Rituximab binds CD20 and FcγRIIb in a cis fashion and activates FcγRIIb. (A) Raji cells were stimulated with tositumomab, rituximab ± AT10, or AT10 alone (all 10 μg/mL) for 2 hours and assessed for phosphorylated FcγRIIb by Western blotting. (B) PKH26-labeled Ramos cells (FcγRIIb⁻, R1) were mixed 1:1 with sorted, high FcγRIIb-expressing Ramos transfectants as described in “Surface fluorescence quenching assay” (R2, left). FcγRIIb⁺ and FcγRIIb⁻ cells were cultured alone, or mixed, and treated with 5 μg/mL Ritux-488 for 6 hours (right) and assessed in the quenching assay. Data are represented as medians ± ranges (n = 3). (C) As before, a low FcγRIIb-expressing CLL sample was PKH26-labeled, mixed 1:1 with a high FcγRIIb-expressing CLL sample and assessed in the quenching assay after treatment with 5μg/mL Ritux-488 for 6 hours. Data are represented as medians ± ranges (n = 3). (D) CLL cells were cultured with Ritux-488 at various densities shown and the quenching assay performed after treatment with 5 μg/mL Ritux-488 for 6 hours as before. (E) Raji cells were cultured at decreasing densities and assessed by Western blotting for phosphorylated FcγRIIb. Representative images from these experiments demonstrate differences in cell proximity. Cells in were viewed while in culture using an Olympus CKX21 inverted microscope with a 10×/0.25 PH lens and 10× magnification was used. Bar represents 100 μm.