Comparable HLA-B*5801–mediated inhibition of NK cell subsets expressing different KIR3DL1 allotypes. Purified NK cells from donors heterozygous for KIR3DL1 allotypes were tested for IFN-γ production in response to 221 or 221-B*5801 in the presence of IL-2 (3000 U/mL). As a control, NK cells were incubated with IL-2 in the absence of any target. NK cells were stained with DX9-PE (anti-KIR3DL1) and CD85j-CyChrome (anti-LILRB1) to gate on the NK subpopulation that expresses KIR3DL1 but not LILRB1. Dead cells and target cells were excluded from the gate. Cells were stained intracellularly with anti-IFN-γ–FITC mAb, and 100 000 cells were analyzed. Results are shown from KIR3DL1⁺ populations. (A) Donor 35 is heterozygous for KIR3DL1*001/*002 alleles and exhibits a unimodal pattern of KIR3DL1 expression as determined by staining with DX9 mAb. Percentages of IFN-γ–producing cells are reported in the right quadrants. (B) Donor 43 is heterozygous for 3DL1*002/*005 alleles and therefore exhibits a bimodal distribution of NK cells expressing KIR3DL1: the low-expressing cells express 3DL1*005 whereas the high-expressing cells express 3DL1*002 or both alleles.⁵¹  IFN-γ–producing cells in each subset are shown in the upper right and lower right quadrants. Donor KIR genotypes are shown in Figure 3.