[多药耐药鼻咽癌细胞系CNE2/DDP中NKG2D配体的表达及其对自然杀伤细胞细胞毒性的影响]

[Expression of NKG2D ligands in multidrug-resistant nasopharyngeal carcinoma cell line CNE2/DDP and their effects on cytotoxicity of natural killer cells].

作者信息

Mei Jia-zhuan, Guo Kun-yuan, Wei Hong-mei, Song Chao-yang

机构信息

Department of Hematology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.

出版信息

Nan Fang Yi Ke Da Xue Xue Bao. 2007 Jun;27(6):887-9.

PMID:17584663

Abstract

OBJECTIVE

To analyze the expression of NKG2D ligands on human nasopharyngeal carcinoma cell line CNE2 and the multidrug-resistant lin CNE2/DDP and investigate its impact on cytotoxicity of natural killer (NK) cells.

METHODS

Expression of NKG2D ligands on the surface of CNE2 and CNE2/DDP cells was analyzed by flow cytometry, and their HLA genotypes, along with inhibitory killer cell immunoglobulin-like receptors (KIRs) expressed on NK cells from 5 healthy donors, were determined by PCR with sequence specific primers. Cytotoxicity of NK cells against CNE2 and CNE2/DDP cells was evaluated by LDH-releasing assay at different effector-to-target ratios (E:T). In blocking experiments, different monoclonal antibodies (mAb) were added to the target cells at the E:T of 20:1 ratio.

RESULTS

The HLA genotypes of CNE2 and CNE2/DDP cells were A2, 24, B18, 35, Cw4, 7, and the inhibitory KIR genotypes of the 5 healthy donors were KIR2DL1, KIR2DL3, KIR3DL1, and KIR3DL2, mismatched with the HLA -class I molecules expressed by the CNE2 and CNE2/DDP cells. The expression of MHC class I chain-related proteins A and B (MICA and MICB) on CNE2 cells was higher than that on CNE2/DDP cells (P<0.01), and ULBP1 and ULBP3 were not detectable. NK cells displayed highly in vitro cytotoxicity against CNE2 and CNE2/DDP cells with a mean cell lysis rate of (10.50-/+2.17)%, (4.98-/+0.95)%; (27.68-/+1.47) %, (15.48-/+2.10) %; (36.99-/+3.13) %, (28.46-/+4.30) %; (55.00-/+2.20) %, (40.95-/+2.21) %, respectively, corresponding to the E:T ratios of 5:1, 10:1, 20:1, and 30:1 (P<0.01). Blocking experiments confirmed that killing of CNE2 and CNE2/DDP cells by NK cells was efficiently inhibited by anti-MICA, anti-MICB, and anti-ULBP2 mAbs, whereas anti-ULBP1 and anti-ULBP3 mAbs had no effects on the cytotoxicity of the NK cells.

CONCLUSION

Expression of NKG2D ligands on CNE2 and CNE2/DDP cells is correlated with NK cell-mediated lysis, and NK cells display higher cytotoxicity against parental CNE2 cells than the multidrug-resistant CNE2/DDP cells.

摘要

目的

分析NKG2D配体在人鼻咽癌细胞系CNE2及多药耐药细胞系CNE2/DDP上的表达情况，并研究其对自然杀伤（NK）细胞细胞毒性的影响。

方法

采用流式细胞术分析CNE2和CNE2/DDP细胞表面NKG2D配体的表达，并用序列特异性引物PCR法检测其HLA基因型以及5名健康供者NK细胞上表达的抑制性杀伤细胞免疫球蛋白样受体（KIR）。采用乳酸脱氢酶释放法在不同效应细胞与靶细胞比例（E:T）下评估NK细胞对CNE2和CNE2/DDP细胞的细胞毒性。在阻断实验中，以20:1的E:T比例向靶细胞中加入不同的单克隆抗体（mAb）。

结果

CNE2和CNE2/DDP细胞的HLA基因型为A2、24、B18、35、Cw4、7，5名健康供者的抑制性KIR基因型为KIR2DL1、KIR2DL3、KIR3DL1和KIR3DL2，与CNE2和CNE2/DDP细胞表达的HLA -Ⅰ类分子不匹配。CNE2细胞上MHC Ⅰ类链相关蛋白A和B（MICA和MICB）的表达高于CNE2/DDP细胞（P<0.01），未检测到ULBP1和ULBP3。NK细胞对CNE2和CNE2/DDP细胞表现出高度的体外细胞毒性，平均细胞裂解率分别为（10.50±2.17）%、（4.98±0.95）%；（27.68±1.47）%、（15.48±2.10）%；（36.99±3.13）%、（28.46±4.30）%；（55.00±2.20）%、（40.95±2.21）%，对应E:T比例为5:1、10:1、20:1和30:1（P<0.01）。阻断实验证实，抗MICA、抗MICB和抗ULBP2单克隆抗体可有效抑制NK细胞对CNE2和CNE2/DDP细胞的杀伤，而抗ULBP1和抗ULBP3单克隆抗体对NK细胞的细胞毒性无影响。