Tn-glycosylated antigens target dermal dendritic cells (dDCs) in vivo. (A) Analysis of macrophage galactose-type lectin expression (bold line) in Langerhans cells (CD11c⁺ major histocompatibility complex [MHC]) II⁺ epithelial-cell adhesion molecule positive [EpCam⁺] CD11b⁺ Langerin⁺ CD103⁻), CD103⁺ dDC (CD11c⁺ MHCII⁺ EpCam⁻ CD11b⁻), and CD103⁻ dDC (CD11c⁺ MHCII⁺ EpCam⁻ CD11b⁺ CD103⁻) from BALB/c mouse skin. Isotype control staining is shown as a gray histogram. (B) CD45⁺ CD11c⁺ cells from the dermis were incubated at 4°C or 37°C with Alexa⁶⁴⁷-labeled antigen glycosylated multiple antigenic glycopeptide (MAG):Tn3-poliovirus (PV; left panel), MUC6:Tn(MCF7) (right panel), or nonglycosylated controls MAP-PV and MUC6, respectively. Antigen binding and uptake were analyzed in the presence or absence of ethylenediaminetetraacetic acid, anti–macrophage galactose-type lectin monoclonal antibody (ERMP23), or control Ig2a (FACS-Aria II; BD Biosciences). (C) Interstitial DCs from dermis were incubated with glycosylated MAG:Tn3-PV or nonglycosylated multiple antigenic peptides-PV and cultured with a PV-specific T-cell hybridoma. IL-2 release by the T-cell hybridoma was analyzed in the 24-hour supernatant by enzyme-linked immunosorbent assay. (D) Alexa⁶⁴⁷- or (E) Alexa⁴⁸⁸-labeled MAG:Tn glycopeptides or nonglycosylated multiple antigenic peptides-PV were injected intradermally into the right ear of mice. (D) Eighteen hours later, antigen capture was analyzed by flow cytometry in CD45⁺ CD11c⁺ Langerin⁻ cells present in the dermis, by comparison with antigen-free dermis of the left ear. (E) Alternatively, cells that have captured labeled antigen were directly analyzed from skin by gating on Langerhans cells (gray line), CD103⁺ (dashed line), and CD103⁻ (bold line) dDCs. Mean fluorescence intensities (MFI) are shown in the boxes. Representative results from 3 experiments are shown.