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目的建立用人B淋巴细胞体外获得大量的同种异基因抗原特异性调节性T(Treg)细胞的方法,以此检测扩增细胞的表型及其免疫无能性和免疫抑制性。方法第1轮扩增将免疫磁珠分选的人CD4+CD25+T细胞和人B淋巴细胞按1∶4体外混合培养,并加入外源白介素-2(IL-2)和抗-CD28;将第1轮扩增得到的Treg细胞用抗-CD3/CD28包被的免疫磁珠和IL-2刺激,做第2轮扩增以获得更多数量的抗原特异性Treg细胞,分为添加和或未添加免疫抑制剂雷帕霉素(RAPA)2组(n=3)。结果经过2轮的扩增后,在第2轮扩增中未添加RAPA组扩增1×103倍,纯度>80%;添加RAPA组扩增0.8×103倍,纯度>90%。添加RAPA组得到的Treg细胞的Foxp3、CT-LA4、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,前者表现出部分免疫反应无能性,后者表现出完全的免疫反应无能性,两者都表现出免疫抑制性功能特征。人B淋巴细胞扩增得到的抗原特异性Treg细胞能够极大地抑制同源抗原引起的免疫反应,而对多克隆刺激的免疫反应抑制能力较弱。结论用人B细胞体外扩增抗原特异性Treg细胞,再通过抗-CD3/CD28包被的免疫磁珠进一步刺激可以体外获得大量的抗原特异性的Treg细胞,加入RAPA后可有效地提高Treg细胞的纯度和免疫抑制力且呈现抗原特异性。
OBJECTIVE: To establish a method for obtaining a large amount of alloantigen-specific regulatory T (Treg) cells from human B lymphocytes in vitro to detect the phenotype of the expanded cells and their immunocompetence and immunosuppression. Methods In the first round of amplification, human CD4 + CD25 + T cells and human B lymphocytes sorted by immunomagnetic beads were mixed in vitro at a ratio of 1: 4 and exogenous interleukin-2 (IL-2) and anti-CD28 were added. Treg cells obtained from the first round of amplification were stimulated with anti-CD3 / CD28-coated immunomagnetic beads and IL-2 for a second round of amplification to obtain a greater number of antigen-specific Treg cells divided into addition and Or no addition of immunosuppressant rapamycin (RAPA) 2 (n = 3). Results After two rounds of amplification, the RAPA group did not amplify 1 × 103 times and the purity was> 80% in the second round of amplification. The amplification of RAPA group was 0.8 × 103 times and the purity was> 90%. The expression level of Foxp3, CT-LA4 and CD39 in Treg cells obtained by adding RAPA group was higher than those without RAPA group, but HLA-DR had little change. Treg cells expanded without RAPA group secreted low levels of IL-2, IL-17, IL-4 and IFN-γ, while Treg cells obtained by adding RAPA group secrete almost none of the above cytokines, the former showed partial immune response impotence, the latter showed complete immune response impotence, two All showed immunosuppressive features. Antigen-specific Treg cells obtained by human B lymphocyte expansion can greatly inhibit the immune response caused by homologous antigens, but have less ability to suppress the immune response induced by polyclonal antibodies. CONCLUSION: Antigen-specific Treg cells can be expanded by human B cells in vitro, and then a large number of antigen-specific Treg cells can be obtained by anti-CD3 / CD28 coated magnetic beads. After adding RAPA, Treg cells Purity and immunosuppressive and antigen-specific.