Distinct Uptake Mechanisms but Similar Intracellular Processing of Two Different Toll-like Receptor Ligand-Peptide Conjugates in Dendritic Cells

Abstract

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam 3 CysSK 4 (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpGϳ conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pamϳconjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin-or caveolindependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpGϳconjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8؉ T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.

Molecular Mechanisms of TLR2-Mediated Antigen Cross-Presentation in Dendritic Cells

“…Moreover, Pam3CysK4-conjugated lipopeptides activated APCs through TLR2, but the internalization of the Pam3CysK4-conjugated lipopeptide was independent of TLR2. Interestingly, both clathrin-and caveolin-mediated endocytosis were necessary for the conjugate's endocytosis (37). Additionally, the different lipid structures of glycolipids with TLR2 agonist activity were found to be cross-presented by BMDCs using different intracellular pathways, but all required TLR2 (40).…”

“…Furthermore, the incorporation of CTL epitopes into synthetic lipopeptides with TLR2 agonist activity has been shown to induce Ag-specific CD8 + T cell responses (34,37). However, there is limited information regarding the TLR2-mediated cross-priming of CTLs and its antitumor effects in vivo.…”

MF59 and Pam3CSK4 Boost Adaptive Responses to Influenza Subunit Vaccine through an IFN Type I-Independent Mechanism of Action

“…Therefore, we cannot exclude that R848 and CpG would boost flu subunit vaccine responses if physically linked to the HA-Ag through direct conjugation or through a delivery system. Indeed, several publications have shown that direct conjugation of TLR2, 7, and 9 agonists to the Ag increases their adjuvanticity (26,29,30).…”

Extracts of Larix Leptolepis effectively augments the generation of tumor antigen-specific cytotoxic T lymphocytes via activation of dendritic cells in TLR-2 and TLR-4-dependent manner

“…Several reports demonstrated that TLR ligand promoted endosomal acidification, proteasomal activity, and TAP translocation in DCs, resulting in the efficient presentation of exogenous antigen and enhancement of CTL activation [58][59][60][61]. Therefore, promotion of cross-presentation would be involved in CTL induction by vaccination with ELL.…”