两种多克隆刺激条件下人诱导性Treg表型的多样性变化

Plasticity of human induced regulatory T cells during two different polyclonal stimulations

目的:研究人诱导性调节性T细胞(iTreg)细胞表型的多样性变化,并比较PMA/Ionomycin和PHA两种多克隆刺激对人iTreg的诱导的异同。方法:用Ficoll密度梯度离心分离出人PBMC,空白对照组直接检测,其余则在细胞培养液(非刺激对照组)、PMA/Ionomycin溶液(PMA/Ionomycin刺激组)或PHA溶液(PHA刺激组)中培养16小时,以流式细胞仪检测CD4+CD25+FoxP3+CD127-、CD4+CD25+FoxP3+IL-2-、CD4+CD25+FoxP3+IL-10+、CD4+CD25+FoxP3+TGF-β+和CD4+CD25+FoxP3+IFN-γ+的表达。结果:空白对照组显示,约4%的CD4+细胞表达CD4+CD25+FoxP3+CD127-,即nTreg。PBMC在细胞培养液培养16个小时,iTreg的表达无明显变化,但经多克隆刺激后,各不同细胞表型的iTreg的表达明显上升(均P<0.01),表明多克隆刺激可以明显诱导人iTreg的产生。PHA对IL-2-和TGF-β+iTreg的诱导比PMA/Ionomycin强(P<0.01),但仅PMA/Ionomycin可以诱导产生CD4+CD25+FoxP3+IFN-γ+iTreg,而PHA不能诱导。刺激后产生的CD4+FoxP3+IFN-γ+PBL表达与CD25水平呈逆相关。结论:多克隆刺激可以诱导产生人iTreg,PMA/Ionomycin和PHA对人iTreg诱导的机制和程度不一样。多克隆刺激后产生的不同细胞表型反映了人iTreg的多样性变化。

Objective：To investigate the plasticity of human induced regulatory T cell（iTreg） phenotypes during polyclonal stimulation by comparing the difference between PMA/Ionomycin and PHA stimulation to the in-vitro induction of human iTreg.Methods：Human PBMC were isolated from human peripheral blood using Ficoll density gradient centrifugation.Human PBMC were incubated in cell culture medium with or without PMA/Ionomycin or PHA.Phenotypes of iTreg were analyzed 0 h and 16 h after initiation of cell culture using four-color fluorescence flow cytometry.Results：In freshly isolated PBMC,approximate 4% CD4＋ T cells expressed CD4＋CD25＋FoxP3＋CD127-,which were regarded as nTreg.The proportion remained stable after 16 h incubation in culture medium,whereas increased strongly during polyclonal stimulation（P0.01）.CD4＋CD25＋FoxP3＋ iTreg co-expressing IL-2-,IL-10＋,or TGF-β＋ were induced either during 16 h PMA/Ionomycin or PHA stimulation（all P0.01）,whereas CD4＋CD25＋FoxP3＋IFN-γ＋ iTreg can only be induced by PMA/Ionomycin stimulation（P0.01） but not PHA stimulation（P=n.s.）.Interestingly,expression of IL-2-iTreg after PHA stimulation were higher than it after PMA/Ionomycin stimulation,similar as of TGF-β＋ iTreg（all P0.01）.Expression of CD4＋FoxP3＋IFN-γ＋ iTreg after PMA/Ionomycin stimulation decreased with CD25 expression.Conclusion：Human iTreg could be induced during polyclonal stimulation in-vitro.PMA/Ionomycin and PHA have different mechanisms and pathway for the induction of Treg.The induction of CD4＋CD25＋FoxP3＋ iTreg subsets co-expressing IL-2-,IL-10＋,TGF-β＋,or IFN-γ＋ during polyclonal stimulation shows the plasticity of human iTreg phenotypes.