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

Sjoerd H. van der Burg,Gosse J. Adema,Selina Khan,Martijn S. Bijker,Hermen S. Overkleeft,Gijsbert A. van der Marel,Jan W. Drijfhout,Ferry Ossendorp,Dmitri V. Filippov,Jimmy J. Weterings,Thorbald van Hall,Cornelis J.M. Melief,Hans J. Tanke

doi:10.1074/jbc.m701705200

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 CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated 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 caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates 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.

Highlights

Toll-like receptors (TLR)3 are germ line-encoded receptors expressed mainly on cells of the innate immune system, such as granulocytes, macrophages, and dendritic cells (DCs)
The generated peptides are transported from the cytosol into the endoplasmic reticulum via the peptide transporter TAP [7], after which the peptides undergo further trimming and are loaded onto major histocompatibility complex (MHC) class I molecules, which translocate to the cell surface, where the peptide is presented to CD8ϩ T-cells
No up-regulation of the cell surface markers CD86 and MHC class II was detected when bone marrow-derived dendritic cells (BMDCs) from TLR2-deficient mice or TLR9-deficient mice were stimulated with Pamϳconjugated peptide or CpGϳconjugated peptide, respectively (Fig. 2). This impaired up-regulation was not due to a general defect in the maturation signaling pathway, because stimulation with LPS could induce up-regulation of CD86 and MHC II in BMDCs derived from both TLR2- and TLR9-deficient mice to a similar extent as observed for BMDCs derived from wild type mice. These results demonstrate that the conjugates are as effective as free TLR ligand in activating the DCs and show that the expression of the cognate TLR is required for activation of DCs by the TLR-L-peptide conjugates

Summary

EXPERIMENTAL PROCEDURES

TLR2-deficient mice were purchased from Jackson Laboratories, whereas the TLR9-deficient mice were obtained from S. Isolated DCs were cultured from mouse bone marrow cells as described elsewhere [17]. D1 cell line, a long term growth factor-dependent immature splenic DC line derived from B6 (H-2b) mice, was cultured as described [18]. PyBOP (benzotriazole-1yl-oxytrispyrrolidinophosphonium hexafluorophosphate) was purchased from MultiSynTech GmbH. Fmoc amino acids were from SENN Chemicals or from MultiSynTech GmbH. Tentagel-based resins were purchased at Rapp Polymere GmbH. All chemicals and solvents used in the solid phase peptide synthesis were from Biosolve. Resins, and solvents used in the solid phase DNA synthesis except Beaucage reagent and Control Pore Glass support used to introduce 3Ј-thiol modification were from Proligo and were used as received. 3Ј-Thiol modifier C3 S-S Control Pore Glass and Beaucage reagent were purchased at Glen Research.

General Methods

RESULTS

DISCUSSION