We questioned whether targeting DCs with OVA-3-sulfo-LeA or OVA-tri-GlcNAc influenced CD4+ T-cell polarization BMN 673 in vivo rather than proliferation. Thereto, naive OVA-specific CD4+CD62Lhigh T cells were co-cultured with neo-glycoprotein-pulsed CD11C+ splenic DCs and 1 wk later production of cytokines related to Th1-, Th2 and Th17-differentiation was analyzed using flow cytometry. We compared this with the profile of T cells differentiated by native OVA pulsed CD11C+ splenic DCs. DCs targeted with either neo-glycoconjugate

generated significantly higher frequencies of IFNγ-producing CD4+ T cells compared to native OVA-loaded DCs (Fig. 4, left panel). By contrast, OVA-3-sulfo-LeA and OVA-tri-GlcNAc either reduced or did not affect the frequency of IL4 or IL17-producing MG-132 ic50 T cells, respectively (Fig. 4, middle and right panel). These data imply that 3-sulfo-LeA- and tri-GlcNAc-glycosylated antigens that target efficiently to the MR on DCs result in induction

of IFNγ-producing effector T cells. As targeting of the MR with OVA-3-sulfo-LeA and OVA-tri-GlcNAc resulted in enhanced cross-presentation to CD8+ T cells, we investigated the intracellular routing of native OVA and OVA-3-sulfo-LeA into BMDCs derived from C57BL/6 and MR−/− mice. To this end, BMDCs were incubated with fluorescent-labeled OVA or OVA-3-sulfo-LeA. Two hours later, cells were washed and co-stained for MR, EEA-1 (endosomal marker) or LAMP-1 (lysosomal marker) and analyzed using confocal microscopy. We observed that OVA and OVA-3-sulfo-LeA science (red) that bind to the MR (green, co-localization with

OVA appears yellow) co-localized with the endosomal marker EEA-1 (blue, co-localization OVA-MR-EEA-1=cyan) (Fig. 5A and B). This co-localization is also clearly observed when fluorescence images are converted into histograms (indicated by arrows). Surprisingly, we observed that co-localization of the MR-bound OVA-3-sulfo-LeA with EEA-1 was higher compared to native OVA. In addition, we assessed that the internalized OVA-3-sulfo-LeA did not co-localize with the lysosomal marker LAMP-1, but only with the MR (data not shown). The uptake of OVA and OVA-3-sulfo-LeA in BMDCs derived from MR−/− was dramatically decreased (Fig. 5C and D). These data correlate with the data on binding and antigen presentation demonstrating that OVA-3-sulfo-LeA targeted to the MR results in increased internalization of antigen to the endosomal compartment to facilitate loading of antigen to MHC class I molecules leading to enhanced cross-presentation to CD8+ T cells. Here, we show that DC-expressed MR is capable of binding sulfated glycans such as 3-sulfo-LeA or GlcNAc besides mannose glycans, present on native OVA.