Tim-3 cross-linking inhibits killing by NK cells. (A-C) Each graph depicts the percentage of specific lysis of mouse P815 target cells mediated by the human NK-cell line NKL (A); NKL cells transduced with human Tim-3 containing a FLAG epitope tag on the N terminus, NKL-FLAG-Tim-3 (B); or freshly isolated PBMCs from a representative donor in the absence or presence of the indicated single or combined mAbs: anti–Tim-3 (clone 344801), anti–Tim-3 (clone 344823), anti-NKG2D, anti-CD94, anti-CD16, or anti-FLAG (C). (D-E) Graphs represent lysis of human 721.221 cells transduced with human CD32 Fc receptors by NKL-FLAG-Tim-3 (D) or freshly isolated PBMCs from a representative donor in the presence of the indicated mAbs: anti–Tim-3 (clone 344801), anti–Tim-3 (clone 344823), or anti-CD56 (E). Anti-CD56 was used as a negative control because it does not affect NK-cell cytotoxicity. (F) Graph represents lysis of mouse P815 target cells mediated by PBMCs stimulated overnight with IL-2 from a representative donor in the absence or presence of the indicated single or combined mAbs: anti–Tim-3 (clone 344801), anti–Tim-3 (clone 344823), and anti-CD16. Results are representative of 3 independent experiments performed in triplicate.