OX40Ig和ICOSIg基因修饰自体脂肪间充质干细胞诱导大鼠肾移植免疫耐受及机制研究

How to induce immune tolerance while avoiding taking immunosuppressants adverse reactions become a serious problem to be solved. The current study has found that mesenchymal stem cell therapy or T-cell costimulatory pathway blockade are to some extent induce immune tolerance, which is associated with Treg differentiation as well as immature DC proliferation. But specific molecular mechanisms of synergistic effect and immune cell differentiation are not yet clear. We have previously shown that simultaneous blockade of OX40 and ICOS pathway may induce antigen-specific T cells anergy. Therefore, we conceive a cell combination with gene therapeutic strategy and put forward a scientific hypothesis that genetically modified adipose-derived mesenchymal stem cells (ADSCs) expressed OX40Ig and ICOSIg can home to sites of transplanted organ, which play a synergistic effect of local masking the immunogenicity as well as modulating immune response to protect the allograft long-term survival. In this study, using a rat orthotopic kidney transplantation model, we investigate the synergistic therapeutic effect of prolonging allograft survival using genetically modified autologous ADSCs infusion. Furthermore, we elucidate the mechanism of local ADSCs therapy combined with T-cell costimulatory blockade induced specific immune tolerance in vitro/vivo from tissue, cellular, molecular level. Therefore, this study provides the theoretical basis and experimental evidence to prolonging allograft long-term survival induced by ADSCs.

如何诱导免疫耐受同时规避服用免疫抑制剂所致的不良反应成为亟待解决的问题。目前研究发现，应用间充质干细胞及阻断共刺激通路均在一定程度上诱导免疫耐受，其机制与Treg分化及耐受性DC增殖相关，但两者联合应用是否产生协同免疫调节作用及参与调控免疫细胞分化的具体分子机制尚不十分清楚。我们前期研究发现，阻断OX40及ICOS信号通路能诱导T细胞产生抗原特异性无反应。我们提出利用取材更为方便的脂肪间充质干细胞（ADSCs）携带OX40Ig及ICOSIg基因在移植器官定植，是否可以在局部发挥细胞治疗与基因治疗二者免疫调节的协同作用保护移植物长期存活。本项目以大鼠原位肾移植模型，观察基因修饰自体ADSCs回输对同种异体移植肾存活时间的影响；并从组织、细胞、分子水平阐明局部细胞治疗联合共刺激通路阻断诱导特异性免疫耐受的体内外作用机制。本研究为临床应用ADSCs诱导移植肾长期存活提供新的理论基础和实验依据。

围绕自体ADSCs携带外源性OX40Ig及ICOSIg缓解肾移植术后急性排斥反应的治疗效果以及二者免疫调节的协同作用，完成了以下方面的研究：（1）构建pcDNA3.1(+)/OX40Ig及pcDNA3.1(+)/ICOSIg，转染大鼠ADSCs。（2）OX40Ig及ICOSIg基因修饰的大鼠ADSCs对大鼠移植肾急性排斥反应的影响。（3）基因修饰的ADSCs对异基因反应性T细胞功能正向调节作用及分子机制。（4）基因修饰的ADSCs对DC反向免疫调节作用及分子机制。结果显示：ICOS-Ig和OX40-Ig在ADSCs细胞中的表达水平均明显升高，表明重组质粒能够成功介导这两种融合蛋白在真核细胞中的表达，转染后成脂、成骨、成软骨细胞诱导结果与转染前相同。体内实验显示，ADSCsOX40Ig-ICOSIg干预能减轻受鼠急性排斥的临床表现，充分利用了ADSCs向损伤组织归巢特性，显著降低急性排斥评分，血清肌酐及24h尿蛋白升高幅度最低，移植后生存时间最长。作用机制可能为：ADSCsOX40Ig-ICOSIg干预可显著降低受体血清、脾脏及肾组织Th1、Th17细胞分化，转录因子表达及细胞因子分泌；诱导Th2、Th3、Treg细胞分化，转录因子表达及细胞因子分泌，同时显著降低移植肾免疫细胞浸润。体外实验显示，ADSCsOX40Ig-ICOSIg协同抑制异基因T细胞的活化和增殖，显著抑制DC表型的成熟，诱导脾脏未成熟DC，减弱DC对同种T细胞刺激能力，并体内诱导供体特异性T细胞的低反应性，即供体特异性免疫耐受。作用机制可能为：降低Th1细胞分化及细胞因子表达；诱导Treg细胞分化。其对异基因T淋巴细胞的抑制作用可能部分通过调节PI3K及p38MAPK信号途径；对mDC分化、成熟的抑制作用可能部分通过调节MAPK信号途径来实现，提示ADSCsOX40Ig-ICOSIg对DC细胞免疫功能存在反向调节作用。该研究将丰富ADSCsOX40Ig-ICOSIg免疫调控的理论知识，并为将来ADSCsOX40Ig-ICOSIg在临床肾移植中的可能应用提供了实验依据。