EBV抗原特异性T细胞体外抗淋巴瘤细胞机制研究

Mechanism of EBV antigen-specific T cells against lymphoma cells in vitro

目的 Epstein-Barr病毒(Epstein-Barr virus,EBV)在淋巴瘤的发生发展中发挥重要作用,EBV相关淋巴瘤预后较差,在临床治疗过程中面临挑战。本研究旨在体外培养获得EBV抗原特异性T细胞,检测其功能及体外抗肿瘤能力。方法采集2015-02-22-2016-05-01北京大学肿瘤医院淋巴瘤科10例结外NK/T细胞淋巴瘤患者外周血,分离获得外周血单个核细胞(peripheral blood mononuclear cell,PBMC),体外培养获得EBV抗原特异性T细胞。检测特异性T细胞比例,测定EBV抗原特异性T细胞对自体淋巴母细胞系(lymphoblastoid cell lines,LCL)杀伤作用以及对来自EB病毒潜伏膜蛋白1(Epstein-Barr virus latent membrane protein1,LMP1)、LMP2和EB病毒核抗原1(Epstein-Barr virus nuclear antigen,EBNA1)不同部位肽段的反应性。结果采集10例EBV阳性NK/T细胞淋巴瘤患者外周血,体外培养发现随时间延长,抗原特异性T细胞比例升高。培养2周后的EBV抗原特异性T细胞,在再次遭遇抗原后,效应细胞比例可以达到(6.837±1.031)%,与阴性对照(1.447±0.566)%相比较,差异有统计学意义,t=5.622,P=0.001。EBV抗原特异性T细胞可以识别已经证实的LMP2表位。抗原特异性T细胞中同时含有EBV抗原反应性CD4和CD8细胞。体外杀伤试验证实,EBV抗原特异性T细胞能够杀伤患者自体来源的EBV病毒转化的B细胞产生淋巴母细胞细胞系(lymphoblastoid cell lines,LCL),但对人类白细胞抗原(human leukocyte antigen,HLA)不相合的异体LCL却不具备杀伤活性,即具有HLA限制性。结论 EBV抗原特异性T细胞有可能用于治疗EBV相关淋巴瘤,为后续开展相关体内研究提供理论基础。

OBJECTIVE Epstein-Barr virus（EBV）plays a critical role in the pathogenesis of some lymphoma types.EBV positive lymphoma patients present with worse prognosis than those EBV negative patients,which is a challenge in clinical practice.This study aims to obtain EBV antigens specific T cells and test the function and anti-tumor ability in vitro.METHODS Peripheral blood was collected from 10 patients with NK/T cell lymphoma at Peking University Cancer Hospital from Feb.22,2015 to May 1,2016.Antigen-specific T cells targeting EBV antigens were obtained from mononuclear cells stimulated by peptides derived from EBV antigens.We detected the ratio of antigen-specific T cells,tested the activity of anti-LCL of T cells,and the reaction to different peptide fragments from latent membrane protein 1,（LMP1）,LMP2,Epstein-Barr virus nuclear antigen（EBNA1）.RESULTS Peripheral blood was collected from 10 patients with EBV positive NK/T cell lymphoma.EBV antigen responded T cells expanded after being stimulated over time（6.837±1.031）% of the cells were effective cells after encountering with antigen in 2 weeks of cell culture,indicating significant difference compared with negative control（t=5.622,P=0.001）.EBV antigen-specific T cells from patients expressing human leukocyte antigen（HLA）-A02 could recognize LMP2 epitopes.We also found that EBV antigen-specific T cells existed in both CD4 positive and CD8 positive T cell subsets.In-vitro killing assay showed EBV antigen-specific T cells can kill autologous EBV transformed LCL,with HLA restriction.CONCLUSION EBV antigen-specific T cells could be used for the treatment of EBV positive lymphoma,which provides basis for further in-vivo studies.