Tim-3 blockade decreases IFN-γ production of activated human NK cells against primary AML tumor cells, but has no effect on degranulation. (A) Western blots were performed on cell lysates of AML (n = 2) and CML (n = 2) tumor cells to evaluate Gal-9 (40 kDa) expression. The Jurkat cell line has endogenous Gal-9 expression and was used as the positive control. The murine stromal cell line EL08-1D2 was used as the negative control. (B-C) PBMCs were incubated overnight in media containing IL-12 (1 ng/mL) and IL-18 (10 ng/mL) and a 5-hour CD107a/IFN-γ assay was performed as described using primary human AML and CML tumor cell targets. The percentage of control intracellular IFN-γ (B) or CD107a (C) production of NK cells when exposed to targets with and without Tim-3 and HLA blocking alone and in combination (calculated for each blocking condition as [% cells IFN-γ⁺ or CD107a⁺ in the presence of target/% cells IFN-γ⁺ or CD107a⁺ without targets] × 100) was determined by FACS analysis (*P < .005, n = 6; error bars represent the SEM).