Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells

Abstract

Toll-like receptors (TLRs) sense microbial products and initiate adaptive immune responses by activating dendritic cells (DCs). As pathogens may contain several TLR agonists, we sought to determine whether different TLRs cooperate in DC activation. In human and mouse DCs, TLR3 and TLR4 potently acted in synergy with TLR7, TLR8 and TLR9 in the induction of a selected set of genes. Synergic TLR stimulation increased production of interleukins 12 and 23 and increased the Delta-4/Jagged-1 ratio, leading to DCs with enhanced and sustained T helper type 1-polarizing capacity. Global gene transcriptional analysis showed that TLR synergy 'boosted' only approximately 1% of the transcripts induced by single TLR agonists. These results identify a 'combinatorial code' by which DCs discriminate pathogens and suggest new strategies for promoting T helper type 1 responses.

Figure 1. Synergistic stimulation of IL-12p70 production by combinations of TLR agonists

Human MoDCs (a), human DCs isolated from peripheral blood (b), mouse bone marrow derived DCs (BMDCs) (c) were cultured with 10 ng/ml LPS, 20 µg/ml poly(I:C), 2.5 µg R848 and 2.5 µg CpG 1826 alone or in combination. IL-12p70 was measured in the 48 h culture supernatant. Data are presented as mean ± SD of three replicates of one representative experiment out of five. (d,e) MoDCs were stimulated with LPS (filled diamonds), R848 (filled squares), poly(I:C) (filled triangles) or combinations of LPS plus R848 (empty squares) or poly(I:C) plus R848 (empty triangles). The TLR agonists were added at the indicated concentrations. IL-12p70 was measured in the 48 h culture supernatant. Data are presented as mean of duplicates of one representative experiment out of four.

Figure 2. Temporal requirements for TLR synergy

(a,b) MoDCs were stimulated with LPS (10 ng/ml) and R848 (2.5 µg/ml) (a) or poly(I:C) (20 µg/ml) and R848 (b). The stimuli were given simultaneously (time 0) or sequentially (dashed line: LPS or poly(I:C) after R848; solid line: R848 after LPS or poly(I:C)) at the indicated interval. IL-12p70 was measured in the 48 h culture supernatant. The data are expressed as percent of the value obtained when the agonists were added simultaneously. Data represent the mean ± SD of three independent experiments.

Figure 3. IFN-γ and CD40L further amplify IL-12p70 production induced by synergistic combinations of TLR agonists

MoDCs were stimulated with 10 ng/ml LPS, 2.5 µg/ml R848, 20 µg/ml poly(I:C) alone or in combinations in the absence (grey histograms), or in the presence of 5 ng/ml IFN-γ (empty histograms) or 2 µg/ml CD40L (filled histograms). IL-12p70 was measured in the 48 h culture supernatant. Data represent the mean ± SD of three replicates of one representative experiment out of three.

Figure 4. Synergistic TLR stimulation is required for maximal upregulation of IL-12p35, IL-23p19 and Delta-4

MoDCs were stimulated with 10 ng/ml ultra pure LPS (filled diamonds), 2.5 µg/ml R848 (filled squares), 20µg/ml poly(I:C) (filled triangles), LPS+R848 (empty squares) or poly(I:C)+R848 (empty triangles). The amounts of IL-12p40, IL-12p35, IL-23p19 (a), and of Delta-4 and Jagged-1 (b) mRNAs relative to 18S rRNA were determined by quantitative real time RT-PCR at the indicated time points. One representative experiment out of three. AU, arbitrary units of the indicated mRNA/18S rRNA x 10⁻³.

Figure 5. TLR synergy enhances and sustains the T_H1 polarizing capacity of DCs

MoDCs were stimulated with 10 ng/ml LPS, 2.5 µg/ml R848 or LPS+R848 for 12 or 48 h, washed and cultured with allogeneic naive CD4⁺ T cells. After 8 days the proliferating T cells were tested for their capacity to produce IFN-γ and IL-4 following stimulation with PMA and Ionomycin. Numbers indicate percent of cells producing intermediate and high amounts of IFN-γ. One representative experiment out of five.

Figure 6. Selective effect of TLR synergy on DC gene expression and on IL-1β release

MoDCs were stimulated with 10 ng/ml ultra pure LPS (filled diamonds), 2.5 µg/ml R848 (filled squares), 20 µg/ml poly(I:C) (filled triangles), LPS+R848 (empty squares) or poly(I:C)+R848 (empty triangles). The amounts of TNF, IL-6, IL-10, COX-2, IFN-β, CXCL10, CCL3, CCR7 (a) and of IL-1β (b) mRNAs relative to 18S rRNA were determined by quantitative real time RT-PCR. AU, arbitrary units of the indicated mRNA/18S rRNA x 10⁻³ (c) IL-1β release was measured in the 48 h culture supernatant and data are presented as mean ± SD of three replicates of one out of three independent experiments.

Figure 7. Effect of TLR synergy on global gene expression is observed only at late time point

MoDCs from three different donors were left untreated (control) or stimulated with 10 ng/ml ultra pure LPS, 2.5 µg/ml R848 or LPS+R848 for 2 or 8 h. Global transcriptional analysis was performed using the Affymetrix methodology. (a,b) Correlation between fold change (relative to control) induced by LPS+R848 and fold change induced by the best agonist (either LPS or R848). The data are from the 2 h (a) and 8 h time points (b). The dashed lines mark five fold changes.

Figure 8. TLR synergy sustains IκB-ζ expression and c-Jun phosphorylation

(a) MoDCs were stimulated with 10 ng/ml LPS (filled diamonds), 2.5 µg/ml R848 (filled squares), 20 µg/ml poly(I:C) (filled triangles), LPS+R848 (empty squares) or poly(I:C)+R848 (empty triangles). IκB-ζ mRNA was determined by quantitative real time RT-PCR at the indicated time points. One representative experiment out of three. (b) Cell lysates of MoDCs stimulated with the indicated agonists were prepared at different time points, separated by SDS electrophoresis and blotted with antibodies to phosphorylated c-Jun (p-Jun), IκB-α, phosphorylated p38 (p-p38) and total p38. L, LPS; R, R848; I, poly(I:C). One representative experiment out of three.