Infection with Epstein-Barr virus (EBV) during adolescence results in an immunopathological disease, Infectious Mononucleosis (IM), in around 25% of cases. A role for Regulatory T cells (Treg) in IM has yet to be established. These suppressive cells may affect the well-characterised cytotoxic T cell (CTL) response to EBV and thus the level of viral persistence and reactivation, potentially creating an environment conducive to the outgrowth of EBV-infected cells and tumour development. The work in this thesis examines the frequency and functional capacity of Treg in primary EBV infection. The results show that the frequency of Treg within the CD4+ T cell population of IM patients was reduced with borderline significance (p=0.05) compared with healthy controls as revealed by fluorescence activated cell sorting. Treg function was confirmed using suppression assays on peripheral blood mononuclear cells (PBMC) from healthy controls but could not be assessed in IM patients due to low cell numbers. EBV-specific Treg function was analysed using Interferon (IFN)-γ ELISPOT assays in which PBMC from IM patients and healthy controls were stimulated with phytohaemagglutinin (PHA) and EBV peptides in the presence or absence of Treg. The IFN-gamma response of PBMC to PHA stimulation was significantly reduced in IM patients compared to healthy controls (p=0.009) but the IFN-gamma response to EBV peptides did not alter, irrespective of the presence or absence of Treg. Investigation of FOXP3 expression by immunohistochemistry provided evidence of Treg presence and preliminary data indicated an increased expression in IM tonsil sections compared with healthy tonsil sections. The proliferative responses and cytokine profiles of healthy controls, as measured by proliferation assays and ELISAs, in response to stimulation with the recall antigen PPD did not significantly alter upon the addition of latent membrane protein (LMP)-1 peptide. In IM patients, the same treatment resulted in a significant reduction in IFN-gamma (p=0.026) but no significant differences in IL-10 production or cell proliferation. The significantly reduced frequency of Treg in peripheral blood of IM patients and abundant FOXP3 expression in IM tonsils provides evidence for a Treg role in primary EBV infection. One plausible explanation is the recruitment of Treg to the site of primary infection by an as yet unidentified EBV-specific mechanism. Clarification of Treg activity in IM may expose opportunities for immunomanipulation during early stages of infection.

Epstein-Barr virus (EBV) is a gamma herpes virus persistently infecting over 90% of the adult population worldwide. It has been aetiologically linked to a number of human malignancies, including more than 90% of post transplant lymphoproliferative disease (PTLD), 50% of Hodgkin’s lymphoma (HL), virtually all undifferentiated nasopharyngeal carcinoma (NPC), and approximately 10% of gastric carcinoma (GC). As EBV infection in healthy individuals is mainly controlled by virus specific cytotoxic T lymphocytes (CTLs), we hypothesise that engineering T cells with chimeric T cell receptors (cTCRs) specific for EBV latent membrane proteins (LMPs) will confer on these cells the ability to target and kill the malignant cells of cancers associated with Epstein-Barr virus. Thus, the aim of this project was generate these engineered T cells and to set up a severe combined immunodeficient (SCID) mouse model in which to test their effectiveness. Three EBV-infected cell lines derived from HL, NPC and GC gave rise to tumours in 11 of 12 (92%), 12 of 12 (100%) and 10 of 10 (100%) SCID mice respectively, when 1x107 cells were injected subcutaneously. Immunohistochemical analysis showed that the HL SCID tumours were CD4-, CD15-, CD20+, CD30+, consistent with a HL Reed-Sternberg cell phenotype, and NPC and GC SCID tumours expressed the epithelial cell marker cytokeratin. Furthermore, all tumours expressed EBVencoded RNAs (EBERs) and LMP1. This was identical to parent cell line expression patterns, and hence growth in vivo did not affect cell phenotype. T cells were successfully transduced with a retroviral vector encoding a CD19-specific cTCR (CD19- cTCR) with a mean transduction rate of 13%±6%. Transduced cells were cytotoxic for HL-derived L591 cells in vitro, with specific lysis of 24%±11% at an effector to target ratio of 20:1. This was significantly higher than specific lysis seen in mock transduced cells (p>0.05). At a tumour inoculation dose of 5x106, in vivo sc transfer of 5x107 CD19-cTCR transduced cells was able to prevent HL tumour development in 6 of 6 (100%) test mice, whereas 17 of 22 (77%) control mice and 2 of 3 (66%) mice treated with unmodified EBV-specific CTLs developed tumours. Moreover, iv transfer of 5x107 CD19-cTCR transduced cells mediated complete regression of HL SCID tumours in 3 out of 6 (50%) mice. Phage display selection experiments to isolate a single chain antibody fragment (scFv) specific for viral LMPs for incorporation in a cTCR were performed. Linear, biotinylated and cyclised biotinylated peptides derived from the external reverse turn loops of LMP2 were used as target antigens. Despite extensive testing, no reactive clones specific for the peptides were identified. The ability of CD19-cTCR transduced cells to specifically lyse HL cells in vitro, and clear tumour burden in vivo, supports a future role for engineered T cells in the treatment of HL. Despite the lack of success in isolating a scFv for LMP2, the use of viral antigen specific, cTCR redirected T cells remains in principle a valuable therapeutic alternative for EBV-associated malignancies. The SCID models for HL, NPC and GC will provide a useful preclinical tool for investigation of their efficacy in vivo.