NKG2D Mediated Cytotoxicities of NK Cells Against Human Nasopharyngeal Carcinoma Cell Line (CNE2) in Vitro

Objective 　To analyze HLA-class I molecules and the expression of NKG2D ligands in human nasopharyngeal carcinoma cell line (CNE2) and their effects on cytotoxicity of natural killer (NK) cells. Methods 　The expression of NKG2D ligands on the surface of CNE2 and K562 cells were analyzed by flow cytometry. The HLA2 class I molecules in CNE2 cells and killer cell immunoglobulin-like receptors ( KIR) expressed by NK cells (isolated from 5 healthy persons) were analyzed by PCR-SSP. Cytotoxicities of NK cells against CNE2 and K562 cells were detected by LDH releasing assay at different effect-to-target cell ratios ( E∶T) . In blocking experiments, different anti-NKG2D ligands monoclonal antibodies (mAbs) were added to the target cells at 20∶1 E∶Tratio. Results 　It was found that MICA, MICB, ULBP2 were expressed by CNE2, ULBP1, ULBP3 were not detectable on CNE2 ; K562 expressed all the NKG2D ligands. There were mismatches between inhibitory KIRs expressed by NK cells and HLA-class I molecules expressed by the CNE2 cells. NK cells displayed highly in vit ro cytotoxicity against K562 and CNE2 cells with anlysis of (29. 02 ±0. 45) %, (10. 50 ±2. 17) %; (44. 43 ±1. 36) %, (27. 68 ±1. 47) %; (57. 82 ±1. 35) %, (36. 99 ±3. 13) %; (71. 24 ±2. 36) %, (55. 00 ±2. 20) % respectively at 5∶1, 10∶1, 20∶1, 30∶1 E∶T ratios ( P = 0. 000) . Blocking experiment s confirmed that killing of K562 by NK cells was efficiently inhibited by anti-MICA mAb, anti-MICB mAb, anti-ULBP1 mAb, anti-ULBP-mAb and anti-ULBP-mAb. anti-MICA mAb. Anti-MICBmAb, anti-ULBP2 mAb could partially inhibit the cytotoxicity of NK cells against CNE2 cells, whereas anti-ULBP1 mAb and anti-ULBP3 mAb could not inhibit the cytotoxicity of NK cells. Conclusion 　Expression of NKG2D ligands is correlated with the cytotoxicity of NK cells. NK2 mediated cytolytic activity may be boosted by engineering cells expressing high levels of activating NKG2D ligands.