UNDERSTANDING THE ROLE OF EPSTEIN BARR VIRUS IN T CELL AND NATURAL KILLER CELL LYMPHOPROLIFERATIONS AND MALIGNANCIES

Epstein Barr virus (EBV) is a common herpesvirus that infects the majority of the human population for the lifetime of each individual. The vast majority of individuals are unaware that they have been infected because the virus is kept under tight control by their immune system. Although EBV is most well known for causing glandular fever, it is also associated with cancers in a small number of individuals. These cancers usually occur in the cells infected as part of the natural life cycle of the virus, B lymphocytes and epithelial cells. However, on rare occasions EBV can also infect cells that are not involved in the lifecycle of the virus, T lymphocytes and Natural killer (NK) cells. Infection of these cell types always results in serious, life-threatening diseases including cancers. These diseases are extremely difficult to treat and have a very poor clinical outcome, usually measured in survival from weeks to months. There is an urgent need to better understand the pathology of these EBV-associated T/NK cell diseases in order to develop new effective, personalized therapies.

Progress into understanding these diseases has been extremely slow, due to the rarity of the diseases and our inability to infect T/NK cells in the lab. However, we have addressed both these issues. We currently have a bank of blood samples from UK patients with EBV-associated T/NK cell diseases ready for analysis. Furthermore we have optimized the culture conditions and have generated matching EBV-positive T/NK cell lines from the patients' samples. Finally, and perhaps most importantly, we have managed to reproducibly infect T cells in our laboratory, which for the first time will allow us to examine how EBV triggers these T/NK cell diseases and induces their transformation into malignancy.

We have four key objectives. (1) Using patient blood, we have identified white blood cells and chemical signals (cytokines) from the microenvironment which are required to help the patients' EBV-infected T/NK cells to grow in culture. We plan to replicate these conditions in the lab to help the newly-infected T/NK cells grow and determine how EBV, the surrounding cells and cytokines contribute to the growth of newly-infected T/NK cells. (2) Once the growth conditions are optimized, we will examine how specific EBV gene products able to stimulate the chronic inflammation observed in the EBV-associated T/NK cell diseases. (3) By replicating the EBV infection and chronic inflammation in our cell cultures, we will examine how EBV and chronic inflammation drive the infected cells to grow indefinitely and dysregulate the anti-EBV immunity. (4) We have established a new analysis tool, which labels the EBV-infected cells with markers for EBV and markers for the cell type. We can expand the range of markers to identify potential new drug targets on the EBV-infected cell to include therapeutic antibodies such as anti-CCR4 (mogamulizumab) currently in phase 2 clinical trial for peripheral T cell lymphoma. Furthermore, we can culture the matching ex-vivo EBV-infected T/NK cells, as above, in the presence of the drug to determine sensitivity to the drug.

This is the first study of its kind to analyse the contribution of EBV to the EBV-associated T/NK cell diseases by combining ex-vivo patient samples and primary infected T/NK cells. We anticipate that by understanding how EBV causes the diseases, either directly or in combination with other cells and cytokines of the surrounding microenvironment, and by monitoring disease progression both in vitro and in vivo, we will develop a better understanding of alternative novel therapeutic options. Finally, the assays we have established to examine the potential new treatments will have wider benefit to the EBV-associated T/NK cell disease patient groups if the study were taken to trial, based on the findings of this study. The results will identify potential drugs for personalized treatment options.

Epstein Barr virus (EBV) is a common herpesvirus that infects the majority of the human population and establishes a silent latent infection in B cells for the lifetime of the infected host. However, under poorly understood circumstances, EBV may ectopically infect T cells and NK cells. This infection always results in overt disease ranging from clonal proliferations of infected cells with hypercytokinemia to aggressive malignancies, all of which are inherently resistant to the current treatment regimens. There is an urgent need to better understand the pathobiology of EBV-associated T/NK diseases in order to develop effective novel therapeutic strategies.

The aim of this research is to understand how Epstein Barr virus (EBV) drives the pathogenesis of these T/NK cell lymphoproliferative diseases. We will concentrate on four main aspects of EBV infection of T/NK cells. First, we will examine the viral and microenvironmental factors that enable the establishment of infection. We established a model of T cell differentiation and exploited the transient expression of CD21 in the T/NK cell precursors to infect these cells with EBV. We will also explore EBV entry into mature T/NK cells via an immunological synapse formed between HLA-matched, EBV-specific T-cells and plasma cells undergoing productive viral replication. Next, using these new infection models and recombinant virus technology, we will determine how EBV drives the replication and clonal outgrowth of T/NK cells and the chronic inflammatory environment characteristic of these diseases. We will then determine how EBV drives the transformation of infected T/NK cells and contributes to immune-inhibition. The viral oncogene LMP1 appears to play a major role in both transformation and immune inhibition and we will examine how. Finally, using a new flow cytometry-based assay to screen cells directly from patient blood, we will examine the EBV-positive cells for expression of potential new therapeutic targets.

EBV is associated with a number of rare lymphoproliferations and malignancies of T cells and NK cells, all of which have an extremely poor prognosis. Two of the major obstacles to our understanding of how EBV causes these diseases are the lack of any system to reliably infect the T cells or NK cells with EBV and the lack of a reliable assay to examine the EBV-positive malignant cells ex-vivo.
We have recently developed a system of infecting primary T/NK cells with EBV, which is critical for our understanding of how EBV drives these diseases. Using this system we can begin to address the fundamental questions including: (1) Identification of the viral glycoproteins essential for EBV entry into T/NK cells. There is increasing academic interest in the development a prophylactic vaccine against EBV. Thus identification of differences in viral glycoproprotein usage between the B cells, epithelial cells and T/NK cells will be critical to the success of any vaccination programme. (2) Identification of the roles played by the virus and the microenvironment in driving EBV-associated T/NK cell diseases. We have no idea of how EBV initiates and drives these diseases or indeed how EBV contributes to the resistance to conventional chemotherapeutics, but this is essential if we are to identify novel therapeutic targets. We have also established an RNA-Flow assay which identifies the EBV infected (pre) malignant cells and analyses the cells for expression of multiple cell-surface and intracellular markers in a flow-cytometry-based format.
Biotechnology companies and academic groups involved in generating anti-herpesvirus vaccines. This study will highlight the virus proteins essential for transmission and indicate whether, during cell-cell transmission the virus can be accessible to the neutralising antibodies.
Scientists working on EBV infection of T/NK cells. Nothing is known of how EBV enters T/NK cells; equally little is known of the pathogenic mechanisms by which EBV causes disease in these cells. The only models we have to examine EBV in these cells are cell lines, which have adapted and changed over time in culture. Reliable infection of primary T/NK cells will aid our understanding not only of viral entry into these cell types, but also how the microenvironment is involved in perpetuating chronic viral diseases.
Clinicians and Biotech. The advent of therapeutic monoclonal antibodies such as rituximab has pioneered the role of such therapeutics for blood-borne cancers. With this in mind, the RNA-Flow assays we have established to examine the presence of EBV in the lymphoid subsets of patient blood enables us to perform complex immune-phenotyping of the EBV-infected cells. Importantly, several clinical trials using the therapeutic anti-CCR4 monoclonal antibodies (Mogamulizumab) are being performed on patients with peripheral T cell lymphoma. Our assays will enable an initial screening of patient blood for CCR-4 and others before the application of targeted chemotherapy with such drugs. Furthermore, given these assays use very little blood, we have been able to isolate and culture the EBV-positive patient cells, giving us scope to determine drug efficacy directly on the malignant patient cells. This is a major step forward in our efforts towards development of novel personalized therapeutic strategies.
Nationally and Internationally. Given the potential interest by biotechnology companies in development of new personalised treatments, and in vaccine development, this study could be an advantage economically to the UK. Diagnosis of EBV-associated disease in non-B cells is devastating, not least because the prognosis is invariably poor. Therefore, this research will benefit patients with EBV-associated T/NK cell cancers both within and outside the UK. This is especially important given the worldwide incidence of haemophagocytic lymphohistiocytosis, chronic active EBV, extranodal NK/T cell lymphoma and aggressive NK leukaemia.