Hyperactivated and exhausted NK cells accumulate in peripheral blood of patients with ALL. Flow cytometry analysis depicting frequencies of CD69⁺ total NK cells and CD56^brightCD69⁺ and CD56^dimCD69⁺ NK cells in PBMCs from healthy donors and B-ALL and T-ALL patients (A; n = 9), as well as in healthy donors and patients with B-ALL (n = 9). (C) Comparison of Ca²⁺ flux in ionomycin-stimulated CD3⁻CD56⁺-gated NK cells in PBMCs from healthy donors (n = 6) and patients with B-ALL (n = 5). (D) Normalized Ca²⁺ flux in 1 representative sample (left panel); area under the curve is shown for 6 healthy donors and 5 patients with B-ALL (right panel). (E) Flow cytometry analysis depicting frequencies of CD69⁺CD94⁺ NK cells in PBMCs from healthy donors and B/T-ALL patients (n = 9 each). (F) CyTOF analysis of the frequencies of cells expressing LAG-3, KLRG1, PD-L2, and Siglec-7 in total NK cells and in the CD56^bright and CD56^dim NK cell subsets in PBMCs from healthy donors (n = 4) and patients with B/T-ALL (n = 9). (G) Representative dot plots of the expression of LAG-3, KLRG1, PD-L2, and Siglec-7 in total NK cell and NK cell subsets in PBMCs from healthy controls and patients with B-ALL and T-ALL. Data are median ± interquartile range. Exact P values were calculated using the Mann-Whitney U test.