FcγRIIb1 in trans does not inhibit the rate of anti-CD20 mAb internalization. (A) CD20-negative FcγRIIb-negative RPMI 8226 cells were transfected with empty vector or human FcγRIIb1 and stable transfectants were selected expressing different levels of FcγRIIb1 as assessed by flow cytometry. Control cells (filled histogram), FcγRIIb1 low cells (dotted line), and FcγRIIb1 high cells (dashed line) were stained with RTX F(ab′)2-A488 (top left panel) and AT10-PE (top right panel). Equivalent staining of Ramos control cells transfected with empty vector (solid histogram) and Ramos FcγRIIb1 high cells (dashed line) stained with RTX F(ab′)2-A488 (bottom left panel) and AT10-PE (bottom right panel) is included for comparison. (B-C) Unlabeled Ramos FcγRIIb1-high cells were mixed 1:1 with PKH26-labeled RPMI 8226 FcγRIIb-negative control, RPMI 8226 FcγRIIb1-low or RPMI 8226-high cells and cultured with 5 µg/mL A488-labeled anti-CD20 mAb for 1 hour. The proportion of total mAb remaining on the cell surface of PKH26-negative Ramos FcγRIIb1 high cells was assessed by flow cytometry after treatment of cells with anti-A488 to quench cell-surface fluorescence. (B) Dot plot showing the gating strategy used to discriminate PKH26-positive RPMI 8226 transfectants from PKH26-negative Ramos FcγRIIb1-high transfectants. (C) Ramos FcγRIIb1-high transfectants cocultured with FcγRIIb-expressing RPMI 8226 and RPMI 8226 FcγRIIb-negative control cells were compared using the Mann-Whitney U test. Horizontal bars represent the median. *P < .05; n = 6.
