One of the most important events .in the development of an immune response is the production of secreted antibodies. The cell biology of this process is well understood in the sense that the antigen-specific B cells are induced to become antibody secreting or plasma cells, usually as a result of T cell 'help'. What is not well understood at all is the role of regulator genes internal to the B cell and what changes are taking place in the overall genetic program. During the previous granting period we used subtractive cDNA cloning to isolate a zinc-finger containing gene, BLIMP-1, which appears to be an excellent candidate to play a major role in this critical segment of B cell maturation. BLIMP-1 is B cell specific and turned on very early in a cytokine inducible B--->plasma cell model (3cl-l). Most importantly, when this gene is transfected into B cell stage lines, either stably or transiently, it triggers many of the hallmarks of plasma-cell induction such as antibody secretion, J chain synthesis and the upregulation of Syndecan-1. We have recently isolated and characterized genomic clones containing the BLIMP-1 gene and gene-targeted disruptions (knock-outs) in embryonal stem cell lines. We propose to continue this work in order to derive a line of mice deficient in BLIMP-1. We will also use the new RAG-2-/- blastocyte injection system to make chimeric mice having B cells lacking BLIMP-1. This will enable us to test whether this gene is crucial for B cell maturation to antibody secreting cells or whether it is one of several pathways. In parallel to this will be antisense oligonucleotide experiments to try and disrupt normal BLIMP-1 expression during T cell help of antigen-specific B cells, using an in vitro system of transgenic cognate B and T cells also developed during this last granting period in collaboration with Dr. Goodnow. We will also develop monoclonal antibodies to BLIMP-1 in order to investigate the exact timing and kinetics of protein expression as well as to assess what post-transcriptional modifications may occur in different in vitro and in vivo circumstances. In addition, we will investigate the control of BLIMP-1 gene expression using standard methods and portions of the genomic clone in order to determine what factors are controlling its induction. In summary, we believe the above studies of the BLIMP-1 gene and its protein product may yield significant new insights into how an antigen-specific B cell matures into an antibody secreting, plasma cell.
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