Importantly, although no signaling-motif is recognized in the cyt

Importantly, although no signaling-motif is recognized in the cytoplasmic tail, the MR has been shown to be essential for cytokine production, both pro- and anti-inflammatory. However, the outcome is dependent on TLR co-triggering by pathogens or synthetic ligands. Mannose-capped lipoarabinomannans from Mycobacterium tuberculosis inhibited LPS-induced pro-inflammatory cytokine production by DCs 18, whereas Candida albicans-derived mannan triggers IL-17 production 19. In this study,

we exploited the feature of the selleck compound library MR to cross-present antigens, aiming to generate more potent activation of tumor-specific T cells. To this end, we selected two glycan ligands of the MR other than mannose, of which one also has a different binding

site than mannose that showed profound binding to bone marrow-derived DCs (BMDCs) and ex vivo purified splenic DCs. These ligands, 3-sulfo-LeA and tri-GlcNAc, were conjugated to the model antigen OVA to examine their potency to enhance antigen presentation in MHC class I and II, as well as Th differentiation. The glycan-binding specificity of the MR is not solely restricted to mannose. Using purified MR-Fc fusion proteins, also sulfated and GlcNAc glycan moieties were shown to bind 7, 9. We investigated whether we could use these GlcNAc and sulfated glycan structures to specifically target antigen to the MR. First, expression of MR on BM-DCs and splenic DCs was confirmed. DCs were either cultured from BM or ex vivo isolated from the spleen from C57BL/6 mice and MR expression was analyzed using Smoothened antagonist flow cytometry. Both, CD11c+ BMDCs and splenic DCs expressed significant levels of MR protein on Methane monooxygenase their cell surface (Fig. 1A), herewith confirming previous

reports 20, 21. Subsequently, binding of GlcNAc and sulfated glycan structures was examined by incubating DCs with biotinylated polyacrylamide (PAA)-conjugated glycans at 4°C. Streptavidin-Alexa488 was used to visualize bound glycans. From Fig. 1, it is clear that BMDCs bind GlcNAc and chitobiose (GlcNAcβ1-4GlcNAc; di-GlcNAc) as well as the sulfated blood group antigens 3-sulfo-LeA [HSO3-3Galβ1-3(Fucα1-4)GlcNAc] and 3-sulfo-LeX [HSO3-3Galβ1-3(Fucα1-3)GlcNAc] (Fig. 1B). The PAA-conjugated control structure glucitol did not bind to BMDCs. Surprisingly, when purified CD11c+ splenic DCs were used, we observed significant binding of PAA-conjugated 3-sulfo-LeA and di-GlcNAc but not of 3-sulfo-LeX. This can either be due to low specificity of MR for 3-sulfo-LeX or the involvement of another glycan-binding receptor on BMDCs with specificity for 3-sulfo-LeX, which is absent on splenic DCs. Together, these results show that sulfated blood group antigens and GlcNAc glycan structures can interact with murine DCs.

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