During B cell development, cell surface molecule expression is modulated and the pattern of expression of these molecules can identify different stages of differentiation or activation of B cells. These molecules also provide signals to the B cell for further differentiation or activation. We have been characterizing a monoclonal antibody that recognizes an activation-induced antigen on B and T lymphocytes that is 16-18 kD. This molecule is upregulated to varying levels on small resting B cells with different stimuli. While this antigen can be detected on the surface of a small percentage of resting B cells (5-20%), we have recently shown that it is present in the cytoplasm of all B cells. Upon stimulation with LPS, this molecule is secreted into the tissue culture medium Determination of the N-terminal amino acid sequence shows homology with Calgranulin B, a calcium-binding protein in the S100 family. The function of these proteins is unknown. The raison d~etre of a B cell is to produce antibodies. The mechanism of antibody generation is largely understood in that different gene segments are combined to create an antibody with specificity for a particular antigen. Variable region genes, however, are not expressed equally. We are studying, a small Vk family, Vk10, which is known to be utilized in a number of antigenic responses. One member of this family has not been detected in functional antibodies and is expressed at levels at least 1000 fold lower than the other family members in the spleen. Rearrangements of this gene are detected in both bone marrow and spleen. A comparison of the promoters between an expressed and the non-expressed Vk10 genes shows a significant difference in their transcriptional efficiencies in pre-B cells, the stage of development when light chains begin to rearrange. We conclude that cells rearranging this gene do not express enough light chain protein to be selected, either negatively or positively. Such cells can undergo receptor editing or can be eliminated by apoptosis. The promoters of the expressed and non-expressed Vk10 genes differ by only three nucleotides in potential regulatory sequences. Site directed mutagenesis of each of the sites improves the efficiency of the inefficient promoter. Gel shift experiments show some differences in the proteins binding to two of these sites.
