Figure 5.

Production of IL-2 by CD4 T cells is required for lenalidomide-increased NK and NKT-like cells proliferation. (a) Whole PBMCs or purified NK cells (>95% of purity) from the same individual (n = 4) were labeled with CFSE and cultured with 1 μM lenalidomide or DMSO for 14 days (in the absence of recombinant IL-2, w/o). The proliferation of NK cells (CD3⁻CD56⁺) was assessed by flow cytometry. The figure shows the cytometric profile of NK cells (gated as CD3⁻CD56⁺) before and after the stimulation with lenalidomide. The numbers represent the percentage of NK cells. The histograms represent the expression of CFSE in NK cells from one representative donor. (b) PBMCs from healthy donors (n = 4) and CLL patients (n = 4) were cultured in the presence of lenalidomide for 14 days and the intracellular production of IL-2 by CD4 T cells (CD3⁺CD4⁺) was analyzed by flow cytometry. The bars represent the mean and the standard deviation of the percentage of IL-2-producing CD4 T cells (*P < 0.05, Mann-Whitney U test). (c) PBMCs from healthy donors (n = 4) and CLL patients (n = 4) were stained with CFSE and cultured with DMSO, IL-2 (50 U/mL), or lenalidomide (1 μM) in presence or absence of cyclosporine A (CsA) (1 μM) or as anti-IL-2 blocking antibody (15 μg/mL) for 14 days. PBMCs treated with IL-2 (50 U/mL) were used as a positive control of proliferation. Baseline peaks of CFSE are the same as in Figures 3(c) and 4(a). The expression of CFSE on NK cells and NKT-like cells (CD3⁺CD8⁺CD56⁺) was analyzed by flow cytometry. One representative healthy donor and one CLL patient are shown.