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J. Biol. Chem., Vol. 282, Issue 29, 21145-21159, July 20, 2007

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

Selina Khan, Martijn S. Bijker¹, Jimmy J. Weterings¹, Hans J. Tanke^¶, Gosse J. Adema^||, Thorbald van Hall^**, Jan W. Drijfhout, Cornelis J. M. Melief, Hermen S. Overkleeft, Gijsbert A. van der Marel, Dmitri V. Filippov, Sjoerd H. van der Burg^**, and Ferry Ossendorp²

From the Departments of Immunohematology and Blood Transfusion, ^¶Molecular Cell Biology, and ^**Clinical Oncology, Leiden University Medical Centre, P. O. Box 9600, 2300 RC Leiden, The Netherlands, Leiden Institute of Chemistry, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands, and ^||Immunology Laboratory, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands

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, Pam₃CysSK₄ (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 CpGconjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pamconjugated 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 CpGconjugates 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.

Received for publication, February 27, 2007 , and in revised form, April 13, 2007.

^* This work has been funded by Netherlands Organization for Scientific Research as a part of the From Molecule to Cell program (to S. H. v B., D. V. F., G. A. M., and F. O.) and by the KWF Kankerbestrijding UL 2003-2817 (to S. H. v B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally.

² To whom correspondence should be addressed. Tel.: 31-71-5263843; Fax: 31-71-5265267; E-mail: f.a.ossendorp{at}lumc.nl.

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