CD20 mAb-mediated lymphoma depletion is complement independent in vivo. (A) Reactivity of CD20 mAbs with spleen B cells in B6 (left panel) and BUB (right panel) mice. Mean fluorescence intensity (MFI) of IgG2a MB20-18 (■) and MB20-18^K322A (□) staining over a range of mAb concentrations. Staining was visualized using PE-conjugated isotype-specific secondary Abs with flow cytometric analysis. Results represent those obtained in 3 or more experiments. (B) In vitro complement-dependent cytotoxicity of A20 cells by MB20 mAbs. Values represent mean (± SEM) percentages of B220⁺ cells that were PI⁺ in 3 or more experiments. (C) Serum C3 levels in B6 and BUB mice determined by ELISA after injection of MB20-18 (■) or MB20-18^K322A (□) mAb (downward ). Results represent 2 different experiments with 4 mice for each group and strain. C3 values in CVF-treated mice are shown (▴, n = 3 mice) in the left panel. Significant differences between sample means are indicated (*P < .05; **P < .001). (D) MB20-18 (■) or MB20-18^K322A (□) mAb depletion of B cells in B6 and BUB mice. Bone marrow (mature IgM⁺B220^hi), blood (B220⁺), spleen (mature CD24⁺CD21⁺B220⁺), and peripheral lymph node (B220⁺) B-cell numbers were determined 7 days after mAb treatment at the indicated mAb doses. Values (± SEM) represent percentages of B cells present in mAb-treated mice relative to control mAb-treated littermates (≥ 2 mice per value). (E) B6 mouse survival following transfer of 10⁵ BL3750 cells subcutaneously on day 0 with MB20-18 (■), MB20-18^K322A (□), or control (○) mAb (100 μg/mouse; n = 6 mice/group) given intravenously on day 1 (downward ). (F) Mouse survival following transfer of 10⁵ BL3750 cells subcutaneously on day 0 with CD20 (MB20-11) or control mAb (250 μg/mouse) given intraperitoneally on day 1 (downward ). CVF or PBS was given on days 0, 3, 5, and 9 (downward ). Mice were treated with CVF and CD20 (■) or control (□) mAb. As controls, PBS was given along with CD20 (•) or control (○) mAb treatments (n = 5 mice/group).