DNA was extracted from whole blood using a Roche MagNAPure 96 or Qiagen® DNeasy Blood & Tissue kit according to the manufacturer’s instructions. KIR3DL1 typing was performed using PCR primers described by Boudreau et al. to detect the major alleles *004 (null), *001, *002 (high), *013 (3DS1) and *005, *007 (low). [25 (link)]. Furthermore, flow cytometry was used to determine the KIR3DL1 expression level by staining cells with anti-CD3-APC-H7 (SK7), anti-CD14-APC-H7 (MϕP9), anti-CD16-BV786 (3G8), anti-CD56-BV711 (NCAM1), anti-CD57-BV605 (NK-1), anti-CD19-APC-H7 (SJ25C1; all from BD Biosciences), anti-NKG2C-Alexa Fluor 488 (134,591), anti-KIR2DL1 Alexa Fluor 700 (143211, both R&D Systems), anti-NKG2A-PE (Z199), anti-KIR2DL1/S1-Pe-Cy7 (EB6B), anti-KIR2DL2/L3/S2-Pe-Cy5.5 (GL 183; all from Beckman Coulter), anti-KIR3DL1-APC (DX9; BioLegend). Stained samples were analyzed on a five laser BD LSR Fortessa SORP instrument. Data was analyzed using FACSDiva (v.8.0.1 or later) and FlowJo (v.10.8.1 or later) software (BD Biosciences). The median frequency of KIR3DL1+ cells among CD16+CD56+ NK cells was 11% (range 7.71–56.5%). The HLA-B and -C allele genotype was determined using LABType SSO Class I Locus Typing Tests from One Lambda as described elsewhere [26 (link)]. Complementary KIR ligand typing for HLA-A Bw4 was performed using the Olerup SSP KIR HLA Ligand kit.
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