同种异基因抗原特异性调节性T细胞的体外扩增

发布时间：2018-05-05 17:39

本文选题：抗原特异性Treg细胞 + 人B淋巴细胞　；参考：《华东师范大学》2012年硕士论文

【摘要】：CD4+CD25+Foxp3+调节性T细胞(Tregs)在维持免疫耐受、抑制自身免疫性疾病病理反应及移植排斥反应的过程中起着重要的作用。但是由于Treg细胞的表型和Treg细胞在外周血中数量甚少,限制了基于Treg细胞治疗方法的临床应用。同时有文献报道扩增得到的Treg细胞的免疫治疗效果大于初始的Treg细胞,因此,开展体外诱导或扩增大量Treg细胞的研究具有重要的意义。目前主要可以通过anti-CD3/CD28抗体包被的免疫磁珠结合IL-2体外广谱扩增Treg细胞,或者利用同种异基因抗原呈递细胞(APCs)通过抗原特异性方式体外扩增抗原特异性Treg细胞。前者扩增得到的Treg细胞的抗原特异性较差,而后者的扩增倍数较少。动物模型研究表明抗原特异性Treg细胞在抑制自身免疫性疾病方面功能强于多克隆Treg细胞。本文综合了目前采用的各种扩增方法,建立了通过同种异基因B细胞诱导产生抗原特异性Treg细胞的方法：即第一轮扩增将免疫磁珠分选的人CD4+CD25+Foxp3+T细胞和人B淋巴细胞按1：4比例体外混合培养并加入外源白介素-2(IL-2)和抗CD28抗体；将第一轮扩增得到的Treg细胞用anti-CD3/CD28抗体包被的免疫磁珠和IL-2刺激,进行第二轮扩增以获得更多数量的抗原特异性Treg细胞,第二轮刺激分为添加或未添加免疫抑制剂雷帕霉素(Rapamycin, RAPA)两组。本研究结果表明经过两轮的扩增后,在第二轮扩增中未添加RAPA组可扩增1×103倍,纯度80%；而添加RAPA组可扩增0.8×103倍,纯度90%。添加RAPA组得到的Treg细胞的Foxp3、CTLA4、CD39表达水平高于未添加RAPA组,但是HLA-DR变化不大。未添加RAPA组扩增得到的Treg细胞分泌低水平的IL-2、IL-17、IL-4和IFN-γ,而添加RAPA组得到的Treg细胞几乎不分泌上述各种细胞因子。体外扩增实验结果表明未添加RAPA组Treg细胞表现出部分免疫反应无能性,而添加RAPA组Treg细胞则表现出完全的免疫反应无能性。经人B淋巴细胞扩增得到的抗原特异性Treg细胞能够有效地抑制同源抗原引起的免疫反应,而对多克隆刺激和异源抗原的免疫反应抑制能力较弱,表明体外扩增的Treg细胞呈现出抗原特异性特征。本文采用人B细胞体外扩增抗原特异性Treg细胞,再通过anti-CD3/CD28抗体包被的免疫磁珠进一步进行刺激可以在体外获得大量的抗原特异性的Treg细胞,并且在加入雷帕霉素后可以有效地提高Treg细胞的纯度和免疫抑制功能。本研究同时表明人B淋巴细胞扩增得到的Treg细胞呈现出抗原特异性的特征。本研究结果为利用第三方无关供者Treg细胞进行免疫治疗的可行性提供了理论依据。另外,本文还探讨了通过调控细胞代谢途径从CD4+CD25-T细胞体外诱导产生抗原特异性Treg细胞的方法。研究表明通过磷酸二酯酶(PDEs)抑制剂提高细胞胞内cAMP水平不仅可以抑制T细胞增殖和IL-2的分泌,同时可诱导产生具有免疫抑制功能的Treg细胞。本文采用PDEs抑制剂Cilostamide和耐受型树突状细胞(tDCs),在分别添加不同的外源细胞因子条件下体外诱导CD4+CD25Foxp3-T细胞产生CD4+CD25+Foxp3+Treg细胞。初步研究结果表明通过添加外源IL-2可以促进Cilostamide诱导产生CD4+CD25+Foxp3+Treg细胞。另外,在IL-2和Cilostamide存在的条件下,通过添加TGF-β可以提高Foxp3的表达水平。因此,在添加TGF-β和IL-2的条件下,Cilostamide和tDCs可以体外诱导产生Treg细胞。本研究结果将有助于进一步有效地利用C ilostamide体外诱生功能性Treg细胞。
[Abstract]:CD4+CD25+Foxp3+ regulatory T cells (Tregs) play an important role in maintaining immune tolerance, inhibiting the pathological response of autoimmune diseases and transplantation rejection. However, the phenotype of Treg cells and the low number of Treg cells in the peripheral blood limit the clinical application of Treg based therapy. The immunotherapy of Treg cells obtained by channel amplification is more effective than the initial Treg cells. Therefore, it is of great significance to develop and amplify a large number of Treg cells in vitro. At present, it is possible to amplify Treg cells in vitro by anti-CD3/CD28 antibody coated immunomagnetic beads combined with IL-2 in vitro, or to present the same allogenic antigen with the same allogenic antigen. The antigen specific Treg cells were amplified by APCs in vitro. The antigen specificity of the former Treg cells was poor and the amplification of the latter was less. The animal model study showed that the antigen specific Treg cells were stronger than polyclonal Treg cells in the inhibition of autoimmune diseases.
In this paper, we synthesized various methods of amplification, and established a method of inducing antigen specific Treg cells by the same allogeneic B cells: first round amplification of human CD4+CD25+Foxp3+T cells and human B lymphocytes in vitro mixed in 1:4 ratio and adding exogenous interleukins -2 (IL-2) and anti CD28 resistance. The first round amplified Treg cells were amplified with anti-CD3/CD28 antibody coated immunomagnetic beads and IL-2 for second rounds of amplification in order to obtain more amounts of antigen specific Treg cells, and the second round of stimulation was divided into two groups of adding or without adding the immunosuppressant, rapamycin (Rapamycin, RAPA). The results of this study showed that two rounds were carried out. After amplification, the number of non RAPA groups in the second round amplification was 1 x 103 times, the purity was 80%, and the addition of RAPA group was 0.8 x 103 times, the purity 90%. added to the RAPA group, the Foxp3, CTLA4, CD39 expression level was higher than that of the non RAPA group, but the HLA-DR changed little. IL-17, IL-4 and IFN- gamma, and the Treg cells obtained from the RAPA group almost did not secrete the various cytokines. In vitro amplification experimental results showed that the Treg cells without the RAPA group showed partial immune response inenergy, while the addition of RAPA group Treg cells showed complete immune response incompetence. The antigen obtained by human B lymphocytes was obtained. Specific Treg cells can effectively inhibit the immune response caused by homologous antigens, but the ability to inhibit the immunoreaction of polyclonal and heterologous antigens is weak, indicating that the Treg cells in vitro are characterized by antigenic specificity.
The amplification of antigen specific Treg cells in vitro by human B cells and the further stimulation of the immunomagnetic beads coated with anti-CD3/CD28 antibody can obtain a large number of antigen specific Treg cells in vitro, and can effectively improve the purity and immunosuppressive function of Treg cells after adding rapamycin. The Treg cells derived from the B lymphocyte of the people of the Ming Dynasty are characterized by antigen specificity. The results of this study provide a theoretical basis for the feasibility of using third party independent donor Treg cells for immunotherapy.
In addition, the method of inducing antigen specific Treg cells from CD4+CD25-T cells by regulating cell metabolic pathways is also discussed. The study shows that the enhancement of intracellular cAMP level by phosphodiesterase (PDEs) inhibitors not only inhibits the proliferation of T cells and the secretion of IL-2, but also induces the production of immunosuppressive function. Treg cells. This paper uses PDEs inhibitor Cilostamide and tolerance dendritic cells (tDCs) to induce CD4+CD25Foxp3-T cells to produce CD4+CD25+Foxp3+Treg cells under the conditions of adding different exogenous cytokines respectively. The preliminary results show that the addition of exogenous IL-2 can promote Cilostamide induced CD4+CD25+Foxp3+Treg to produce CD4+CD25+Foxp3+Treg. In addition, under the condition of IL-2 and Cilostamide, the expression level of Foxp3 can be enhanced by adding TGF- beta. Therefore, Cilostamide and tDCs can induce Treg cells in vitro under the condition of adding TGF- beta and IL-2. The results of this study will help to further effectively use C ilostamide to induce functional Treg cells in vitro.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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