This is a proposal to study the regulation of B lymphocyte activation at the molecular level, and particularly to gain insight into control mechanisms governing cell proliferation. A major focus of this study is the identification of the gene product defective in a lipopolysaccharide (LPS) non-responsive mouse line. This inbred of mice, C3H//HeJ, is genetically deficient in LPS- induced polyclonal spleen cell activation. This recessive mutation maps to a single locus (lps) on mouse chromosome 4. It is our long range goal to identify this gene and its product, and ultimately to restore competency for LPS responsiveness in mutant lymphocytes by gene re-introduction. Resting splenic lymphocytes respond to a variety of cell surface stimulation by undergoing general metabolic activation, maturation of immunoglobulin production from a surface receptor to a secreted molecule, and entry into the cell cycle. LPS is a potent polyclonal (antigen-independent) B cell activator, stimulating a high proportion (30%) of splenic B cells. B cell responses induced by LPS have common pathways of metabolic and cellular responses exhibited by antigen-induced B cell activation, and these events are currently poorly characterized at the molecular level. We have identified immunochemically a molecule (p-lps) which is unique to LPS-responsive B cells, and absent in LPS non- responsive B cells or spleen cells from C3H/HeJ mice. We have generated monoclonal antibodies (MoAb) which recognize this molecule, and use these antibodies to successfully screen a spleen cell gtII cDNA library. We propose to rigorously test these cDNA clones for their linkage to the lps allele by several distinct strategies. These anti-p-lps MoAb are valuable reagents to use in biochemically characterizing the p-lps molecule. We will determine its pattern of expression in various tissues and lymphocyte subpopulations. As well, we will study potential modification, both post-transcriptionally and post-LPS stimulation. We will investigate the association of p-lps with other molecules, with a particular focus on determining the involvement of p-lps in signal transduction pathways operating in B cell activation. The study of the lps gene and its product will yield insight into the molecular basis by which B cells are activated and lymphocyte proliferation responses induced in the immune response. This contributes to studies on abnormal B cell growth control in autoimmunity or neoplastic disease. This is a model system for an approach of gene complementation to restore inherited immune defects.
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