多色流式细胞术检测人外周血BCG特异性效应型记忆γδT细胞的表型特征

Phenotypic analysis of BCG-specific effector memory γδ T cells in PBMCs by multi-color flow cytometry

目的检测卡介苗(BCG)刺激后,PPD+正常人外周血中分泌IFN-γ的TCRγδ亚群及其表型特征。方法分离PPD+正常人外周血单个核细胞(PBMCs),用多色流式细胞术检测TCRγδ亚群;BCG刺激PBMCs后检测γδT细胞IFN-γ分泌水平,并分析BCG特异性γδT细胞表型特征。结果依据PBMCs中TCRγδ和δ2的表达,可将γδT细胞分为3个亚群,即δ2high、δ2low和TCRγδ中非δ2T细胞(non-δ2-γδT)细胞。其中,δ2high和δ2lowT细胞几乎全部(>98%)为CD45RO+,而non-δ2-γδT细胞中只有少部分表达CD45RO,其余大部分不表达CD45RO。δ2high和δ2lowT细胞表型类似,主要为CD45RO+CD62L-CD27-,而non-δ2CD45RO+细胞主要为CD62L-CD27+,non-δ2CD45RO-细胞几乎全部为CD62L-CD27-。BCG刺激PBMCs后,可诱导γδT细胞分泌IFN-γ,且主要为δ2high和δ2low细胞,而non-δ2-γδT几乎不产生IFN-γ。进一步分析BCG特异性γδT细胞的表型特征,结果表明,δ2high-CD45RO+IFN-γ+和δ2lowCD45RO+IFN-γ+细胞主要为CD62L-CD27-和CD62L-CD27+,为效应型记忆细胞。结论 BCG刺激PPD+正常人外周血PBMCs后,主要诱导δ2T细胞分泌IFN-γ,且该细胞表现出CD45RO+CD62L-效应型记忆细胞特征,可能在预防结核早期感染中发挥重要作用。

This study aimed to evaluate IFN-γ-producing γδ T cell subsets in PBMCs from PPD＋normal donors after stimulation with BCG.We isolated PBMCs from PPD＋ normal individuals and assessed γδ T cell subsets by multi-color flow cytometry.And the phenotype of IFN-γ-producing γδ T cells was further analyzed.According to the expression of TCRγδ and δ2,γδT cells could be delineated as δ2high,δ2low and non-δ2-γδ T cells.Furthermore,δ2high and δ2low cells were mostly（98%） CD45RO＋,only small percentages of non-δ2-γδ T cells expressed CD45RO.Further analysis indicated that δ2high and δ2low cells were CD45RO＋CD62L-CD27-,whereas non-δ2CD45RO＋T cells were mostly CD62L-CD27＋ and non-δ2CD45RO-were CD62L-CD27-.Following stimulation with BCG,δ2high and δ2low cells expressed IFN-γ,whereas non-δ2 γδ T cells did not.Further analysis also indicated that δ2highCD45RO＋IFN-γ＋ and δ2lowCD45RO＋IFN-γ＋ were mostly CD62L-CD27-or CD62L-CD27＋,suggestive of effector memory cells.In conclusion,our study demonstrated that BCG could induce δ2 T cells to produce IFN-γ.And phenotypic analysis indicated that IFN-γ＋δ2 T cells are effector memory cells,which probably play an important role during TB infection.