肿瘤新抗原肽诱导献血者PBMC特异反应性T细胞  

Specific reactive T cells from allo-blood donors'PBMC induced by tumor neoantigen peptides

作　　者：杨颖[1] 路丽明[2] 李勤[1] 郭忠慧[1] 杨启修[1] 张嘉敏[1] 赵俸涌[1] 王晨[1] 朱自严[1] YANG Ying;LU Li-Ming;LI Qin(Shanghai Blood Center,Shanghai 200051)

机构地区：[1]上海市血液中心输血研究所,200051 [2]上海交通大学医学院上海市免疫学研究所

出　　处：《临床输血与检验》2022年第5期565-573,共9页Journal of Clinical Transfusion and Laboratory Medicine

基　　金：中国输血协会威高重点(No.CSBT-MWG-2020-01);上海市卫健委面上项目(No.202040503)资助。

摘　　要：目的观察异基因献血者来源PBMC(peripheral blood mononuclear cells)是否可被来自于肿瘤患者测序来源的新抗原肽诱导为特异反应性T细胞。方法取健康献血者新鲜外周血白膜层,分离获得PBMC后,粘附获得单核细胞,同时冻存PBL(peripheral blood lymphocytes)。单核细胞以每三天加入细胞因子GM-CSF和IL-4进行细胞诱导培养,7天后收获成熟DC(dendritic cells),24孔铺板后分别装载16条肿瘤新抗原肽,其中13条为本项目组前期研究所得,5 h后加入4~10倍细胞数量已复苏的PBL共培养,每三天加入IL-2,对照为不加抗原肽(DC/PBL),加入PHA(Phytohemagglutinin,植物血凝素)PBL孔作为阳性对照;第14天、第21天分别再行新抗原肽脉冲刺激,第22天留取上清液用ELISA方法测量IFN-γ浓度,剩余细胞用流式细胞仪分析CD3^(+)IFN-γ^(+)/CD8^(+)IFN-γ^(+)T细胞占比。结果发现10/13中的每条多肽至少可诱导3份PBL分化为分泌IFN-γ反应性CD3^(+)或CD8^(+)T细胞,同步ELISA测得上清液中IFN-γ浓度也升高,以上结果具有一定的相关性(CD3^(+)或CD8^(+)T细胞r值分别为0.66、0.58,P<0.05)。结论我们成功地建立了用来自肿瘤患者的肿瘤新抗原肽诱导健康献血者的PBMC分化为肿瘤新抗原肽特异性的T细胞。Objective To observe whether neoantigen peptides sequenced from tumor patients can induce allo-donor sourced PBMC to be specific reactive T cell.Methods PBMC were separated from healthy donors'buffy coat,then monocytes were collected using adhesion method,remaining PBL was kept frozen in-80℃.GM-CSF and IL-4 were added to induce monocytes into dendritic cells every three days.At Day 7 mature dendritic cells were harvested and plated in 24 well-plates,then these plates were loaded with 16 neoantigen peptides as one peptide in each well,13/16 peptides were designed by our team.Five hours later PBLs recovered from-80℃were added into these plates to be co-cultured with these treated DC in ratio of 4~10∶1.No peptide wells(DC only)were negative controls,and PBL only wells were prepared for adding PHA later as positive control.IL-2 was replenished in these plates every three days.Peptides were pulsed twice more at Day 14 and Day 21 respectively.Supernatant of each well was collected at Day 22 to measure IFN-γconcentration using ELISA methods,remaining cells were analyzed using Flowcytometry to measure the percentages of CD3^(+)IFN-γ^(+)or CD8^(+)IFN-γ^(+)Tcells.Results 10 of 13 peptides can induce more than 3 kinds of PBL to proliferate into reactive CD3^(+)or CD8^(+)T cells,similar increases were also found in IFN-γconcentration(r=0.66,0.58 for CD3^(+),CD8^(+)respectively,P<0.05)in supernatant by ELISA method.Conclusions Our research data demonstrate these neoantigen peptides sequenced from tumor patients can induce allo-blood donors'PBMC proliferate to neoantigen specific reactive T cells.

关 键 词：外周血单个核细胞 树突状细胞 肿瘤新抗原 反应性T细胞 过继免疫治疗 

分 类 号：R392[医药卫生—免疫学]