Tim-3 is expressed on resting NK cells and is up-regulated on activation. (A-C) PBMCs and purified PB NK cells were stained with anti-CD56, anti-CD3, and anti-Tim-3 mAbs. (A) Tim-3 expression on resting lymphocyte populations was determined by FACS analysis and a representative donor is shown in flow plot A. (B) PBMCs were enriched for CD56⁺/CD3⁻ NK cells using a negative immunomagnetic bead depletion strategy. NK cells were incubated overnight in basal media or media containing IL-15, IL-2, IL-7, or IL-21 at the indicated concentrations. Tim-3 MFI was determined by FACS analysis (n = 8). (C) PBMCs were enriched for CD56⁺/CD3⁻ NK cells using a negative immunomagnetic bead depletion strategy. NK cells were incubated overnight in basal media or media containing IL-12 alone, IL-18 alone, IL-12 and IL-18 (concentration ratio of 1:1), and IL-12 and IL-18 (concentration ratio of 1:10) at the indicated concentrations. Tim-3 MFI was determined by FACS analysis (n = 8).