The enhanced cross-presentation was independent of TLR-signaling and inducible at low concentrations of antigen. Furthermore, the addition of 3-sulfo-LeA or tri-GlcNAc
to OVA protein enhanced the frequency of IFN-γ-producing CD4+ T cells, illustrating Th1 skewing. Previous studies showed that the MR specifically binds high mannose, fucose and GlcNAc residues via the carbohydrate recognition domains (CRD) 7, 24. Of the eight CRDs, CRD4-5 are sufficient to generate the affinity of the whole receptor for natural ligands. Moreover, the MR contains an N-terminal CR domain, demonstrated to bind novel sulfated saccharides 9, 25. In this study, we show that murine DC-expressed MR strongly binds to sulfated blood antigens such as 3-sulfo-LeA and GlcNAc. When these glycans were
conjugated to OVA, increased binding and uptake of the neo-glycoconjugates was see more detected compared to native OVA, which itself is mannosylated. Interestingly, 3-sulfo-LeA and tri-GlcNAc bind to different sites of the MR. Whereas tri-GlcNAc binds to the CRD, 3-sulfo-LeA binds the MR via the CR domain 8–10. Nevertheless these sulfated glycans exert similar potentiating Selleck JNK inhibitor effects. When chemically conjugated to OVA, these novel MR-specific ligands direct antigen more potently to the MR and enhance cross-presentation of antigens to CD8 T cells when compared to native OVA. This enhancement in cross-presentation is predominantly mediated by the MR as cross-presentation was greatly reduced in MR−/− splenic DCs. The fact that cross-presentation of the neo-glycoconjugates by MR−/− BMDCs was not completely abolished may be explained by binding Y27632 of these glycans to other receptors, such as SIGNR1 and SIGNR3 26, although their presence on myeloid DCs has not been formally shown. Although we could exclude the involvement of SIGNR1 since
SIGNR1−/− DCs did not show any reduced antigen binding and uptake (data not shown), we cannot completely exclude the involvement of other lectin receptors or processes such as pinocytosis in the uptake of these neo-glycosylated proteins. Thus, we concluded that the MR is predominantly involved in the enhanced induction of antigen presentation, due to this glycan modification. The potentiating effect of tri-GlcNAc may lie in its higher affinity for the MR than mannose resulting in increased responses 7. Since 3-sulfo-LeA binds the CR region instead of the CRD, it cannot compete with mannose. However, binding to the CR region might be with stronger affinity than of mannose to the CRD, although to our knowledge a direct comparison between these ligands and regions has not been described. CR-ligand binding may elicit stronger responses than CRD-ligand binding. This is underlined by the fact that the response to OVA-3-sulfo-LeA is stronger than to native OVA.