title: 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 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.
authors.affiliation: Department of Immunohematology and Blood Transfusion, Molecular Cell Biology, and Clinical Oncology, Leiden University Medical Centre, P. O. Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Selina
authors.initials: S
authors.lastname: Khan
authors.forename: Martijn S
authors.initials: MS
authors.lastname: Bijker
authors.forename: Jimmy J
authors.initials: JJ
authors.lastname: Weterings
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Tanke
authors.forename: Gosse J
authors.initials: GJ
authors.lastname: Adema
authors.forename: Thorbald
authors.initials: T
authors.lastname: van Hall
authors.forename: Jan W
authors.initials: JW
authors.lastname: Drijfhout
authors.forename: Cornelis J M
authors.initials: CJ
authors.lastname: Melief
authors.forename: Hermen S
authors.initials: HS
authors.lastname: Overkleeft
authors.forename: Gijsbert A
authors.initials: GA
authors.lastname: van der Marel
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Filippov
authors.forename: Sjoerd H
authors.initials: SH
authors.lastname: van der Burg
authors.forename: Ferry
authors.initials: F
authors.lastname: Ossendorp
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D022461:Caveolins / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D018899:CpG Islands; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D004705:Endocytosis; D006680:HLA Antigens / Q000737:chemistry; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D013601:T-Lymphocytes / Q000378:metabolism; D051193:Toll-Like Receptors / Q000378:metabolism*
chemical_list: D022461:Caveolins; D002966:Clathrin; D006680:HLA Antigens; D008024:Ligands; D010455:Peptides; D051193:Toll-Like Receptors