转录因子EBF1在间充质干细胞调控B细胞分化治疗抗体介导排斥反应中的作用及机制

Antibody-mediated rejection (AMR), induced by donor specific antibody (DSA) derived from B lymphocytes, is an important risk factor for renal allograft dysfunction. AMR in kidney transplantation is refractory under current available therapeutic regimens. We have completed and reported the first study in which the efficacy and safety of mesenchymal stem cells (MSCs) on AMR in kidney transplantation was approved (Am J Transplant 2014). The subsequent ex vivo experiments showed that MSCs decreased DSA level as well as the proportion of plasma B cells, and increased both the proportion of IL-10(+) regulatory B cells (Bregs) and the expression of early B-cell factor 1 (EBF1). Our collaborator was the first to confirm the significant effect of EBF1 on the peripheral differentiation of B lymphocytes (Genes Dev. 2012). We therefore proposed the hypothesis that it is through the regulation of EBF1 that MSCs can direct the differentiation of B lymphocytes to IL-10(+) Bregs instead of DSA-secreting plasma cells. In this program, we intend to place emphasis on clarifying the role of EBF1 in the regulation of peripheral B cell differentiation by MSCs, exploring the upstream signaling pathways and identifying the key role of Bregs in the treatment of AMR. This project will provide new theoretical explanations and experimental evidence for an in-depth understanding of the cellular and molecular mechanisms of MSCs in the treatment of AMR.

B细胞产生供体特异性抗体(DSA)介导的排斥反应(AMR)是导致移植肾远期失功的重要危险因素,治疗棘手且疗效不佳。我们首次明确了间质干细胞(MSCs)治疗肾移植AMR的疗效和安全性(Am J Transplant. 2014)。进一步实验中,MSCs能明显降低浆细胞比例、DSA水平,且增加IL-10+调节性B细胞(Bregs)比例,B细胞转录因子EBF1表达升高。合作者率先阐明EBF1对外周B细胞分化的重要作用(Genes Dev. 2012),这启发我们提出假设:MSCs通过EBF1调控B细胞向IL-10+Bregs而非向浆细胞分化。本项目拟通过体内外实验,结合EBF1条件性基因敲除技术,重点阐明EBF1在MSCs调节外周B细胞分化中的作用,探讨参与其中的上游信号通路,明确Bregs在AMR治疗中的关键作用,能为深入认识MSCs治疗AMR的细胞与分子机制提供新的理论解释和实验依据。

在前期工作基础上，本课题进一步研究MSCs 调控外周 B 细胞分化（特别是 IL-10+Bregs）的作用并研究EBF1在其中的作用及分子机制。通过流式分析肾移植 AMR 患者接受 MSCs 治疗前后不同时间点的外周血 B 细胞亚群变化及EBF1的表达水平，课题组首先明确了其与IL-10+Bregs相关性。随后成功建立 B 细胞活化与分化的体外培养体系以及小鼠肾移植 AMR实验动物模型和Ebffl/flRERTCre小鼠体系，通过体外与体内实验明确了转录因子EBF1对于MSCs调控B细胞向IL10+Breg细胞的免分化具有关键作用，EBF1沉默后的B细胞向IL10+Breg分化比例减少，而浆细胞分化比例增高。同时本课题也发现MSCs上调B细胞中EBF1表达的过程可能依赖于IL-7/JAK/STAT5 信号通路。本课题资助下，还研究了供体来源游离 DNA、可溶性 CD146 和 Baff 在肾移植 ABMR诊断中的辅助作用。课题进展顺利，完成了合同书主要研究任务。以上研究结果发表 SCI 论文15篇，美国移植学会年会（ATC）口头报告 1 次，尚余部分结果仍在进行资料整理和论文的撰写。