Lenalidomide promotes time-dependent CD154 transmembrane protein expression. (A) CD19⁺ CLL cells were incubated with lenalidomide 0.5μM and vehicle control. CD154 expression was analyzed by flow cytometry after 48 hours of treatment. The graph shows fold change in percentage (%) of CD154⁺ cells in the lenalidomide-treated group compared with vehicle control (n = 30, P < .001). (B) CD19⁺ CLL cells were incubated with lenalidomide 0.5μM and vehicle control. CD154 expression was analyzed by flow cytometry after 24, 48, and 72 hours. The graph shows fold change in percentage (%) of CD154⁺ cells in the lenalidomide-treated group compared with vehicle control (n = 5, P = .006). (C) The panel shows 48-hour results for CD154 expression from a representative experiment. Black line denotes lenalidomide-treated cells and dark gray line denotes vehicle-treated CLL cells. Control IgG1k-PE is shown (light gray line). (D) CLL B cells were treated with vehicle (V) or 0.5μM lenalidomide (L) and after 48 hours, cell-surface proteins were biotinylated, and lysates were prepared. For each condition, 400 μg of protein lysate was subjected to immunoprecipitation using anti-CD154 or anti-IgG1 antibodies. The 39- and 32-kDa bands, corresponding to the 2 transmembrane forms of CD154, are indicated with arrows. Immunoblot shown is representative of 2 patient samples of 5 tested. Error bars represent SD. *Statistical significance.