HFRS患者汉滩病毒核蛋白特异性T细胞克隆及其靶细胞的建立

目的　建立肾综合征出血热 (HFRS)患者汉滩病毒 (HTNV)核衣壳蛋白 (NP)特异性CTL克隆 ,为HTNVNPT细胞表位鉴定及HFRS患者T细胞免疫功能研究奠定基础。方法　采用Fi coll密度梯度离心法分离HFRS患者外周血单个核细胞 (PBMC) ,用灭活HTNV和IL 2体外刺激 ,有限稀释法建立T细胞克隆 ,流式细胞术鉴定克隆表型。并用EB病毒 (EBV)转化B淋巴细胞 ,建立B淋巴母细胞样细胞系 (B LCL) ,以含HTNVS基因的重组痘苗病毒感染B LCL作靶细胞 ,以CTL克隆作效应细胞进行细胞毒杀伤试验 ,测定T细胞克隆的抗原特异性。结果　T细胞克隆能特异性识别表达NP的B LCL。在 5名患者中 ,3名有较高的杀伤率 ,并自 2名患者建立了 5株HTNV特异性CTL克隆 ,其表型为CD8 均大于 6 0 %。结论　成功建立了HFRS患者HTNVNP特异性CTL克隆及其靶细胞。NP是HFRS患者HTNV特异性CTL应答的主要靶抗原之一。

Establishment of Hantaan virus-specific T cell clone and its target cells in patients with hemorrhagic fever with renal syndrome

ObjectiveTo establish T cell clone which is specific for nucleocapsid protein (NP) of Hantaan virus (HTNV) in patients with hemorrhagic fever with renal syndrome (HFRS) and to lay a foundation for the study of T cell epitope and T cell immunology. MethodsPeripheral blood mononuclear cells (PBMCs) of patients with HFRS separated by Ficoll-Hypaque density gradient centrifugation were stimulated in vitro with inactivated HTNV and recombinant human IL-2. T cell clones were established with limiting dilution technique and clone phenotype was identified by flow cytometer. PBMCs infected by EB virus (EBV) were used to establish immortalized B lymphoblastoid cell lines (B-LCLs). B-LCLs infected by rVV were used as the target cells of HTNV-specific CTL clones. The antigenic specificity of T cell lines was identified by cytotoxicity assays. ResultsB-LCLs expressed NP were recognized by HTNV-specific T cell clones. Of the 5 individuals tested,three had high specific lysis rate. Five T cell clones were obtained from two patients and CD8 T cell clone takes more than 60%. ConclusionHTNV NP- specific T cell clones and their target cells have been established. NP is potentially a major target of the CTL response during HTNV infection.