慢性病毒感染中CD8T细胞功能耗竭的分子机制

发布时间：2018-01-18 15:20

本文关键词：慢性病毒感染中CD8T细胞功能耗竭的分子机制　出处：《上海交通大学》2012年博士论文　论文类型：学位论文

【摘要】：背景和目的：慢性病毒感染后，CD8T细胞增殖分化成为病毒特异性的效应CD8T细胞，由于慢性病毒持续存在，效应CD8T细胞免疫功能逐渐降低，增殖能力下降，细胞存活率下降，并且不能分化形成记忆性CD8T细胞，这一现象已经被学术界广泛研究发现，并且称此为T细胞功能耗竭,大部分发生在慢性感染和肿瘤中。目前关于CD8T细胞功能耗竭的机制尚不完全清楚。我们的前期研究结果显示，表观遗传修饰在记忆性CD8T细胞的分化形成以及功能发挥中具有重要作用。因此本研究中，我们将首次从表观遗传修饰角度探讨慢性病毒感染中CD8T细胞功能耗竭的分子调控机制，重点是组蛋白乙酰化调控与病毒慢性感染中功能耗竭性CD8T细胞的关系，从而以此为理论基础进一步研究延缓或者逆转慢性病毒感染后CD8T细胞功能的耗竭。方法：利用淋巴细胞行脉络丛脑膜炎病毒(lymphocytic choriomeningitisvirus-LCMV)不同病毒株感染小鼠建立小鼠感染模型：急性病毒株LCMV-Arm（Arm）感染小鼠建立急性感染，LCMV-clone13（Cl-13）感染小鼠建立慢性感染。通过与Arm急性病毒感染比较，分析Cl-13慢性感染过程中，病毒特异性CD8T细胞组蛋白乙酰化的表达特点、功能意义及与疾病进展的关系，从表观遗传学角度分析慢性感染中抗原特异性CD8T细胞免疫功能耗竭的分子机制。应用四聚体染色技术检测表位特异性CD8T细胞的频率；流式细胞仪检测病毒特异性CD8T细胞的表型特征；体外多肽刺激淋巴细胞，流式细胞仪检测CD8T细胞产生细胞因子的能力；流式细胞染色以及染色质免疫共沉淀(chromatinimmunoprecipitation, ChIP）实验研究CD8T细胞组蛋白乙酰化表达水平；体内和体外实验探讨组蛋白乙酰化酶抑制剂丙戊酸钠（valproic acid sodium,VPA）是否可以逆转耗竭性CD8T细胞的免疫功能；流式细胞染色和实时定量PCR检测VPA处理后CD8T细胞的功能状态；细胞过继转移实验研究VPA体外处理后的功能耗竭性CD8T细胞是否可以在体内存活并且发挥更强的免疫功能。结果：1)与Arm急性致病病毒株比较，Cl-13慢性病毒株感染小鼠后，病原特异性CD8T细胞免疫功能逐渐降低，分泌细胞因子IFN-γ, TNF-α以及IL-2能力下降，并最终进入耗竭状态。2) Cl-13感染中，流式细胞仪染色分析显示，，病毒特异性CD8T细胞组蛋白乙酰化水平在感染初期高表达，随着感染的进程，逐渐低表达。ChIP结果显示，在慢性病毒感染初期，ifng-promoter和ifng-enhancer两基因位点结合更多乙酰化的组蛋白；在功能耗竭期，两基因位点结合乙酰化的组蛋白水平显著降低。3) Cl-13感染中，不仅病毒特异性CD8T细胞功能耗竭，整体CD8T细胞免疫功能也下降，表现为分泌细胞因子的能力降低，并且整体CD8T细胞组蛋白乙酰化水平也降低。4)体外VPA处理耗竭性CD8T细胞后，其组蛋白乙酰化水平升高，分泌IFN-γ等细胞因子的能力提高。5)体外VPA处理后的耗竭性CD8T细胞过继转移入正常小鼠体内后，存活率升高，并且分泌细胞因子能力升高，表明这群细胞免疫功能增强，成为具有功能的记忆性CD8T细胞。6)体内VPA治疗Cl-13病毒感染后小鼠，病毒特异性CD8T细胞组蛋白乙酰化水平升高，耗竭性CD8T细胞延缓形成。7)体内或者体外VPA处理耗竭性CD8T细胞后，ChIP实验结果显示，ifng-promoter和ifng-enhancer两基因位点结合乙酰化的组蛋白水平显著升高。结论：Cl-13慢性病毒株感染后，CD8T细胞免疫功能渐进性耗竭；Cl-13感染过程中CD8T细胞功能缺陷与其不能进行必需的组蛋白乙酰化重塑有关；慢性感染过程中CD8T细胞功能缺陷可以通过增加组蛋白乙酰化而得以逆转。本研究首次从表观遗传学角度阐明了病毒慢性感染后CD8T细胞功能耗竭的机制，从而为病毒慢性感染的免疫调节治疗提供理论基础与实验依据。
[Abstract]:Background and objective: chronic virus infection, proliferation and differentiation of CD8T cells into virus specific CD8T cells that are due to chronic virus persistence, CD8T cell immune function effect is gradually reduced, the proliferation ability decreased, cell survival rate decreased, and cannot differentiate into memory CD8T cells, this phenomenon has been widespread academic research found, and called for the function of T cell depletion, mostly in chronic infections and tumors. The mechanism of CD8T cell depletion is not completely understood. Our preliminary results show that epigenetic modification plays an important role in the formation of differentiation of memory CD8T cells and function in the study. We will, for the first time from the perspective of epigenetic modification to explore the molecular mechanism of chronic viral infection in CD8T cell depletion, especially histone acetylation and disease control Based on the relationship between functional depletion CD8T cells in chronic infection, we further study the depletion of CD8T cell function after delaying or reversing chronic viral infection.
Methods: for lymphocyte choriomeningitis virus (lymphocytic choriomeningitisvirus-LCMV) with different strains of mice infected mice to establish infection model: acute virus strain LCMV-Arm (Arm) infection in mice acute infection, LCMV-clone13 (Cl-13) infection in mice to establish chronic infections. By comparison with Arm acute viral infection, analysis during chronic Cl-13 infection, virus specific CD8T cell histone acetylation expression characteristics, functional significance and the relationship with disease progression, from the perspective of epigenetics analysis of molecular mechanisms of chronic infection of antigen-specific CD8T cell immune function exhaustion. Four application of dimer staining technique for the detection of epitope specific CD8T cell phenotype frequency; detection of virus specific CD8T flow cytometry; in vitro peptide stimulated lymphocytes, cytokine production of CD8T cells were detected by flow cytometry Capacity; flow cytometry staining and chromatin immunoprecipitation (chromatinimmunoprecipitation, ChIP) CD8T cells histone acetylation levels; in vivo and in vitro experiments of histone deacetylase inhibitor valproic acid (valproic acid sodium, VPA) could reverse immune function of CD8T cell exhaustion; the functional status of CD8T cells VPA processing flow cytometry staining and real-time PCR cells; adoptive transfer function is exhausted CD8T cell VPA in vitro after treatment and can play a stronger immune function in vivo.
Results: 1) compared with Arm acute pathogenic strains, Cl-13 strains of mice infected with chronic virus, pathogen specific CD8T cell immune function decreased, secretion of cytokines IFN-, TNF- alpha and IL-2 decreased, and eventually into the exhaustion of Cl-13 infection,.2) staining and flow cytometry analysis showed that virus specific CD8T cells with high expression level of histone acetylation in the early stage of infection, with the infection process, has low expression of.ChIP showed that in the early stage of chronic viral infection, ifng-promoter and ifng-enhancer genes with more acetylation of histone; in the functional exhaustion period, genes with acetylated histones were significantly reduced.3) Cl-13 infection, not only virus specific CD8T cell function, CD8T cell immune function overall also decreased, decreased secretion of cytokine, and whole cell CD8T Histone acetylation levels also decreased.4) in vitro VPA depletion of CD8T cells, increased the level of histone acetylation, ability to secrete IFN- and other cytokines in vitro after VPA treatment increased.5) depletion of adoptive transfer of CD8T cells into normal mice, the survival rate increased, and the ability to increase the secretion of cytokines, showed that these cells enhanced immune function, as has the function of memory CD8T cells in VPA.6) for the treatment of Cl-13 virus infection in mice, increased virus specific CD8T cell histone acetylation, depletion of CD8T cells delayed the formation of.7) in vivo or in vitro depletion of CD8T cells after VPA treatment, ChIP results showed that the ifng-promoter and ifng-enhancer genes with acetylated histone levels increased significantly.
Conclusion: Cl-13 chronic virus infection, CD8T cell immune function, progressive exhaustion; the process of Cl-13 infection CD8T cell function defect and not histone acetylation remodeling required; CD8T cell function defect during chronic infection can be reversed by increasing histone acetylation. This is the first study from the perspective of epigenetics the chronic virus infection after CD8T cell depletion mechanism, which is the chronic virus infection immune regulation and provide a theoretical basis and experimental basis for the treatment.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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