Cytolytic mechanisms and T-cell receptor Vbeta 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 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.
Vanhoutte
Victoria J
VJ
Clinical and Molecular Virology, University of Edinburgh, Summerhall, Edinburgh, UK.
McAulay
Karen A
KA
McCarrell
Erin
E
Turner
Marc
M
Crawford
Dorothy H
DH
Haque
Tanzina
T
D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002455:Cell Division; D016207:Cytokines / Q000096:biosynthesis; D003602:Cytotoxicity, Immunologic; D020031:Epstein-Barr Virus Infections / Q000276:immunology*; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping; D054353:Perforin / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D016207:Cytokines; D016693:Receptors, Antigen, T-Cell, alpha-beta; D054353:Perforin; D053804:Granzymes