Lysosome-Dependent Activation of Human Dendritic Cells by the Vaccine Adjuvant QS-21

Abstract

The adjuvant properties of the saponin QS-21 have been known for decades. It is a component of the Adjuvant System AS01 that is used in several vaccine candidates. QS-21 strongly potentiates both cellular and humoral immune responses to purified antigens, yet how it activates immune cells is largely unknown. Here, we report that QS-21 directly activated human monocyte-derived dendritic cells (moDCs) and promoted a pro-inflammatory transcriptional program. Cholesterol-dependent QS-21 endocytosis followed by lysosomal destabilization and Syk kinase activation were prerequisites for this response. Cathepsin B, a lysosomal cysteine protease, was essential for moDC activation in vitro and contributed to the adjuvant effects of QS-21 in vivo. Collectively, these findings provide new insights into the pathways involved in the direct activation of antigen-presenting cells by a clinically relevant QS-21 formulation.

Keywords: adjuvant; cathepsins; cytokine; dendritic cells; lysosome; saponins; vaccine.

Figure 1

QS-21 directly activates human dendritic cells. (A) moDCs were stimulated with increasing concentrations of QS-21, and IL-6, TNF, and IL-8 were quantified in the culture supernatant after 24 h. Statistical significance was determined by a Friedman test followed by Dunn’s multiple comparisons. The means and SEM from four donors are shown. (B) moDCs were stimulated with QS-21 (10 µg/ml) or MPL (1 µg/ml) for 24 h, and surface marker expression was evaluated by flow cytometry. Representative histograms of HLA-DR and CD86 MFI and bar graphs representing the mean and SEM of cumulative data from eight donors are shown. (C) moDCs were stimulated with QS-21 (10 µg/ml) or liposomes for 24 h and CD86 and HLA-DR expression were evaluated by flow cytometry (D–F) moDCs from four donors were stimulated with QS-21 or MPL for 4 h and gene expression was analyzed by microarray. (D) Volcano plot of microarray data. Each point represents one gene plotted by log2 fold change (FC) vs. medium against −log10 of the p-value (average of four donors). The horizontal bar represents a p-value of 0.05. The light gray points represent genes with FC < 2 and FC > 0.5 and black points represent FC > 2 or FC < 0.5. (E) Scatter plot analysis of microarray data. Log transformed FC vs. medium of QS-21 is plotted against FC vs. medium of MPL (average of four donors). The dotted lines represent FC = 1, the solid black line represents FC_QS-21 = FC_MPL and the red lines represent a FC_QS-21/FC_MPL ratio of +2 or −2. (F) Hierarchical clustering and heatmap representation of differentially expressed genes (FC vs. medium >2 or <0.5) for the four donors. The color-coded scale representing fold change vs. medium (blue = downregulated vs. medium, red = upregulated vs. medium) is indicated at the top of the chart. (G) InnateDB analysis of pathway over-representation of significantly upregulated genes (p-value < 0.05 and fold change > 2) for cells stimulated with QS-21 (left) or MPL (right) using the hypergeometric algorithm and Benjamini–Hochberg correction for p-values. (H) moDCs were stimulated with QS-21 (10 µg/ml) or MPL (1 µg/ml) for the indicated durations, and IL-8, IL-1β, IL-6, and TNF transcript abundance was measured by qPCR. Data are from one donor representative of at least three independent experiments. (I) moDCs (n ≥ 5) were treated with Z-VAD-fmk (10 µM) and stimulated with QS-21 (10 µg/ml) or MPL (1 µg/ml), and IL-8, TNF, and IL-1β in the supernatant were measured by ELISA. (J) moDCs (n ≥ 5) were treated with recombinant IL-1 receptor antagonist (Anakinra—10 µg/ml) and stimulated with QS-21 (10 µg/ml) or IL-1β (10 ng/ml) and IL-8 was measured by ELISA. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 2

QS-21 is internalized by cholesterol-dependent endocytosis. (A) Imaging: THP-1 cells were incubated with 10 µg/ml BODIPY-QS-21 for 1 h at either 4°C or 37°C, and fluorescence was visualized by vital confocal microscopy. (B) Surface stripping: THP-1 cells (n = 3) were incubated at 4°C for 1 h with 10 µg/ml ¹⁴C-QS-21, then extensively washed (Ctrl) or further stripped at 4°C by trypsin or heparin sulfate. (C) Endocytosis: Control or ATP-depleted THP-1 (n = 3) cells were incubated at 37°C for 4 h with 10 µg/ml ¹⁴C-QS-21, then stripped by trypsin as at (B). Parallel cells were incubated at 4°C. Statistical significance was determined by a one-way ANOVA followed by Tukey’s multiple comparisons test. (D) Cholesterol dependence: THP-1 cells (n = 2–3) were cholesterol depleted or not by treatment with 2.5 mM methyl-β-cyclodextrin (MβCD) for 4 h, extensively washed, then incubated with ¹⁴C-QS-21 for 1 h or ¹²⁵I-transferrin or ¹²⁵I-cholera toxin/B subunit (CTxB) for 15 min, surface-stripped by trypsin and counted. Statistical significance was determined by a two-way ANOVA followed by Sidak’s multiple comparisons test. (E,F) moDCs (n = 8) were treated with the indicated concentration of MβCD for 1 h, extensively washed and stimulated with QS-21 or MPL for 4 h. IL-6 (E) and TNF (F) mRNA abundance was measured by qPCR and normalized to β-actin expression. Statistical significance was determined by a Wilcoxon matched-pairs signed-rank test. The asterisks depict significant differences between 5–10 mM MβCD and 0 mM MβCD for QS-21 stimulated cells.

Figure 3

Syk activation and signaling is indispensable for response to QS-21 in moDCs. (A,B) Immunoblot analysis of cytoplasmic extracts of moDCs stimulated with QS-21, MPL, or Zymosan (Zym) for the indicated durations. Blots were probed sequentially with pY323-SYK (A) or pY352-SYK (B), followed by total Syk. (C–E) moDCs were treated with Bay 61-3606 (10 µM) for 1 h and stimulated with QS-21 (10 µg/ml) for 24 h. IL-6 (C), TNF (D), and IL-8 (E) were quantified in the supernatant by ELISA. Statistical significance was determined by a Wilcoxon matched-pairs signed-rank test. (F–H) moDCs were transduced with lentiviruses coding for shRNAs targeting GAPDH or Syk, and Syk (F), IL-6 (G), and TNF (H) mRNA expression were measured by qPCR and normalized against β-actin expression. Statistical significance was determined by a Wilcoxon matched-pairs signed-rank test. (I) moDCs were seeded on coverslips, treated with Bay 61-3606 (10 µM) for 1 h and stimulated with 10 µg/ml QS-21 for 2 h. NF-κB p65 was visualized by immunofluorescence and nuclear p65 was quantified using ImageJ software. Data are representative of three experiments. Statistical significance was determined with a non-parametric Mann–Whitney t-test.

Figure 3

Syk activation and signaling is indispensable for response to QS-21 in moDCs. (A,B) Immunoblot analysis of cytoplasmic extracts of moDCs stimulated with QS-21, MPL, or Zymosan (Zym) for the indicated durations. Blots were probed sequentially with pY323-SYK (A) or pY352-SYK (B), followed by total Syk. (C–E) moDCs were treated with Bay 61-3606 (10 µM) for 1 h and stimulated with QS-21 (10 µg/ml) for 24 h. IL-6 (C), TNF (D), and IL-8 (E) were quantified in the supernatant by ELISA. Statistical significance was determined by a Wilcoxon matched-pairs signed-rank test. (F–H) moDCs were transduced with lentiviruses coding for shRNAs targeting GAPDH or Syk, and Syk (F), IL-6 (G), and TNF (H) mRNA expression were measured by qPCR and normalized against β-actin expression. Statistical significance was determined by a Wilcoxon matched-pairs signed-rank test. (I) moDCs were seeded on coverslips, treated with Bay 61-3606 (10 µM) for 1 h and stimulated with 10 µg/ml QS-21 for 2 h. NF-κB p65 was visualized by immunofluorescence and nuclear p65 was quantified using ImageJ software. Data are representative of three experiments. Statistical significance was determined with a non-parametric Mann–Whitney t-test.

Figure 4

QS-21 accumulates in lysosomes and promotes their destabilization. (A) Differentiated THP-1 cells were pulsed with ¹⁴C-QS-21 for 4 h followed by an overnight chase, and postnuclear particles were resolved by density gradients fractionation. Na+/K+-ATPase identifies the plasma membrane and N-acetyl-β-hexosaminidase activity identifies lysosomes. The abscissa axis represents the different fractions. The ordinate reflects enrichment (C/C_i > 1) or depletion (C/C_i < 1) vs. initial abundance (C_i = 1), indicated by dotted lines. (B) Differentiated THP-1 cells were co-incubated with BODIPY-QS-21 and Lysotracker Red then analyzed by vital confocal microscopy. (C) moDCs were incubated with 1 µg/ml acridine orange (AO), washed, and stimulated for 2 h with QS-21 (10 µg/ml). AO fluorescence was quantified by flow cytometry and a 610 nm filter. Cells with decreased AO fluorescence are indicated with the double arrow. One representative donor of 3 is shown. (D) moDCs were incubated with Lucifer Yellow, 10 kDa dextran-Alexa633 and 40 kDa dextran-Texas Red for 16 h then stimulated for QS-21 for 4 h. The localization of fluorescent markers was observed by confocal microscopy. The proportion of cells with punctate (lysosomal), diffuse (cytoplasmic) or both (punctate + diffuse) signal for the different fluorescent markers was determined with ImageJ. The data are representative of four donors.

Figure 5

QS-21-mediated dendritic cell activation and Syk phosphorylation depend on lysosomal maturation. (A,B) moDCs were treated with bafilomycin A1 (BafA1—250 nM) or NH₄Cl (10 mM) for 1 h and stimulated with QS-21 (10 µg/ml) or MPL (1 µg/ml) for 4 h (A) or 24 h (B). TNF and IL-6 mRNA (A) and protein (B) were quantified by qPCR (n = 8) and ELISA (n = 11), respectively. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test. (C) moDCs were treated with BafA1 for 1 h and stimulated with QS-21 (10 µg/ml) or zymosan as a positive control (Zym—50 µg/ml) for 2 h. Phospho-Syk (Y352), Syk, and β-actin were detected by sequential western blotting. One representative donor of 2 is shown. (D) moDCs were incubated with Lucifer Yellow, 10 kDa dextran-Alexa633, and 40 kDa dextran-Texas Red for 16 h then pretreated with BafA1 or DMSO (vehicle) and stimulated with QS-21 for 4 h. The localization of fluorescent markers was observed by confocal microscopy. The proportion of cells with punctate (lysosomal), diffuse (cytoplasmic), or both (punctate + diffuse) signals for the different fluorescent markers was determined with ImageJ. The data are representative of two donors.

Figure 6

Cathepsin B activity is required for an optimal response to QS-21. (A) moDCs were incubated with CA-074 Me (cathepsin B inhibitor), Z-FA-FMK (cathepsin B inhibitor), Z-FF-FMK (cathepsin B/L inhibitor), or pepstatin A (cathepsin D, cathepsin E, and pepsin inhibitor) for 1 h and stimulated with QS-21 for 24 h. TNF release into the supernatant was measured by ELISA. Statistical significance was determined with a Friedman test followed by Dunn’s multiple comparison test. (B) moDCs were pretreated with CA-074 Me or Z-FA-FMK for 1 h and stimulated with QS-21 or MPL for 6 h. IL-6 and TNF mRNA levels were quantified by qPCR and normalized to β-actin mRNA levels. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test. (C,D) moDCs were transduced with lentiviruses coding for shRNAs targeting GAPDH (shGAPDH) or cathepsin B (shCtsb), and cathepsin B (C), IL-6 and TNF (D) mRNA expression were measured by qPCR and normalized against β-actin expression. Statistical significance was determined by a Wilcoxon matched-pairs signed-rank test.

Figure 7

QS-21 elicited antigen-specific CD4 and CD8 T cell responses are dependent on cathepsin B. (A) Monocyte (CD11b⁺ Ly6C⁺ Ly6G⁻), neutrophil (CD11b⁺ Ly6C^int Ly6G⁺), and dendritic cell (CD11c^hi MHCII^hi) recruitment to the draining lymph node 24 h post intramuscular immunization with PBS or QS-21 assessed by flow cytometry. (B) WT and cathepsin B (Ctsb)-invalidated mice were immunized at day 0 and day 14 with hepatitis B surface (HBs) antigens or with HBs antigens adjuvanted with QS-21. At day 21, median cytokine production of HBsAg-specific splenic CD4 and CD8 T cells were evaluated by intracellular staining following ex vivo restimulation with antigenic peptides (Ag: n = 6, QS-21: n = 20). (C) IFN-γ and IL-2 production by HBs-specific spleen cells were measured by ELISA following restimulation with HBs peptides (Ag: n = 3, QS-21: n = 10). (D) Anti-HBs IgG1 and IgG2c titers in the serum at day 21 were measured by ELISA (Ag: n = 6, QS-21: n = 26). Each dot represents one mouse, and the horizontal bar represents the geometric mean. Statistical significance was determined by a non-parametric Mann–Whitney t-test. The data represent the pooled results of two independent experiments. (E) Proposed model: QS-21 is endocytosis via a cholesterol-dependent mechanism. It then traffics to lysosomes and induces their destabilization. This can induce inflammasome activation and Syk- and cathepsin B-dependent cell activation and cytokine production in moDCs. Lysosomal destabilization could also affect antigen processing and antigen translocation to the cytosol.