14 with OVA/gE/AS01 (0.5/2.5/2.5 µg) per injection site and DT treatment. (J) Total VZV gE specific IgG levels in the serum of Itgax-DTR BM chimeric mice 21 days after i.m. immunization at d0 and challenge at d14 with OVA/gE/AS01 (0.5/2.5/2.5 µg) per injection site and DT treatment. Data are representative of 4 independent experiments (n = 2–5 mice per group). Error bars indicate mean ± SEM (B–J). Data analyzed with a One-way ANOVA and Sidak’s multiple comparison test *p < 0.05, **p < 0.01, ns, non-significant.

Figure 4

Both Flt3 and CCR2 dependent cells are required for optimal adaptive response driven by AS01. (A) Schematic representation of CD45.1 WT: CD45.2 Ccr2^-/- BM chimeras. (B) Normalized CD45.1/CD45.2 ratio relative to B cells of cell subsets in the dLN 24 h after i.m. immunization with OVA/gE/AS01 (0.5/2.5/2.5 µg) per injection site. (C) Absolute number of total cDCs, monocytes and neutrophils in the dLN of WT, Flt3^-/-, and Ccr2^-/- mice 24 h after i.m. immunization with OVA/gE (0.5/2.5 µg) formulated in 2.5 µg AS01 or buffer per injection site. (D) Absolute number of different cDC subsets in the dLN of WT, Flt3^-/-, and Ccr2^-/- mice 24 h after i.m. immunization with OVA/gE (0.5/2.5 µg) formulated in 2.5 µg AS01 or buffer per injection site. (E) Percentage OVA_257–264 MHCI-Tetramer⁺ CD8⁺ T cells in the spleen of WT, Flt3^-/-, and Ccr2^-/- mice 21 days after i.m. immunization with OVA/gE (0.5/2.5 µg) formulated in 2.5 µg AS01 or buffer per injection site. Same set-up as in Figure 3E without DT treatment. (F) Percentage IFN-γ and/or IL2 producing CD8⁺ T cells upon OVA restimulation in the spleen of WT, Flt3^-/-, and Ccr2^-/- mice 21 days after i.m. immunization with OVA/gE (0.5/2.5 µg) formulated in 2.5 µg AS01 or buffer per injection site. Same set-up as in Figure 3E without DT treatment. (G) Percentage IFN-γ and/or IL2 producing CD4⁺ T cells upon VZV gE restimulation in the spleen of WT, Flt3^-/-, and Ccr2^-/- mice 21 days after i.m. immunization with OVA/gE (0.5/2.5 µg) formulated in 2.5 µg AS01 or buffer per injection site. Same set-up as in Figure 3E without DT treatment. (H) Total VZV gE specific IgG levels in the serum of WT, Flt3^-/-, and Ccr2^-/- mice 21 days after i.m. immunization with OVA/gE (0.5/2.5 µg) formulated in 2.5 µg AS01 or buffer per injection site. Same set-up as in Figure 3E without DT treatment. (I–M) Cytokine and chemokine levels in serum of WT, Flt3^-/-, and Ccr2^-/- mice 24 h after i.m. immunization with OVA/gE (0.5/2.5 µg) formulated in 2.5 µg AS01 or buffer per injection site. Shown individually for CXCL9 (J), CXCL10 (K), CCL2 (L), and IFN-γ (M). (A–M) Data are representative of 4 independent experiments (n = 2–5 mice per group). Error bars indicate mean ± SEM. Data analyzed with a Two-way ANOVA (B–D) or One-way ANOVA (E–M) and Sidak’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ns, non-significant.