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Title: The characterisation of ex vivo generated Epstein-Barr virus-specific cytotoxic T-cell lines
Author: Vanhoutte, Victoria J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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Post-transplant lymphoproliferative disease (PTLD) is an Epstein-Barr virus (EBV)- associated disease, which occurs in up to 10% of transplant patients and can be fatal in up to 70% of cases. PTLD has been successfully treated with cellular adoptive immunotherapy using EBV-specific cytotoxic T-cell lines (CTLs). However, little is known about the development of these CTLs. This project therefore sought to establish how the CTLs developed in terms of their growth, phenotype and T-cell receptor (TCR) repertoire diversity. It also sought to establish which cytolytic molecules were present in the CTLs and which pathways they employed to effect their cytotoxic potential. The phenotype and the cytolytic proteins of the CTLs were investigated by fluorescence activated cell sorting and western blotting. The TCR repertoire diversity was established by spectratyping analysis of the complementarity determining region 3 of the variable β chain of the TCR. Cytotoxic activity and pathways were assessed using chromium release assays. The results show that the CTLs developed phenotypically into mature T-cells within the first four weeks of culture, with an increase in CD45RO and CD69 expression and a decrease in CD45RA, CD62L, CD27 and CD28 expression. The CTLs also expanded most during that period and had viability in excess of 60%. The growth and viability of the polyclonal CTLs decreased continuously after four weeks of culture. The TCR repertoire of the CTLs remained diverse during the course of culture. Granzyme B, perforin and Fas ligand (FasL) were all detected in the CTLs. There was significantly more granzyme B in CD8+ T-cells than in CD4+ T-cells (p=0.0016) and there was also a significantly greater proportion of CD8+ T-cells expressing granzyme B than CD4+ T-cells (p=0.0023). There was no significant difference in the proportion of CD4+ T-cells and CD8+ T-cells expressing FasL or perforin, or in the amounts of FasL or perforin found in the respective cell types. Granulysin was detected in 33% of the CTLs tested. Cytotoxicity was significantly inhibited by concanamycin A (p = 0.0007) and ethylene glycol-bis tetraacetic acid (p < 0.0001) indicating that the principal cytotoxic pathway employed by the CTLs was a calcium and perforin-mediated exocytosis pathway leading to the release of granzyme B. These findings suggest that CTLs develop into mature CTLs in the early stages of culture without a reduction in TCR repertoire diversity. These findings also suggest that both CD4+ T-cells and CD8+ T-cells contribute to the overall cytotoxicity of the CTLs using a perforin-mediated cytotoxic granule exocytosis pathway. It is hoped that this basic characterisation of CTLs will reveal features that can potentially enhance the clinical effects of CTLs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available