体外扩增的人调节性T细胞抑制小鼠骨髓移植急性移植物抗宿主病

发布时间：2019-04-26 00:12

【摘要】：调节性T细胞(Tregs)在免疫调节和维持外周免疫耐受方面起重要作用。由于从供者外周血采集分离到的Tregs的数量有限,新鲜分离的Tregs需要在体外进行扩增以获得临床应用所需的Tregs细胞数。已有文献报道人Tregs可以在体外有效扩增,但是扩增后的Tregs是否能够在体内有效抑制急性移植物抗宿主病(aGVHD)还不清楚。本文从脐带血中分离CD4+CD25+细胞以及从成人外周血中分离CD4+CD25+CD127-细胞进行体外扩增,比较扩增后Tregs的免疫抑制功能。脐带血和外周血Tregs通过两轮多克隆刺激或第一轮同种异基因抗原刺激加第二轮多克隆刺激进行体外扩增。两轮扩增结束后检测Tregs的表型、分泌的细胞因子以及抑制功能。采用C57BL/6小鼠骨髓和脾细胞移植经致死性照射的DBA/2小鼠建立aGVHD模型。Tregs治疗组用体外扩增的脐带血或外周血Tregs过继性输注治疗aGVHD模型小鼠。分析比较Tregs治疗组和对照组小鼠生存率、体重、GVHD症状评分、组织病理学标本、血清细胞因子、Th细胞亚群。脐带血和外周血Tregs多克隆扩增两轮后平均分别扩增1.5×103和1.8×103倍,而采用第一轮同种异基因抗原特异性扩增加第二轮多克隆扩增后的平均扩增倍数分别是5×102和4.7×102。扩增的Tregs高表达Foxp3、CD39、CTLA-4,低表达CD127、IL-2、IFN-Y,并且维持了免疫反应无能性。采用两轮多克隆扩增的脐带血Tregs不仅能够抑制广谱扩增的效应T细胞(Tresp)增殖,而且还能够抑制HLA抗原不相合的树突状细胞激活的效应T细胞增殖。当两轮多克隆扩增的脐带血或外周血Tregs输注aGVHD模型小鼠后,分别有87.5%和16.7%的小鼠存活期超过63天,而aGVHD模型小鼠在18天里全部死亡。第7天时扩增脐带血Tregs治疗组小鼠体内IFN-γ和TNF-a含量显著低于aGVHD模型小鼠。接着Tregs治疗组小鼠体内IFN-γ、TNF-α、IL17的含量迅速降低并维持在低水平,而TGF-β的含量持续上升。小鼠体内免疫细胞亚群的变化与小鼠体内血清细胞因子含量变化是同步的：Th1细胞百分比从第7天的30%下降到第14天的10%并维持在低水平；小鼠CD4+Foxp3+细胞(Tregs)比例持续上升而IL-17阳性细胞(Th17)则从高水平(约40%)降低到2%以下。在Tregs治疗组小鼠体内,脐带血Tregs表面的CD39分子能够抑制小鼠T细胞的扩增,而分泌的TGF-β则诱导Treg/Th17平衡向Tregs方向偏移。采用两轮多克隆扩增的脐带血Tregs比外周血Tregs具有更强的免疫抑制功能,能够通过表达CD39分子和分泌TGF-β减轻小鼠同种异基因骨髓移植aGVHD症状,表明脐带血Tregs具有临床应用治疗aGVHD的潜力。本文建立的从脐带血和外周血扩增Tregs的方法能够在体外获得大量的满足临床治疗需求的多克隆或同种异基因抗原特异性的调节性T细胞。
[Abstract]:Regulatory T cell (Tregs) plays an important role in immunomodulation and maintenance of peripheral immune tolerance. Due to the limited number of Tregs isolated from donor peripheral blood, fresh isolated Tregs needs to be amplified in vitro to obtain the number of Tregs cells needed for clinical application. It has been reported that human Tregs can be effectively amplified in vitro, but it is not clear whether the amplified Tregs can effectively inhibit the (aGVHD) of acute graft-versus-host disease in vivo. CD4 CD25 cells isolated from umbilical cord blood and CD4 CD25 CD127- cells isolated from adult peripheral blood were amplified in vitro to compare the immunosuppressive function of amplified Tregs. Umbilical cord blood and peripheral blood Tregs were amplified in vitro by two rounds of polyclonal stimulation or the first round of allogeneic antigen stimulation plus the second round of polyclonal stimulation. After two rounds of amplification, the phenotype, cytokine secretion and inhibitory function of Tregs were detected. The aGVHD model of C57BL/6 mice was established by transplantation of bone marrow and spleen cells into DBA/2 mice after lethal irradiation. In the Tregs treatment group, aGVHD model mice were treated with expanded umbilical cord blood or peripheral blood Tregs adoptive infusion in vitro. The survival rate, body weight, GVHD symptom score, histopathological specimens, serum cytokines and Th cell subsets in the Tregs treated group and the control group were analyzed and compared. The average amplification of Tregs in cord blood and peripheral blood was 1.5 脳 10 ~ 3 and 1.8 脳 10 ~ 3 times respectively after two rounds of polyclonal amplification, while the average amplification multiple of 5 脳 10 ~ 2 and 4. 7 脳 10 ~ 2 were 5 脳 10 ~ 2 and 4. 7 脳 10 ~ 2 respectively after the first round of alloantigen specific amplification and the second round of polyclonal amplification. The amplified Tregs expressed high expression of Foxp3,CD39,CTLA-4, and low expression of CD127,IL-2,IFN-Y, and maintained the inpotency of immune response. Two rounds of polyclonal amplification of Tregs in cord blood not only inhibited the proliferation of wide-spectrum amplified effector T cells (Tresp), but also inhibited the proliferation of effector T cells activated by dendritic cells with different HLA antigens. When two rounds of polyclonal amplification of cord blood or peripheral blood Tregs were infused into aGVHD model mice, 87.5% and 16.7% of the mice survived for more than 63 days, respectively, while the aGVHD model mice all died within 18 days. On the 7th day, the levels of IFN- 纬 and TNF-a in the Tregs treatment group were significantly lower than those in the aGVHD model mice. Then, the contents of IFN- 纬, TNF- 伪 and IL17 in Tregs group decreased rapidly and maintained at low level, while the content of TGF- 尾 continued to increase. The changes of immune cell subsets in mice coincided with the changes of serum cytokines in mice: the percentage of Th1 cells decreased from 30% on the 7th day to 10% on the 14th day and maintained at a low level. The percentage of (Tregs) in mouse CD4 Foxp3 cells continued to increase, while the percentage of IL-17 positive cells (Th17) decreased from 40% to less than 2%. In the Tregs treatment group, the CD39 molecules on the Tregs surface of cord blood could inhibit the expansion of mouse T cells, while the secreted TGF- 尾 induced the shift of Treg/Th17 balance to Tregs. The cord blood Tregs amplified by two rounds of polyclonal amplification has stronger immunosuppressive function than the peripheral blood Tregs, and can alleviate the aGVHD symptoms of allogeneic bone marrow transplantation in mice by expressing CD39 molecule and secreting TGF- 尾. It is suggested that umbilical cord blood Tregs has the potential of clinical application in the treatment of aGVHD. The method established in this paper to amplify Tregs from cord blood and peripheral blood can obtain a large number of polyclonal or alloantigen-specific regulatory T cells in vitro to meet the needs of clinical treatment.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

【相似文献】