for Cross-Presentation Targeting Antigen to Human Dendritic Cells over Receptor-Specific Antibodies in No Advantage of Cell-Penetrating Peptides

Induction of CTL responses by dendritic cell (DC)-based vaccines requires efﬁcient DC-loading strategies for class I Ags. Coupling Ags to cell-penetrating peptides (CPPs) or receptor-speciﬁc Abs improves Ag loading of DCs. In contrast to CPPs, receptor-speciﬁc Abs deliver conjugated Ags to DCs with high speciﬁcity, which is advantageous for in vivo strategies. It has, however, been speculated that CPPs facilitate uptake and endosomal escape of conjugated Ags, which would potently enhance cross-presentation. In this study, we directly compare the in vitro targeting efﬁciency of a humanized D1 Ab directed against the human DC surface receptor DC-SIGN hD1 to that of three CPPs. The three CPPs colocalized within endosomes when targeted to human monocyte-derived DCs simultaneously, whereas hD1 was present in a different set of endosomes. However, within 75 min after uptake CPPs and hD1 colocalized extensively within the lysosomal compartment. Ab-mediated targeting of class I-restricted peptides to DC-SIGN enhanced cross-presentation of the peptides, while only one of the CPPs enhanced peptide presentation. This CPP and hD1 enhanced cross-presentation with equal efﬁciencies. Thus, we found no evidence of CPP speciﬁcally favoring the delivery of conjugated Ag to the DC class I presentation pathway. Given the speciﬁcity with which Abs recognize their targets, this favors the use of DC receptor-speciﬁc Abs for in vivo vaccination strategies. The Journal of Immunology, 2008, 180: 7687–7696. the of DC-SIGN is ligand speciﬁc or de-pendent on variations in intracellular DC-SIGN routing between different cell types. Our experiments show that SA-polyR accu-mulates in DCs more rapidly than hD1, although this does not result in increased Ag presentation. Likely, this is a consequence of the distinct mechanisms responsible for internalizing CPPs and the targeting Ab. Upon uptake by DCs, internalized SA-Tat, SA-DPV3, and SA-polyR constructs showed a high degree of colocalization. Many types of endocytosis have been implicated in the uptake of CPPs by cells, including clathrin-dependent endocytosis (47), caveolin-dependent endocytosis (38, 48), and raft-dependent macropinocytosis (49). These contrasting ﬁndings might be explained by variations in experimental setup, such as the type of cargo attached to the CPP and the cell type used. Our data suggest that Tat, DPV3, and polyR use identical or at least largely overlapping endosomal routes to enter DCs. Internalized hD1 Ab was localized in a different set of endosomes than SA-polyR, demon-strating that proteins endocytosed via DC-SIGN follow a different route. These ﬁndings are supported by two recent publications showing that DC-SIGN ligands enter cells via a clathrin-dependent (50) while the CPP polyR enters cells via a clathrin-independent, ﬂotillin- and dynamin-dependent internalization process (51). In our study we showed that both endosomes carrying SA-polyR and hD1 eventually delivered their antigenic cargo to the lysosomal compartment.Theaimofour study was to compare the efﬁciency of DC cross-presentation by using different targeting strategies. The results do not predict whether this level of cross-presentation is sufﬁcient for cross-priming of CTL responses in vivo. To initiate a proliferative response, naive T cells require a higher amount of MHC-peptide complexes on the APC surface than primed T cells (52) and, possibly,