调节性T细胞功能稳定性及其在免疫治疗中的作用机制研究

发布时间：2018-10-31 14:29

【摘要】：CD4+CD25+Foxp3+调节性T细胞(Tregs)在维持机体免疫平衡中起着重要作用,而Foxp3在Treg的表达水平与Treg的功能直接相关。基础与临床的多项研究已证实Tregs能够治疗多种疾病,包括自身免疫性糖尿病,实验性关节炎等。目前,如何培养稳定,有效的Tregs功能是其研究的热点。有研究小组发现Tregs在炎症环境下不稳定,容易失去Foxp3表达,同时失去Foxp3的前Tregs表达大量的IL-17,从而加重疾病反应。因此,本课题旨在分析和探究能够获得稳定Tregs的最佳方案。并检测与Tregs稳定性关系重大的相关标志物及其相关的信号通路。本课题的成功对Tregs I临床治疗具有一定的普遍意义并为移植术后免疫耐受的诱导提供新的思路。总课题分为下面三个部分：第一部分TGF-β诱导的CD4+Foxp3+iTregs在急性移植物抗宿主病中的作用和机制研究。研究背景：诱导型调节性T细胞(induced regulatory T cells, iTregs)被认为可以用来治疗自身免疫性疾病。然而最新研究发现其在急性移植物抗宿主病模型中疗效不明显,其原因在于iTregs功能不稳定,在体内容易转化为其他细胞,从而失去免疫抑制功能。本文旨在探讨iTregs在急性移植物抗宿主病中功能及稳定性的影响。研究目的：我们通过研究iTregs在急性移植物抗宿主病模型中的作用,鉴别不同iTregs培养方案对其细胞功能的影响。方法：我们运用TGF-β, IL-2诱导iTregs治疗两种不同的小鼠急性移植物抗宿主病模型。在一些实验中CFSE标记的Tregs被注入小鼠体内来监测Tregs体内的稳定性。抗CD25抗体被用来拮抗Tregs,观测拮抗Tregs后,移植物抗宿主病模型的生存率。最后,我们还监测了不同方案的iTregs的体外与体内稳定性和功能。结果：iTregs具有较强的保护急性移植物抗宿主病的功能,这种保护作用是通过iTregs抑制供者CD4细胞,CD8细胞的扩增以及供者CD8细胞的杀伤作用,保护受者B细胞来实现的。在体内实验中,我们观测到iTregs能够保持稳定并持续发挥功能,最后我们的研究还发现anti-CD3/CD28磁珠诱导的iTregs比anti-CD3包板诱导的iTregs具有更强的免疫调节功能。结论：只有运用正确的诱导扩增方案,才能够培养出状态稳定,具有强大抑制功能的iTreg s,从而保护急性移植物抗宿主病。第二部分全反式维甲酸对肝脏移植术后患者iTreg s的作用研究研究目的：我们通过全反式维甲酸作用肝脏移植术后患者iTregs,分析全反式维甲酸对iTregs功能的影响。方法：我们运用TGF-β, IL-2与全反式维甲酸从肝脏移植术后患者提取的幼稚T细胞中的诱导iTregs,检测Foxp3表达,体外扩增功能。通过CFSE标记的T细胞与iTregs共培养,观察全反式维甲酸作用的iTregs的体外抑制功能。同时,建立小鼠异种移植物抗宿主病模型,并由尾静脉回输iTregs观察iTregs对异种移植物抗宿主病的保护作用以及相关细胞因子表达情况。结果与结论：全反式维甲酸能够增强患者iTregsFoxp3的表达以及扩增倍数,同时提高iTregs的体内与体外功能。本研究为iTregs在临床治疗的实际问题提供了新的解决方案。第三部分人CD39hi nTreg亚群的稳定性和功能研究及其相关机制分析研究背景：自然调节性T细胞(natural regulatory T cells, nTregs)被认为可以用来治疗自身免疫性疾病。然而研究表明其在炎症环境下易转化为Th1, Th17细胞,从而失去免疫抑制功能,CD39是与Tregs功能相关的表面标志,可能与Tregs的稳定性有关。研究目的：我们通过研究CD39hi nTregs与CD39low nTregs在炎症环境下的表型和稳定性差异,为临床运用具有功能的调节性T细胞诱导免疫耐受提供实验依据。方法：分选出人CD4+CD25hrightnTreg并在体外培养扩增7天,流式分选出CD39hi nTreg和CD39low nTreg加入IL-1β或者IL-6继续培养1至3天,将获得的nTregs进行体内外抑制功能检测,利用免疫印迹法检测STAT1, p-STAT1, STAT3, p-STAT3的表达水平,并分析Foxp3启动子区域的变化。体内实验采用不同类型nTregs回输异种移植物抗宿主疾病。结果：CD39hi nTregs比CD39low nTregs具有更加强大的免疫抑制功能,在体外实验中,CD39hi nTregs能够保持Foxp3表达,具有较强的抑制T细胞扩增的能力；而CD39low nTregs快速丢失Foxp3表达,并转化为Th1, Th17细胞。在体内实验中,CD39hi nTregs能够保护异种移植物抗宿主病的体重变化,提高生存率。最后,IL-2能够显著提高CD39low nTregs的体内功能和稳定性,这种作用与STAT1, STAT3磷酸化和DNA甲基化水平有关。结论：nTregs的稳定性与CD39表达有关,Tregs体外扩增后分选CD39hinTregs能够提高细胞功能和稳定性。
[Abstract]:CD4 + CD25 + Foxp3 + regulatory T cells play an important role in maintaining immune balance of body, while Foxp3 is directly related to the function of Treg. A number of studies based on clinical and clinical studies have demonstrated that Tregs can treat multiple diseases, including autoimmune diabetes, experimental arthritis, and so on. At present, how to cultivate stable and effective Treggs function is the hot spot of its research. A study team found that Tregs was unstable in inflammatory conditions, easily lost Foxp3 expression, while losing Foxp3 's preTregs to express a significant amount of IL-17, thus worsening the disease response. Therefore, the purpose of this study is to analyze and explore the best solution that can obtain stable Treggs. and to detect relevant markers that are significant in relation to the stability of the Tregs and their associated signal paths. The successful treatment of Treggs I has a certain general significance and provides a new idea for the induction of immune tolerance after transplantation. The total subjects were divided into three parts: the role and mechanism of CD4 + Foxp3 + iTregs induced by the first part of retinoic acid in acute graft versus host disease. Background: Inducible regulatory T cells (iTregs) are believed to be useful in the treatment of autoimmune diseases. However, the latest study found that it was not effective in the acute graft-versus-host disease model because the iTregs function was unstable and easily converted into other cells in vivo, thus losing the immunosuppressive function. The purpose of this paper is to investigate the effect of iTregs on the function and stability of acute graft versus host disease. Objective: To investigate the effect of iTregs on cell function in acute graft versus host disease model. Methods: We used IL-2 and IL-2 to induce iTregs to treat two different models of acute graft versus host disease in mice. In some experiments, the CFSE-labeled Tregs were injected into mice to monitor the stability of the Treggs body. Anti-CD25 antibody was used to antagonize Treggs and observe the survival rate of graft versus host disease model following Treggs observation. Finally, we also monitored the in vitro and in vivo stability and function of iTregs of different protocols. Results: iTregs has a strong function of protecting against host disease of acute graft. This protective effect is achieved by inhibiting the proliferation of CD4 cells, CD8 cells and the killing effect of donor CD8 cells through iTregs. In vivo experiments, we observed that iTregs can maintain stability and continue to function. Finally, our study also found that the iTregs induced by anti-CD3/ CD28 beads had a stronger immune modulatory function than the anti-CD3 envelope-induced iTregs. Conclusion: Only the correct induction amplification protocol can be used to develop the iTreg s with stable state and strong inhibitory function, thus protecting the acute graft anti-host disease. Objective: To study the effect of all-trans retinoic acid on iTreg s function in patients with liver transplantation, and to analyze the effect of all-trans retinoic acid on iTregs function. Methods: We detected the expression of Foxp3 in naive T cells extracted from patients with liver transplantation by means of anti-retinoic acid, IL-2 and all-trans retinoic acid, and detected the function of Foxp3 expression and in vitro amplification. In vitro inhibition of trans-retinoic acid on iTregs was observed by co-culture of CFSE-labeled T-cells with iTregs. At the same time, the anti-host disease model of mouse xenografts was established, and the protective effect of iTregs on the host disease of xenografts and the expression of relevant cytokines were observed from the tail vein. RESULTS & CONCLUSION: All trans retinoic acid can enhance the expression of iTregsFoxp3 and the amplification factor of iTregsFoxp3, and improve the in vivo and in vitro function of iTregs. This study provides a new solution for the practical treatment of iTregs. The stability and functional studies of CD39hi nTreg subpopulations of the third part and their related mechanisms are discussed in this paper. The natural regulatory T cells (nTregs) are considered to be useful in the treatment of autoimmune diseases. However, the study shows that it is easily transformed into Th1 and Th17 cells in an inflammatory environment, thus losing the immunosuppressive function, and CD39 is a surface marker related to the Tregs function, possibly related to the stability of Treggs. Objective: We study the phenotype and stability of CD39hi nTregs and CD39low nTregs in inflammatory environment, and provide experimental basis for clinical use of regulatory T cells with function to induce immune tolerance. Methods: Human CD4 + CD25hrightnTreg was selected and cultured in vitro for 7 days. CD39hi nTreg and CD39low nTreg were selected to add IL-1 MBP or IL-6 for 1 to 3 days, and the obtained nTgs were detected in vivo. The expression levels of STAT1, p-STAT1, STAT3, p-STAT3 were detected by Western blot. and the change of Foxp3 promoter region was analyzed. In vivo experiments were performed using different types of nTregs back-to-lose xenografts against host diseases. Results: CD39hi nTregs has a stronger immunosuppressive function than CD39low nTregs. In vitro experiments, CD39hi nTregs can maintain Foxp3 expression, has stronger ability to inhibit T cell amplification, and CD39low nTregs rapidly lost Foxp3 expression and transformed into Th1 and Th17 cells. In vivo experiments, CD39hi nTregs can protect the body weight of xenografts against host disease and improve survival. Finally, IL-2 can significantly improve the in vivo function and stability of CD39low nTregs, which is related to STAT1, STAT3 phosphorylation and DNA methylation level. Conclusion: The stability of nTregs is related to the expression of CD39, and the sorting of CD39hinTreggs after Treggs in vitro can improve cell function and stability.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392.4

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