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


CONCLUSIONS
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.
Mei
Jia-zhuan
JZ
Department of Hematology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. mjzhuan@163.com
Guo
Kun-yuan
KY
Wei
Hong-mei
HM
Song
Chao-yang
CY
D000906:Antibodies / Q000494:pharmacology; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000276:immunology; D018432:Drug Resistance, Multiple; D005434:Flow Cytometry; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000276:immunology / Q000378:metabolism*; D009303:Nasopharyngeal Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction; D054340:Receptors, KIR / Q000235:genetics
D000906:Antibodies; D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; C527085:KLRK1 protein, human; C099187:MHC class I-related chain A; C098080:MICB antigen; D055655:NK Cell Lectin-Like Receptor Subfamily K; D054340:Receptors, KIR