Antibody synthesizing B lymphocytes constitute key mediators of the immune response (Cooper 1987). To date, research on human B lymphocytes has concentrated on topics including the extracellular signals influencing B cell function (Gordon and Guy 1987, Kishimoto and Hirano 1988, O'Garra et al. 1988), the surface molecules expressed on B lymphocytes that are important for B cell activation or for interactions with other effector cells of the immune system (Clark and Lane 1991), or the cells and factors affecting B cell maturation (Kincade et al. 1989). However, intracellular mechanisms regulating B growth and differentiation are not well understood at present. In particular our knowledge of the contribution of individual genes to normal and malignant B cell function is limited, in part due to the lack of a gene transfer and expression system for these cells.
To investigate gene function in normal and malignant B lymphocytes, a gene transfer and expression system for B lymphocytes was developed in the course of this thesis (chapter 2) and a number of expression vectors containing oncogene sequences were designed (chapter 3). Unlike normal B cells, the malignant B lymphocytes of B-CLL represent a homogeneous B cell population of high purity with large B cell numbers available for studies. For these reasons gene transfer experiments were initiated with cell material derived from patients with B-CLL (chapter 4). Although several different oncogene constructs were transfected into B-CLL cells alone and in combination, no continuous growth of B-CLL cells, as initially intended, was observed following gene transfer. One explanation for the failure to transform B-CLL cells with oncogenes could be that the sequences used are simply not able to transform B-CLL cells, or that other, untested genes and combinations are needed to achieve growth transformation of the malignant cells.
One oncogene that would be appropriate to study in the system I have described is bcl-2. The gene product of the bcl-2 proto-oncogene appears to prolong survival of cells by preventing apoptosis, rather than inducing constant proliferation like classical oncogenes (Korsmeyer 1992a). Data derived from a transgenic mouse model using a construct representing the t(14;18) translocation, demonstrated that bcl-2 gene activation is directly
involved in B cell neoplasia (McDonnel et al. 1989). The transgenic mice were initially characterized by an expanded polyclonal resting B cell pool with approximately 97% non cycling B lymphocytes. After a latency period, aggressive monoclonal lymphomas evolved. More than 50% of the tumours had acquired secondary translocations which involved the myc gene and the Ig heavy chain gene locus (McDonnel and Korsmeyer 1991). Recently published data show that in the majority of B-CLL cases high levels of bcl-2 transcripts are present (Mariano et al. 1992). Therefore, in analogy to bcl-2 transgenic mice, high levels of bcl-2 may support the slow and progressive accumulation without obvious proliferation of neoplastic cells observed in vivo. In cultured cells, however, bcl-2 levels may be down-regulated or unstable due to the lack of specific nutrients or growth factors in the medium, leading to the induction of programmed cell death. High and constitutive bcl-2 expression levels following gene transfer may therefore support survival and, in combination with transfected c-myc, growth of B-CLL cells in vitro.
Although transfection of B-CLL cells did not result in growth transformation, a number of distinct effects of transfected c-myc on B-CLL cells were observed. Most prominently and consistently, prolonged survival of c-myc transfected B-CLL cells compared to controls was found following gene transfer (chapter 4) and in addition, phenotypic changes were detected. Subsequent analysis revealed that IFN-y was secreted into the tissue culture medium by cells transfected with c-myc, but not controls, and IFN-y was shown to be a survival promoting cytokine for the CD5+ B lymphocytes of CLL (chapter 5).
IFN-y is an important immune modulator with multiple effects on a variety of cells of the immune system including T cells, monocytes/macrophages and B lymphocytes (Balkwill 1989, Murray 1988, Trichineri and Perussia 1985). At present, there are no reports demonstrating a role for IFN-y in supporting survival of other (non-CD5^) normal and malignant B lymphocytes: IFN-y treatment does not prevent initiation of apoptosis in cultured germinal centre B cells (Liu et al. 1991) or in malignant B lymphocytes of acute lymphoblastic leukaemia (Dr. D. Campana, personal communication). Therefore, IFN-y
may exclusively support the survival of a specific B cell subtype, in particular CD5^ B cells. Experiments with separated B cell subpopulations would allow determination of the effects of IFN-y on normal CD5^ B lymphocytes, and other B cell subtypes.
At present it is unclear whether IFN-y acts directly on B-CLL cells or via induction of other growth factors which would include any of the cytokines known to be synthesized by B-CLL cells such as IL-1 (Morabito et al. 1987), IL-6 (Blondi et al. 1989) and TNF (Cordingley et al. 1988, Digel et al. 1989). It has been reported that IFN-y augments expression of the CD23 antigen on the surface of B-CLL cells (Fournier et al. 1992) and elevated levels of the cleaved, soluble form of CD23 are also found in sera of B-CLL patients (Sarfati et al. 1988). The cleaved form of CD23 together with IL-1 a prevent germinal centre B cells from initiating apoptosis (Liu et al. 1991). The effects of IFN-y on CD23 expression in B-CLL cells and the consequences of CD23 alone or in combination with other growth factors on the inhibition of apoptotic cell death in B-CLL could be studied using a similar approach as detailed in chapter 5.
In normal peripheral blood, IFN-y is predominantly produced by activated T lymphocytes and NK cells (Young and Hardy 1990). That the malignant B lymphocytes of B-CLL contribute to the synthesis of this cytokine may be the reason why elevated levels of IFN-y were detected in sera from B-CLL patients (see chapter 5). The events triggering synthesis of IFN-y in B-CLL are unknown, but as demonstrated with transfection experiments conducted in chapter 4, stimuli activating transcription of the c-myc gene may well be involved in the production of this cytokine by B-CLL cells. Determining the nature of such a stimulus (or stimuli) could be useful for the understanding of the mechanisms involved in the neoplastic process of B-CLL, and may ultimately lead to novel forms of treatment. More immediately, growth factors known to down-regulate IFN-y synthesis by peripheral blood lymphocytes such as IL-4 (Peleman et al. 1989) and IL -10 (Vieira et al. 1991) may diminish B-CLL cell survival, and could be of therapeutic value. These effects could readily be tested in the system I have described.