Quantification of NK Cell Populations in PBMCs

To quantify various NK cell populations in the PBMCs, 5 × 10⁵ cells were stained with various combinations of fluorophore-conjugated monoclonal antibodies (mAbs). The Gating process of CD3⁻CD16^brCD56^dimNK cells was shown in Figure S1, according to the previous report (36 (link)). Stained cells were fixed with 4% paraformaldehyde and examined by FACSCalibur (BD Biosciences), and the data analyzed Cell Quest Pro software (FlowJo, LLC, Ashland, OR). A total of 50,000 lymphoid events were acquired in each sample. Cells were phenotypically analyzed by staining for 20 min with the following fluorophore-conjugated anti-human mAbs: fluorescein isothiocyanate (FITC)-αCD3, PerCP eFluor710-αCD16, phycoerythrin (PE)-CD56, eFlour660-CD107a, APC-CD158 (KIR2D L1/S1/S3/S5), APC-CD158e1 (KIR3DL1), APC-CD159a (NKG2A), APC-CD226 (DNAM-1), APC-CD244 (2B4), APC-CD314(NKG2D), AF647-CD337 (NKp30), and APC-anti-HLA-E (all from BioLegend, eBioscience, and Miltenyi Biotec). Positive staining populations were determined by comparison with isotype controls. To evaluate HLA expression on CD45⁺CD34⁺ myeloid progenitor cells, the cells were stained with PerCP Cy5.5-CD45 (BioLegends), PE-CD34 mAbs (BD Biosciences), and APC-anti-HLA-E mAb, and examined by flow cytometry.