Lymphoma is the fifth most common cancer in Australia. This thesis includes investigations of T-cell immunology to enhance immunotherapeutic strategies for lymphoma and identification of novel biomarkers to better interpret treatment efficacy.
The presence of Epstein-Barr virus (EBV) within the malignant cells of several types of lymphoma implicates EBV in the pathogenesis, but also offers important targets for immunotherapeutic strategies. Focusing on two distinct EBV-associated lymphomas with different EBV expression profiles, (Post-transplant lymphoproliferative disorders (PTLD) and classical Hodgkin lymphoma (cHL)), relevant EBV-specific T-cell immunity was investigated to better understand pathogenesis and enhance therapeutic strategies.
EBV+ PTLD is known to arise as a result of impaired EBV-specific T-cell immunity. Restoration of this immunity by adoptive T-cell therapy can induce remission in some, but not all, cases. The EBV latent protein EBV-nuclear antigen-1 (EBNA1) is unique in being expressed in all cases of EBV+ PTLD. However, there is no data on EBNA1-specific T-cells in PTLD. EBNA1 was previously thought to be immunologically silent, but recent data demonstrates otherwise. By assaying EBNA1-specific T-cells capable of proliferation, IFN-γ release and CD107a/b degranulation, we demonstrate that EBNA1-specific CD4+ and CD8+ functional T-cells can be expanded in EBV+ PTLD patients. EBNA1-specific CD4+ T-cells predominated while the magnitude of EBNA1-specific CD8+ T-cells was influenced by human leukocyte antigen class I alleles. The majority of PTLD patients had a polymorphism in an EBNA1 epitope compared to the commonly used laboratory strain, and CD8+ T-cell recognition was greatly enhanced when EBNA1 peptides from appropriate viral strains were used. These results indicate that EBNA1-specific Tcells should be included in adoptive immunotherapy for PTLD and expansion protocols should utilize antigenic sequences from relevant EBV strains.
In EBV+ cHL, EBV expression is restricted to sub-dominant latent proteins (EBNA1, latent membrane protein (LMP) 1/2) whereas in EBV+ PTLD immuno-dominant latent-proteins (e.g. EBNA3A/3B/3C) are often expressed. In EBV+ cHL, a decreased frequency of HLA-A*02 has been observed in large patient cohorts, while studies of EBV+ PTLD have had inconsistent results. We analysed 269 PTLD/ cHL patients and confirmed reduced HLA-A*02 susceptibility to EBV+ cHL but found no HLA-class I associations to EBV+ PTLD. In order to understand the functional basis for these observations, we firstly assayed global LMP1/2A ex-vivo CD8+ T-cell immunity in newlydiagnosed cHL patients. HLA-A*02 EBV+ cHL elicited higher immunity than non-HLA-A*02 EBV+ cHL, whereas immunity in EBV-ve cHL patients was equivalent. We then investigated the diversity of HLA-class I restricted responses to different EBV latent proteins. Ex-vivo peptide specific CD8+ T-cells were quantified in healthy EBV-seropositive participants using 61 HLA-class I LMP1/2A and EBNA3A/3B/3C-derived peptides. Dominant EBNA3A/3B/3C-specific CD8+ Tcell responses were stimulated by peptides presented by numerous HLA-class I alleles, whereas LMP1/2A-specific peptide responses were largely confined to HLA-A*02. Furthermore, in HLAA* 02 heterozygotes, HLA-class I deficient cell-lines transfected with HLA-A*02 were superior in expanding LMP2A-specific CD8+ T-cells than those transfected with other HLA-class I alleles. These findings demonstrate that HLA-class I impacts EBV-latent antigen-specific CD8+ T-cell hierarchy. This results in differential susceptibility to the development of EBV+ lymphomas expressing only sub-dominant latent-antigens, such as EBV+ cHL, but not EBV+ lymphomas that express immuno-dominant latent-antigens as in EBV+ PTLD. Thus, immunotherapeutic approaches that also generate T-cell immunity against sub-dominant antigens through several HLAclass I allele are more likely to be effective for EBV+ cHL.
Current treatment strategies for lymphoma include chemotherapy and radiotherapy which are associated with high treatment related complications such as secondary cancer, cardiopulmonary complications, stroke and infertility. Lymphoma patients with a rapid response to initial treatment have the best outcomes, and may benefit from a truncated treatment regimen. Furthermore, in those patients with refractory disease, prolonged exposure to first-line agents can induce chemo-resistance and unnecessary toxicity. The current challenge for clinicians is to tailor treatments to eradicate malignancy with minimal side-effects, and to simultaneously identify those patients in whom alternate strategies should be instituted early. Tumour burden and treatment response are currently assessed by computerized tomography (CT) or positron emission tomography (PET) scans. Although invaluable, these methods have their limitations and interpretation can be difficult. In
three prospective studies, we performed an in depth characterization of circulating DNA, protein and microRNA disease response biomarkers for lymphoma, including several that we identify for the first time. We describe the differential kinetics of these biomarkers throughout out therapy, identify those that reflect interim treatment response and define associations with current prognosticators. In addition, these studies compare tumour-specific, tumour-infiltrate-specific and non-specific markers throughout therapy. Unlike radiological assessment, the blood biomarkers we identify are practical for frequent testing. Methods used to quantify these markers include qRT-PCR and ELISA, both commonly used techniques in diagnostic laboratories.
This thesis reports a comprehensive study of EBV-associated lymphoma including functional evidence of novel strategies to treat and prevent lymphoma as well as identifying innovative methods to monitor treatment response and assist interpretation of PET/CT.