Isolation of CD158b⁺ (KIR 2DL2/3) and CD158a⁺ (KIR 2DL1) NK populations. (A) NK cells were enriched by negative selection from PBMCs of a healthy donor (KR0350) homozygous for HLA-C group 1 (C-G1), then expanded in vitro using irradiated EBV-LCL feeders. Two weeks later, CD3^–/CD56⁺/CD158b⁺ NK cells were isolated by flow sorting, then expanded for another 2 weeks to remove any remaining surface-bound antibodies. (B) In a similar fashion, CD158a⁺ NK cell populations were isolated by flow sorting from healthy donor MORRC homozygous for HLA-C group 2 (C-G2). Percentages of CD3^–/CD56⁺/CD1586b⁺ cells are shown. (C) At a 20:1 E/T ratio, NK cells enriched for CD158b (80% positive) lysed a significantly higher percentage of KIR-incompatible EBV-LCL, melanoma, and RCC cells homozygous for HLA-C group 2 (KIR incompatible) compared with HLA-C group 1 homozygous KIR-matched tumor targets. (D) These cells lysed a significantly higher percentage of EBV-LCL and melanoma cells homozygous for HLA-C group 1 (KIR incompatible) compared with the HLA-C group 2 homozygous KIR-matched targets. Mean values +SD of 3 samples are shown.