不同的冻存方法对1型糖尿病患者胰岛抗原特异性T细胞反应的影响

Effect of cryopreservation method on islets specific T cell responses in type 1 diabetic patient

目的:明确何种冻存方案更利于保持1型糖尿病患者的外周血单个核细胞(peripheral blood mononuclearcells,PBMCs)对胰岛抗原特异性的T细胞反应,更接近于新鲜细胞的状态。方法:本研究系国际免疫学协会-T细胞工作组(IDS-TCW)牵头的国际标准化研究Freezing Study I。中南大学糖尿病中心选取5例新发的T1D患者和年龄、性别相匹配的5例正常对照。所有对象新鲜分离的PBMCs分为3份,一份进行亚型细胞的流式细胞分析和酶联免疫斑点实验(enzyme linked immunospot assay,ELISPOT),另两等份分别采用低温和室温冻存方案冻存;1个月后分别按低温和室温方案复苏,以台盼蓝染色判定细胞活力;并行流式细胞分析术和ELISPOT检测。ELISPOT中4种抗原[包括人GAD65、内对照、Pediacel(巴斯德五合一疫苗)和PMA]均由IDS-TCW中心化实验室盲法标记,盲法测定。以新鲜PBMCs为参照,比较两种冻存方案下PBMCs的复苏率和活力、各亚型细胞比例和ELISPOT检测结果。结果:1)室温冻存下的PBMCs复苏率和细胞活力均略高于低温冻存方案,但二者差异无统计学意义(分别为61.2%vs 60.1%,77.5%vs 74.9%;P>0.05);2)相比新鲜细胞,两种冻存方案均会造成一定程度的单核细胞的损失[(3.2±1.1)%(低温)和(3.0±0.9)%(室温)vs(7.0±1.1)%(新鲜);P<0.05];而CD4+T,CD8+T,B细胞,NK细胞和NKT细胞比例均无明显变化(P>0.05);3)ELISPOT检测结果显示:对多克隆刺激物PMA诱发的T细胞分泌IFN-γ的反应,在新鲜和冻存复苏细胞间无明显差异;记忆性抗原Pediacel和胰岛抗原GAD65刺激下的特异性T细胞反应在冻存复苏细胞均明显差于新鲜细胞,T1D患者新鲜细胞的GAD特异性T细胞反应的SI为5.1,均高于低温(1.3)和室温(1.4)冻存复苏的刺激指数(SI)(均P<0.05);而且仅新鲜细胞的GAD-反应性T细胞的SI和斑点数均明显高于正常对照(5.1 vs 0.9和8.1 vs 0.1,P<0.05),而两种方案下的冻存细胞的SI在患者和对照未发现差异。结论:需要开展更多的研究,在T1D患者中鉴定出一种更接近于新鲜PBMCs反应的冻存方案和T细胞测定方法。

Objective: To explore the better freezing protocol to preserve peripheral blood monuclear cells(PBMCs),islets antigen-specific T cells responses compared with freshly isolated samples in type 1 diabetic(T1D) patients.Methods: The T cell Workshop Committee of the Immunology of Diabetes Society(IDS-TCW) organized the Freezing Study I and we were one of the 9 centers in the world to participate in the study.According to the two standardized T cell freezing protocols(warm and cold) to freshly isolated PBMCs in terms of recovery,viability,cell subset composition(FACS) and performance in Enzyme-linked immunospot(ELISPOT) assays,we chose 5 newly onset T1D patients and 5 age and sex matched healthy controls.Besides the protocols,all the freezing reagents and antigens were also centralized.The antigens used in ELISPOT were labeled blindedly.Results: 1) Although warm frozen-thawed(W) samples had a slightly higher recovery rate(61.2% vs 60.1%,P>0.05) and viability(77.5% vs 74.9%,P>0.05) as compared with the cold frozen ones(C),the difference was not significant.2) Both protocols led to a relative loss in monocytes as compared with the fresh samples(F) [3.2±1.1%(C) and 3.0±0.9%(W) vs 7.0±1.1%(F),both P<0.05],while other subsets including CD4+T,CD8+T,B cells,NK cells and NKT cells didn’t.3) Freezing and fresh samples showed similar IFN-γ secretion responses to polystimuli in ELISPOT.Irrespective of the freezing protocol,recall antigen Pediacel and islet antigen-reactive responses were both lower in the frozen cells compared with fresh PBMCs.The stimuli index(SI) of GADspecific T cell response in the fresh samples from T1D patients was 5.1,higher than that of frozen samples with either cold protocol(1.3) or warm one(1.4)(both P<0.05).Only fresh samples from T1D showed significantly higher GAD-specific T cell responses than the healthy controls no matter in SI(5.1 vs 0.9,P<0.05) or spot forming cells(8.1 vs 0.1,P<0.05),whereas the frozen samples did not show such difference.Conclusion: More studies are needed to verify a freezing method to bring comparable islets antigen specific T cell responses in T1D patients to fresh PBMCs.