Vanhoutte Victoria J, McAulay Karen A, McCarrell Erin, Turner Marc, Crawford Dorothy H, Haque Tanzina
Clinical and Molecular Virology, University of Edinburgh, Summerhall, Edinburgh, UK.
Immunology. 2009 Aug;127(4):577-86. doi: 10.1111/j.1365-2567.2008.03035.x.
Ex-vivo-generated Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable beta-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0.0001) and ethylene glycol-bis tetraacetic acid (P < 0.0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-gamma and TNF-alpha. Granzyme B, perforin and Fas ligand were detected in CD8(+) and CD4(+) cells in all CTL; however, a greater proportion of CD8(+) than CD4(+) T cells expressed granzyme B (P < 0.0001) and more granzyme B was detected in CD8(+) T cells than in CD4(+) T cells (P = 0.001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.
体外产生的爱泼斯坦-巴尔病毒（EBV）特异性细胞毒性T淋巴细胞（CTL）已被用于EBV相关淋巴瘤的细胞过继性免疫治疗。在此，我们研究了通过每周用EBV转化的自体淋巴母细胞系（LCL）刺激产生的正在生长的和已建立的CTL的表型、细胞溶解机制、多功能性和T细胞受体（TCR）使用情况。我们的结果表明，表型成熟的CTL在培养的前4周内形成，CD45RO和CD69增加，而CD45RA、CD62L、CD27和CD28表达减少。TCR可变β链的谱型分析显示，在培养过程中TCR库保持多样化。抗霉素A（P < 0.0001）和乙二醇双四乙酸（P < 0.0001）可显著抑制CTL的细胞毒性，表明钙和穿孔素介导的伴有颗粒酶B释放的胞吐途径是主要的细胞毒性机制。在用自体LCL再次刺激时，CTL主要产生干扰素-γ（IFN-γ）或肿瘤坏死因子-α（TNF-α），尽管有一些多功能细胞同时产生IFN-γ和TNF-α。在所有CTL的CD8(+)和CD4(+)细胞中均检测到颗粒酶B、穿孔素和Fas配体；然而，表达颗粒酶B的CD8(+) T细胞比例高于CD4(+) T细胞（P < 0.0001），且在CD8(+) T细胞中检测到的颗粒酶B比CD4(+) T细胞中的更多（P = 0.001）。Fas配体或穿孔素表达未观察到这种差异。我们的结果为体外产生的CTL的基本特征提供了见解。
