Cyclophosphamide administration induces killing of proliferating NK cells. (A) Flow cytometry histograms depicting Ki-67 expression measured on T (blue) and NK cells (red) from the leukapheresis (LP) graft and in the PB of a representative patient (OSR #1) at different time points after HSCT, as indicated. Percentages indicate frequencies of Ki-67–positive cells. (B) Ki-67 positivity in NK (red circles) and T (blue squares) cells from the graft (day 0), or circulating in the PB of patients after HSCT with PT-Cy (n = 17). (C) Ki-67 positivity in NK cells from the graft (day 0) or in the PB of patient transplanted at OSR (red circles: n = 10) or JHU (white circles: n = 7). (D) Level of proliferation of donor-derived NK cells (red circles) and residual host NK cells (gray circles) measured with Ki-67 intracellular staining in 3 representative patients. Donor- or host-derived NK cells were discriminated by immunophenotypic analysis using differential expression of the mismatched HLA-A*02 allele. (E) Scatter plot depicting the mean percentage of ALDH⁺ cells detected by flow cytometry among NK (red circles), T (blue squares), and stem (orange diamonds) cells present within the infused graft, or in the patient PB 3 days after transplant, in 4 representative patients. Note that for 2 of 4 patients, CD34⁺ stem cells were no more detectable in the patient PB at day 3. (F) In vitro assay of mafosfamide-induced cell death. The LP product of 6 patients was stimulated with IL-15 at day 0 and treated with different doses of mafosfamide at day 3. The graphs report the number of viable (AnnexinV⁻) NK cells, T cells, and HSCs detected by flow cytometry before the treatment (day 3) and after administration of different doses of the drug (day 5; light pink bars: untreated; dark pink bars: 2.5 μg/mL; light red bars: 7.5 μg/mL) and after irradiation as positive control (day 5, red bars). Unless otherwise specified, shown in all panels are average values ± SEM.