卡介苗与ESAT-6激活的γδT细胞表达抗原提呈细胞表型和功能的研究

The research phenotypic expression and function of antigen-presenting cells in human γδT cells activated by BCG and Esat-6

目的:探讨卡介苗(BCG)与ESAT-6激活的人外周血γδT细胞是否具有抗原提呈细胞的表型和功能。方法:用Ficoll密度梯度离心法分离人外周血单个核细胞(PBMC)后,经尼龙毛柱法分离获得T细胞。将T细胞分别用卡介苗与ESAT-6刺激扩增γδT细胞后用流式细胞仪分选纯化γδT细胞,并检测其抗原提呈细胞表型。PBMC用贴壁法制备单核细胞后,诱导培养分化为成熟DC细胞,用流式细胞仪检测其成熟程度。T细胞用CFSE标记后分别按四种方法培养:①与结核分枝杆菌分泌性抗原(Mtb-Sag)共同培养;②与Mtb-Sag孵育2小时后的卡介苗激活的γδT细胞共同培养;③与Mtb-Sag孵育2小时后的ESAT-6激活的γδT细胞共同培养;④与Mtb-Sag孵育2小时后的成熟DC细胞共同培养;培养后检测T细胞及CD4+T细胞的增殖情况。结果:卡介苗与ESAT-6激活的γδT细胞CD80、CD86、HLA-DR的表达水平都增加,且后者显著高于前者。卡介苗与ESAT-6激活的γδT细胞培养组的T细胞增殖总数及CD4+T细胞的增殖有所增加;而后者显著高于前者,与成熟DC细胞培养组的增殖情况相似。结论:用卡介苗与ESAT-6激活的人外周血γδT细胞都具有抗原提呈细胞的表型和抗原提呈作用;但后者显著高于前者。

Objective：To study the phenotype and antigen-presenting functions of human peripheral blood γδT cells stimulated by Bacille Calmette-Guerin（BCG） and the early secreted antigenic target-6（ESAT-6）.Methods：Mononuclear cells from human peripheral blood were separated with the Ficoll density gradient centrifugation method,and the T cells were gained by the nylon wool column method.The T cells were treated and stimulated by BCG and ESAT-6 to increase the number of γδT cells,and the γδT cells were separated and purified via Flow Cytometry.The antigen-presenting phenotype of the γδT cells was detected by Flow Cytometry.The mononuclear cells had been separated from PBMC by adherent methods,induced to differentiate into mature dendritic cells（DC）,and then the maturity of DCs were estimated by Flow Cytometry.The T cells marked with CFSE were cultured in four groups：①The secreted antigen of Mycobacterium tuberculosis（Mtb-Sag）,②the γδT cells activated by BCG and together for 2 hours,③the γδT cells activated by Esat-6 and Mtb-Sag together for 2 hours,④ the mature dendritic cells which had been incubated with Mtb-Sag for 2 hours.At last the proliferation of the T cells and the CD4＋ T cells was detected.Results：The CD80,CD86 and HLA-DR expression levels of the γδT cells activated by BCG and ESAT-6 were increased,but the levels of the γδT activated by Esat-6 were significantly higher than those of γδT activated by BCG.In both the two groups,the total number of T cells and CD4＋ T cells increased.However,the number of the cells in Esat-6 group was much higher,as shown when the T cells were cultured with mature dendritic cells.Conclusion：The human peripheral blood γδT cells activated by BCG and ESAT-6 have antigen-presenting cells’ phenotype and antigen presentation functions,and the antigen presentation functions are much stronger in the Esat-6 group.