Tn-glycosylated antigens target human dermal DCs. Representative results from 3-5 experiments are shown in panels A, B, and D; panel C corresponds to a single experiment. Unstained cells are shown as dashed line. (A) Human monocytes, MoDCs, CD34⁺-derived Langerhans cells, and dermal-like DCs were tested for macrophage galactose-type lectin expression using macrophage galactose-type lectin antibodies (bold line) or isotype control antibodies (shaded gray; FACS-Aria II, BD Biosciences). (B) Alternatively, prior to FACS analysis, these cell types were incubated at 4°C, as indicated, with Alexa⁴⁸⁸-labeled glycosylated multiple antigenic glycopeptide (MAG):Tn3-TT (bold line), or unglycosylated multiple antigenic peptides (MAP):TT (shaded gray), or medium (dashed line; FluoProbes Protein Labeling Kit, Interchim). (C) Monocyte-derived DCs were incubated at 4°C with Alexa⁶⁴⁷-labeled MUC6-Tn (bold line), MUC6 (shaded gray), or medium (dashed line). (D) Incubation of MAG:Tn3-TT with dermal-like DCs was performed in the presence (shaded gray) or absence (bold line) of ethylenediaminetetraacetic acid to evaluate divalent cation-dependency binding to dermal DCs.