Figure 2
Figure 2. Enhancement of CD20 capping is specific for rituximab and B cells. (A) Daudi/CD20-GFP cells were incubated alone; or in the presence of 10 μg/mL rituximab (Rtx) or 10 μg/mL mouse CD20-targeting antibody 2H7; or preincubated with 20 µg/mL CD32-blocking antibody followed by incubation with 10 μg/mL rituximab (Rtx+CD32); or incubated with 20 μg/mL of a monovalent version of rituximab (Rtx mono). Number of cells with polarization of CD20 was scored. Graph represents mean ± SEM of 3 independent experiments. More than 160 cells were analyzed per condition. (B) Fluorescent and bright-field images of Jurkat/CD20-GFP cells untreated (left panels) or incubated with 10 μg/mL rituximab with uniform distribution of CD20 or CD20 capped on 1 side (middle and right panels, respectively). (C) Capping of CD20 was quantified on images of Jurkat/CD20-GFP cells incubated in the absence or presence of 10 μg/mL rituximab. Graph represents mean ± SEM of 3 independent experiments. A total of 112 to 118 cells were analyzed per condition. (D) Amount of CD20 accumulated in the cap in Jurkat/CD20-GFP cells pretreated with 10 μg/mL rituximab was compared with the amount of CD20 accumulated in the cap in primary B cells pretreated with 10 μg/mL rituximab. Data were analyzed by unpaired t test (2-tailed). A total of 21 to 31 cells from 3 or 4 experiments were analyzed per condition. (E) Daudi/CD20-GFP (CD20 WT) or Daudi cells expressing a mutant form of CD20-GFP (CD20 mut) were left untreated or incubated with rituximab and then fixed and analyzed by laser scanning confocal microscopy. The proportion of cells with polarized CD20 is shown. Graph represents mean ± SEM of 3 independent experiments. Data were analyzed by unpaired t test (2-tailed). More than 200 cells were analyzed per condition. (F) The amount of CD20 accumulated in the cap in Daudi cells expressing a mutant form of CD20-GFP (CD20 mut) pretreated with 10 μg/mL rituximab was compared with the amount of CD20 accumulated in the cap in Daudi/CD20-GFP (CD20 WT) cells pretreated with 10 μg/mL rituximab. A total of 31 cells from 2 or 3 experiments were analyzed per condition. Data were analyzed by unpaired t test (2-tailed). **P < .01; ***P < .001. Ab, antibody.

Enhancement of CD20 capping is specific for rituximab and B cells. (A) Daudi/CD20-GFP cells were incubated alone; or in the presence of 10 μg/mL rituximab (Rtx) or 10 μg/mL mouse CD20-targeting antibody 2H7; or preincubated with 20 µg/mL CD32-blocking antibody followed by incubation with 10 μg/mL rituximab (Rtx+CD32); or incubated with 20 μg/mL of a monovalent version of rituximab (Rtx mono). Number of cells with polarization of CD20 was scored. Graph represents mean ± SEM of 3 independent experiments. More than 160 cells were analyzed per condition. (B) Fluorescent and bright-field images of Jurkat/CD20-GFP cells untreated (left panels) or incubated with 10 μg/mL rituximab with uniform distribution of CD20 or CD20 capped on 1 side (middle and right panels, respectively). (C) Capping of CD20 was quantified on images of Jurkat/CD20-GFP cells incubated in the absence or presence of 10 μg/mL rituximab. Graph represents mean ± SEM of 3 independent experiments. A total of 112 to 118 cells were analyzed per condition. (D) Amount of CD20 accumulated in the cap in Jurkat/CD20-GFP cells pretreated with 10 μg/mL rituximab was compared with the amount of CD20 accumulated in the cap in primary B cells pretreated with 10 μg/mL rituximab. Data were analyzed by unpaired t test (2-tailed). A total of 21 to 31 cells from 3 or 4 experiments were analyzed per condition. (E) Daudi/CD20-GFP (CD20 WT) or Daudi cells expressing a mutant form of CD20-GFP (CD20 mut) were left untreated or incubated with rituximab and then fixed and analyzed by laser scanning confocal microscopy. The proportion of cells with polarized CD20 is shown. Graph represents mean ± SEM of 3 independent experiments. Data were analyzed by unpaired t test (2-tailed). More than 200 cells were analyzed per condition. (F) The amount of CD20 accumulated in the cap in Daudi cells expressing a mutant form of CD20-GFP (CD20 mut) pretreated with 10 μg/mL rituximab was compared with the amount of CD20 accumulated in the cap in Daudi/CD20-GFP (CD20 WT) cells pretreated with 10 μg/mL rituximab. A total of 31 cells from 2 or 3 experiments were analyzed per condition. Data were analyzed by unpaired t test (2-tailed). **P < .01; ***P < .001. Ab, antibody.

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