Abstract

As part of the humoral immune system, the B cell receptor (BCR) is expressed on B lymphocytes and later, after modification, secreted as an antibody. It is synthesized from a rearranged immunoglobulin (Ig) heavy (H)-chain gene and a rearranged kappa or lambda light (L)-chain gene. This thesis investigates B cell development as a function of H- and L chain gene rearrangement in the so called nuclear transfer mouse. At the beginning of this work the exact germline configuration of the Ig lambda locus was unknown even in normal mice. Hence, a physical map of the mouse lambda L chain and related loci was created: The lambda locus was found to stretch over three sections (Vlambda2-VlambdaX; Jlambda2-Clambda2-Jlambda4-Clambda4 and Vlambda1-Jlambda3-Clambda3-Jlambda1-Clambda1), spanning 179,346 bases on chromosome 16. Furthermore, the surrogate L-chain gene VpreB2 was located 1,077,001 bases downstream of the lambda locus; VpreB1 is 2,180,618 bases 3` of the lambda locus and the lambda5 gene is located 4,667 bases downstream of the VpreB1 locus. Even though the diploid B cells have two H-chain alleles and two alleles for each L-chain locus, kappa and lambda, antibodies are only expressed from one H-chain allele and one L-chain allele. This phenomenon is called allelic exclusion. Seemingly contradicting allelic exclusion, a distinctive feature of the nuclear transfer mouse is the Ig gene configuration with one H and two kappa gene rearrangements. In this work, it was determined that both kappa alleles of the mouse are productive, i.e., in-frame. For a functional analysis, mice with the H chain transgene in combination with one or both kappa chain genes were generated, some of them on a RAG deficient background. In the absence of RAG, endogenous gene rearrangement is not possible, nor is editing of preformed Ig transgenes. In the absence of RAG, the H chain combined with either kappa chain led to a functional BCR on mature B cells. The antibodies containing either kappa chain were also detected in the serum of mice, and one of the two HxL combinations was found to be self-reactive. In general, B cells destroy receptor autoreactivity by editing, i.e., by replacing the self-reactive L-chain allele while preserving allelic exclusion. In RAG sufficient nuclear transfer mice with the autoreactive BCR, however, editing failed to destroy self-reactivity. Instead, a second kappa allele was recombined, in addition to the pre-existing one. Breaking allelic exclusion, the surviving B cells recreated dual receptor expression, as presumably was the case in the original B cell that gave rise to the nuclear transfer mouse. These results indicate that receptor editing does not necessarily destroy the self-reactive allele; and that in normal mice autoimmune antibodies may be fellow travelers in B cells contravening allelic exclusion. RAG mediates recombination and editing in normal mice, but recombination activity is unnecessary in mice with transgenic Ig genes. Hence, expression of RAG in Ig transgenic mice before Ig genes synthesis may give rise to an artifact that just looks like editing. To address this possibility, RAG expression was analyzed in mice with the H and different L chains using the green fluorescent protein (GFP) as a substitute marker. In mice with the non-autoreactive BCR, very few GFP+ B cells were found. However, in Ig wild-type B cells and cells with the autoreactive BCR, GFP was widely expressed. These results suggest that in most cells the transgenic Ig loci are expressed before RAG is synthesized; furthermore, that the autoreactive receptor induces BCR editing by re-expressing RAG.