Higher levels of active TGF-β1 are associated with impaired antileukemic responses of NK cells in the bone marrow of patients with relapsed AML. (A-B) Flow cytometry analysis indicating the percentage of 7-AAD⁺ HL60 cells (target cells) cocultured for 5 hours with NK cells isolated from the bone marrow of AML patients with early relapse (red; n = 13) or without relapse (blue; n = 21). NK cell: target cell ratio = 5:1. (C-G) Flow cytometry data of the proportion of (C) IFN-γ⁺ TNF-α⁺ NK cells, (D) IFN-γ⁺ CD107a⁺ NK cells, (E) IFN-γ⁺ granzyme B⁺ NK cells, and (F) granzyme B⁺ CD107a⁺ NK cells among the total NK cells purified from the bone marrow of AML patients with early relapse (red; n = 13) or without relapse (blue; n = 21) after coculture with HL60 cells for 5 hours. (H) Flow cytometry analysis showing NKG2D expression on NK cells isolated from the bone marrow of AML patients with early relapse (red; n = 22) or without relapse (blue; n = 28). (I) Spearman rank correlation analysis shows the relationship between active TGF-β1 levels and the proportion of different immune cell types and indicated molecules as shown by the Spearman correlation coefficients (r) and P values. Data in B, G, and H were analyzed by 2-tailed unpaired t-test; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Data are represented as means ± standard deviation. (J-K) Representative immunohistochemistry images show the staining intensity for CD56, granzyme B, and IFN-γ in the bone marrow biopsy samples from AML patients with early relapse (J) or without relapse (K). Scale bars, 100 μm.