Single-KIR⁺ NK cells after haploidentical HSCT are highly variable in frequency and hyporesponsive, independently from predicted NK alloreactivity. (A,B) CD107a mobilization of single-KIR2DL3⁺ NK cells in response to the 721.221 BLCL in patients at 75 to 150 days or more than 2 years after haploidentical HSCT, and their HSC donors, was analyzed as described in “CD107a mobilization assay.” Donors carried the KIR2DL3 gene but not KIR2DL2 or KIR2DS2, thus allowing for specific detection of KIR2DL3 by use of the mAb GL183. Out of a first gate on CD56⁺ lymphocytes, a subsequent gate was selected for cells positive for KIR2DL3 and negative for CD3, NKG2A, KIR2DL1/2DS1, and KIR3DL1 (single-KIR2DL3⁺ NK cells, highlighted gates in each density plot). CD107a mobilization by this subset of cells was assessed after a 6-hour incubation with high-dose rhIL-2 in the presence (black profile in histograms) or in the absence (gray profile in histograms) of the 721.221 HLA class I–deficient BLCL. Shown are one representative nonalloreactive (A) and one alloreactive (B) donor-recipient pairs. Frequency of KIR2DL1 (C) or KIR2DL3 (D) single-positive NK cells in donors (gray symbols) and in patients at 3 to 5 months following haploidentical HSCT (black symbols), in the presence or absence of predicted NK alloreactivity (triangles and circles, respectively). (E) Mean percentage of CD107a mobilization, with SE, by total CD56⁺, CD56^bright, CD56^dim, single-KIR2DL1⁺, or single-KIR2DL3⁺ NK cells in patients at days 75 to 150 after haploidentical HSCT () or in their donors (). Asterisks indicate a P value of .05 or less from a Wilcoxon test for paired data.