Identification of alloreactive NK cells and their cytolytic effect against AML. This experiment shows the susceptibility of 2 representative AMLs (ML2 and ML11) to lysis mediated by KIR/HLA-class I mismatched (alloreactive) NK cells derived from different allogeneic donors. In both cases, one of the donors (donor A and C, respectively) were siblings with a haploidentical HLA haplotype, while the others (donor B and D) were unrelated. In panels A and B, IL-2–cultured NK cell populations were analyzed for the expression of informative KIRs and CD94/NKG2A. In panel A, mAb against KIR3DL1 (AZ158, IgG2a) was followed by FITC-conjugated anti-IgG2a second reagent (x-axis), whereas a mixture of mAb to KIR2DL1 (EB6b, IgG1), KIR2DL2/3 (GL183, IgG1), and NKG2A (Z270, IgG1) was followed by PE-labeled anti-IgG1 antisera. In this case, the potentially alloreactive population was represented by cells expressing only KIR3DL1 (lower right quadrant), and the percentage is indicated. In panel B, NK cell populations from donors C and D were analyzed for KIR2DL1 (EB6b, IgG1) on the y-axis and the mixture of KIR2DL2/3 (CH-L, IgG2b), KIR3DL1 (AZ158, IgG2a), and NKG2A (Z199, IgG2b) on the x-axis, followed by appropriate isotype-specific fluorochrome-conjugated second reagents. In this case, the expected alloreactive cells were those expressing KIR2DL1 only (upper left quadrant; percentage is indicated). In panels C and D, the various NK cell populations cultured for 5 days in IL-2 were tested in a cytolytic assay against ⁵¹Cr-labeled ML2 (C) or ML11 (D) AML (black symbols). Selected B-EBV cell lines carrying appropriate groups of HLA alleles, AC (HLA-B^Bw6, HLA-C^Lys80/C^Asn80), and BM15 (HLA-B^Bw4, HLA-C^Asn80) were also used as target cells in panels C and D, respectively (open symbols). Effector cells were represented by the familial donors (triangles) and by the unrelated donors (circles).