Cell surface CLEC9A and DEC205 are internalized into BDCA3⁺ mDCs after receptor triggering. (A) CLEC9A internalization is induced in immature freshly isolated BDCA3⁺ mDCs by receptor triggering with αCLEC9A antibodies. (B) DEC205 internalization is induced by receptor triggering with αDEC205 antibodies on immature freshly isolated BDCA3⁺ mDCs (squares) and BDCA3⁺ mDCs matured overnight with 2 μg/mL poly I:C and 4 μg/mL R848 (triangles). BDCA3⁺ mDCs were labeled with αCLEC9A or αDEC205 antibodies on ice. Surface expression of antigen and antibody after incubation at 37°C was analyzed by labeling with Alexa 488–conjugated secondary antibodies. Data are expressed as percentage of surface expression at 4°C. Data shown are mean ± SD of 3 independent experiments. **P < .01; ***P < .001 compared with incubation at 4°C (t = 0). #P < .05; ##P < .01 immature vs mature BDCA3⁺ mDCs. (C) Confocal analysis of CLEC9A internalization in freshly isolated BDCA3+ mDCs. BDCA3+ mDCs were stained on ice with rabbit polyclonal αCLEC9A antibodies. Internalization at 37°C was allowed for 45 minutes, followed by staining with biotinylated goat anti–rabbit and Alexa 488–conjugated streptavidin (red). Extracellular MHC class II was stained with mouse anti–human HLA-DR/DP, followed by Alexa647-conjaged goat anti–mouse (green).