FcγRIIb participates in internalization of rituximab. (A) The quenching assay in Figure 1A was repeated after treating CLL cells for 6 hours with Alexa-488–labeled F(ab′)2 or IgG molecules of rituximab (both 5 μg/mL). *P < .05, Wilcoxon test. (B) The same assay was performed with Tosit-488 or Ritux-488 (both 5 μg/mL) ± addition of 10 μg/mL blocking AT10 in CLL cells. Medians ± ranges are shown (n = 6; **P < .001, Wilcoxon test). (C) Flow cytometric quantification of FcγRIIb expression in healthy B cells and various B-cell tumors using AT10-PE. Medians are shown (*P < .05, **P < .001, Mann-Whitney test). (D) FcγRIIb expression obtained in panel C correlated strongly with internalization of rituximab obtained in Figure 1 (after culture with Ritux-488 for 6 hours) across all lymphoma subtypes and normal B cells (Spearman r value = -.74; 95% confidence interval between −.83 and −.61; P < .0001). (E) Ramos cells transfected with FcγRIIb were sorted by flow cytometry to express low, medium and high levels of FcγRIIb and assessed in the quenching assay alongside mock-transfected cells after treatment with Tosit-488 or Ritux-488 (both 5 μg/mL) for 6 hours. Data are represented as medians ± ranges, n = 3. (F) The quenching assay was repeated with Tosit-488 or Ritux-488 (both 5 μg/mL) on Rx3 cells (which lack BCR expression), Ramos cells, mock-transfected Rx3 cells and FcγRIIb-transfected Rx3 cells afterincubation for 6 hours. Median values are shown, **P = .0079, Mann-Whitney test.
