Active TGF-β1 inhibits effector function, mTORC1 activity, and mitochondrial respiration of bone marrow−derived NK cells ex vivo. (A) Flow cytometry analysis showing the percentage of 7-AAD⁺ HL60 cells (target cells) when cocultured for 5 hours with control (dimethyl sulfoxide) NK cells, 10 ng/mL latent TGF-β1-treated NK cells, and 10 ng/mL active TGF-β1-treated NK cells to estimate cytotoxicity. NK cells: target cells ratio = 5:1; n = 20. (B-C) Flow cytometry data indicating the proportion of IFN-γ⁺TNF-α⁺ NK cells, IFN-γ⁺granzyme B⁺ NK cells, IFN-γ⁺ CD107a⁺ NK cells, and granzyme B⁺ CD107a⁺ NK cells within the total population of control NK cells, latent TGF-β1-treated NK cells, and active TGF-β1-treated NK cells that were cocultured with HL60 cells for 5 h. n = 20. (D-E) Flow cytometry analysis showing the proportion of (D) pSMAD2/3⁺ and (E) pS6⁺ NK cell populations in the control (black) and active TGF-β1-treated (red) BMNK cells. n = 15. (F-G) Oxygen consumption rates (OCRs) of control and active TGF-β1-stimulated NK cells under basal conditions and in response to oligomycin (Oligo), the mitochondrial decoupler FCCP, and rotenone + antimycin (R + A). (G) Estimation of OCR values (OXPHOS activity) under basal conditions (left) and maximum respiration rates after FCCP uncoupling (right). OCRs were analyzed for 9 donors per group. (H-I) Flow cytometry analysis indicating the proportion of (H) tetramethylrhodamine methyl ester⁺ and (I) MitoTracker Green⁺ NK cells in the control (black) and active TGF-β1-treated (red) groups of BMNK cells. n = 15. Data were analyzed by 1-way analysis of variance with Tukey multiple comparisons test (A,C) or 2-tailed paired t-test (E, I); ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. The data are represented as means ± standard deviation.