document: We addressed the question whether carbohydrate coupling increased antigen uptake by DCs via C-type lectin receptor targeting. Therefore, the antigens were labeled with pHrodo Red dye (Invitrogen), a dye that specifically fluoresces as pH decreases from neutral to acidic, as provided in endosomes/lysosomes of cells. In vitro characterization of the cellular uptake of neoglycocomplexes using bone marrow derived dendritic cells (BMDCs) demonstrated superior ingestion of mannan-conjugates MN–Ova and MN–Pap (Supplementary Fig. S4A-D,F). This was confirmed in vivo by intradermal needle-injection of labeled antigen into the ear pinnae of mice. Antigen uptake and transport to the ear dLNs were measured after 24h by FACS analysis. DCs in cervical LNs were identified according to their high expression of MHC class II (Fig. 3A) and additionally characterized by CD8α, CD11b, and CD11c expression and uptake of pHrodo-labeled antigen (Fig. 3B, D–F). The results showed significantly elevated numbers of pHrodo+MHCIIhigh DCs for mannan conjugates MN–Ova and MN–Pap (and to a lesser degree for MD–Pap) in comparison to the unmodified antigens (Fig. 3C). Both carbohydrates targeted antigen preferentially to CD8α− DCs, as indicated by an increase in CD8α−/pHrodo+ DCs compared to unmodified antigens (Fig. 3E and F). Nevertheless, whether the antigens were taken up in situ by dermal DCs or by LN resident APCs via the afferent lymphatics could not be fully elucidated. Histology revealed that antigen-loaded cells in the dLNs were already present 30min after intradermal injection (Supplementary Fig. S4G), suggesting both mechanisms.
doc_bio_tags: O O O O O O O O B O O O O O O O O O O B O O O B I I B O O O O B O O O O O O O O O O O O O O O O O O O O O B O B I I I I B O O B O O B O B O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B B O B O O O O O B O O O O O O O O O O O O O O O B O B O O O O O O B O O O O O B O O O B O B O O B I O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O
extractive_keyphrases: antigen antigens bmdcs bone marrow derived dendritic cells carbohydrates cd11b cd11c cd8α cd8α− dcs facs analysis fluoresces ingestion intradermal injection invitrogen md–pap mn–ova mn–pap neoglycocomplexes phrodo red dye
abstractive_keyphrases: antigen-loaded cells cd8α−/phrodo+ dcs c-type lectin receptor targeting endosomes intradermal needle-injection lysosomes mannan-conjugates mn–ova and mn–pap phrodo-labeled antigen phrodo+mhciihigh dcs whether carbohydrate coupling increased antigen uptake