KIR2DS1⁺ alloreactive NK cells produce more IFN-γ than KIR2DS1^– alloreactive NK cells. Resting NK cells were incubated with immature or LPS- or A fumigatus–pulsed HLA-C1–missing, HLA-C2/C2 DCs. Cells were stained with the following mAb combination: anti-CD56-PE-Cy7, anti-KIR2DL2/L3/S2-FITC, anti-KIR2DL1-PE, anti-KIR3DL1/S1-PE, anti-KIR3DL1/L2-PE, anti-KIR2DS4-PE, anti-NKG2A-PE, anti-KIR2DL1/S1-APC, and anti-IFN-γ-PerCp-Cy5.5. (A) Gating strategy that identified potentially alloreactive NK cells against HLA-C1–missing, HLA-C2/C2 cell targets. Left panel: identification of KIR2DL3⁺ NK cells within CD56^dim lymphocytes. Middle panel: identification of potentially alloreactive NK cells, which expressed KIR2DL3 and did not express KIR2DL1, KIR3DL1/S1, KIR3DL2, KIR2DS4, and NKG2A receptors. Right panel: identification of KIR2DL3⁺ potentially alloreactive NK cells that expressed KIR2DS1 (right quadrant) or did not express KIR2DS1 (left quadrant) (a minimum of 300 events were analyzed). (B) IFN-γ production by KIR2DS1^– alloreactive NK cells. (C) IFN-γ production by KIR2DS1⁺ alloreactive NK cells. (D) Frequencies of IFN-γ⁺ cells in KIR2DS1^– (squares) and KIR2DS1⁺ (triangles) alloreactive NK cells in 9 HLA-C1⁺ individuals. Each symbol represents NK cells from 1 individual. Bars represent median frequency of IFN-γ⁺ cells. Differences between the groups were tested by Wilcoxon matched-pairs test.