Forty years of research in immunobiology have provided a sound yet rudimentary understanding of the most fundamental of all events in acquired immunity: clonal selection. Our proposed studies will focus on the cardinal elements that direct clonal development of B cell memory: somatic mutagenesis of antibody V genes, cell proliferation and selection. We will test the hypothesis 1) that somatic mutagenesis of antibody genes and selection for affinity improvements both occur within a specific subset of germinal center B cells that bear surface immunoglobulin, and 2) that positively selected cells enter a proliferation phase in which mutation ceases and immunoglobulin is not expressed. A major goal of our work is to isolate the mutating B cell. Our studies will be performed on B cells isolated ex vivo that are participating in an exceptionally well-defined immune response to the hapten p-azophenylarsonate, and that express a single combination of antibody variable (v) gene segments (VH, D, JH, VK, JH). A strength of the model is that these cells belong to lineages destined for the memory compartment. The feasibility of this proposal derives from recent advances in our laboratory that permit the isolation of single memory lineage cells from immune spleen, the direct sequence analysis and expression of their H and L V genes and V gene transcripts, and the production of single cell cDNA libraries. Germinal centers are sites for memory B cell development for the genesis of several common B cell neoplasias and for recruitment of autoreactive B cells. Results of these studies will advance our limited knowledge of regulatory processes that control mutation and proliferation in B cells by providing basic information at an unprecedented level of resolution. They will also position us to elucidate the mechanism of the only recognized physiologic mutator in the Animal Kingdom.
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