可作为细胞治疗制品的CD8调节性T细胞的体外诱导扩增方法  

Anin vitromethod to induceand expand stable CD8^+ regulatory T cells for cell therapy

作　　者：孙娟[1,2] 霍小娜 杨懿铭 蒋雪玉[1] 朱蓓蓓[2] 李振华 谢如锋[1] 高跞[1] 刘李栋[1] 范华骅[1] 朱永明[1] 杨洁[1] SUN Juan;HUO Xiaona;YANG Yiming;JIANG Xueyu;ZHU Beibei;LI Zhenhna;XIE Rufeng;GAO Li;LIU Lidong;FAN Huahua;ZHU Yong-ming;YANG Jie(Blood Engineering Laboratory,Shanghai BlOod Center,Shanghai,China,200051;Life Science De-partment,East China Normal University.)

机构地区：[1]上海市血液中心血液工程研究室,上海200051 [2]华东师范大学生命科学学院

出　　处：《中国输血杂志》2018年第9期938-942,共5页Chinese Journal of Blood Transfusion

基　　金：上海市公共卫生三年行动计划重点学科建设项目(15GWZK0501);上海市卫生和计划生育委员会项目(20154Y0079,201640096,20154Y0192)资助

摘　　要：目的建立1种可用于细胞治疗制品的CD8调性T细胞（CD8＋Reularory T cell,Treg）的体外诱导扩增方法,并检测所获Treg细胞性质及功能的稳定性。方法通过免疫磁珠从人外周血PBMC中分选出CD8＋T细胞,在抗人-CD3/CD28磁珠与外源性IL-2激活下,联合使用不同组合的诱导因子：转化生长因子1（TGF-β1）、雷帕霉素（Rapamycin,RAPA）和全反式维甲酸（All-trans retinoic acid,ATRA）,多克隆诱导扩增出CD8＋Treg细胞,比较不同诱导组CD8＋Treg细胞扩增能力、表型、细胞内因子分泌、抑制功能等情况,筛选出最合适的诱导组细胞,进行体外多轮重刺激扩增并检测其功能及性质的稳定性。结果对不同组CD8＋Treg细胞扩增数量、表型、细胞内因子分泌情况分析得出：TGF-β1和雷帕霉素联合诱导组细胞高表达Foxp3和CD103,扩增能力强,对同种异基因CD4效应T细胞抑制功能可达90%以上、炎性因子IL-2和IFN-γ分泌细胞比例低、在炎性条件下不分泌IL17A和分泌很低量的IFN-γ。体外进行四轮重刺激,扩增倍数最低可达10000倍,且扩增所获Treg细胞活性高,Foxp3表达稳定,各轮抑制功能无显著差别。对Treg抑制作用机制研究发现,Treg通过竞争性消耗内源性IL-2起到部分抑制功能。结论 CD8＋Treg细胞体外扩增诱导方法的建立将填补了目前CD8＋Treg培养方法上的空白。体外大量扩增的CD8＋Treg细胞具有治疗自身免疫病、GVHD等疾病的应用潜力,可作为1种细胞治疗制品运用于临床。同时,通过利用原本在输血中废弃的PBMC,诱导出功能稳定的CD8＋Treg细胞,在一定程度上提高了血液的利用效率和促进了成份输血的进程。Objectives To establish a method to induce and expand a large amount of CD8＋regulatory T cell（ Treg） in vitrofor cell therapy. Evaluate its stability of inhibition and identify the phenotypes. Methods CD8＋T cells were purified from human PBMC and stimulated with anti-CD3/CD28 beads and IL-2. Induction with TGF-β1 and/or Rapamycin and/or All-trans Retinoic Acid was then performed. We deduced the best induction combination by comparing the proliferation,phenotype,cytokinessecretion and inhibition status in different induction setup groups. Induced CD8＋Tregs were stimulated in multiple rounds to obtain sufficient cells and their stability of inhibition and phenotypes were tested.Results Through analysis of proliferation,phenotype,cytokines secretion and inhibition ability of different induction groups,the combination of TGF-β1 and rapamycin was found to trigger a superior CD8＋Treg induction outcome. This method led to sufficient CD8＋Treg proliferation,high Foxp3 and CD103 expression,critical inhibition ability of allogenic CD4＋T cells,low percentage of IL-2 and IFN-γ secreting cells whilemaintained stable under inflammation conditions.Four rounds of in vitrostimulation cultivatedCD8＋Tregs expansion beyond 10000 fold. In addition,obtained Tregs presented high cell activity,stable Fox3 expression and inhibition ability after each induction round. Moreover,a CD8＋Treg suppression-function mechanism assay revealed that CD8＋Tregs exerted partial inhibition ability through competitive consumption of endogenous IL2. Conclusions This induction and expansion method is superior to current methods of CD8＋Treg culture in vitro. CD8＋Tregscould be induced and expanded sufficiently,andwere potentially capable of treating autoimmune diseases and GVHD in the future. Moreover,purified CD8＋T cells from discarded PBMC can generated functional CD8＋Tregsto improveblood utilization and thus improve component blood transfusion to a certain degree.

关 键 词：CD8调性T细胞 诱导扩增 成份输血 

分 类 号：R457.1[医药卫生—治疗学]