Quantifying EBV-specific CD8? T cell immunity in patients with post transplant lymph proliferative disease (PTLD) and other EBV-driven malignancies.

Epstein-Barr virus (EBV) specific T cells control EBV-driven proliferation of infected B cells in vivo. Deficient EBV immunity predisposes to post-transplant lymphoproliferative disease (PTLD) and EBV-positive Hodgkin's disease (HD). Treatment for PTLD is reduction in immunosuppression (RIS) allowing EBV immune recovery. Current methods of monitoring RIS include immunophenotyping for increases in MHC Class IICD8 T cells, but this is not a specific measure of EBV immunity. A method was developed for enumerating functional EBV-specific T cells using an intracellular cytokine staining (ICS) assay to measure interferon-gamma (IFN-gamma) production after brief in vitro stimulation with EBV antigens. Analysis of EBV-positive PTLD patients showed a temporal relationship between emergence of EBV-specific IFN-gamma'CD8 T cells and PTLD regression in response to RIS. The increase paralleled the rise in MHC class IICD84 T cells. Analysis of tumour infiltrating lymphocytes (TILs) from EBV-positive HD patients showed presence of EBV-specific IFN-gamma+CD4 and IFN-gamma4CD8+ T cells. Although this result indicates EBV-positive HD is not a consequence of deficient EBV immunity, the observed accumulation of CD4+ T cells may represent immune suppressive cells. The ICS assay was demonstrated as a rapid and reliable method for detecting functional EBV immunity, but requirement for several million PBMCs can limit utility. Quantitative RT-PCR (qRT-PCR) was used to measure IFN-gamma gene transcription by T cells stimulated with EBV antigen and showed comparable results to ICS using fewer cells. Clinical assessment of EBV status of tumours is hampered by lack of a single monoclonal antibody able to detect all EBV antigens. A novel monoclonal antibody, RFD3, was used to stain EBV-positive tissue and detected all forms of EBV latency. Results correlated with current diagnostic techniques.. In summary, this thesis describes development and evaluation of novel approaches for detecting EBV and measuring EBV-specific T cell immunity that have potential for clinical and research applications