dNK expressing other NKRs (LILRB1, KIR2DL2/L3/S2, KIR3DL1, and KIR2DS4) were gated out from the analysis, except where stated. (A) CD107a staining was measured in the 4 dNK subsets after incubation with 221-C2 target cells (n = 25). Horizontal bars indicate mean for each group. (B) Analysis similar to that in A, but performed in the presence of the HLA class I blocking mAb 6A4 or isotype control (con) (n = 6). (C) Effect of other NKRs on KIR2DS1 responses to 221-C2 targets. After culture with 221-C2 targets, KIR2DS1⁺ dNK were gated into 3 subsets: KIR2DS1sp with no other NKRs expressed (2DS1sp NKR⁺), KIR2DS1sp that do coexpress other NKRs apart from KIR2DL1 (2DS1sp NKR⁺) and dp cells without other NKRs (dp NKR^–). Frequency of CD107a⁺ dNK was compared between the 3 different subsets (n = 24). (D) Effect of donor’s HLA-C genotype on KIR2DS1 response. Degranulation of KIR2DS1sp NKR^– subset in response to culture with 221-C2 targets was compared between donors who had at least one HLA-C2 allele (C2/X, n = 19) and donors homozygous for HLA-C1 (C1/C1, n = 13). (E) Degranulation of the KIR2DS1sp NKR^– subset from matched pbNK and dNK was measured after coculture with 4 different 221-C2 clones expressing different surface levels of the HLA-C2 allele Cw*0602 (Supplemental Figure 6). Clone 1 is 221-parent. Horizontal bars indicate means for 4 separate donors. (F) Degranulation of the KIR2DS1sp NKR^– subset in response to the same clone of 221-C2 target cells by pbNK and dNK from the same donor (n = 18). *P < 0.05 and **P < 0.01, Mann-Whitney test (A, C, and D). *P < 0.05 and **P < 0.01, Wilcoxon paired samples test (B and F). *P < 0.05, Student’s paired t-test (E).