Lenalidomide inhibits CLL-cell proliferation. (A-B) CFSE-labeled CLL cells from 3 different patients were cocultured with Fibroblasts_CD154 and IL-4/IL-10 for 48 hours and exposed to increasing single doses of lenalidomide or DMSO as vehicle control for 7 days, at which point, the cells were harvested and analyzed by flow cytometry for proliferation. (A) CFSE profiles are presented for cells of a representative patient. (B) The fraction of dividing CLL cells present with increasing doses of lenalidomide were determined using FlowJo software, by establishing the nondividing cells based on unstimulated, CFSE-labeled CLL cells. Data from 3 patients are presented. One-way ANOVA and Tukey’s multiple comparison test were used to determine statistically significant differences from day 1. *P < .05; ***P < .001 (mean ± SEM; n = 3). (C) CLL cells from 3 different patients were stimulated with IL-4/IL-10 in the presence of Fibroblasts_CD154 and exposed to increasing doses of lenalidomide or DMSO as control. Cell-cycle analysis was performed by flow cytometry after 6 days using PI staining, as described in “Methods.” Percentage of cells in G₀/G₁, S, or G₂/M were assessed using the cell-cycle analysis tool from FlowJo software and are presented as boxes indicating the median, minimum, and maximum values of cell proportions in each phase of the cell cycle. One-way ANOVA and Tukey’s multiple comparison test were used to determine statistically significant differences from control. *P < .05; **P < .01; ***P < .001 (mean ± SEM; n = 3). (D) CLL cells from 3 patient samples were cocultured on Fibroblasts_CD154 in the presence of IL-4 and IL-10, and increasing doses of lenalidomide from day 2 of coculture. The fraction of viable CLL cells were measured after 7 days of treatment by flow cytometry using 7-AAD. Live cells were identified as 7-AAD–negative cells (mean ± SEM; n = 3).