Antibody responses require the specific proliferative expansion of a small number of B cell clones in germinal centers (GC). During the first funding period we found that in addition to somatic mutation, antibody genes can undergo V(D)J recombination in the GC. B cells in GCs were found to express the recombinase activating genes RAG1 and RAG2, and contain V(D)J recombination intermediates. Experiments with gene targeted mice showed that mature B cells that express RAGs can alter the specificity of the antibody they express, and that secondary V(D)J recombination is regulated by signals from the antigen receptor. We and others showed that strong BCR crosslinking inhibits RAG expression whereas antigens that bind weakly to the BCR stimulate further recombination. Based on these experiments several functions were suggested for V(D)J recombination in GCs including: 1. editing of self reactive receptors that arise by somatic mutation; 2. rescuing cells that have lost receptors; 3. improving the affinity of low affinity receptors; 4. induction of cell death. But despite potential impact of secondary recombination on the antibody response, the role of secondary V(D)J recombination in GCs has not been determined. The longrange goal of the proposed research is to elucidate the physiologic function of secondary V(D)J recombination in the antibody response. The working hypothesis is that recombination is induced in B cells with low affinity receptors and may contribute to affinity maturation. The first part of the project will make use of a RAG2-green fluorescent protein indicator mouse strain to define which mature B cells are induced to express RAGs in the periphery and how RAG expression is regulated. The second part of the project will be to try to measure the contribution of secondary recombination to antibody responses and editing during immune responses in vivo. The third part of the project will aim at trying to define how RAG1 is regulated in vivo. These studies have significant potential implications for understanding how B cells produce antibody responses and maintain tolerance in vivo.
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