体外工程化修饰供体器官和细胞并快速表达致凋亡性蛋白（FasL）在诱导肾移植免疫耐受中的研究

FasL-induced apoptosis plays an important role in immune homeostasis and tolerance to self-antigens, which led to intense studies for its use to induce tolerance to self and alloantigens for the treatment of autoimmunity and allograft rejection. However the delivering system of FasL in vivo remains unsolved due to the considerable limitations of gene therapy in expression efficiency, safety and clinical practice, etc. We recently developed a novel protein display technology called ProtEx, to transiently display a chimeric form of FasL on the surface of splenocytes, islets and vascular endothelium system, which proved to be efficient to induce tolerance to allogeneic grafts. These results were published in Immunity, Circulation and The Journal of Immunology. Also we found in the preliminary study that in vitro engineered renal allograft exhibited significantly prolonged survival. To further investigate its feasibility in kidney transplantation mode, we propose to engineer to display FasL on the surface of the kidney, as well as certain transferable cells (splenocytes, bone marrow cells and peripheral blood mononuclear cells) from the donor, possibly combing a short period cover of rapamycin, to optimize the protocol of inducing tolerance in an allogeneic kidney transplantation model in rodents, possibly extending to non-human primates. If tolerance is achieved, mechanistic studies include the apoptosis of Teff, phagocytosis of apoptotic bodies, secretion of TGF-β and IL-10, followed by the conversion of inducible Treg will be fully explored. The results from this project will provide a novel, safe, and clinically applicable approach for the prevention of rejection after kidney transplantation.

转基因技术在表达率、安全性和实际操作上有许多不足，限制了经典的致凋亡性蛋白Fas配体(FasL)诱导器官移植耐受的转化应用。本课题组自主研发了新型的体外器官、组织和细胞工程化修饰技术-ProtEx，可使膜型FasL快速高效的表达在血管内皮系统、胰岛和脾细胞表面，并以此为基础成功诱导移植耐受(发表在Immunity, Circulation, J Immunol)。我们前期研究发现，用ProtEx体外修饰供肾可明显延长其移植后存活。本课题将在以上基础上首次联合应用工程化修饰的供肾、供体细胞(如脾、外周血单个核或骨髓细胞)，和/或短期联合雷帕霉素，在包括灵长类动物的异体肾移植模型上探讨诱导免疫耐受的可行性和临床前景；并从效应淋巴细胞凋亡、继而巨噬细胞分泌TGF-β、IL-10，从而诱导外周产生调节性T细胞(iTreg)等方面深入探讨作用机制；能为该技术向临床转化提供可靠的实验证据和理论基础。

转基因技术在表达率、安全性和实际操作上有许多不足，限制了经典的致凋 亡性蛋白Fas配体（FasL）诱导器官移植耐受的转化应用。本项目利用课题组自主研发的新型体外器官、组织和细胞工程化修饰技术—ProtEx，使大鼠肾脏血管内皮细胞、脾细胞、骨髓细胞和外周血单个核细胞表面快速高效表达膜型FasL，均能延长大鼠移植肾存活时间，其中SA-FasL修饰的供体脾细胞或骨髓细胞联合短期小剂量雷帕霉素诱导了大鼠肾移植免疫耐受（存活超过100 d），非人灵长类肾移植存活至今已超过6个月，仍在观察中；进一步研究发现，受体小鼠脾脏及引流淋巴结中CD4+CD25+Foxp3+ Treg细胞比例明显升高，DTR清除B6.Foxp3GFP.DTR受体小鼠的Foxp3+ Treg细胞后，移植物均被排斥，说明Tregs在本方案诱导移植耐受中发挥重要作用。研究过程中，围绕供体脾细胞输注（DST）、移植耐受诱导以及联合免疫抑制剂的优化和移植免疫状态监测等必要技术问题，完成多项连续性研究工作并在专业期刊发表SCI论文5篇；培养博士生毕业2名，其中美国路易维尔大学-中山大学联合培养1名。本项目为膜型SA-FasL嵌合蛋白向肾移植临床转化应用提供了重要的实验证据和理论基础。