Studies on Peyer's patch T cell hybridomas
The initial objective of this thesis was to generate Peyer's patch T cell hybridomas producing lymphokines that regulate IgA-secreting B lymphocytes. Unprimed Peyer's patch cells were fused with BW5147. Karyotype analysis and fluorescent staining of the thy-1.2 marker confirmed the generation of hybridomas. It was envisaged that these hybridomas would be tested for their effects on IgA production by LPS-stimulated B cells. However, when the panel of hybridomas was available for testing there were technical difficulties with this assay. Sendai virus-primed Peyer's patch T cells were used in a subsequent fusion, which was screened using an antigen-specific in vitro helper assay. A number of hybridomas stimulated the production of anti-Sendai antibodies by primed B cells. The same hybridomas secreted IL-2 on stimulation by syngeneic spleen cells in the absence of added virus. Recombinant IL-2 replaced the hybridomas in stimulating primed B cells in the helper assay. These studies showed that several of the hybridomas had apparent auto-reactivity. This was not due to viral contamination of the animal stocks since spleen cells from isolator-reared syngeneic mice gave similar results. The genes responsible for stimulating the hybridomas were mapped to the I-region of the MHC. It was important to elucidate whether these T cells were truly auto-reactive or whether they were in fact in vitro artefacts. Hybridomas adapted to grow in serum free medium and subsequently tested for their response to syngeneic cells in the abscence of serum, did not produce IL-2. The addition of foetal calf serum restored the response. The component of foetal calf serum which is necessary for the stimulation of the hybridomas has been partially purified. It can be separated from the main serum protein components by HPLC on a DEAE ion exchange column. It is eluted by high salt which suggests that it is highly acidic or is bound strongly by hydrophobic interactions. The material is trypsin sensitive. It is labile at 4o C and is unstable to freezing and thawing and this has hampered its further purification. The mode of action of the component has been studied using pulsing experiments and it has been shown to act on the stimulator cells and not on the hybridomas. The data suggests that the hybridomas do not recognise a self-antigen alone, but rather that they recognise a component of the xenogeneic serum as antigen with self-MHC restriction. However it has not been formally excluded that the foetal calf serum component stimulates the expression or processing of an auto-antigen.