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

Lead Research Organisation: University of Birmingham
Department Name: Cancer Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Cancer Research UK Career Development Fund
Amount £20,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 01/2019
 
Description Nottingham University Hospital Charity
Amount £12,154 (GBP)
Funding ID 1950/03/17 
Organisation Nottingham University Hospitals NHS Trust 
Department Nottingham University Hospitals Charity
Sector Charity/Non Profit
Country United Kingdom
Start 03/2017 
End 03/2018
 
Description Project grant
Amount £386,241 (GBP)
Funding ID MR/N023781/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 11/2016 
End 10/2019
 
Description Understanding the role of Epstein Barr virus in Haemophagocytic lymphohistiocytosis
Amount $50,000 (USD)
Organisation Histiocytosis Association, Inc. 
Sector Private
Country United States
Start 04/2019 
End 03/2020
 
Description Understanding the role of Epstein Barr virus in T/NK cell malignancies
Amount £22,000 (GBP)
Organisation Gregor Mckay Fund 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2018 
End 05/2021
 
Description Wellcome Trust ISSF Critical Data Award
Amount £12,500 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2017 
End 11/2018
 
Title PrimeFlowRNA 
Description Fluorescence in-situ hybridisation for viral mRNA transcripts in combination with cell surface staining. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The PrimeFlow RNA has enabled us to identify the lymphocyte subset infected by EBV in a very small number of PBMC directly from patients suffering from EBV-associated T cell and NK cell lymphoproliferations or malignancies. Because we are using very small cell numbers, we can perform experiments we have not previously been able to perform, including: Isolation and culture of cells containing EBV and perform drug sensitivity assays using novel drugs. Immunophenotype the EBV-infected cells and the non-infected cells to determine the difference between the two. Perform RNAseq on EBV-infected vs non-infected T cells or NK cells to determine how EBV changes the cellular gene expression profile. Determine why the immune system does not recognise EBV infected cells. Viral gene expression profile. This assay has really opened up the field of research to enable us to determine exactly how EBV is causing these diseases and if we can identify potential new treatments. 
URL http://eu.ebioscience.com/knowledge-center/application/flowrna/primeflow-rna-assay-principle.htm
 
Description Development of novel antibody drug conjugates to treat Epstein Barr virus (EBV)-associated lymphoproliferative diseases and malignancies. 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have identified expression of CD38 on two cell types we believe are driving EBV-associated lymphoproliferative diseases; the EBV-infected T cells or NK cells plus the myeloid-derived suppressor cells. We have shown CD38 antibody cannot kill the EBV-infected cells by cross-linking the antibody and have shown the patient NK cells may not be functional so may not be able to kill the EBV-infected cells by ADCC. Therefore we have teamed up with Dr Miraz Rahman to develop an anti-CD38 antibody-drug conjugate (ADC). Once this has been achieved, we will evaluate the performance of the drug on both cell types, firstly cell lines then primary cells directly from the patient blood.
Collaborator Contribution Dr Rahman will be developing the ADCs. His team will be performing the chemistry to link two highly toxic chemicals to the anti-CD38 antibody.
Impact No current outcomes. Disciplines are virology, immunology and medicinal chemistry.
Start Year 2018
 
Description Identification of virally-mediated inhibition of apoptosis 
Organisation The Walter and Eliza Hall Institute of Medical Research (WEHI)
Country Australia 
Sector Academic/University 
PI Contribution My research team have established cell lines from patients with CAEBV and ENKTL. We have used these and previously established cell lines to show that the current treatment for ENKTL and ANKL is completely ineffective against the cell lines. The currently used treatments usually stimulate apoptosis, including the DNA damaging agents such as etoposide. We have begun to determine how EBV contributes to the resistance to the current treatments and have demonstrated which viral genes are responsible for the resistance to apoptosis. To further these studies, our PhD student took the cell lines to our collaborators in the WEHI to set-up an in vivo mouse model.
Collaborator Contribution In collaboration with Dr Gemma Kelly and Professor Andreas Strasser, we have used the EBV-associated Extranodal NK/T cell lymphoma cell lines to identify a new class of drug that can successfully kill the cell lines in vivo. Work is currently being performed in their lab to establish a good mouse model whereby we can treat the mice with ENKTL and CAEBV with these new drugs. We have also performed a large drug screen to determine if the cell lines are sensitive to any other classes of drugs currently being trialed or used in clinical practice. This has identified the JAK-STAT inhibitors as potential candidates. Work is continuing to finish two papers.
Impact (1) Identification of a novel class of drugs with in vivo efficacy. (2) Identification of other classes of drugs with apparent in vitro efficacy. Work is being finished which should lead to two papers.
Start Year 2014
 
Description Understanding the role of Epstein Barr virus (EBV) in Haemophagocytic Lymphohistiocytosis (HLH). 
Organisation Royal Children's Hospital Melbourne
Country Australia 
Sector Hospitals 
PI Contribution The contributions we have made are two-fold: (1) We have developed a rapid flow-cytometry based in situ hybridisation assay to identify lymphocyte subsets infected with EBV directly on small samples of blood. We are aiming for this assay to be developed as a clinical diagnostic assay to rapidly diagnose EBV-associated HLH, to monitor patients during treatment and to examine patient outcome following treatment. (2) We have begun to analyse how EBV drives the differentiation of myeloid-derived suppressor cells.
Collaborator Contribution Our collaborator, Dr Sharon Choo, immunology consultant, looks after EBV-associated HLH patients. They have tried to use a previously published DAKO assay to diagnose their patients but this has proven to be unreliable and s very subjective. Our collaborators are coming to my lab to learn the technique to establish it in their hospital. They will collect blood samples and perform the assay. They will also send us the blood samples to confirm and validate their findings.
Impact We have only just received the grant so there are currently no outputs. The disciplines are virology and immunology.
Start Year 2018
 
Description Understanding the role of Myeloid-derived suppressor cells in EBV-associated T/NK cell lymphoproliferative diseases. 
Organisation University Duisburg-Essen
Country Germany 
Sector Academic/University 
PI Contribution This collaboration started in August 2019. We recently sent a paper for publication detailing the vast expansion of MDSC in the blood of patients with EBV-associated T/NK cell lymphoproliferative diseases. In this we showed the ex-vivo MDSCs effectively inhibited the growth of T cells and despite the presence of EBV-specific cytotoxic T cells, the virus-infected cells were able to persist as the MDSCs inhibited their function. We have gone on to examine how the EBV-infected cells are able to activate the neutrophils, drive their expansion and give them immune-suppressive functions.
Collaborator Contribution Prof Sven Brandau is a world expert in the field of neutrophils and myeloid-derived suppressor cells. Prof Sven Brandau supported my postdoc, Dr Paul Collins, in his lab to learn how to isolate and stimulate neutrophils and to learn how to do T cell suppression assays. Neutrophils are very difficult to work with because they are notoriously short-lived and very easy to non-specifically activate. His help and collaboration will aid the identification of how EBV drives the expansion of MDSCs in the blood of the patient groups we are studying.
Impact No outcomes have yet been achieved. This includes immunology and virology.
Start Year 2019
 
Description CR-UK lab tour 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 25 CR-UK fundraisers from TK Max attended my lab for an afternoon of talks and lab tours. The participants were particularly interested in the EBV-associated T/NK cell diseases and the gastric organoid work.
Year(s) Of Engagement Activity 2018
 
Description Participation in CR-UK lab tours 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Supporters
Results and Impact We hosted a lab tour for Cancer Research UK fundraisers. We hosted 3 research stations so the attendees could see what we do, look at 3 different techniques and have a go themselves. As usual, there was lots of discussion and everyone reported it was informative, interesting and helpful.
Year(s) Of Engagement Activity 2019