The longterm goal of this project is to elucidate the mechanism of B cell development that leads to a diverse immune repertoire. A major emphasis of this proposal is to further delineate changes in the B cell repertoire during fetal, adult, and late stages of life and how such changes are regulated. At issue is what aspects of the evolving repertoire can be explained by genetic factors, selective factors, or lineage relationships. The fetal Ig repertoire is highly restricted in terms of both V exon usage and junctional diversity. At 15 days of gestation only a handful of VH and VK exons are used repeatedly. Making use of this remarkable restriction, experiments are planned to test dominant V exons for potential genetic advantages compared with V exons that are used randomly or are underutilized. Recombination substrates will be used to measure strength of recombination signal sequences and coding end disadvantages. With the kappa locus, fetal B cells are even restricted in terms of particular VK- JK combinations, some of which recombine by inversion. Recombination substrates will be used to compare the efficiency of inversion using VK exons that are over- or under-utilized as well as to compare inversion vs deletion mechanisms. Because of the extent of restriction in V exon usage, it will be possible to examine the effects of selection on repertoire development. Two models are planned. B cells from bcl-2-Ig transgenic mice appear to be defective in apoptosis as a result of deregulated expression of bcl-2 and exhibit repertoire skewing. This will serve as a model for B cell expansion in the absence of negative selection. Bcl-2 mice with xid background will also be tested to clarify lineage relationships. Particular VK and VH exons will be followed in these mice at several stages of development. The motheaten mouse model will be used since it is likely that the defect is associated with a lack of effective CD40 ligand- CD40 interactions. Therefore, motheaten mice will be used as a prototype for lack of T-dependent positive selection leading to follicular lymphocytes. These experiments should provide important new insights into the regulation of the B cell repertoire. Understanding the generation of diversity is fundamental to questions of how the immune response works and what goes wrong in disease states such as autoimmunity.
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