Figure 7.

Binding of mAb FA2B10 to CLEC12A induces receptor internalization in blood DC subsets. (A) PBMCs were labeled with biotin-conjugated anti-CLEC12A mAb FA2B10, followed by 0- to 60-min incubation at 37°C. Surface expression of bound anti-CLEC12A mAb was analyzed by flow cytometry following labeling with streptavidin BV421. cDC subsets were identified according to the expression of CD14, CD172a, and CADM-1 markers; pDCs were identified based on the expression of CD4. Results are representative of three independent experiments. (B) CLEC12A internalization was calculated relative to the surface expression in cells kept at 4°C based on the mean fluorescence intensity. Data are shown as mean+SD of three independent experiments. (C) Analysis by confocal microscopy of CLEC12A internalization in pDCs. Cells were stained with biotinylated anti-CLEC12A mAb FA2B10, followed by labeling with Alexa Fluor 488-conjugated streptavidin (green). Then, cells were incubated at either 37°C, for allowing internalization, or 4°C, to prevent it, and subsequently stained with anti-CD4 mAb 74-12-4, and Alexa Fluor 594-conjugated anti-mouse IgG2b (red). Yellow arrowheads show anti-CLEC12A green labeled complexes inside cell; bluish arrowheads point to surface located anti-CLEC12A.