骨髓瘤RPMI 8226细胞体外逃逸NK细胞免疫杀伤机制的初步研究

Mechanism of immune escape of multiple myeloma cell RPMI 8266 from cytotoxicity of natural killer

目的:探讨骨髓瘤细胞RPMI 8266逃逸NK细胞免疫杀伤的机制。方法:流式细胞仪检测K562和8266细胞表面MICA/B、ULBP1～3和HLA-Ⅰ类分子的表达。4hLDH释放法测定效靶比20∶1时阻断前后NK细胞对2种细胞杀伤活性变化。观察效靶比20∶1时NK细胞对药物处理后RPMI 8226细胞杀伤活性变化、RPMI 8226细胞表面NKG2D配体和HLA-Ⅰ类分子的表达及NK细胞对RPMI 8266细胞的克隆形成率的影响。结果:K562细胞高表达MICA/B和ULBP 1～3分子;RPMI 8266细胞表达HLA-Ⅰ类分子,2株细胞NKG2D配体表达差异有统计学意义,P<0.001。单抗分别阻断MICA/B、ULBP 1～3分子后,效靶比为20∶1时NK细胞对K562细胞的杀伤活性明显降低,P值均<0.05;对RPMI 8266细胞的杀伤活性基本无变化,P值均>0.05。抗W6/32单抗封闭8266细胞表面HLA-Ⅰ类分子后,NK细胞对K562细胞的杀伤活性无上升(P=0.721),而对RPMI 8266细胞的杀伤活性上升,P=0.000。1/2IC50的三氧化二砷处理后RPMI8226细胞ULBP2、3配体升高(P=0.000),NK细胞对8266细胞的杀伤活性与对照组相比差异有统计学意义,P=0.001;1/2IC50硼替佐米处理后RPMI 8226细胞表面各NKG2D配体无变化,P>0.05。NK细胞对K562细胞克隆形成抑制率为(63.48±6.78)%,对RPMI 8226细胞为(23.71±2.39)%,P=0.000。结论:RPMI8266细胞逃逸NK细胞免疫杀伤机制可能与该细胞高表达HLA-Ⅰ类分子,低表达NKG2D的配体MICA/B和ULBP1～3分子有关。

OBJECTIVE：The mechanism of immune escape of multiple myeloma cell line PRMI-8266 from cytotoxicity of natural killer（NK）cell was studied.METHODS：The hypersensitive cell K562 was controlled,after attacked by NK cell at 20∶1,the cytotoxicity of NK cell and proteins expression of NKG2D ligands on K562 and RPMI 8266 cell line was detected by flow cytometry.RPMI 8226 cells was treated by arsenic trioxide and brotezomib（proteasome inhibitor）,cytotoxicity of NK cells against pre-treated RPMI 8226 cells at 20∶1 ratio was assayed by LDH releasing kit.Expression of HLA class Ⅰ molecules and NKG2D ligands on the surface of pre-treated RPMI 8226 cells were analyzed by flow cytometry.RESULTS： For K562 cell,MICA/B and ULBP 1-3 were high expressed.There were HLA-Ⅰ moleculer on membrane of RPMI 8266 cell.There were statistical differences between them（P0.001）.After blocking MICA,MICB and ULBP 1-3 by mAbs,the Cytotoxicity of NK cells against K562 cell at 20∶1 ratio was much lower than before（P 0.05）.But for RPMI 8266,there were no significant difference between before and after blocking experiment（P0.05）.But for blocking HLA-Ⅰ,the result were reversely.There was no significant difference of cytotoxity of NK cell against K562 cell between before and after blocking HLA-Ⅰ by W6/32（P=0.721）,but for RPMI 8266 it was contrarily（P=0.000）.The cytotoxicity of allo-NK cells against RPMI 8226 cells after treated by 1/2 IC50 arsenic trioxide,the expression of ULBP2 and ULBPE were higher than untreated and without the change of HLA-Ⅰ.It was statistically（P=0.001）.Cytotoxicity of NK cells against RPMI 8226 cells treated with brotezomib had not affected and no change of NKG2D ligands（P0.05）.The inhibiton of clone formation of K562 and RPMI 8226 before and after attacked by Nk cell were（63.48±6.78）% and（23.71±2.39）%（P=0.000）.CONCLUSION：Aberrant expression of NKG2D ligand and high expression HLA-Ⅰ molecules may contribute to multiple myeloma cell immune escape from cytotoxicity of NK cell.