Cytolytic mechanisms and T-cell receptor V beta usage by ex vivo generated Epstein-Barr virus-specific cytotoxic T lymphocytes

Victoria J. Vanhoutte, Karen A. McAulay, Erin McCarrell, Marc Turner, Dorothy H. Crawford, Tanzina Haque

Research output: Contribution to journalArticlepeer-review

Abstract

P>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 center dot 0001) and ethylene glycol-bis tetraacetic acid (P < 0 center dot 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 center dot 0001) and more granzyme B was detected in CD8(+) T cells than in CD4(+) T cells (P = 0 center dot 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.

Original languageEnglish
Pages (from-to)577-586
Number of pages10
JournalImmunology
Volume127
Issue number4
DOIs
Publication statusPublished - Aug 2009

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