体外扩增的Vγ9Vδ2 T细胞对肺癌细胞的杀伤作用

In Vitro Amplification of Vγ9Vδ2 T Cells Has Growth Inhibiting Activity in Lung Cancer Cells

目的探讨体外扩增外周血Vγ9Vδ2 T细胞对肺癌细胞系的杀伤作用及其与程序化死亡分子(PD-1)的关系。方法通过固相化抗体扩增法扩增肺癌患者外周血γδT细胞,并通过流式细胞术进行亚型分析和Vγ9Vδ2 T细胞表面PD-1表达情况分析。通过乳酸脱氢酶(LDH)杀伤实验检测来自肺癌患者外周血扩增的Vγ9Vδ2 T细胞对肺癌细胞系NCI-H446和A549细胞的杀伤功能。采用中和抗体封闭PD-1作用和FasL途径、穿孔素/颗粒酶途径的阻断剂(CMA)封闭穿孔素-颗粒酶途径后,通过杀伤实验检测Vγ9Vδ2 T细胞杀伤功能的变化。流式细胞术检测Vγ9Vδ2 T细胞表面CD107a的表达情况,以观察Vγ9Vδ2 T细胞脱颗粒情况。通过激光共聚焦实验检测Vγ9Vδ2 T细胞裂解性颗粒极化情况。结果采用anti-pan-TCRγδ mAb固相化扩增肺癌患者外周血单个核细胞14 d后,γδT细胞比例可增加至(82.5±13.4)%,其中Vγ9Vδ2 T细胞比例为(40.1±8.51)%,Vγ9Vδ1 T细胞比例为(42.4±7.92)%。Vγ9Vδ2 T细胞对肺癌细胞系NCI-H446细胞和A549细胞均具有杀伤效果。阻断PD-1作用后Vγ9Vδ2 T对肺癌细胞的杀伤能力增强(P<0.01)。以CMA处理Vγ9Vδ2 T细胞,Vγ9Vδ2 T细胞对肺癌细胞的杀伤作用降低(P<0.01);而采用FasL中和抗体封闭Fas-FasL途径后,Vγ9Vδ2 T细胞对肺癌细胞的杀伤作用无变化(P>0.05)。阻断PD-1作用后,Vγ9Vδ2 T细胞脱颗粒方面能力无变化(P>0.05),而裂解性颗粒极化水平增加(P<0.01)。结论体外扩增的肺癌患者外周血Vγ9Vδ2 T细胞对肺癌细胞系存在一定杀伤能力,PD-1可抑制Vγ9Vδ2 T细胞对肺癌细胞系的杀伤,主要是通过抑制Vγ9Vδ2 T细胞内裂解性颗粒极化而发挥抑制作用。

Objective To investigate the killing effect of peripheral blood Vγ9Vδ2 T cells expanded in vitro on lung cancer cell lines and its relationship with PD-1(programmed death-1). Methods γδ T cells from peripheral blood of lung cancer patients were amplified by solid phase antibody amplification,and subtype analysis and PD-1 expression on the surface of Vγ9Vδ2 T cells were performed by flow cytometry. LDH killing assay was to detect the killing function of Vγ9Vδ2 T cells amplified from the peripheral blood of lung cancer patients against lung cancer cell lines NCI-H446 and A549 cells. Changes in the killing function of Vγ9Vδ2 T cells were detected by killing assays using neutralizing antibodies to block PD-1 action and Fas L pathway,and CMA was used to block perforin-granzyme pathway. Flow cytometry was performed to detect the expression of CD107 a on the surface of Vγ9Vδ2 T cells to observe the degranulation of Vγ9Vδ2 T cells.Vγ9Vδ2 T cell lysogenic particle polarization was detected by laser confocal assay. Results The proportion ofγδ T cells increased to(82.5±13.4)%,Vγ9Vδ2 T cells was(40.1±8.51)%,Vγ9 Vδ1 T cells was(42.4±7.92)%after 14 days of amplification of individual nuclear cells in peripheral blood of lung cancer patients using anti-pan-TCRγδ m Ab solid phase. Vγ9Vδ2 T cells from peripheral blood of lung cancer patients amplified in vitro had a significant killing effect on both NCI-H446 cells and A549 cells of lung cancer cell lines. Different degrees of PD-1 expression were present on the surface of Vγ9Vδ2 T cells in the peripheral blood of lung cancer patients amplified in vitro,and the killing ability of Vγ9Vδ2 T against lung cancer cells was significantly enhanced(P<0. 01)after blocking PD-1 action. The killing effect of Vγ9Vδ2 T cells on lung cancer cells was significantly reduced(P<0.01)after treating Vγ9Vδ2 T cells with CMA and blocking the perforin-granulase pathway,while the killing effect of Vγ9Vδ2 T cells on lung cancer cells was not significantly changed(P>0. 05)after inhibiting the Fas-Fas L pathway with Fas L neutralizing antibody. After blocking the action of PD-1,the ability of Vγ9Vδ2 T cells to degranulate did not increase significantly(P>0.05),while the level of cleavage granular polarization increased significantly(P<0.01).Conclusion This study confirms that Vγ9Vδ2 T cells from peripheral blood of lung cancer patients amplified in vitro have a certain killing capacity on lung cancer cell lines,and the mechanism of PD-1 inhibition of Vγ9Vδ2 T cells’ killing capacity on lung cancer cell lines is mainly through inhibiting the polarization of cleavage particles within Vγ9Vδ2 T cells.