In vitro mechanisms of TCR-like mAbs-mediated antitumor activity. (A) Representative annexin-V staining histograms of BLCLs treated with TCR-like mAbs. BLCLs were incubated with 10 or 100 μg/mL mAbs and were harvested at 6 or 24 hours for detection of surface phosphatidylserine via annexin-V staining. Camptothecin (1 μM) was used as a positive control. (B) Immunofluorescence microscopy showing an EBV BLCL (green) in contact with a macrophage (red) (middle), as well as BLCL engulfment by a macrophage (bottom). Images were obtained using Olympus IX81 inverted microscope MetaMorph software at 40× objective. Scale bars, 10 μm. (C) Representative dot plots from phagocytosis assay. BLCLs were preincubated with the mAbs for 24 hours (pretreatment) or without pretreatment before CFSE labeling, further TCR-like mAbs staining, and subsequent coculturing with macrophages. Phagocytosed cells were defined as the CD11b-PE⁺ CFSE⁺ population. (D) Immunofluorescence microscopy images visualizing the presence of CFSE-labeled BLCLs within macrophages (as indicated by white arrows) under the stated conditions. Scale bars, 20 μm. (E, F) Tabulated percentages of CD11b⁺ CFSE⁺ cells and phagocytic index from phagocytosis assays. Results represent the average of 2 independent experiments, expressed as mean ± SD. *P < .05; **P < .01 (unpaired Student t test). (G, top) Illustration denoting the point mutation from aspartate to alanine at position 265 of the antibody Fc region, abrogating Fc receptor binding. (Middle) Representative SDS-PAGE gels of D265A antibody variants treated with nonreducing (−dithiothreitol) or reducing (+dithiothreitol) conditions. (Bottom) Histogram plots comparing the unmodified mAb and D265A antibody variants of the 3 TCR-like mAbs. (H) Representative flow cytometry plots from phagocytosis assays using the respective unmodified and D265A mAbs.