可溶性HLA-G1与NK-92细胞表面ILT2及KIR2DL4受体相互作用的研究

 目的 探讨可溶性 HLA-G1（sHLA-G1）对人 NK-92 细胞杀伤活性的抑制与细胞表面免疫球蛋白样转录分子 2（ILT2）和杀伤细胞免疫球蛋白样受体 2DL4（KIR2DL4）受体的关系。 方法 ①通过原核表达技术获得 sHLA-G1 重组蛋白（重组蛋白），并采用蛋白质印迹法进行鉴定。②取 NK-92 细胞，加入终浓度 20 μg/ml 的重组蛋白分别培养 10、30 min，再分别加入抗 HLA-G1/G5、抗ILT2 和抗 KIR2DL4 抗体，采用流式细胞术检测各组 NK-92 细胞表面 sHLA-G1 和 ILT2、KIR2DL4 受体表达阳性率；以 NK-92 细胞单独培养作为对照组。③以人白血病 K562 细胞为靶细胞，以经不同方式处理的 NK-92 细胞为效应细胞，效靶比为5:1，共同培养 2 h，采用流式细胞术检测 NK-92 细胞对 K562 细胞的杀伤率。NK-92 细胞处理方式为单纯重组蛋白处理（分别加入终浓度为 0、10、20 μg/ml 的重组蛋白培养 30 min）和表面受体封闭 + 重组蛋白处理（分别加入抗 ILT2、抗 KIR2DL4、抗 LT2 + 抗 KIR2DL4 抗体培养 30 min，再分别加入终浓度为 0、10、20 μg/ml 的重组蛋白培养 30 min）。 结果 ①蛋白质印迹分析表明所获重组蛋白为带有组氨酸标签的特异蛋白。②NK-92 细胞与 20 μg/ml 重组蛋白共培养 30 min 后，sHLA-G1 表达阳性率明显高于而 ILT2、KIR2DL4 受体表达阳性率均明显低于对照组（均 P < 0.05）。③以终浓度 0、10、20 μg/ml 的重组蛋白处理的 NK-92 细胞对 K562 细胞的杀伤率分别为 39.79% ± 2.00%、27.79% ± 0.75%、21.36% ± 0.67%（两两比较，均 P < 0.01）；单独封闭 ILT2 受体，杀伤率分别为 23.09% ± 1.63%、21.13% ± 0.38%、18.42% ± 0.47%（两两比较，均 P < 0.01）；单独封闭 KIR2DL4 受体，杀伤率分别为 30.74% ± 0.44%、26.03% ± 0.38%、21.15% ± 0.35%（两两比较，均 P < 0.01）。 结论 sHLA-G1 通过与 NK-92 细胞表面 ILT2 和 KIR2DL4 受体直接结合而抑制 NK-92 细胞的杀伤活性。

Interactions between sHLA-G1 and its receptors ILT2, and KIR2DL4 on NK-92 cells

Objective To investigate the inhibitory effect of soluble HLA-G1 (sHLA-G1) on the cytolytic function of NK-92, and the relation of sHLA-G1 receptors, ILT2 and KIR2DL4, to the inhibition. Methods ①Recombinant sHLA-G1 was obtained by prokaryotic expression and verified by Western blot. ②NK-92 cells were treated with the recombinant sHLA-G1 at 20 μg/ml for 10 or 30 min, and then added with, anti-HLA-G1/G5 mAb, anti-ILT2 Ab, and anti-KIR2DL4 mAb. NK-92 without treatment served as a positive control. The expression of sHLA-G1, ILT2, and KIR2DL4 on the surface of NK-92 cells was analyzed with flow cytometry. ③Human leukemia cells K562 was used as target cells and the NK-92 cells treated with recombinants sHLA-G1 as the effecters. The effecters and target cells was co-cultured for 2 h at a ratio of 5:1. Then, flow cytometry was used to detect the death rate of K562 cells. Before the co-culture, the effecter, NK-92 cells were divided into two groups. In one group, the NK-92 was treated with 0, 10, or 20 μg/ml recombinant sHLA-G1 for 30 min; and in the other group, it was first incubated with anti-ILT2 Ab, or anti-KIR2DL4 mAb, or both, for 30 min, and then co-cultured with 0, 10, or 20 μg/ml recombinant sHLA-G1 for 30 min. Results ①The recombinant protein was verified as having a histidine tag by Western blot. ②Compared with the control, the positive rate of sHLA-G1 expression was significantly increased and that of the ILT2 and KIR2DL4 was significantly decreased in the NK-92 cells treated with 20 μg/ml recombinant sHLA-G1 for 30 min (P < 0.05). ③After being co-cultured with 0, 10, or 20 μg/ml recombinant sHLA-G1-treated NK-92, the death rate of the K562 reached 39.79% ± 2.00%, 27.79% ± 0.75%, or 21.36% ± 0.67% (one-to-one compared, P < 0.05). When the ILT2 was blocked, the death rate was 23.09% ± 1.63%, 21.13% ± 0.38%, and 18.42% ± 0.47% (one-to-one compared, P < 0.05); and when the KIR2DL4 was blocked, it was 30.74% ± 0.44%, 26.03% ± 0.38%, 21.15% ± 0.35% (one-to-one compared, P < 0.05). Conclusion sHLA-G1 can inhibit the cytolytic function of NK-92 by directly binding to ILT2 and KIR2DL4.