Apoptosis induced by anti-CD20 mAbs. (A) Raji cells were either preincubated with ethyleneglycoltetraacetic acid (1.5 mM, Sigma-Aldrich) or not for 15 minutes at room temperature before addition of different rituximab variants (10 μg/mL). The cells were immediately split into 3 aliquots. Goat anti–human κ F(ab′)2 fragment (20 μg/mL) was added to one aliquot, goat anti–human κ F(ab′)2 fragment (20 μg/mL) plus excessive CaCl2 to the second, and medium to the third. After 16 hours of incubation, apoptotic cells were scored by single staining of annexin V–Fluos on a flow cytometer. (B) Intracellular calcium mobilization initiated by anti-CD20 mAb with or without hyper-cross-linking. Fluo-3–loaded Raji cells were either preincubated 1mM ethyleneglycoltetraacetic acid or not for 15 minutes at room temperature before addition of distinct rituximab variants (10 μg/mL). The cells were immediately split into 3 aliquots. Goat anti–human κ F(ab′)2 fragment (20 μg/mL) was added to 1 aliquot and medium to the second. The third aliquot was incubated with goat anti–human κ F(ab′)2 fragment for 5 minutes, followed by the addition of excessive calcium. Samples were analyzed using a FACScan flow cytometer with excitation at 480 nm. Data are mean ± SD (n = 3). (C-D) Effect of ZVAD (C) and VDVAD (D) on the apoptosis induced in 16 hours by inhibitor alone, 10 μg/mL rituximab, and triple variant in the presence or absence of cross-linker (goat anti–human κ F(ab′)2 fragment, 20 μg/mL) in Daudi cells. Inhibitors (ZVAD and VDVAD) were added over a range of different concentrations for 2 hours before the addition of mAbs. Data are mean ± SD of at least 3 experiments.
