Studies in our laboratory have focused on defining the nature of the B cell defects in the disease, Common Variable Immunodeficiency (CVI), a primary acquired human immunodeficiency state characterized by hypogammaglobulinemia (low levels of immunoglobulin in the serum), and impaired functional antibody response. Our previous studies of purified B cells from CVI patients show that although the cells have a normal capacity to proliferate and on the mRNA level, express equivalent amounts of C&#61549;, C&#61543;, and C&#61537;1 mRNA message in comparison to normal controls, they manifest differentiation defects at multiple levels beyond immunoglobulin transcript expression. For example, when compared to normal B cells, circulating CVI B cells contain reduced numbers of surface bearing IgG and IgA cells with a commensurate increase in surface bearing IgM B cells, suggesting an in vivo defect in isotype switch. In addition, these cells fail to undergo differentiation into immunoglobulin producing cells. During this period we have initiated the study of CVI B cells with respect to their expression of transcription factors and cofactors known to be necessary for B cell switching and terminal differentiation. In particular, we have focused on the protein Bob-1, a co-factor that enhances Ig promoter activity through its association with the octamer-binding proteins, Oct-2 and Oct-1. It was previously reported that mice lacking the Bob- 1 protein as a result of gene targeting have a phenotype similar to that of patients with CVI. For example, mice lacking the Bob- 1 gene in vivo are unable to produce Bob-1 protein and manifest defects in B cell differentiation following antigenic challenge but still exhibit early immunoglobulin gene expression and germ- line switching in comparison to controls. It is the similarity to the CVI disease state that focused our attention on the possible role of Bob-1 in CVI. In initial studies, we prepared RNA from purified B cells of patients and controls following stimulation with SAC, IL-2, and IL-10 and amplified the RNA by RT-PCR using primers specific for the Bob-1 gene. CVI patient and control RNA gave rise to identical signals indicating no gross abnormality in the expression of Bob-1 RNA. We then evaluated the ability of CVI cDNA to be translated into Bob-1 protein and found via Western blotting that both patient and control cDNA yielded a protein band of appropriate size and identical intensity. Finally, using protein translated from patient and control cDNA, we tested the ability of patient and control Bob-1 protein to bind to Oct-2 from the nuclear extracts of stimulated B cells from patients and controls. We found using a supershift assay that Bob-1 from both patients and controls were able to retard the migration of Oct-2 in an equivalent manner, indicating that Bob- 1 from CVI B cells are able to bind normally to Oct-2. Presently, we are constructing a Bob-1 fusion protein from CVI and control cDNA in order to assay the ability of Bob-1 to be phosphorylated at certain amino acid residues. It has been previously published that when certain amino acids which undergo phosphorylation are mutated in the Bob-1 protein, the ability of Bob-1 to enhance gene transcription is greatly reduced. We postulate that although CVI cDNA is able to produce Bob-1 protein, certain regions of the protein which are essential for enhancing gene transcription may be defective and through the use of an in vitro kinase assay, we will determine if CVI Bob-1 is able to phosphorylate the regions necessary for the mediation of gene transcription.