The immortalisation of B-lymphocytes with Epstein-Barr virus

Flanagan, James Michael. (2002). The immortalisation of B-lymphocytes with Epstein-Barr virus PhD Thesis, School of Population Health, The University of Queensland.

       
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Author Flanagan, James Michael.
Thesis Title The immortalisation of B-lymphocytes with Epstein-Barr virus
School, Centre or Institute School of Population Health
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Tom Sculley
Norbert Kienzle
Kenia Krauer
Total pages 226
Language eng
Subjects 11 Medical and Health Sciences
Formatted abstract

Epstein-Barr virus (EBV) is a herpes virus with a DNA genome of about 172 kbp. At least 90% of the world's population is infected with EBV and it is the causative agent of infectious mononucleosis and has also been associated with a number of tumours including Burkitt's lymphoma (from which the virus was originally isolated), nasopharyngeal carcinoma, Hodgkin's disease, post-transplant lymphoproliferative disease and immunoblastic B cell lymphomas. Infection of B-lymphocytes with EBV, in vitro, leads to persistent latent infection, expression of a restricted set of latently expressed viral genes and continuous cell proliferation. This continuous cell proliferation is believed to be the major predisposing factor in the development of EBV associated tumours. Although the latent EBV genes have been shown to have functions in cellular growth and in cell cycle progression, their specific roles in immortalisation have not yet been fully characterised. Experiments involving mini- EBV genomes have shown that there is no contribution from the viral particle towards the immortalisation process, and that only naked DNA is required. It is also proposed that only five of the latent genes (EBNA 1,2,3,6 and LMP 1) are essential for the immortalisation process.

 

The first aim of these studies thesis was to construct two plasmids expressing the EBV latent genes essential for immortalisation with the purpose of transfecting and immortalising primary B-lymphocytes. Each of the latent genes essential for immortalisation were cloned into two episomal vectors designed to be co-dependant on each other within the mammalian cell system. The results presented show that expression of each of the latent genes could be demonstrated in an EBV negative BL cell line DG75. These two plasmids were then transfected into primary B-lymphocytes with the aim of transforming the B-lymphocytes. However, primary B-lymphocytes transfected with these plasmids were not transformed. The process of transformation is very complex and the most likely reason why these plasmids could not transform B-lymphocytes could be due to the timing and regulation of the expression of each of the latent genes.

 

The remainder of this work was aimed at further characterising the cytostatic effects of LMP 1 and the role of LMP 1 in transformed B-lymphocytes and EBV associated tumours. The first experimental approach to investigate the cytostatic effects of LMP 1 was to express EBNA 2 in the EBV-positive Burkitt's lymphoma cell line Mutu I resulting in up-regulation of LMP 1 expression in these cells. Stable EBNA 2 expressing Mutu I cell lines were established by transfecting an episomal vector containing the EBNA 2 gene under the cadmium chloride inducible metallothionein promoter. The expression of LMP 1 was monitored by RT-PCR and immunoblotting and was found to be up-regulated after 12 hours of EBNA 2 expression. The results demonstrate that after 72 hours of EBNA 2 expression, LMP 1 was expressed at levels higher than in the group III cell line Mutu III, however it did not induce cytostasis as expected. These results suggested that expression of EBNA 2 in the Mutu I cell line induced a shift to a group III phenotype and that overexpression of LMP 1 did not induce cytostasis due to expression of other EBV latent genes.

 

The second experimental approach to investigate the cytostatic effects of the LMP 1 protein used transient transfection with LMP 1 expression plasmids and also analysis of the proliferation of cells when cultured with an LMP 1 derived peptide. Transfection of the Burkitt's lymphoma cell line DG75 with LMP 1 expression plasmids, showed that the EBNA 3 family of proteins prolonged the expression of LMP 1 suggesting they may be involved in overcoming the LMP 1 induced cell cycle arrest. The LMP 1 peptide, Ac-LALLFWL, derived from a membranespanning region known to be involved in cytostasis, was used to show that it could inhibit proliferation of primary T- and B-lymphocytes as well as EBV negative but not EBV positive cell lines. This peptide was shown to cause cytostasis by blocking cells in the Gi phase of the cell cycle. The G1 phase cell cycle arrest, however, was not overcome by the EBNA 3 gene family suggesting that other EBV proteins may have been required to alleviate this effect. These results showed that LMP 1 may utilise more than one mechanism to inhibit proliferation.

 

Finally immunofluorescence confocal microscopy as well as immuno-electron microscopy was used to determine the subcellular localisation of LMP 1 within the cell. Using these methods it was shown that LMP 1 is a membrane protein that not only associates with the plasma membrane but also to an intracellular compartment and that it may co-localise with MHC class II molecules in their endosomal compartment. The EM data has also shown the existence of LMP 1 positive extracellular vesicles known as exosomes. Exosomes are thought to be involved in the immune response and LMP 1 has been shown, in this thesis and by others, to inhibit the proliferation of T- lymphocytes, therefore the LMP 1 positive exosomes could be a means by which LMP 1 could be secreted and inhibit the immune response.

 

Overall these studies have revealed that B-lymphocyte immortalisation by EBV is a complex process and that transfection of primary B-lymphocytes with plasmids expressing the latent genes EBNAs 1, 2, 3, 4 and 6 as well as LMP 1 was not sufficient to transform these cells. Secondly this study has shown that LMP 1 can inhibit the proliferation of EBV negative T- and B-lymphocytes and that LMP 1 is secreted in small extracellular vesicles called exosomes. This thesis proposes a novel role for LMP 1 in immune evasion by being secreted in exosomes and inhibiting the proliferation of infiltrating lymphocytes surrounding EBV positive, LMP 1 expressing tumour cells.

Keyword B cells
Epstein-Barr virus

 
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