Treg和双基因修饰的imDC诱导肝移植免疫耐受的相互作用机制

Immunosuppressive drugs have led to overall improvements in allograft and patient survivals. However,the improved short-term survival rates have come at a cost: these drugs induce many indirect and direct side effects and pose an increased risk of life-threatening complications. These data emphasize the critical role of the recipient's alloimmune response as a major determinant of transplant outcome and highlight the need to develop novel strategies to induce immunologic donor-specific tolerance, defined as a lack of a destructive immune response toward the graft in the presence of generalized immune competence. Regulatory T cells (Treg) and tolerogenic DCs are instrumental in maintaining self-tolerance. In vitro manipulation of these 2 protagonists of the immune response is now emerging as a therapeutic tool to induce tolerance to allotransplantation. Although studies have focused on the development of immature DCs (imDC) as a tool to induce transplant tolerance. Several studies have addressed the significance of Tregs in human transplantation. TGF-β1 and IL-10 signaling seem to be essential for the maintenance of immune homeostasis and suppressing autoimmunity, they are considered to be a key factor in immune tolerance. We do not yet understand when, why and how the human immune system decides to accept the graft. The immune response against the graft involves multiple mechanisms and factors that intervene at different time points and levels. It might therefore not be reasonable to limit an immunotherapeutic intervention to only one target, and, especially in humans, combined strategies appear to have more chances of success. .In this study, we investigated the effects of imDC co-transfected with IL-10 and TGF-beta 1 genes and Treg on inducing immune tolerance. We will discuss interactive mechanisms underlying Treg、imDC、cytokines-induced tolerance to allografts.This study was designed to explore the effects of IL-10 and TGF-β1 genes co-transfected imDCs combined with Treg in vitro and in vivo immune response in rat liver transplantation. Liver transplantation was induced using the classic methods from DA rats to Lewis rats. IL-10 and TGF-β1 genes co-transfected imDCs and Treg were injected intravenously for treatment 5 days after the liver transplantation. Th1/Th2 cytokines were detected by ELISA and MHC-II molecules, costimulatory molecules were identified by flow cytometry. T lymphocyte proliferation assay and protein were performed. HE staining were performed to detect the hepatic function and inflammation. Survival time was observed..Understanding the interactive mechanisms underlying Treg、imDC、cytokines-induced tolerance to allografts will afford new opportunities to harness Treg and imDCs function in clinical outcomes. Furthermore, the study of spontaneously tolerant patients will discover an immunologic "tolerance signature" that should help the development of reproducible nontoxic strategies that will promote tolerance in all patients.

新型免疫抑制剂使移植器官早期失功的危险性降到较低水平，如何诱导受体抗原特异性免疫低下状态或免疫耐受仍是器官移植追求的理想目标。尽管不成熟树突状细胞（immature dendritic cells, imDC）在诱导免疫耐受中起决定性作用，但调节性T细胞（regulatory T cell, Treg）及相关细胞因子共同参与了移植免疫耐受的过程，它们之间是否存在协同作用及其具体机制有待进一步研究证实。课题组在前期构建IL-10和TGF-β1真核表达载体转染imDC后，显示其在大鼠肝脏的移植模型中具有保护作用。本项目基于前期的研究基础上，深入探讨双基因修饰的imDC是否具有诱导并调节Treg细胞的作用及其可能的作用机制；同时利用已建立的大鼠肝移植模型，将脾脏来源的Treg和双基因共修饰imDC，输入肝移植大鼠体内，探讨二者是否具有协同诱导移植免疫耐受的作用，以期实现寻找持久耐受理想方法。

肝移植已成为许多终末期肝病的治疗手段，新型免疫抑制剂使移植器官早期失功的危险性降到最低，但长期生存并不乐观，移植排斥问题尚未解决。因此，如何诱导移植物受体的抗原特异性免疫低下状态或免疫耐受成为同种器官移植追求的目标，已成为该领域的中心课题。. 研究内容：在体外及大鼠肝移植模型中探讨IL-10和TGF-β1双基因修饰后的imDC和Treg相互作用诱导免疫耐受的机制及效果，寻找能高效持久发挥免疫耐受作用的最佳方法。研究方案（1）原代培养大鼠骨髓来源DC，并对DC进行形态、功能和表型鉴定；（2）构建携带IL-10和TGF-β1基因的两种慢病毒载体，比较脂质体法、电穿孔法和慢病毒载体法将外源性基因导入DC的效率；（3）磁珠分选大鼠脾脏淋巴细胞得到CD4+CD25-T细胞和Treg，分别与IL-10、TGF-β1基因单独或共修饰后的DC共培养，分析Treg生成增殖情况及机制；（4）构建DA→Lewis大鼠肝移植模型，单独或联合输注Treg和基因修饰前后DC，观察并检测大鼠生存期、肝功能、血清细胞因子、排斥评分等。. 主要研究结果：（1）原代培养DC具备典型树突状细胞形态学特征，CD86和MHC-II在未成熟DC（imDC）表面呈低表达，在成熟DC（mDC）呈高表达，OX-62在两者表达无明显差别；mDC能促进淋巴细胞增殖，而imDC对其作用较弱。（2）构建携带IL-10和TGF-β1基因的两种慢病毒载体，经基因测序、RT-PCR、western blot法证实慢病毒载体构建成功。用慢病毒载体感染DC的细胞融合率可达90%以上。 （3）imDC、双基因单独或共修饰的imDC均可诱导iTreg细胞分化，双基因共修饰组作用最强，mDC和空载体慢病毒DC组作用不明显。（4）大鼠肝移植模型中，imDC、双基因修饰DC和Treg单独输注均能诱导肝移植免疫耐受，各种DC和Treg共同输注组的作用增强，双基因修饰DC和Treg联合组的作用最强。. 结论：本研究成功进行DC体外培养，构建携带IL-10和TGF-β1基因慢病毒载体，比较三种转染方法，证实慢病毒载体转染效率最高。基因修饰后的imDC生物学特性发生相应改变，诱导Treg生成和增殖能力增强。共同输注双基因修饰的DC和Treg能有效延长肝移植大鼠生存期，并缓解移植物排斥反应，是诱导肝移植免疫耐受的有效方法。