Immature dendritic cells efficiently phagocytosed B-CLL cells killed by CD47-induced caspase-independent pathway.

Freshly isolated B-CLL cells were cultured in the presence of soluble or immobilized CD47 mAb (10 μg/mL), control mAb (10 μg/mL), or HC (5 × 10⁻⁴ M). (A) Cells were double-stained with FITC-labeled annexin-V and PI (upper graphs) or with DiOC₆(lower graphs) and analyzed by flow cytometry. Shown are percent of dead cells (annexin-V⁺ or DiOC$6low$⁠). (B) Soluble (lane 1) or immobilized (lane 2) CD47 mAb-treated cells were lysed, and Western blot analysis was performed for detection of caspases 3, 7, 8, and 9 cleavage products (as indicated by *). Asterisks indicate “expected molecular weight” of caspase cleavage products; arrowheads, molecular weight of procaspases. (C) Caspase 3 activity was measured after 48 hours by flow cytometry using the cell permeable fluorogenic substrate DEVDase. (D) B-CLL cells were stained in red with PKH26 linker before treatment overnight with soluble or immobilized CD47 mAb or HC. Cells were then cocultured for 3 hours with iDCs at a 10:1 ratio. The mixture was analyzed by flow cytometry for red fluorescence (FL2) after gating on iDCs. Shown are percent of iDCs that have phagocytosed red dead cells. One representative experiment of 3 is shown.