Alemtuzumab binds neutrophil CD52 and induces complement-mediated lysis. (A) Unseparated leukocytes were stained with various dilutions of alemtuzumab. Gating on lymphocytes (R1 in panel i) and neutrophils (R2 in panel i), the flow cytograms show that alemtuzumab binds neutrophil CD52 but requires higher concentrations for detection (iii; 3-4, 100-300 μg/mL), compared with lymphocyte binding (ii; 1-4, 1-300 μg/mL). (B) Gating on a purified neutrophil population (i) stained with CD16 (ii) and alemtuzumab (iii) confirms the binding of alemtuzumab to neutrophils. (C) Neutrophils and mononuclear cells were lysed in a dose-dependent manner in the presence of alemtuzumab with either purified rabbit complement (i) or autologous serum (ii) as the complement source. The anti–HLA class I antibody W6/32 induced cell death in the presence of rabbit complement (i), but not autologous serum (ii), due to the inhibition of autologous complement activation by complement regulators (such as decay accelerating factor and CD59) expressed on PBMCs and neutrophils. In the autologous serum condition, alemtuzumab itself served as the positive control antibody, as its ability to activate human complement is not inhibited by complement regulators. Open histograms represent staining with alemtuzumab; shaded histograms represent staining with an isotype-matched control mAb.