TLR3-specific double-stranded RNA oligonucleotide adjuvants induce dendritic cell cross-presentation, CTL responses, and antiviral protection

Abstract

Maturation of dendritic cells (DC) to competent APC is essential for the generation of acquired immunity and is a major function of adjuvants. dsRNA, a molecular signature of viral infection, drives DC maturation by activating TLR3, but the size of dsRNA required to activate DC and the expression patterns of TLR3 protein in DC subsets have not been established. In this article, we show that cross-priming CD8α(+) and CD103(+) DC subsets express much greater levels of TLR3 than other DC. In resting DC, TLR3 is located in early endosomes and other intracellular compartments but migrates to LAMP1(+) endosomes on stimulation with a TLR3 ligand. Using homogeneous dsRNA oligonucleotides (ONs) ranging in length from 25 to 540 bp, we observed that a minimum length of ∼90 bp was sufficient to induce CD86, IL-12p40, IFN-β, TNF-α, and IL-6 expression, and to mature DC into APC that cross-presented exogenous Ags to CD8(+) T cells. TLR3 was essential for activation of DC by dsRNA ONs, and the potency of activation increased with dsRNA length and varied between DC subsets. In vivo, dsRNA ONs, in a size-dependent manner, served as adjuvants for the generation of Ag-specific CTL and for inducing protection against lethal challenge with influenza virus when given with influenza nucleoprotein as an immunogen. These results provide the basis for the development of TLR3-specific adjuvants capable of inducing immune responses tailored for viral pathogens.

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 3 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 co-stained for TLR3 and LAMP-1. Scale bars represent 10μm. Indicated squares are shown magnified on the right of each panel. Bottom magnification, TLR3 alone; middle magnification, vesicle marker alone; top magnification, 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 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 CD24^high and CD11b^high FL-DC subsets from WT and TLR3^−/− mice were stimulated with 25 bp, 60 bp, 90 bp, 139 bp 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 3 independent experiments. * designates significant differences compared to untreated control (*P<0.05; **P<0.01; ***P<0.001), whereas † indicates 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 co-cultured with CFSE-labeled OT-I CD8+ T cells. Proliferation was detected by CFSE dilution. Panel (A) shows representative histograms of proliferating T cells induced by DC that had been treated with OVA and the indicated stimulants. (B) Shows number of proliferating T cells; n=6 from two independent experiments, means + SEM * designates significant differences compared to OVA alone control (***P<0.001), whereas † indicates 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 seven days 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 3 mice per group (mean + SEM). * designates significant differences compared to control (**P<0.01; ***P<0.001), whereas † 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 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 naïve 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 naïve and immunized mice (*P<0.05; **P<0.01; ***P<0.001).