同种异基因抗原特异性调节性T细胞的体外扩增  

Ex vivo expansion of human alloantigen-specific CD4^+CD25^+ regulatory T cells

作　　者：曹国番[1,2] 郝俊[1,2] 李振华[1,2] 杨燕[3] 杨懿铭[4] 杨洁[4] 任亚娜[4] 谢如峰[4] 范华骅[4] 钱开诚[4] 

机构地区：[1]华东师范大学生命科学学院 [2]上海市血液中心研究生培养基地,上海200062 [3]复旦大学 [4]上海市血液中心输血研究所

出　　处：《中国输血杂志》2012年第4期318-323,共6页Chinese Journal of Blood Transfusion

基　　金：上海市科委基金(11140902902)

摘　　要：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 ex vivo expand a higher amount of functionally active human alloantigen-specific Foxp3^＋ CD4^＋ regulatory T cells from allogeneic B cells, and determine their anergic and suppressive possibilities. Methods In the primary stimulation cycle, the magnetic bead-sorted CD4^＋ CD25^＋ T cells were cultured with allogeneic B cells at B-to-T cell ratios of 4：1 in the presence of exogenous IL-2 and costimulatory anti-CD28 antibody. To obtain the highest number of tregs with direct alloantigen-specificity,the B cell-expanded tregs were expanded polyclonally by using anti-CD3 and anti-CD28 antibodies in combination with IL-2 in the presence or absence of rapamycin. Results After the primary and secondary expansions ,the naive tregs could be expanded 1× 10^3 fold into alloantigen-specific CD4^＋ CD25^＋ Foxp3^＋ regulatory T cells in the absence of rapamycin with the percentage of B-expanded tregs 〉 80% ;however, an 0. 8× 10^3 fold with the percentage of B cell-expanded tregs 〉 90% could be obtained in the presence of rapamycin;B cell-expanded tregs cultured in the presence of rapamycin expressed higher level of Foxp3, CTLA4 and CD39 as compared to B cell-expanded tregs cultured in the absence of rapamycin. But the level of HLA-DR was expressed equally regardless to the presence or absence of rapamycin or not. B cell-expanded tregs cultured in the absence of rapamycin expressed low levels of IL-2, IL-17, IL-4 and IFN- γ- However,B cell-expanded tregs cultured in the presence of rapamycin almost did not express IL-2 ,IL-17 ,IL-4 and IFN-γ. The former remains partially anergie, and the latter anergie. They were all suppressive. B cell-expanded Tregs were capable of imposing potently inhibitive alloproliferation of responder cells elicited by specific alloantigens, and less potential to inhibit nonspecific immune response. Conclusion The highest number of functionally active human alloantigen-specific CD4^＋ CD25^＋ Foxp3^＋ regulatory T cells could be obtained after ex vivo stimulation with a

关 键 词：TREG细胞 抗原特异性 人B淋巴细胞 体外扩增 抗-CD3/CD28 免疫磁珠 免疫抑制力 

分 类 号：R593.2[医药卫生—内科学] R457.2[医药卫生—临床医学]