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.

Identification of Teleost Skin CD8α+ Dendritic-like Cells, Representing a Potential Common Ancestor for Mammalian Cross-Presenting Dendritic Cells

“…These findings support the hypothesis that mammalian cross-presenting DCs, including murine lymphoid CD8 + DCs, murine tissue CD103 + DCs, and the human CD141 lineage, share a common ancestor that has been conserved through evolution in teleost fish. Previous evidence that led other investigators to formulate the hypothesis includes the expression of common transcription factors, such as Batf3 (20,21) and IRF8 (18,19), the sensitivity to TLR3 ligands, and the use of CLEC9A and XCR1 (18,19,(22)(23)(24)29). We were able to identify CLEC9A and XCR1 homologs in trout EST databases, but we showed that the skin CD8 + DC-like subpopulation expressed both Batf3 and IRF8, distinctive transcription factors essential in the development of cross-presenting DCs.…”

“…Additionally, both populations use the CLEC9A lectin to recognize necrotic cells (22) and express the chemokine receptor XCR1 (23). They also express higher levels of TLR3 than do other DC subtypes and, consequently, are responsive to virus-derived intracellular dsRNAs (24). In humans, a discrete CD141 + (BDCA-3 or thrombomodulin) DC population in blood also exhibits a crosspresenting function (25).…”

“…Multiple lines of evidence point to a common cross-presenting DC lineage for mice lymphoid CD8 + DCs and tissue-migratory CD103 + DCs (17)(18)(19)(22)(23)(24). In humans, blood CD141 + cells also cross-present Ags and exhibit markers in common with mice CD8 + DCs (23,(25)(26)(27), suggesting a common origin for all mammalian cross-presenting cells (29).…”

The TLR3/TICAM-1 Pathway Is Mandatory for Innate Immune Responses to Poliovirus Infection

“…This pathway is involved in type I IFN induction, as in the IPS-1 pathway, but cells expressing TLR3 are limited. The TLR3 distribution profile by flow cytometry confirms its expression in myeloid cells in mice (30). The TICAM-1 pathway converges with the IPS-1 pathway via the molecular complex of IRF-3-activating kinases (38), and therefore activation of the TICAM-1 pathway induces type I IFN and other IFNinducible genes (39).…”

“…2). Thus, CD8a + CD11c + DC, which reportedly express TLR3 (30), are the source of type I IFN in PV-infected PVRtg mice.…”