Dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and T‐cell activation

Abstract

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T-cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre-)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte-derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4 + and CD8 + T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasomeand transporter associated with Ag processing-dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8 + T-cell activation.Keywords: Antigen presentation/processing r Cellular immunology r CD8 + T cells r Dendritic cells Additional supporting information may be found in the online version of this article at the publisher's web-site lower efficiency compared to SLP-loaded DCs (Fig. 1B). Prestimulation of DCs with the TLR4 ligand LPS had no effect on the MHC class I presentation of OVA-protein but improved Ag presentation of SSP-OVA 8aa (data not shown) and long peptide Ag (Fig. 1C). HLA-B7-restricted presentation by human monocyte-derived DCs (MoDCs) of HIV-derived protein and SLPs was also studied. We were unable to detect cytokine production by CD8 + T cells cocultured with GAG-protein-loaded DCs. In contrast, SLP-GAG 22aa induced significant CD8 + T-cell activation (see Fig. 2 and below).Together, these data show that cross-presentation of SLPs is superior to that of proteins as examined with both mouse and human DCs. Rapid Ag presentation of SLPs by murine and human DCsThe efficiency of SLP-processing was assessed by studying the time required for DCs to present Ag on MHC class I (H2-K b )molecules. Murine DCs were incubated with a single concentration of SLPs, synthetic short peptides (SSPs) or protein for the indicated time periods. The minimal peptide, SSPs, was rapidly presented to CD8 + T cells resulting in strong activation already after 1 h. DCs loaded with SLP also activated CD8 + T cells 1 h after Ag loading but with lower potency. We excluded that SLPs were cleaved extracellularly, processed, and loaded on MHC class I and II molecules by incubating paraformaldehyde (PFA) fixed cells with the peptide Ag and observed no cross-presentation (data not shown). DCs loaded with 10 μM OVA-protein failed to induce significant CD8 + T-ce...

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