Staining of cells for microscopy analysis was performed with the following mAbs: EB6 for KIR2DL1 (mouse IgG1, 10 μg/ml), GL183 (mouse IgG1, 10 μg/ml) for KIR2DL3, Z199 (mouse IgG2b, 10 μg/ml) for NKG2A (all from Beckman Coulter), DX9 (mouse IgG1, 10 μg/ml) (R&D Systems) for KIR3DL1, and HP-F1 (mouse IgG1, 5 μg/ml) (eBioscience) for LILRB1. Because the mAbs EB6, GL183, and Z199 cross-react with KIR2DS1/L3, KIR2DL2/S2, and NKG2C, respectively, we detected inhibitory receptors of interest by using flow cytometry to screen clonal NK cells that lacked expression of the confounding receptors. To do this, we used competitive staining (53) with the generic mAbs mentioned earlier and specific mAbs against KIR2DL1, 143211 (mouse IgG1, 10 μl per test); KIR2DL3, 180701 (mouse IgG2a, 10 μl per test); and NKG2C, 134591 (mouse IgG1, 5 μl per test) (all from R&D Systems), antibodies that cannot be used for immunocytochemistry. For example, competitive staining with the mAb 143211 (which binds to KIR2DL1 and blocks staining by EB6) and the mAb EB6 (which binds to KIR2DL1/S1/L3*005/10) enabled the identification of clones that expressed KIR2DL1 (143211+ EB6) and did not coexpress KIR2DS1/L3*005/10 (143211+ EB6+) (fig. S1A). The antireceptor mAbs used for microscopy were directly conjugated to Alexa Fluor 647 (Thermo Fisher Scientific). Staining was specific, because NK cell clones lacking these receptors showed no staining (fig. S1B). For all batches of antibody, the ratio of fluorophore to mAb (degree of labeling) was measured by absorption and calculated according to the manufacturer’s instructions (mean, 5.0/mAb; SD, 1.68). mAb against CD56 (HCD56, mouse IgG1, 1 μl per test; BioLegend), KIR2DL2/3 (DX27, mouse IgG2a, 5 μl per test; BioLegend), and KIR3DL1 (177407, mouse IgG2a, 10 μl per test; R&D Systems) were also used for flow cytometry.

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