Cross-Presentation of Synthetic Long Peptides by Human Dendritic Cells: A Process Dependent on ERAD Component p97/VCP but Not sec61 and/or Derlin-1

Abstract

Antitumor vaccination using synthetic long peptides (SLP) is an additional therapeutic strategy currently under development. It aims to activate tumor-specific CD8+ CTL by professional APCs such as DCs. DCs can activate T lymphocytes by MHC class I presentation of exogenous antigens - a process referred to as “cross-presentation”. Until recently, the intracellular mechanisms involved in cross-presentation of soluble antigens have been unclear. Here, we characterize the cross-presentation pathway of SLP Melan-A16–40 containing the HLA-A2-restricted epitope26–35 (A27L) in human DCs. Using confocal microscopy and specific inhibitors, we show that SLP16–40 is rapidly taken up by DC and follows a classical TAP- and proteasome-dependent cross-presentation pathway. Our data support a role for the ER-associated degradation machinery (ERAD)-related protein p97/VCP in the transport of SLP16–40 from early endosomes to the cytoplasm but formally exclude both sec61 and Derlin-1 as possible retro-translocation channels for cross-presentation. In addition, we show that generation of the Melan-A26–35 peptide from the SLP16–40 was absolutely not influenced by the proteasome subunit composition in DC. Altogether, our findings propose a model for cross-presentation of SLP which tends to enlarge the repertoire of potential candidates for retro-translocation of exogenous antigens to the cytosol.

Long-Peptide Cross-Presentation by Human Dendritic Cells Occurs in Vacuoles by Peptide Exchange on Nascent MHC Class I Molecules

“…This conclusion contrasts with a number of previous reports supporting a cytosolic pathway (8)(9)(10)(11). Our proposed model implying a key role of suboptimally loaded HLA-I molecules provides a potential explanation for those discrepancies.…”

“…Contrary to these studies, Ménager et al (8) did evaluate the effect of TAP inhibition on the cross-presentation of a long HLA-A2-restricted Melan-A peptide. They did so using a synthetic peptide corresponding to the N-terminal 35-amino acid residues of TAP inhibitor ICP47.…”

“…We have observed competition effects between long peptides for cross-presentation. Because the TAP inhibitor used by Ménager et al (8) was, in essence, a long peptide, it is expected to compete with the long peptide for cross-presentation. Therefore, it is likely that the inhibition observed was due to competition rather than TAP inhibition.…”

“…Therapeutic vaccines based on long peptides have shown clear promise in preclinical models (6), and recently showed clinical efficacy in patients with human papillomavirus-induced neoplasia (7). Contrasting with the advanced clinical development of long-peptide vaccination, relatively little is known about the pathway used for cross-presentation of long peptides (8)(9)(10)(11).…”

Spatial separation of the processing and MHC class I loading compartments for cross-presentation of the tumor-associated antigen HER2/ neu by human dendritic cells

“…After proteasomal processing some peptides require further trimming by aminopeptidases such as the cytosolic tri-peptidil peptidase II (TPPII) 30 and ER aminopeptidases (ERAPs). 37,38 TPPII may have a slight impact on the presentation of HER2/neu 369-377 , as its inhibition caused a slight decrease in the HLA-A*201/HER2/neu 369-377 complexes at the DCs surface. ERAPs however, appear to play no role in the processing of HER2/neu for cross-presentation, as we found no HLA-A2/ HER2/neu 369-377 complexes in the ER.…”