Lenalidomide induces CD20 internalization. (A) Cells were incubated with 50 μg/mL rituximab to block extracellular CD20, and then washed 3 times, fixed and stained with CD20-PE antibody, and analyzed by flow cytometer. In 6 of 8 patients, CD20 antigen down-regulation on the CLL cell surface is accompanied by increased intracellular fluorescence (P = .006). (B) Binding and internalization of anti-CD20 in CLL cells were examined by laser scanning confocal microscopy. CLL cells were incubated with lenalidomide for 48 hours at 37°C. Cells were washed, fixed, permeabilized, and stained for CD20 (green), concanavalin A (red), and DRAQ5 (blue). The images were examined by laser scanning confocal microscopy. (C) Lenalidomide treatment does not alter total level of CD20 protein. CD19⁺ cells were incubated with lenalidomide (0.5 μM) or vehicle control. CD20 total protein level was analyzed by Western blot after 48 and 72 hours of treatment. Results are shown from 2 representative patients who showed down-modulation of surface CD20 expression.