Fig 2: After the addition of CD96 antagonists, the function of NK cells in the coculture system decreased significantly. (A) Cell fluorescence staining assay to detect the adhesion of NK cells to DSCs before and after the addition of antagonists (400× magnification). (B) Cell fluorescence staining to detect the adhesion of NK cells to HTR8 cells before and after the addition of antagonists (400× magnification). (C) Number of dNK cells adhered to the surface of DSCs. (D) Number of dNK cells adhered to the surface of HTR8. (E) Flow cytometry to detect the effect of adding antagonists on the expression of NK cell adhesion molecules. (F) Flow cytometry to detect the expression intensity histogram of three cytokines. (G) Flow cytometry was used to detect the average fluorescence intensity of the three cytokines and count their differences. MFI: mean fluorescence intensity. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Fig 3: Low-dose palmitic acid upregulates the surface expression of CD155 on trophoblast cells and CD96 on decidual natural killer (dNK) cells, resulting in decreased expression of cytotoxic cytokines in dNK cells. Moreover, it leads to an increase in inhibitory cytokine IL-10 and adhesion molecules ICAM1, VCAM1, and SELE. These changes signify the transition of dNK cell function towards an immune-tolerant phenotype. Additionally, there is a significant decrease in the expression of the proliferation marker Ki-67 in dNK cells. These findings suggest the potential involvement of these factors in the regulation of early-pregnancy maternal–foetal immune tolerance.

Fig 4: Palmitic acid may inhibit NK cell activity through CD96 in dNK cells. (A) Effect of palmitic acid on CD155 expression in HTR8 T cells. (B) Effect of palmitic acid on CD155 expression in HTR8 cells cocultured with dNK cells. (C) Effect of palmitic acid on CD96 expression in dNK cells. (D) Effect of palmitic acid on CD96 expression in dNK cells cocultured with HTR8 cells. (E) ROS content of dNK cells under different conditions. (F) Ratio of JC-1 polymer/monomer of dNK cells under different conditions. (G) Histogram of the expression intensity of the three adhesion molecules detected via flow cytometry. (H) The mean fluorescence intensity of the three adhesion molecules was measured via flow cytometry, and the differences were calculated. (I) Histogram of the expression intensity of the three adhesion cytokines detected via flow cytometry. (J) The mean fluorescence intensity of the three cytokines were measured via flow cytometry, and the differences were calculated. PA: palmitic acid; MFI: mean fluorescence intensity. ns: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Fig 5: Expression of CD96 and CD155 at the decidual interface in normal pregnancy: (A) Gating strategies for NK cells, T cells, and total immune cells. (B) Histogram of differences in the expression of CD96 and TIGIT in different cell subsets in decidual and endometrial tissues of normal pregnancy. (C) Differences in the average fluorescence intensity of CD96 expression in different subpopulations of cells. (D) Differences in the mean fluorescence intensity of TIGIT expression in different cell subpopulations. (E) Entrapment gating strategies for stromal cells in deciduous tissue and endometrial tissue. (F) Histogram of differences in the expression of CD155 and CD112 in DSCs and ESCs. (G) Statistics of the difference in the expression of CD155 and CD112 in DSCs and ESCs. DSCs: deciduous stromal cells; ESCs: endometrial stromal cells. TL: total lymphocyte; DSC: decidual stromal cell; ESC: endometrial stromal cell; MFI: mean fluorescence intensity. ns: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001.