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Cytolytic mechanisms and T‐cell receptor Vβ usage by ex vivo generated Epstein–Barr virus‐specific cytotoxic T lymphocytes

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 β‐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‐γ (IFN‐γ) or tumour necrosis factor‐α (TNF‐α) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN‐γ and TNF‐α. 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.