Determination of NK cell licensing status

NK cells were identified as CD45+CD56+CD3- lymphocytes, and divided into CD56^bright and CD56^dim subsets based on CD56 and FcγRIIIa (CD16) expression. Analyses were restricted to NKG2A⁻CD56^dim NK cells to eliminate potentially confounding effects of CD94/NKG2A on KIR-based licensing. To establish human NK cell licensing status, non-overlapping NKG2A-CD56^dim NK subsets [triple positive (3/3 KIR); double positive (2/3 KIR); single positive (1/3 KIR); triple negative (0/3 KIR)] were gated based on KIR2DL2/2DL3 (ligand human leukocyte antigen (HLA)-C1), KIR2DL1 (ligand HLA-C2), and KIR3DL1 (ligand HLA-Bw4) expression.^6,7 Licensed subsets contained at least one self-KIR (KIR with its cognate HLA ligand present in that individual).