Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells.
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.
Khan
Selina
S
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.
Bijker
Martijn S
MS
Weterings
Jimmy J
JJ
Tanke
Hans J
HJ
Adema
Gosse J
GJ
van Hall
Thorbald
T
Drijfhout
Jan W
JW
Melief
Cornelis J M
CJ
Overkleeft
Hermen S
HS
van der Marel
Gijsbert A
GA
Filippov
Dmitri V
DV
van der Burg
Sjoerd H
SH
Ossendorp
Ferry
F
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*
D022461:Caveolins; D002966:Clathrin; D006680:HLA Antigens; D008024:Ligands; D010455:Peptides; D051193:Toll-Like Receptors