We have been studying the in vitro differentiation of human peripheral blood B lymphocytes (B-cell) into immunoglobulin-secreting cells. Current model of B-cell differentiation is: (1) antigens and B cell stimulating factor (BSF) activate resting B cells to B-blasts; (2) B-blasts develop receptors for growth factors (BCGF and interleukin 2) that support their proliferation; (3) B-blasts also develop receptors for other lymphokines (e.g. interferon-Delta and maturation factors, etc.) that induce Ig secretion. To understand B-cell activation and differentiation, it is necessary to characterize biochemically the structure of BCGF, mode of their action, and the nature of BCGF receptors. We plan to attack these questions (1) by purifying BCGF to homogeneity; (2) by cloning complementary DNA for BCGF; (3) by characterizing their immunological function; (4) by characterizing biochemically receptors for BCGF. In the last two years, we have also developed a simple and reproducible method for quantifying BCGF activities. This assay method is particularly well suited for purifying BCGF. To purify BCGF, we will use high and fast performance liquid chromatography in addition to conventional methods designed for protein purification. To clone cDNA for BCGF, we will use a highly efficient mammalian-cell expression screening system that has been recently used for identifying the cDNA for human granulocyte colony stimulating factors. We have also developed several lines of murine monoclonal hybridomas that inhibit BCGF activity specifically. This monocloanl antibody will be used to isolate and characterize BCGF receptors. The availability of purified BCGF produced by a mammalian cell line will allow detailed studies on the molecular mechanism of action in B-cell differentiation and on the pathophysiology of immunodeficiency diseases caused by aberration of B-cell function. Furthermore, purified BCGF will have tremendous potential as a therapeutic reagent.
