协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

Objective To induce the tolerance to cardiac allograft by combined blockade of OX40/OX40L and CD28/B7 co-stimulatory signaling and donor specific spleen cell transfusion in rat mod-els of pre-existent allogenic reactive memory T lymphocytes. Methods Lewis rats that underwent 3 days of adoptive transfer of donor specific CD8+ memory T cells, separated by immunomagnetic bead separation kit, received either separate or combined transfusion of AdCTLA4Ig, AdOX40Ig, donor spleen cells (DST) and transplantations of hearts of DA rats at the same time. Cardiac allografts were taken out 48 h after transplantation for histological analysis and cytokines expression ,and survival time of cardiac allografts with different treatments was observed. Results Compared with AdCTLA4Ig, AdOX40Ig and DST groups, AdCTLA4Ig + AdOX4OIg + DST group showed lower cardiac pathological grade with much lower expression level of IL-2 and IFN-γ,and much longer heart survival time. Conclusion Combined blockade of OX40/ OX40L and CD28/B7 and donor specific spleen cell transfusion could induce rat cardiac graft tolerance.     

协同信号途径阻断和供体脾细胞输注诱导预存同种记忆性T淋巴细胞大鼠心脏移植物的耐受

目的 联合阻断OX40/OX40L和CD28/B7双协同信号途径和供体特异性脾细胞输注,诱导预存同种反应性记忆T淋巴细胞大鼠对同种心脏移植物的耐受.方法 建立大鼠预存同种反应性记忆T淋巴细胞的移植模型,采用免疫磁珠法分选CD8+CD44-记忆性T细胞;对过继转移供体特异性CD8+记忆性T细胞3 d的Lewis大鼠分别/合并输注AdCTLA4Ig、AdOX40Ig、供体脾细胞(DST),同时移植来至DA大鼠的心脏;48 h后取出心脏进行组织学分析、细胞因子表达分析,同时观察不同处理移植心脏存活时间.结果 AdCTLA4Ig+AdOX40Ig+DST组分别与AdCT-LA4Ig,AdOX40Ig和DST比较,心脏组织病理级别较低,白细胞介素(IL)-2及干扰素(IFN)-γ的表达水平也明显比其他组低很多,而心脏存活时间显著延长.结论 联合阻断OX40/OX40L和CD28/B7和供体特异性脾细胞输注能较好地诱导大鼠心脏移植物耐受.     

阻断OX40和CD40协同共刺激信号延长小鼠胰岛移植物存活时间

目的 研究阻断OX40/OX40L和CD40/CD154L协同共刺激通路对小鼠胰岛移植物存活的影响及其机制.方法 以DBA/2小鼠为供者,C57BL/6小鼠为受者,制作胰岛移植模型.受鼠分为4组.(1)对照组,注射IgG; (2)抗OX40组,注射抗OX40L单克隆抗体;(3)抗CD154组,注射抗CD154单克隆抗体;(4)联合治疗组,注射抗OX40L单克隆抗体和抗CD1 54单克隆抗体.记录各组胰岛移植物平均存活时间(MST).将CD154敲除小鼠处死,取其脾脏T淋巴细胞,体外检测活化T淋巴细胞表面OX40的表达;在活化T淋巴细胞中加入不同浓度的抗OX40L单克隆抗体,体外检测T淋巴细胞增殖情况.结果 对照组胰岛移植物MST为19 d,抗CD154组胰岛移植物MST为48 d(P＜0.05);抗OX40组胰岛移植物MST为22 d,与前两组相比较,差异无统计学意义(P＞0.05);联合治疗组胰岛移植物MST＞ 150 d,高于另外3组(P＜0.05).66％的胞表达OX40,较初始T淋巴细胞的表达率高(2％,P＜0.05);加入抗OX40L单克隆抗体后,T淋巴细胞增殖受抑制且呈剂量依赖性.结论 阻断OX40/OX40L和CD40/CD154L双通路可诱导小鼠胰岛移植物长期存活, 其发挥作用的关键机制是抑制了T淋巴细胞的增殖.     

siRNA干扰OX40/OX40L通路对小鼠移植心脏的免疫保护作用

目的 观察真核表达载体介导siRNA对小鼠移植心脏的免疫保护作用.方法 在C57/BL6J→BALB/C组合[主要组织相容性复合体(MHC)不合]、DBA/2→BALB/C组合[次要组织相容性复合体(mHC)不合]中分别于小鼠心脏移植术后第1、4天胸腺注射生理盐水,pRNAT-CTL阴性对照载体和pRNAT-OX40 siRNA,观察移植心脏存活情况.术后第7天取移植心脏行病理学检查,取血清检测心肌酶谱,取脾脏行逆转录.聚合酶链反应(RT-PCR)和Western blot检测OX40mRNA表达和OX40膜蛋白表达.结果 在C57/BL6J→BALB/C组合中(MHC不合),pRNAT-OX40siRNA组移植心脏生存时间较生理盐水组和阴性对照组无明显延长,移植心脏病理学检查无明显改善.而在DBA/2→BALB/C组合中(mHC不合),pRNAT-OX40 siRNA组小鼠移植心脏生存时间较生理盐水组和阴性对照组显著延长,病理改变显著减轻,乳酸脱氢酶(LDH)、谷草转氨酶(AST)、肌钙蛋白(Tn)以及脾脏OX40 mRNA表达和OX40膜蛋白表达与空白对照组和阴性对照组比较明显下降.结论 在DBA/2→BALB/C组合中(mHC不合),单独阻断OXdO/OX40L通路对小鼠移植心脏有保护作用.在C57/BL6J→BALB/C的组合中(MHC不合),单独阻断OX40/OX40L通路未发现对移植心脏的保护作用.     

RNAi对大鼠T淋巴细胞CD28和OX40基因表达的联合抑制作用

目的 通过RNAi技术干扰T细胞CD28、CD134基因表达后,诱导获得抑制性T细胞(Ts);探讨Ts免疫学特性.方法 设计针对目标基因的siRNA,转染大鼠T淋巴细胞,FCM检测CD28、CD134水平,混合淋巴细胞反应(MLR)检测转染后的T淋巴细胞对异体淋巴细胞的增殖能力的影响,逆转录-聚合酶链反应(RT-PCR)及酶联免疫吸附试验(ELISA)法检测细胞因子水平.结果 siRNA转染大鼠T淋巴细胞后,抑制CD28、CD134分子的表达,siRNA转染24 h后,siRNA组CD28、CD134表达受到抑制[转染后及转染前表达分别为(22.35±4.37)%及(34.76±3.51)%(P<0.05)],T淋巴细胞分泌细胞因子IL-10水平增高,转染组及未转染组表达分别为(77.15±12.60)ng/L及(37.56±5.93)ng/L(P<0.01).而转染组及未转染组的IL-2表达分别为(2.79±0.51)及(4.35±1.11)ng/L(P<0.05);干扰素(IFN)-γ表达分别为(277.15±14.8)、(682.7±53.5)ng/L(P<0.05).结论 siRNA可以特异性抑制大鼠T淋巴细胞共刺激分子CD28、CD134基因表达,抑制了IL-2、IFN-γ的表达,提高了IL-10细胞因子表达水平,从而产生了免疫耐受效应.Ts细胞具有抗原特异性,而且在外源性rrIL-2存在时,不能逆转Ts细胞的功能.     

Blockade of both CD28/B7 and OX40/OX40L co-stimulatory signal pathways prolongs the survival of islet xenografts

CTLA4Ig, a recombinant fusion protein composed of the extracellular domain of human CTLA4 and the constant region of human IgG1, inhibits the interaction of CD28/B7 pathway by binding the B7 molecule. OX40Ig, a recombinant fusion protein composed of the extracellular domain of human OX40 and the constant region of human IgG1, abrogates the interaction of OX40/OX40L pathway by binding the OX40L on APCs. So blockade of CD28/B7 or OX40/OX40L co-stimulatory pathways alone in mice with CTLA4Ig or OX40Ig can result in finitely prolonging the survival of islet grafts (43.2 +/- 4.81 and 67.7 +/- 7.74 days, respectively). In this study, a novel replication-defective adenovirus containing both of the CTLA4Ig and OX40Ig genes, AdCTLA4Ig-IRES-OX40Ig, was constructed by homologous recombination and injected into the streptozocin-rendered diabetic BalB/c mouse recipients (H-2d) through the tail vein, at the same day, the freshly isolated islets from Lewis rats (RT-1) were transplanted under the left kidney capsule of the recipients. The results showed that the mean survival time of the islet xenografts in the AdCTLA4Ig-IRES-OX40Ig-treated diabetic mice was significantly prolonged (100.3 +/- 14.94 days), while those in the untreated or AdEGFP-treated mice were rejected in normal fashion (6.7 +/- 0.94 and 7.0 +/- 1.0 days, respectively). In conclusion, utilizing AdCTLA4Ig-IRES-OX40Ig in vivo which can simultaneously express CTLA4Ig and OX40Ig proteins can improve the survival of Lewis-->BalB/c islet xenografts.     

RNA干扰阻断OX40/OX40L共刺激通路对CD4+CD25+T调节细胞的影响

目的 探讨慢病毒介导的RNA干扰技术诱导小鼠树突状细胞OX40L基因沉默对调节性T细胞的影响. 方法 设计针对小鼠OX40L基因的RNA于扰序列,通过外源筛靶筛选出干扰效果最佳的序列.以293T为包装细胞,制备含OX40L的siRNA序列的慢病毒载体OX40L-RNAi-LV和阴性对照载体NC-GFP-LV.采用磁式分选器分离培养骨髓来源的小鼠树突状细胞(DCs),以MOI为25进行转染,分别将转染OX40L-RNAi-LV(实验组)、转染NC-GFP-LV(阴性对照组)和未转染(空白对照组)的DCs与磁式分选器分选得到的CD4+CD25+T调节细胞共培养,6 d后通过流式细胞仪检测T调节细胞的增殖和凋亡情况. 结果外源筛靶筛选出干扰效果最佳的RNA干扰序列(靶序列为GCTCATACAAGAATGAGTA),OX40L蛋白表达的抑制率为73.1%.感染复数为25时,慢病毒载体感染DCs的效率为86.4%.DCs与CD4+CD25+T调节细胞共培养后,实验组的凋亡细胞比例为8.7%,显著低于阴性对照组(20.1%)和空白对照组(19.8%),F=244.22,P=0.000;而增殖细胞前体频率为38.3%,明显高于阴性对照组(24.5%)和空白对照组(22.9%),F=95.40,P=0.000.结论 小鼠OX40L的siRNA慢病毒载体可以有效降低树突状细胞OX40L的表达,对体外培养的CD4+CD25+T调节细胞具有显著地促进增殖、减少凋亡的作用.