Comprehensive immunity essential for protection from invading pathogens and the surveillance of cancerous instigation is dependent on the daily development of B cells within the bone marrow. Discrete stages of proliferation and differentiation of B lymphopoiesis are regulated by the coordinate expression of lineage and stage specific genes. Dysregulated expression of these genes can lead to the development of B cell immunodeficiency, autoimmunity or leukemia. Although studies have demonstrated several mechanisms relevant to B cell development, this process still remains incompletely defined. Therefore, my objective is to identify novel gene regulators of B cell development, which would have major implications toward the understanding and treatment of many human diseases. Through the recent advances in genome wide analysis techniques the Immunological Genome Project has detailed the expression pattern of genes in individual B cell developmental stages of mice. Using custom comparisons of these publically available datasets, I have discerned a portfolio of novel genes displaying unique expression patterns during B cell development. Though some of these genes have been defined in models of non-lymphoid development, none have been described in lymphopoiesis. Additionally, through the success of a proof of concept experiment using short hairpin RNAs (shRNAs) to knockdown the expression of specific genes, I have demonstrated a method for determining the biological relevance of individual genes to early B cell proliferation. Adaption of this shRNA method for high-throughput analysis will allow me to determine the functional role of each novel gene association to individual stages of B cell development. I hypothesize that within my list of candidate genes, are genes that represent novel mechanisms of early B cell proliferation and differentiation. By constructing a focused shRNA library I will target each candidate gene to test their involvement with signaling from the IL-7 receptor (IL-7R) and the pre-B cell receptor (BCR), both of which are known to direct early B cell proliferation and differentiation. I will also elucidate non-IL-7R and non-pre-BCR dependent mechanisms of B cell development through an innovative in vivo adoptive transfer study utilizing the targeted shRNA library. Finally, I will confirm the biological relevance and pathogenic potential of candidate genes through experiments designed to over-express genes of interest in an in vivo model. Establishment of novel gene regulators of B lymphopoiesis, particularly the proliferative events of early B cells, will provide substantial insight into the complete mechanisms driving B cell derived pathologies.
Discrete expression of lineage and stage specific genes is critical for the proper regulation of B cell development and the prevention of B cell immunodeficiencies and leukemia. Through innovative experimental systems, I will define the roles of several novel gene associations with the proliferation and differentiation events of B lymphopoiesis. Successful completion of these projects will elucidate new mechanisms of B cell development and possible therapeutic targets for B cell derived pathologies.
