人外周血中γδT细胞亚群、表型和细胞因子产生的研究

Subpopulations, phenotypes and cytokine productions of human γδT cells from peripheral blood mononuclear cells

目的进一步比较人外周血中γδT细胞及Vδ1^+、Vδ2^+、Vδ1^-Vδ2^-细胞频率、表型及功能方面异同,进一步了解γδT细胞在免疫应答中的作用。方法分离人外周血单个核细胞(PBMCs),流式细胞术检测TCRγδ、Vδ1、Vδ2、CD4、CD8、CD45RO、CD62L、Granzyme A。PMA+Ionomycin刺激前后检测细胞因子IFN-γ、IL-17A及转录因子T-bet、Ro Rγt水平。磁珠富集、流式分选和ELISA检测纯化细胞IFN-γ产生。结果根据γδT细胞受体(TCRγδ)δ链,将γδT细胞分为Vδ1^+,Vδ2^+,Vδ1^-Vδ2^-3群,TCRγδ高低表达与Vδ1、Vδ2表达相关。Vδ1^+、Vδ2^+细胞约有20%~40%细胞表达CD8,低表达CD4,而Vδ1^-Vδ2^-细胞约有60%表达CD8和20%表达CD4。约6%的Vδ1^+和30%的Vδ1^-Vδ2^-细胞表达CD45RO,而约有80%的Vδ2^+细胞表达CD45RO。刺激后,γδT细胞3个亚群均表达IFN-γ和IL-17,Vδ2^+细胞表达最高,3个亚群差异有显著性,与纯化细胞结果相符。3个亚群细胞均高表达转录因子T-bet和Ro Rγt,高表达颗粒酶A。结论人外周血中Vδ1^+、Vδ2^+及Vδ1^-Vδ2^-细胞在频率、表型及功能方面均有显著差异,均可产生高水平细胞因子IFN-γ,该结果将为γδT细胞进一步研究打下良好基础。

The study is aimed to investigate the differences between VS1＋, V82＋ and VS1 -V82- ceils in frequencies, subpopulations, phenotypes and biological functions. We isolated peripheral blood mononuclear cells from healthy donors and detected the surface makers （TCRγδ, VS1, V82, CD4, CD8, CD45RO, CD62L）, intracellular cytokines （IFN-γ, IL-17A）, transcription factors （T-bet, RoRγt）, and Granzyme A by flow cytometry directly or after stimulation with PMA plus Ionomycin. The cytokines produced by purified cells stimulated with PMA plus Ionomycin were detected by ELISA. According to the expression of TCRγδ, γδT ceils could be divided into three groups, and the level of TCRγδ expression was related to the expression of Vδ1 and V82. About 20%-40% of Vδ1＋ and Vδ1＋ cells express CD8 molecules, but not express CD4 molecules, whereas 60% of Vδ1- Vδ2 - γδT cells express CD8 and 20% of them express CD4 molecule. Approximately 6% of Vδ1＋ and 80% of Vδ2＋ cells express CD45RO, whereas about 30% of Vδ1 - Vδ2 - VδT cells express CD45RO. After stimulation, all γδT cells express high levels of IFN-γ and IL-17, and the V82＋ T cells expressed the highest levels of ctokines （P〈 0.01）, compared with other subsets, which was consistent with the results of purified cells detected by ELISA. Further studies had shown that Granzyme A and transcription factors T-bet and RoRγt were highly expressed in Vδ1＋, Vδ2＋ and VM- Vδ2＋ cells. In conclusion, although Vδ1＋, Vδ2＋ and Vδ1- Vδ2- γδT cells has similarity in high secretion of eytokines （IFN-γ）, there are significantly differences in their frequencies, phenotypes and productions of cytokines. These results will be a good foundation for further study in γδT cells.