Figure 5.
Direct lymphoma cell killing by CD20 antibodies of the IgA2 isotype. (A) Homotypic aggregation by CD20 antibodies was analyzed by phase-contrast microscopy using SU-DHL-4 target cells. Antibodies were added at 10 µg/mL for 24 hours. Representative images of 3 independent experiments are shown. (B) Induction of direct cell death induced by CD20 monoclonal antibody was analyzed by DiOC6 staining. Ramos cells were used as target cells, and antibodies were added at a fixed concentration (10 µg/mL) for 6 hours. (C) Lysosomal cell death was quantified by LysoTracker Red staining on Ramos cells after incubation with CD20 antibodies (10 µg/mL) for 6 hours. (D) Redistribution of CD20 to lipid rafts was analyzed by flow cytometry on Ramos cells. For discrimination between nonraft and lipid raft distribution, cells were treated with Triton X-100 after incubation with 10 µg/mL antibody for 6 hours. Residual fluorescence indicated Triton insolubility of formed lipid rafts. SU-DHL-4 cells were treated with the indicated CD20 antibodies at 10 µg/mL for 24 hours in the presence of the pan caspase inhibitor Z-VAD-FMK (E) or the cytoskeleton inhibitor LatB (F). Subsequently, cells were stained with annexin V and 7-aminoactinomycin to assess cell death by flow cytometry. (F) Lysosomal cell death was quantified by LysoTracker Red staining on Ramos cells after incubation with CD20 antibodies (10 µg/mL) for 6 hours. (G) Three OBI variants (IgG1, IgA2, and IgA2-V11L) were compared with regard to direct cell death induction. Lysosomal cell death was quantified by LysoTracker Red staining of Ramos cells after incubation with the antibodies (10 µg/mL) for 6 hours. Results of 3 independent experiments are shown as mean ± SEM, with the exception of (G), for which n = 1. *Significant difference specific antibody vs untreated/isotype control. #Significant difference lgG1 vs lgA2. XSignificant difference between with vs without inhibitor (Z-VAD-FMK or LatB). Significant differences (P ≤ .05) were calculated using ANOVA. Original mangnification x20 for panel A. AV, annexin V; ctrl., control; n.s., not significant; w/o, without; 7AAD, 7-aminoactinomycin.

Direct lymphoma cell killing by CD20 antibodies of the IgA2 isotype. (A) Homotypic aggregation by CD20 antibodies was analyzed by phase-contrast microscopy using SU-DHL-4 target cells. Antibodies were added at 10 µg/mL for 24 hours. Representative images of 3 independent experiments are shown. (B) Induction of direct cell death induced by CD20 monoclonal antibody was analyzed by DiOC6 staining. Ramos cells were used as target cells, and antibodies were added at a fixed concentration (10 µg/mL) for 6 hours. (C) Lysosomal cell death was quantified by LysoTracker Red staining on Ramos cells after incubation with CD20 antibodies (10 µg/mL) for 6 hours. (D) Redistribution of CD20 to lipid rafts was analyzed by flow cytometry on Ramos cells. For discrimination between nonraft and lipid raft distribution, cells were treated with Triton X-100 after incubation with 10 µg/mL antibody for 6 hours. Residual fluorescence indicated Triton insolubility of formed lipid rafts. SU-DHL-4 cells were treated with the indicated CD20 antibodies at 10 µg/mL for 24 hours in the presence of the pan caspase inhibitor Z-VAD-FMK (E) or the cytoskeleton inhibitor LatB (F). Subsequently, cells were stained with annexin V and 7-aminoactinomycin to assess cell death by flow cytometry. (F) Lysosomal cell death was quantified by LysoTracker Red staining on Ramos cells after incubation with CD20 antibodies (10 µg/mL) for 6 hours. (G) Three OBI variants (IgG1, IgA2, and IgA2-V11L) were compared with regard to direct cell death induction. Lysosomal cell death was quantified by LysoTracker Red staining of Ramos cells after incubation with the antibodies (10 µg/mL) for 6 hours. Results of 3 independent experiments are shown as mean ± SEM, with the exception of (G), for which n = 1. *Significant difference specific antibody vs untreated/isotype control. #Significant difference lgG1 vs lgA2. XSignificant difference between with vs without inhibitor (Z-VAD-FMK or LatB). Significant differences (P ≤ .05) were calculated using ANOVA. Original mangnification x20 for panel A. AV, annexin V; ctrl., control; n.s., not significant; w/o, without; 7AAD, 7-aminoactinomycin.

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