Primary Tim-3⁺ NK cells specifically produce IFN-γ in response to rhGal-9. (A) Purified PB NK cells were incubated overnight in media containing IL-12 (1 ng/mL) and IL-18 (10 ng/mL) and intracellular IFN-γ production was determined by FACS analysis. One representative donor is shown in flow plots A. (B) Resting NK-cell populations CD56^Bright, CD56^DIM/Tim-3⁻, and CD56^DIM/Tim-3⁺ were sorted and incubated overnight in media containing IL-12 (1 ng/mL) and IL-18 (10 ng/mL). Each sorted, activated population was exposed to 10nM rhGal-9 with and without β-lactose (30mM) blocking and the percentage of control intracellular IFN-γ production (calculated as [% cells IFN-γ⁺ at 10nM/% cells IFN-γ⁺ at 0nM] × 100) was determined within the Tim-3^neg and Tim-3^hi fractions via FACS analysis (*P < .005, n = 5). (C) IL-12 (1 ng/mL) and IL-18 (10 ng/mL) activated NK cells were sorted into Tim-3⁺ and Tim-3⁻ cell populations and exposed to rhGal-9 (0nM, 10nM, and 20nM) with and without blocking using anti-Tim-3 mAb. The percentage of control intracellular IFN-γ production (calculated as [% cells IFN-γ⁺ at 10nM or 20nM/% cells IFN-γ⁺ at 0nM] × 100) determined via FACS analysis (*P < .05, n = 5; error bars represent SEM).