Inhibition of TGF-β1 signaling restores antileukemic activity of NK cells. (A-C) Flow cytometry analysis showing the percentage of 7-AAD⁺ primary AML blasts and Annexin V ⁺ primary AML blasts (target cells) when cocultured for 5 hours with NK cells purified from the bone marrow of AML patients with early relapse. The BMNK cells were pretreated with dimethyl sulfoxide (solvent control) or galunisertib (10 μM) for 24 hours before coculture. NK cell: target cell ratio = 5:1. (D-E) Flow cytometry analysis indicating the proportions of IFN-γ⁺ granzyme B⁺ NK cells, IFN-γ⁺ TNF-α⁺ NK cells, granzyme B⁺ CD107a⁺ NK cells, IFN-γ⁺ CD107a⁺ NK cells among control and galunisertib-treated NK cells isolated from the bone marrow of AML patients with early relapse. n = 15. (F-G) Flow cytometry analysis showing the proportion of pSMAD2/3⁺ NK cells (upper left), pS6⁺ NK cells (upper right), tetramethylrhodamine methyl ester⁺ NK cells (lower left), and MitoTracker Green⁺ NK cells (lower right) among control and galunisertib-treated NK cells isolated from the bone marrow of AML patients with early relapse. n = 15. (H) Experimental scheme: NCG mice were injected with 5 × 10⁵ HL60 cells stably expressing luciferase into the tail vein. After confirmation of engraftment by BLI on day 7, 2.5 × 10⁶ NK cells were transferred to all the mice via tail vein in combination with vehicle control, 5 × 10⁵ GARP⁺CD4⁺ T cells, 5 × 10⁵ GARP⁺CD4⁺ T cells in the presence of latent TGF-β1 (50 μL, 5 ng/mL; IP; QW), active TGF-β1 (50 μL, 5 ng/mL; IP; QW) in combination with vehicle control, 5 × 10⁵ GARP⁺CD4⁺ T cells in the presence of latent TGF-β1 and galunisertib at 75 mg/kg twice daily (BID) by oral gavage for 21 days, or active TGF-β1 in combination with galunisertib (75 mg/kg; twice daily for 21 days) for treatment. AML burden was monitored by BLI at the indicated time points. (I) BLI of AML burden. (J) AML burden was quantified as the average value of the total flux (p/s). n = 6 mice per group. (K) Kaplan-Meier survival curve of mice bearing HL60 cell−derived tumors. Statistical significance was determined by log-rank Mantel-Cox test. n = 6 mice per group. Data in B, C, E, and G were analyzed by 2-tailed paired t-test. Data in J were analyzed by 1-way analysis of variance with Tukey multiple comparisons test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Data are represented as means ± standard deviation.