(A) Raji cells were incubated with various mAbs (10 μg/ml) for 4 to 6 hours, at which point HA was assessed by light microscopy. The number of plus signs indicates the strength/extent of adhesion as assessed semiquantitatively by microscopic visualization. 20 hours later, samples were assessed for the extent of cell death following staining with AnV-FITC (AnV) and propidium iodide (PI) and flow cytometry. Bars represent the mean cell death (AnV- and PI-positive cells) + SEM from 3 to 7 independent experiments. Representative data are shown in B. A typical “apoptotic” plot is shown for reference following treatment of Raji cells with 4 Gy irradiation. Tos, tositumomab; Ritux, rituximab; NT, no treatment. (C) Raji cells were treated with various actin inhibitors prior to the addition of anti-CD20 or HLA-DR mAbs, and cell death was assessed 4 hours later as previously described. (D) Inhibitor of actin cytoskeleton at noncytotoxic concentrations protects cells from long-term cytotoxicity evoked by both anti-CD20 and HLA-DR mAbs. Prior to the addition of various mAbs (5 μg/ml), Raji cells were treated for 45 minutes with cytochalasin D (CytoD; 0.1 μM). Cell viability was assessed 24, 48, and 72 hours later using Cell Proliferation Kit II (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate [XTT] assay; Roche). Plot represents mean metabolic activity relative to nontreated control from 2 independent experiments run in 3 technical replicates. Data shown represent mean + SEM. (E) Raji cells were treated with DMSO or the actin inhibitor latrunculin B (LatB; 10 μM) prior to the addition of Tos or L243 (10 μg/ml) and assessed for HA 4 to 6 hours later. Original magnification, ×20. Together, these data clearly demonstrate that cell death evoked by both anti-CD20 and HLA-DR mAbs is dependent upon adhesion and that both these processes are dependent upon actin.