GARP⁺CD4⁺ T cells induce TGF-β1 activation that inhibits NK cell-mediated antitumor effects both in vitro and in vivo. (A-C) Flow cytometry data indicating the percentage of 7-AAD⁺ HL60 cells (target cells) cocultured for 5 hours with BMNK cells pretreated with dimethyl sulfoxide (DMSO; solvent control; black), latent TGF-β1 (10 ng/mL; blue), latent TGF-β1 (10 ng/mL) plus GARP^-CD4⁺ T cells (purple), GARP⁺CD4⁺ T cells (green), or latent TGF-β1 (10 ng/mL) plus GARP⁺CD4⁺ T cells (red) to estimate cytotoxicity. The NK cells: target cells ratio = 5:1; the ratio of NK cells: GARP^-CD4⁺ T cells or GARP⁺CD4⁺ T cells was 5:1. n = 10. (D-G) Flow cytometry analysis showing the proportion of (D) CD107a⁺IFN-γ⁺ NK cells, (E) granzyme B⁺ CD107a⁺ NK cells, and (F) granzyme B⁺IFN-γ⁺ NK cells within the total BMNK cell population that was pretreated with either DMSO (solvent control; black), latent TGF-β1 (10 ng/mL; blue), latent TGF-β1 (10 ng/mL) plus GARP⁻CD4⁺ T cells (purple), GARP⁺CD4⁺ T cells (green), or latent TGF-β1 (10 ng/mL) plus GARP⁺CD4⁺ T cells (red) and cocultured with HL60 cells for 5 hours. n = 10. (H) ELISA results indicating the levels of active TGF-β1 in the supernatants of coculture experiments described in D-G. (I-J) Flow cytometry analysis showing NKp30 (I, left) and NKG2D (I, right) expression levels on BMNK cells pretreated with DMSO (solvent control), latent TGF-β1 (10 ng/mL), latent TGF-β1 (10 ng/mL) plus GARP⁻CD4⁺ T cells, GARP⁺CD4⁺ T cells, or latent TGF-β1 (10 ng/mL) plus GARP⁺CD4⁺ T cells. n = 10. (K) Experimental design: NCG mice were injected into the tail vein with 5 × 10⁵ HL60 cells stably expressing luciferase. After confirmation of engraftment by bioluminescence imaging (BLI) on day 7, 2.5 × 10⁶ NK cells were transferred to all the mice via tail vein in combination with the injection of PBS control (50 μL, IP; QW), 5 × 10⁵ GARP⁺CD4⁺ T cells, latent TGF-β1 (50 μL, 5 ng/mL; IP; QW), or 5 × 10⁵ GARP⁺CD4⁺ T cells in the presence of latent TGF-β1. AML burden was monitored by BLI at the indicated time points. (L) BLI of AML burden. (M) AML burden was quantified as the average value of the total flux (p/s). n = 6 mice per group. (N) 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. The data in B, C, G, H, J, and M were analyzed by 1-way analysis of variance with Tukey multiple comparisons test; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. The data are represented as means ± standard deviation.