Article Figures & Data
Figures
- FIGURE 1.
High TLR3 expression in cross-presenting DC. Cells were fixed, permeabilized, and stained for surface markers and TLR3. The same populations of cells from TLR3−/− mice were used as negative controls. A, TLR3 expression in splenic T cells (CD3ε+), NK cells (DX5+), B cells (B220+), peritoneal macrophages (MΦ, CD11b+), and DC (CD11c+). B–D, TLR3 expression in (B) CD11c-enriched splenic DC subsets, (C) FL-DC subsets, (D) GM-DC, and (E) CD11c-enriched LN DC subsets. All cells were electronically gated for CD11c expression and further gated as indicated in the figure. Histograms are representative of at least three experiments.
- FIGURE 2.
Intracellular localization of TLR3 in FL-DC. A, Unstimulated or pIC-stimulated FL-DC were fixed, permeabilized, and stained intracellularly for the ER-resident protein calnexin, early endosome marker EEA-1, or late endosome/lysosome marker LAMP-1 (red), together with anti-TLR3 (green), and examined by confocal microscopy. B, FL-DC were stimulated with 25, 60, 90, 139, or 540 bp dsRNA ONs, and costained for TLR3 and LAMP-1. Scale bars, 10 μm. Boxed areas in left panels are shown magnified in the three right panels. Bottom right panels, TLR3 alone; middle right panels, vesicle marker alone; top right panels, TLR3 and vesicle markers merged.
- FIGURE 3.
TLR3 dependence of activation of splenic and bone marrow-derived DC. CD86 expression of WT, MDA5−/−, and TLR3−/− splenic DC, FL-DC, and GM-DC after 24 h of treatment with dsRNA (540 bp), pIC, or medium alone (Ctrl). Data shown are mean + SEM of triplicate DC cultures and are representative of four independent experiments.
- FIGURE 4.
DC activation increases with dsRNA length. FACS-sorted CD24high and CD11bhigh FL-DC subsets from WT and TLR3−/− mice were stimulated with 25, 60, 90, 139, and 540 bp dsRNA with pIC or were left untreated (Ctrl). After 24 h, CD86 upregulation and cytokine production were determined. pIC and dsRNA ON concentrations were at or near the plateau on dose-response curves. Values depicted are means + SEM from three independent experiments. Asterisks designate significant differences compared with untreated control (*p < 0.05; **p < 0.01; ***p < 0.001), whereas daggers indicate significant differences between WT and TLR3−/− DC (†p < 0.05; ††p < 0.01; †††p < 0.001).
- FIGURE 5.
dsRNA size dependently induces cross-presentation. WT and TLR3−/− FL-DC were incubated overnight with OVA alone (Ctrl) or OVA plus dsRNA ONs with specified length, pIC, or CpG, then were cocultured with CFSE-labeled OT-I CD8+ T cells. Proliferation was detected by CFSE dilution. A, Representative histograms of proliferating T cells induced by DC that had been treated with OVA and the indicated stimulants. B, Number of proliferating T cells are shown; n = 6 from two independent experiments; means + SEM. Asterisks designate significant differences compared with OVA alone control (***p < 0.001), whereas daggers indicate significant differences between T cells stimulated with WT and TLR3−/− DC (††p < 0.01; †††p < 0.001).
- FIGURE 6.
In vivo CTL induction by dsRNA. WT and TLR3−/− mice were immunized with OVA alone (Ctrl) or OVA plus the indicated dsRNA ON or pIC. A, Percentages of OVA-specific (pentamer-positive) CD8+ T cells 7 d after immunization. B, Representative histograms showing in vivo cytotoxicity assay. Six days after immunization, mice were injected with OVA peptide- coated target cells labeled with high amounts of CFSE and control cells labeled with low amounts of CFSE. One day later, cytotoxicity was detected as a loss in CFSE bright cells relative to CFSE dull cells. C, Percent specific lysis. Data are from at least three mice per group (mean + SEM). Asterisk designates significant differences compared with control (**p < 0.01; ***p < 0.001), whereas dagger indicates significant differences between WT and TLR3−/− mice (†p < 0.05; †††p < 0.001).
- FIGURE 7.
dsRNA ONs serve as effective adjuvants for an influenza nucleoprotein vaccine. WT mice were primed and boosted with rNP plus pIC or 540 bp dsRNA (A) or rNP plus 90, 139, or 540 bp dsRNA ONs (B). Controls (dashed lines) include untreated naive mice, mice treated with rNP alone, and mice treated with 540 bp dsRNA alone. Three weeks after the boost, mice were challenged with 10 LD50 of influenza virus, and survival was monitored thereafter. Asterisks indicate a significant difference in the percentage of survival between naive and immunized mice (*p < 0.05; **p < 0.01; ***p < 0.001).
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