Epstein-Barr virus and human B cell immortalisation : mechanisms and applications
(1997)
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- Epstein-Barr virus (EBC) is a herpes virus that has a tropism for human B lymphocytes and is frequently found in tumours. The endemic form of Burkitt’s lymphoma (BL) is a tumour for which there is much evidence for a causative role of EBV. B lymphocyte infection with EBV in vitro induces the outgrowth of immortal lymphoblastoid cell lines (LCL), which can be exploited as a source of human monoclonal antibodies. <BR> The understanding of molecular interactions between EBV and target cells is a prerequisite for the development of new treatments for EBV-positive tumours. It is well established that, in LCLs, the virus expresses a set of 9 proteins, including the viral nuclear antigens, EBNA-1, -2, -3A, -3B, -3C and the leader protein. Paradoxically, in BL, EBNA-1 is the only viral protein expressed. This difference is due to inactivity of the Cp promoter, responsible for the transcription of EBNAs in LCLs. <BR> the reason for the absence of Cp activity in BL is unknown. As it had been suggested tha the activity of the Cp promoter could be moiulated by glucocorticoids, we compared the response to glucocorticoids in BL and in LCLs. We found that glucocorticoid-dependent promoters had a lower activity in cell lines derived from BL than in LCLs and that the glucocorticoid receptor was present in BL cells at a much lower level than in LCLs. Transfection of an expression vector for the glucocorticoid receptor into BL cell lines resulted in outgrowth of cells which had activated EBNA-2 expression, suggesting that GR mau indeed play an important role in Cp activation. <BR> However, in other experiments, we were not able to demonstrate a clear role for the glucocorticoid receptor in the regulation of the Cp promoter. Introduction of an exogenous EBV into BL cells allowed transcription from the Cp promoter of the exogenous virus, suggested that in BL, Cp activity was prevented by methylation. The role of glucocorticoids during infection of mature B lymphocytes was also investigated. Infection of peripheral blood infections of mature B lymphocytes was also investigated. Infection of peripheral blood lymphocytes with EBV in the presence of the glucocorticoid antagonist RU486 made no detectable difference to the efficiency of EBV immortalisation and the cell lines that grew out used Cp as normal. It was therefore concluded that the glucocorticoid receptor is not a key determinant of Cp activity during primary infection. <BR> The ability of EBV to immortalise human B lymphocytes in vitro can be exploited to generate cell lines producing human monoclonal antibodies. We used that strategy to analyse at the clonal level the inhibitor antibodies against factor VIII (FVIII) in haemophilia A patients. Cell lines producing high-affinity human IgG monoclonal antibodies were established by immortalising B lymphocytes from haemophilia A patients with inhibitor antibodies. <BR> We used one monoclonal antibody, BO2CAA, to carry-our a detailed study of the kinetics and mechanisms of inactivation of FVIII. The epitope recognised by this antibody overlapped the factor VIII phospholipid-binding site and residues in volved in von Willebrand factor (vWF) binding on the C2 domain. Accordingly, BO2CAA prevented FVIII interactions with both phospholipids ad vWF. Strikingly, FVIII complexed to vXF was protected from inactivation by BO2CAA but this protection was reduced with time. Comparison of the association and dissociation constants of the vWF- and BOC2C11-FVIII complex indicates that BO2C11 slowly but irreversible binds FVIII as the latter dissociates from vWF to which it is bound in plasma. These observations are expected to favour the development of new strategies for the prevention and treatment of the immune response to FVIII. However, the modality of such treatments and their clinical efficacy remain to be determined.
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Jacquemin, M. (1997). Epstein-Barr virus and human B cell immortalisation : mechanisms and applications. https://hdl.handle.net/2078.5/111412