The second wave of NK cells that appear after PT-Cy displays an immature phenotype. (A) Flow cytometry histograms depicting expression of indicated maturation markers on CD56⁺ CD3⁻ NK cells from a representative patient (OSR #1) at the indicated time points (LP: red; day 3: orange; day 15: green; day 30: blue). (B) The expression of the described maturation markers measured by flow cytometry in NK cells from the graft (day 0) and from patients longitudinally sampled after HSCT with PT-Cy (n = 10). For each marker, a normal reference interval (mean ± SD) measured in NK cells from 5 healthy subjects is displayed (gray box). (C) Heat map of the average NK cell maturation marker expression at different time points, with unsupervised hierarchical clustering, for 10 OSR patients analyzed by multiparametric flow cytometry at the indicated time points. (D-E) Multidimensional single-cell analysis of the maturation status of NK cells harvested from 5 healthy controls (HC), from leukaphereses (LP, n = 10) and from 10 patients at different time points after HSCT. The panel D bidimensional maps were obtained from flow cytometric data using the bh-SNE algorithm show all analyzed NK cell events, with coloring denoting the expression of each maturation marker, as indicated. The same data are depicted in panel E, where NK cells from a selected time point are displayed separately.