The mechanism of B cell differentiation and the development of the immune repertoire will be studied under defined conditions using an in vitro fetal organ culture system and long term lymphocyte cultures. B cell development and diversification in culture will be assessed by a number of criteria including 1) selection of VH genes by in situ hybridization using radiolabeled VH gene family probes, 2) frequency of hapten-responsive B cells to a number of haptens using the splenic focus assay, 3) acquisition of B cell surface markers, 4) development of predominant clonotypes by idiotypic analysis and VH gene selection, 4) degree of clonal expansion by measuring increased frequencies of already existing clonotypes, and 5) degree of diversification by examining the rate of appearance of clonotypes normally acquired late in ontogeny. Since these experiments are performed in the absence of circulation and cell migration, newly developing B cells that are uninfluenced by their environment are examined providing a unique opportunity to analyze genetic vs environmental factors affecting repertoire expression. A number of factors will be tested for their effect on B cell differentation and diversification including growth stimuli, anti-idiotypes, anti-light chain, and anti-pre B cell marker reagents. Moreover, using the criteria above, the metabolic requirements of diversification will be assessed by adding various metabolic inhibitors to the cultures. In addition, the importance of age and compartmentalization on B cell development will be further analyzed by comparing the B cell precursor pools from fetal liver, fetal spleen, and adult bone marrow. Finally, the selection of VH genes in the expressed vs available repertoire will be compared and the importance of chromosomal location of VH gene families in relation to their use in the functional repertoire evaluated.
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