The Tim-3 ligand Gal-9 increases IFN-γ production in the Tim-3–transduced NK92 cell line. (A) NK92 cell lines were stimulated with rhGal-9 for 4 hours in the presence of a blocking Tim-3 mAb or isotype control Ab and intracellular IFN-γ production was determined by FACS analysis (*P < .01, NK92 Tim-3 vs NK92 native [n = 5] and NK92 Tim-3 vs NK92 Tim-3 blocked [anti–Tim-3 mAb, n = 5]). (B) The full-length human Gal-9 gene was cloned into a MSCV vector and the Raji cell line was transduced as described. Gal-9 protein expression (40 kDa) was confirmed by Western blot analysis. 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. (C) NK92 cell lines were incubated in the presence of Raji eGFP and Raji Gal-9 target cell lines for 5 hours and intracellular IFN-γ production was determined via FACS analysis (*P < .001, n = 5; error bars represent SEM).