IL-21 plus CD40 signaling induces proliferation in CLL cells. (A) CFSE-stained CLL cells were cultured after various stimulations as indicated. After 6 days, the percentage of divided cells was calculated with FlowJo. Results are depicted as mean ± standard deviation (SD) from samples 5A, 12A, and 15. (B) CFSE-stained CLL cells were cultured with CD40L-expressing fibroblasts in the absence or presence of IL-21 (25 ng/mL) or IL-2 (50 U/mL). Proliferation was assessed after 5 days, and the percentage of divided cells is depicted as individual values for 5 patients (samples 6B, 7, 8, 10, 28), together with the average value. (C) CFSE-stained CLL cells were cultured with 3T3 control line or with CD40L-expressing 3T3 in absence or presence of different concentrations of IL-21 for 5 days. The percentage of divided cells is depicted as mean ± SD, for samples 12B, 16A, and 29B. (D-E) CFSE-stained CLL cells were cultured with CD40L-expressing fibroblasts in the absence or presence of IL-21 (25 ng/mL) or CpG (1 μg/mL). The percentage of divided cells (panel C) and division index (panel D) after 5 days are depicted as individual values for 9 patients (samples 4, 5C, 6C, 13, 17, 25, 26, 29A, 31), together with the average value. (F) CFSE-stained CLL cells were cultured with CD40L-expressing fibroblasts in the absence or presence of IL-21 (25 ng/mL), and the stimulation was renewed after 3 and 7 days. Proliferation was assessed at day 3, 7, and 10. Results are shown as representative histograms from sample 12B. (G) In the cultures from panel C, cell death was assessed after 5 days. Results are shown as the percentage of cell death (DiOC6⁻ PI^+/−), mean ± SD.