Incubation with soluble Gal-9 increases NK cell function and decreases surface expression of Tim-3. (A) Dot plots represent the percentages of CD107a + NK cells and the MFI of Tim-3 on NK cells from 8 healthy individuals upon pre-activation with 1 ng/mL of IL-15 and IL-18 overnight followed by stimulation with either 0.9 μg/mL of soluble Gal-9 for another 16 h or K562 target cells at an effector:target ratio of 10:1 for 6 h. Representative primary flow cytometry panels show CD107a upregulation (upper panel) and Tim-3 expression (lower panel) on NK cells that were left unstimulated, or were activated as indicated. (B) Unstimulated or Gal-9-activated NK cells were divided into Tim-3bright, Tim-3dim and Tim-3low/neg so that the bright and the low/neg subpopulations each consistently represents about 25% of the bulk NK cells, and subsequently analyzed for CD107a expression. Representative primary flow panel shows an example of subdivision of NK cells according to Tim-3 expression. Percentages of positive NK cells and median fluorescence intensity are indicated. Histograms display CD107a upregulation in each subset following incubation with soluble Gal-9. Horizontal lines indicate the median percentages. Statistically significant difference reached when p < 0.05 is indicated.