Binding of hD1 to DC-SIGN and internalization by DCs. (A) iDCs were treated with 10 μg/mL hD1 (gray shaded curve), 100 μg/mL AZN-D1 (open dotted curve), or pretreated with 100 μg/mL AZN-D1 followed by 10 μg/mL hD1 incubation (open solid curve), followed by incubation with an Alexa Fluor 647–labeled goat anti–human IgG antibody. Binding of hD1 was analyzed by flow cytometry. (B) iDCs were incubated with AZN-D1 (•), hD1 (○), or AZN-L19 (□) at 4°C for one hour, and transferred to 37°C. Cells were fixed at various time points, and stained with Alexa Fluor–labeled secondary antibodies with or without prior permeabilization. The mean fluorescence was determined by flow cytometric analysis, and the amount of internalized antibody was plotted as a percentage of the amount of total cell-associated antibody. Data represent experiments performed in triplicate ± SD. (C) Internalization of hD1 was confirmed by CSLM. iDCs were incubated with hD1, AZN-D1, or their isotype controls 5G1.1 and mouse IgG1 (mIgG) for one hour at 37°C. Cells were stained with Alexa Fluor 647–labeled secondary antibodies (blue), followed by microscopic analysis. The image represents the middle focal plane of the DCs, with iris set at 2 nm. Original magnification, ×600.