人胶质瘤细胞U251逃逸NK细胞免疫杀伤机制的初步探讨

Aberrant Expression of NKG2D Ligand and High Expression HLA-Ⅰ Molecules May Contribute to Immune Escape from Natural Killer Cell-mediated Immune Surveillance of Malignant Gliomas

目的探讨人神经胶质瘤细胞U251逃逸同种异体NK细胞免疫杀伤的机制。方法以K562细胞为对照,应用LDH释放法检测不同效靶比时NK细胞体外杀伤U251细胞的活性。用RT-PCR检测K562和U251细胞MHC-Ⅰ类链相关分子A和B(MICA/B)、人巨细胞病毒糖蛋白UL16结合蛋白(ULBP1～3)基因,用流式细胞仪检测两细胞MICA/B、ULBP1～3和HLA-Ⅰ分子的表达情况。效靶比20:1时用单抗分别阻断K562和U251细胞表面MICA、MICB、ULBP1、ULBP2、ULBP3和HLA-Ⅰ类分子,观察NK细胞对其杀伤活性的变化。结果同一效靶比时NK细胞杀伤U251细胞的活性明显低于杀伤K562细胞的活性,两者之间差异有统计学意义(P<0.05);K562和U251细胞均表达基因MICA/B和ULBP1～3,但U251细胞仅低表达ULBP2分子。用单抗封闭MICA/B和ULBP1～3分子后,NK细胞对K562细胞的杀伤活性明显降低,对U251细胞的杀伤活性无明显改变。封闭HLA-Ⅰ类分子后NK细胞对U251细胞的杀伤活性明显上升,对K562细胞的杀伤活性无明显改变。结论U251细胞逃逸NK细胞免疫杀伤机制可能是由于U251细胞高表达HLA-Ⅰ类分子,不表达NKG2D的配体MICA/B和ULBP1～3。

Objective To explore the mechanism of immune escape from natural killer（NK）cell-mediated immune surveillance of malignant gliomas. Methods We took K562 cells cytolysis sensitively to NK cell as positive control, cytotoxicities of NK cells isolated from 5 healthy volunteers against U251 cells were analyzed by LDH releasing assay at different effector- to-target cell ratios（E：T）. The genes and proteins expression of NKG2D ligands on K562 and U251 cell line were respectively measured by RT-PCR and flow cytometry. In blocking experiments, mAbs of different NKG2D ligands and HLA- I molecules were added to the target cells at E： T of 20： 1. Results Cytotoxicity of NK cells against K562 cells was much higher than that against U251 cells at the same ET ratio. There was a significant difference between them. The genes of NKG2D ligands were positive in K562 and U251 cells, All the proteins of NKG2D ligands were expressed on K562 cell surface, but noly ULBP2 molecule was found on U251 cell surface. In blocking experiments, the cytotoxicity of NK cells against K562 cell was partially inhibited, that against U251 cell was not influenced when mAbs of different NKG2D ligands were added; the cytotoxicity of NK cells against U251 cell was dramatically upgraded, that against K562 cell was not influenced when mAb of HLA- I molecules was added. Conclusion Aberrant expression of NKG2D ligand and high expression HLA- I molecules may contribute to immune escape from NK cell-mediated immune surveillance of malignant gliomas.