PT-Cy eliminates single-KIR⁺NK cells and thus dampens NK cell–mediated alloreactivity. (A) Flow cytometry histogram depicting proliferation measured by Ki-67 expression in total NK cells (red), in single KIR⁺ NK cells predicted to be alloreactive (green), and in single-KIR⁺ NK cells predicted to be nonalloreactive (purple) in a representative patient (OSR #10) immediately before PT-Cy administration (day 3 after HSCT). (B) Time course of Ki-67 positivity in total NK cells (red circles), single-KIR⁺ NK cells predicted to be alloreactive (green triangles), or single-KIR⁺ NK cells predicted not to be alloreactive (purple triangles) from the graft (day 0) or circulating in the PB from a representative patient (OSR #10) after HSCT. (C) Percentage of Ki-67 positivity in total NK cells from 3 PBSC grafts (red), in the subset of single-KIR⁺ NK cells predicted to be alloreactive (green), or in the subset of single-KIR⁺ NK cells predicted not to be alloreactive (purple) upon 3 days of exposure to IL-15, and after subsequent addition of mafosfamide to the culture medium. (D) Percentage of proliferating cells, measured through CTV dilution, among total NK cells from 3 PBSC grafts (red), among the subset of single-KIR⁺ NK cells predicted to be alloreactive (green), and among the subset of single-KIR⁺ NK cells predicted not to be alloreactive (purple) upon 3 days of exposure to IL-15 and after subsequent addition of mafosfamide to the culture medium. (E) Frequency of predictably alloreactive single-KIR⁺ NK cells within the graft and in PB NK cells in patients 30 days after HSCT, measured in 8 donor-recipient pairs with KIR-ligand mismatches. (F) Target cell death, expressed as Annexin V positivity (AnnV⁺), measured on K562 cells, OCI/AML cells, or primary leukemic cells after incubation at a 10:1 effector:target ratio with NK cells purified from patient PB day 30 after HSCT with PT-Cy (n = 8, red dots) or from their respective donors PB (n = 8, black dots). (G-H) Progression-free survival (G) and overall survival (H) in patients who received PT-Cy–based haploidentical HSCT from donor with (green line, n = 41) or without (purple line, n = 58) predicted NK cell alloreactivity. Tick marks represent censoring for live patients. Unless otherwise specified, data are shown in all panels as mean values ± SEM.