表观遗传学甲基化调控Treg/Teff平衡缓解心脏移植排斥反应的作用及机制研究

It is crucial for inducing allograft immune tolerance to finding the target, which induces generation of Treg and stabilizes suppressive function of Treg, and also supresses the function of T effector cell (Teff). Our previous research supported by Youth Funding of NSFC firstly comfirmed that the ‘default’ generated neonatal regulatory T cells were hypomethylated at conserved non-coding sequence 2 and promote long-term cardiac allograft survival. The recent research suggested that GSK3βand miR-148a influenced T cell function by DNA methyltransferase regulating epigenetic methylation modification in TCR signal pathway, and histone methylation inhibitor (DZNep) could induced apoptosis of activating T cell by allogeneic antigen in the bone marrow transplant, but the obvious specific mechanisms of epigenetic demethylation was unclear. So our hypothesis is that ‘default’ generation of Treg was recovered by regulating demethylation of Treg-related gene, while the apoptosis of T effector cells was promoted by inhibition of histone methylation, and finally the long-term immune tolerance was induced. The project will aim to firstly study that how the DNA methyltransferase regulates T cell activation between neonate and adult mouse, and then to find the strategy of recovering the ‘default’ generation of Treg in adult based on above, and secondly aim to study the influence of DZNep on the pro-apoptosis and mechanism of T effector cell activated by cardiac graft. Starting with above epigenetic methylation modification, allograft immune tolerance finally is induced by regulating balance between Treg cell and Teff cell.

诱导移植物免疫耐受的关键是找到既能诱导Treg和稳定抑制功能又能抑制效应性T细胞（Teff）功能的靶点。我们前期研究证实：新生期Foxp3 CNS2区高度去甲基化默认性生成的Treg显著延长心脏移植物存活时间。近来研究示GSK3β和miR-148a可通过DNA甲基化酶调控TCR信号通路中甲基化修饰影响T细胞功能，同时应用组蛋白甲基化抑制剂DZNep可诱导骨髓移植中Teff凋亡，但具体机制不明。据此提出假说：通过调控Treg相关基因去甲基化，恢复Treg默认性生成，同时抑制组蛋白甲基化促进Teff凋亡可诱导移植物免疫耐受。本项目拟研究：1.GSK3β和miR-148a如何通过调控DNA甲基化转移酶影响新生与成年小鼠T细胞活化，并由此找到恢复Treg默认性生成的策略；2.DZNep对心脏移植物活化Teff的促凋亡及机制。由此以表观遗传学甲基化为切入点调控Treg/Teff平衡诱导移植物免疫耐受

课题组在国家自然科学基金的资助下，通过大量的实验研究，发现了表冠遗传学甲基化修饰在器官移植免疫反应中起重要作用，抑制甲基化修饰，特别是抑制组蛋白甲基化修饰可诱导心脏移植物长期存活，为诱导器官移植耐受提供新的干预靶点。现已经基本完成了相关实验，正在整理撰写相关成果。主要研究成果主要有一下几点。. 移植物耐受是器官移植领域的终极目标，特别是在实体器官移植领域，诱导宿主对移植物耐受是使移植患者获益的最佳方式。避免因免疫抑制引起的感染、肿瘤等风险。故本课题组通过抑制提供活性甲基的蛋氨酸循环中关键酶S腺苷同型半胱氨酸水解酶（SAHH）（不可逆SAHH抑制剂ADOX 和可逆性SAHH抑制剂DZ2002）可明显延长移植物存活时间，进一步在心脏移植模型中对针对移植物过继性输注的TEa细胞做单细胞高通量测序结果发现调控组蛋白甲基化的Ezh2在器官移植免疫反应中起重要作用，进一步使用Ezh2抑制剂干预发现其可诱导心脏移植物长期存活，且其机制是不影响移植物抗原特异性T细胞的活化，而诱导其活化后的耗竭状态。我们将进一步构建了CD4T细胞内条件性敲除EZH2小鼠，探究了T细胞内Ezh2在移植免疫反应中调控通路及具体机制。. 现阶段移植物血管病变（CAV）是现阶段影响移植术后的最主要问题，鲜有较好的解决方案，亟需相关研究，攻克CAV造福移植患者。目前，免疫抑制药物治疗靶点主要集中在淋巴细胞，特别是CD4T细胞，对CAV的保护作用有限。课题组研究表明CD40-TRAF6i（CT6i）通过影响APCs细胞功能，进而影响CD4 T细胞分化，减轻急性排斥反应。我们进一步构建了B6.C-H-2bm12KhEg (H-2bm12)C57BL/6(H-2b)小鼠心脏移植慢性排斥模型。CT6i能够改善减轻该模中移植物血管病变（CAV）。为了进一步探究其潜在的应用价值，我们在小鼠心脏移植急性排斥反应模型中应用CT6i和CsA联合抑制。结果表明联合抑制显著延长了同种异体心脏移植物存活时间，与CTLA-4Ig处理组相比减轻了移植物血管病变。