Tim-3 expression on NK cells is increased by maturation and cytokine stimulation. (A-B) Plots from a representative donor show the surface expression of Tim-3, on CD56^brightCD16^– (A) and CD56^dimCD16⁺ (B) NK-cell subsets after culture overnight without stimulation (gray shade) or with IL-2, IL-12 and IL-18, IFN-α, IL-15 and IL-12 (black line for all these stimulations), and IL-15 (dotted line). (C-D) CD19⁻CD14⁻CD3⁻CD56^brightCD16⁻ NK cells either lacking or expressing Tim-3 were sorted and incubated for 24 hours with either no stimulation or IL-15. (C) Histograms show Tim-3 expression of sorted cells from a representative donor after culture without (gray shade) or with IL-15 stimulation (black line). (D) Plots depict CD56 and Tim-3 coexpression on sorted CD56^brightCD16^– Tim-3⁺ (left) and Tim-3⁻ (right) NK cells without (top) or with (bottom) IL-15 stimulation. These experiments were repeated 3 times with different unrelated healthy donors.