Leukaemic engraftment after pre-culture with NK cells and expression. a, Depletion of NKG2DL+ AML cells after in vitro co-culture with pNKCs. Flow cytometric quantification of NKG2DL+ AML cells (no. 10, 13, 15, 17, 35 and 36) after co-culture with pNKCs for 16 h at the indicated effector/target ratios. A Kruskal–Wallis test was used to test for statistical significance. b–d, Differential recognition of CD34+ (LSC-enriched) versus CD34− (non-LSC) subpopulations of AML cells. pNKCs were cultured with sorted CD34+ or corresponding CD34− AML cells (one dot represents one patient sample, n = 7 cases of AML). Analyses of pNKC-mediated cytotoxicity (b), percentages of CD69+ and CD107a+ among total pNKCs (c) and IFNγ, perforin, granzyme B and TNF concentrations in supernatants (d). Two-sided unpaired t-tests (b, c), and Kruskal–Wallis tests (d) were used for statistical analyses. e, f, Leukaemic engraftment in mice transplanted with AML cells derived from in vitro cultures with or without NK cells. Mice transplanted with equal numbers of AML cells (n = 3 mice per condition and patient as indicated) derived from control cultures without NK cells and from anti-NKG2D + pNKC co-cultures did not show leukaemic repopulation in peripheral blood and bone marrow at time points at which engraftment was detected in mice transplanted with corresponding AML cells from mock + pNKC conditions (shown in Fig. 2e). As shown here, these mice engrafted later. Leukaemic burden as quantified by flow cytometry (e) at the delayed time points when leukaemia was detected in these control groups (f). The time points of engraftment of AML cells cultured with unblocked pNKCs (mock + pNKCs) are shown side-by-side to illustrate that, for each patient sample, this condition showed the fastest engraftment. A two-sided Student’s t-test was used for statistical analysis. g–l, Expression of additional immunomodulatory molecules in LSCs compared to non-LSCs, before and after co-culture with NK cells. Analyses of surface expression of CD112, CD155 and B7-H6 (each in n = 23 cases of AML), CD80, CD86 and PD-L1 (each in n = 25 cases of AML), CD47 (n = 14 cases of AML), and HLA-ABC (n = 75 cases of AML) after co-staining of bulk AML cells for NKG2DLs (g), CD34 (h), CD155 (i) and CD112 (j) on NKG2DL− and NKG2DL+ AML cells after co-culture with the indicated ratios of pNKCs compared to cultures without pNKCs (‘AML alone’), and indicated GSEA analyses of gene-expression datasets from sorted LSCs and non-LSCs isolated as indicated (k, no. 1, 6, 7, 8 and 12; l, no. 7, 8 and 12). Statistical analyses in g–j were performed using a two-sided Mann–Whitney U test. Centre values represent mean, error bars represent s.d. (a–e) or s.e.m. (g–j). One dot represents one patient sanple (a–d, g–j) or one mouse (e). Nominal P value and normalized enrichment score (k, l). Source Data