分泌型类纤维蛋白原蛋白2功能片段在调节性T细胞高表达诱导移植免疫耐受的机制研究

Regulatory T cells (Treg) play pivotal role in both the induction and maintenance of transplant tolerance. Two major problems limiting the clinical usage of Tregs are the tremendous dosage requirement and the plasticity of their phenotype. Our previous studies identified a novel effector molecule of Treg, secreted fibrinogen like protein 2 (sFGL2), which bind to the low affinity FcγRIIB/III receptors. Interactions of sFGL2 with the inhibitory FcγRIIB prevent the maturation of dendritic cells, decrease the proliferation of effector T cells, and induce the apoptosis of B cells, thus regulating both the cellular and humoral immunity. Treatment with sFGL2 recombinant protein significantly prevents the graft rejection in both skin and heart allogenic transplantation models. sFGL2 transgenic mice "sFGL2(TG)" have been developed by us, and in a MHC fully mismatched heart transplant model, 50% of sFGL2(TG) recipients accepted allograft without any immunosuppression, suggesting the great potential of sFGL2 in inducing transplant tolerance. However, at natural status, sFGL2 exists as a tetrameric macromolecular glycoprotein and therefore is not appropriate for clinical treatment, due to the potential antigenicity and toxicity. In this project,we plan to make several short cDNA constructs covering different structural regions of sFGL2 by polymerase chain reaction. Mammalian cells will be transformed by these vectors to produce distinct types of truncated sFGL2 protein, which will be characterized by a series of biochemical and biological assays to determine their molecular weight, glycosylation status, solubility, stability, affinity to receptors, half life, antigenicity, toxicity, and regulatory activity. The purpose of this project is to identify the best candidate of truncated sFGL2 for clinical therapy with reduced molecular size but sufficient immunoregulatory activity. In addition, Tregs will be expanded in vitro and transfected with sfgl2 recombinant lentivirus to over-express the truncated sFGL2 protein selected. The regulatory efficiency of these "super" Tregs, Treg(sFGL2), will be validated by the lymphoproliferation- inhibitory assay in vitro and tolerance induction in a mouse cardiac allograft model in vivo. Variable techniques including molecular cloning, ectopic gene expression, flow cytometry analysis, real-time PCR, immunohistochemistry, and immunofluorescence microscopy will be applied to investigate the effect of these truncated sFGL2 protein and Treg(sFGL2) cells in inducing transplant tolerance. This study will not only reveal the functional domain of sFGL2, further decipher the mechanisms of its immunoregulatory activity, but also reduce the antigenicity and improve its therapeutic potentials. Treatment with these Treg(sFGL2) in vivo may significantly improve its stability and reduce the dosage requirement, providing a more efficient, economic and safer way of Treg cell therapy.

临床应用调节性T细胞（Treg）诱导移植免疫耐受的主要问题是细胞需求剂量巨大而且在体内功能不稳定。我们前期发现提高表达一种新型免疫调节分子-分泌型类纤维蛋白原蛋白2(sFGL2),可以显著增强Treg免疫调节效能。但是天然状态下sFGL2为四聚体大分子糖蛋白，由于存在较强免疫原性，不宜用作活体治疗。本项目拟利用分子克隆在哺乳动物细胞表达覆盖sFGL2不同结构区域的截短蛋白, 应用体外功能试验筛选出分子量缩小但保留免疫调节活性的sFGL2功能片段。进而通过重组慢病毒转染将sFGL2功能片段高表达于体外扩增的Treg,在小鼠心脏异位移植模型中探索sFGL2功能片段高表达的Treg细胞对移植免疫耐受的诱导作用。本研究不仅可以进一步定位sFGL2功能区域,揭示其免疫调节机制,而且可以降低机体对sFGL2的异源性免疫反应,提高其活体应用潜能,为减少临床Treg治疗剂量并增强其稳定性提供新方案。

临床应用调节性T 细胞（Treg）诱导移植免疫耐受的主要问题是细胞需求剂量巨大而且在体内功能不稳定。我们前期发现提高表达一种新型免疫调节分子-分泌型类纤维蛋白原蛋白2(sFGL2),可以显著增强Treg免疫调节效能。过表达sFGL2在MHC完全非匹配小鼠心脏移植模型中可以促进免疫耐受。但是天然状态下sFGL2为四聚体大分子糖蛋白，由于存在较强免疫原性，不宜用作活体治疗。..本项目拟利用分子克隆在哺乳动物细胞表达覆盖sFGL2 不同结构区域的截短蛋白, 应用体外功能试验筛选出分子量缩小但保留免疫调节活性的sFGL2 功能片段。进而拟将sFGL2功能片段高表达于体外扩增的Treg, 在小鼠心脏异位移植模型中探索sFGL2功能片段高表达的Treg 细胞对移植免疫耐受的诱导作用。本研究不仅可以进一步定位sFGL2 功能区域, 揭示其免疫调节机制,而且可以降低机体对sFGL2的异源性免疫反应,提高其活体应用潜能,为减少临床Treg 治疗剂量并增强其稳定性提供新方案。..但在实际试验中，我们发现sFGL2截短蛋白表达纯化异常困难。具体表现在真核系统表达效率较低，不同批次转染表达阳性率有所不同，在进一步纯化提取过程中仍有相当损耗。进一步尝试在大肠杆菌中表达sFGL2截短蛋白虽然能达到一定丰度，但表达蛋白溶解度很低，可能与包涵体的形成以及原核系统中蛋白翻译后修饰功能缺陷有关。通过添加标签增加蛋白溶解度后，在切除蛋白标签提纯过程中又出现大量降解的问题。最后我们尝试体外合成的方法生产了两条多肽，但在动物试验中未能证实其诱导免疫耐受的作用。..虽然本项目未能按预期发现sFGL2 功能片段，我们成功地建立了小鼠心脏移植耐受和排斥模型。同时发现应用TLR3激动剂预处理脂肪干细胞可以提高sFGL2表达，并发挥一定的免疫调节作用, 为应用sFGL2诱导免疫耐受的提供新的思路和方向。