Exploring the effects and mechanisms of CD4⁺CD25⁺foxp3⁺ regulatory T cells in regulating NK cytotoxicity through NKG2D in acute myeloid leukemia.

Immunomagnetic bead method was used to isolate peripheral blood NK cells and CD4⁺CD25⁺ cells from acute myeloid leukemia (AML) and healthy controls. The expression of foxp3 and NKG2D, and the relationship between the proportion of CD4⁺CD25⁺foxp3⁺ cells and the cytotoxicity of NK cells were analyzed. Spearman method was used to analyze the correlation between CD4⁺CD25⁺foxp3⁺ cell ratio and NK cell cytotoxicity as well as NGK2D. After overexpressed foxp3 in AML peripheral blood lymphocytes, flow cytometry was used to detect the expression of NKG2D, IFN-γ and CD107a, LDH assay was used to detect the toxicity of NK cell. The correlation between NKG2D and foxp3 gene expression in AML patients was analyzed by GEPIA2 database, while the genes with high correlation of NKG2D expression in AML transcriptome data was analyzed by TCGA database. After treating NK cells with IL-2 (10 μg/mL) and IL-15 (10 μg/mL) for 24 hours, 24 hours after treated with IL-2 (10 μg/mL) and IL-15 (10 μg/mL) in NK cells, flow cytometry was used to detect the expression of NKG2D and CD107a, enzyme-linked immunosorbent assay was used to detect the secretion of IFN-γ by NK cells, LDH assay to detect NK cell toxicity. Statistical analysis of inter group differences and variable correlations using t-tests and other methods.

Compared to the control group, the expression of NKG2D in NK cells in the AML group was significantly reduced (MFI: 942.25 ± 117.17 vs 3245.28 ± 367.43, P < 0.001). The expression of foxp3 is positively correlated with NKG2D (r = 0.590, P < 0.001), and the percentage of NKG2D expression is positively correlated with the proportion of CD4⁺CD25⁺foxp3⁺ cells in peripheral blood lymphocytes (r = 0.708, P = 0.002). The activity of peripheral blood NK cells in AML patients is also positively correlated with the proportion of CD4⁺CD25⁺foxp3⁺ cells (r = 0.655, P = 0.006). Compared to the OE-NC group, the expression level of NKG2D (MFI: 2115.37 ± 269.53 vs 914.28 ± 103.36), the cell killing ability (58.24 % ± 3.17 %vs 24.18 % ± 2.35 %), the NK cell toxicity evaluation indicators CD107a and the level of IFN-γ in the OE-foxp3 group cells were significantly increased (45.43 % ± 1.28 %vs 18.24 % ± 1.49%), (24.16 % ± 1.17 %vs 16.28 % ± 0.95 %) (all P < 0.001). TCGA data analysis found that the expression of NKG2D was highly correlated with genes such as CD3G, IL-3, IL-2, CD3E, IL-10, IL-17, IL-7R, IL-15, EZH2, etc. Flow cytometry analysis showed that after treatment with IL-2 and IL-15, the expression of surface receptors NKG2D and CD107a on NK cells was significantly increased, and IFN-γ level was increases, NK cell toxicity is enhanced.

In peripheral blood of AML, the surface receptor NKG2D of NK cells is lower than that of control, and CD4⁺CD25⁺foxp3⁺ regulatory T cells regulate NK cell toxicity through the expression of NKG2D effecting by IL-2 and IL-15.