托法替尼对HMBPP特异性γδT细胞功能影响的研究

Effects of tofacitinib on the functions of HMBPP-specific γδT cells

目的 研究类风湿性关节炎治疗药物托法替尼对抗原特异性 γδT细胞的抑制作用.方法 选用4-羟基-3-甲基-2-丁烯基焦磷酸盐(4-hydroxy-3-methyl-2-butenyl pyrophosphate,HMBPP)作为抗原刺激人 γδT细胞,利用流式细胞术和酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测托法替尼对HMBPP特异性 γδT细胞细胞因子产生、细胞活化、细胞增殖及转录因子磷酸化的影响.结果HMBPP可刺激外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)中 γδT细胞产生干扰素(interferon,IFN)-γ及肿瘤坏死因子(tumor necrosis factor,TNF)-α,IFN-γ 的产生呈时间剂量依赖性,TNF-α在12 h达到高峰.向培养体系中加入药物托法替尼后,γδT细胞IFN-γ、TNF-α的产生显著降低.流式细胞术分析的结果表明,HMBPP刺激PBMCs后,主要为Vδ2+γδT细胞产生IFN-γ 和TNF-α.且托法替尼还可以抑制HMBPP诱导的Vδ2+γδT细胞活化,抑制CD25表达,Vδ2+γδT细胞中CD25阳性比例在第一天及第三天分别由3.85%、5.37%降至1.66%、1.70%,而不抑制CD69表达.托法替尼还可以抑制HMBPP诱导的Vδ2+γδT细胞增殖,细胞分裂比例从16.1%降至8.06%.此外,研究其机制表明托法替尼抑制HMBPP诱导的Vδ2+γδT细胞中信号传导及转录激活因子(signal transducers and activators of transcription,STAT)1和4分子磷酸化,抑制细胞因子的产生和功能.结论 托法替尼抑制HMBPP特异性 γδT细胞细胞因子产生、细胞活化、细胞增殖及转录因子磷酸化.提示托法替尼除抑制Th1细胞的功能外,还可抑制γδT细胞的功能,为其临床应用提供理论基础.

Objective To investigate the inhibitory effects of Tofacitinib on 4-hydroxy-3-methyl-but-enyl pyrophosphate(HMBPP)-specific γδT cells. Method We used HMBPP as an antigen to stimulate γδT cells,in order to study the inhibitory effects of tofacitinib on the production of cytokines,activation,proliferation and phosphorylation of STATs in HMBPP-specific γδT cells. Results Stimulation with HMBPP could induce the production of interferon(IFN)-γ and tumor necrosis factor(TNF)-α in γδT cells. The production of IFN-γshowed a dose-dependent trend,while TNF-α reached a peak at 12 hours after culture. After the addition of To-facitinib into the culture system,the expression of IFN-γ and TNF-α in γδT cells was significantly reduced. Flow cytometry analysis showed that the cytokine-expressing cells in PBMCs cells after stimulation with HMBPP were Vδ2 + γδT cells,and this subpopulation of cells could produce IFN-γ and TNF-α. Upon further investigations, we found that Tofacitinib inhibited the activation of Vδ2 + γδT cells induced by HMBPP,and mainly suppressed the expression of CD25 from 3. 85% and 5. 37% to 1. 66% and 1. 70% at the first and the third day respectly, but not CD69 molecular. Furthermore,CFSE-labeled cell culture showed that tofacitinib inhibited HMBPP-in-duced proliferation of Vδ2 + γδT cells from 16. 1% to 8. 06% . The results from flow cytometry showed that tofac-itinib inhibited HMBPP-induced phosphorylation of signal transducers and activators of transcriptions (STATs) in Vδ2 + γδT cells,which might be the main mechanism of the inhibition effects by tofacitinib in γδT cells. Con-clusions Tofacitinib could inhibit the production of cytokines,activation,proliferation and phosphorylation of STATs in HMBPP-specific γδT cells. Our results showed that tofacitinib could inhibit the functions of γδT cells, except inhibition on Th1 cells,which provided a theoretical basis for clinical applications.