Rituximab and FcγRIIb internalize together into lysosomes. (A) CLL cells were untreated, or treated with rituximab for 30 minutes on ice, washed, and then stained with AT10-PE and analyzed by flow cytometry. Representative histograms from 3 patients are shown. (gray-filled: isotype control; black line: AT10 expression of untreated cells; gray line: AT10 expression of rituximab treated cells). (B) CLL cells were incubated with Tosit-488 or Ritux-488 (5 μg/mL) for 2 hours and then FcγRIIb expression detected using AT10-PE. Data are represented as medians ± ranges (n = 6; *P = .0313, Wilcoxon test). (C) Untreated CLL cells were cultured for 6 hours, then fixed and permeabilized with saponin. Intracellular FcγRIIb was detected using AT10-647 (blue) and the cells analyzed by confocal microscopy. (D,E) Cells from the same CLL sample as in panel C were cultured with Ritux-488 (green) for 30 minutes (D) or Tosit-488 (green; E) for 6 hours and then treated as described in panel C for detection of intracellular FcγRIIb with AT10-647. In addition the Tosit-488 cultured cells were also so stained with biotinylated LAMP-1 and streptavidin-546 (red) to detect lysosomes. (F) CLL cells were treated with Ritux-488 for 6 hours and assessed as in panel E. 2 representative cells are shown. The top cell shows unambiguous colocalization between Ritux-488 and FcγRIIb, but no colocalization with LAMP-1. The bottom cell shows colocalization of all 3 mAb. In each case the bright field image is shown from the same cell. A Leica TCS-SP5 laser scanning confocal microscope with 10× eye piece, 100× objective oil immersion lens was used in each case. Bar represents 5 μm.