We propose continued investigations on the molecular mechanisms by which antibodies to IgM suppress bacterial lipopolysaccharide (LPS)-driven differentiation of mouse B lymphocytes to immunoglobulin secreting cells. In this experimental system suppression is not immunologically specific but is selective for differentiation-associated gene products. Either anti-mu or anti- kappa antibodies inhibit transcription of mRNA for both mu and kappa chains and for the immunoglobulin J chain, while mRNA for the A beta chain of Ia is not suppressed. Suppression can be induced by brief exposure to anti-mu antibodies and is not reversible in culture. Inhibitors of RNA or protein synthesis, if present during the pulse with anti-mu, abrogate suppression. These data suggest that suppression may be mediated by a trans- acting protein(s) which inhibits transcription of several differentiation-related genes. Membrane and metabolic requirements for suppression induction will be studied in greater detail using inhibitory drugs and insoluble anti-mu and anti-delta. Effects of anti-mu and anti- delta on proliferation and differentiation induced by 8- mercaptoguanosine will be studied. We propose to construct suppressed phenotype (SP) hybridomas by fusing a HAT sensitive B lymphoma M12.4, clone 4.5 to anti-mu treated cells. A preliminary experiment has yielded exclusively non- immunoglobulin secreting hybridomas from such a fusion, while a parallel fusion with LPS-stimulated cells generated immunoglobulin secreting hybridomas. To test SP hybridomas for stable production of the putative repressor protein, we will introduce selectable markers in order to use them as fusion partners. By fusing a """"""""suppressor"""""""" hybridoma to normal cells and to several immunoglobulin-secreting cell lines or hybridomas we will study the specificity of suppression. Supernatants and lysates from suppressor hybridomas will be evaluated for suppressor activity in LPS-stimulated cultures. If a bioassay for soluble suppressor factor can be developed we will undertake purification and characterization of the molecule. This model of anti-mu induced suppression of B cell differentiation appears to be analogous to the type of B cell tolerance which is called clonal energy. This research is also relevant to the general question in cell biology of how receptor- ligand interactions at the cell surface influence gene expression.
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| Lawton, A R; Summerlin, V S; Dooley, J S (1990) Hybridomas derived from lipopolysaccharide-activated anti-mu-treated B cells have active suppression of IgM secretion, demonstrable by secondary fusion. J Immunol 145:3177-80 |
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| Rollins-Smith, L A; Lawton, A R (1988) Regulation of B-cell differentiation: anti-mu antibodies have opposite effects on differentiation stimulated by bacterial lipopolysaccharide and 8-mercaptoguanosine. J Mol Cell Immunol 4:9-19 |
