We are studying the immunoglobulin isotype switch induced by interleukin-4 (IL-4) in human and murine B lymphocytes. In the human system we are focussing on the switch to IgE (epsilon heavy chain), the isotype of allergy. Our earlier evidence for a sequential mu-gamma-epsilon switch in a cloned cell line prompted us to explore this phenomenon in normal lymphocytes switching in culture under the influence of IL-4. From such cells we have cloned nine composite switch regions reflecting mu-gamma- epsilon sequential switch and have determined that all of these involve the switch region of gamma. This determination required the complete sequences of the switch regions from all four of the human gamma genes, which we have not obtained. These studies of composite switch regions on the chromosomal DNA are being complemented by analysis of the reciprocal products amplified from circular DNA. In the murine system we are collaborating on several studies of IL-4 induced switching to IgG1. We have employed our Digestion-Circularization-Polymerase-Chain- Reaction(DCPCR) assay to show that the percent of cytokine-induced cultured splenocytes expressing surface IgG1 can be accounted for by the amount of DNA switch recombination detectable in the cultured cells, so non-recombinational switching mechanisms (e.g. trans-splicing) need not be postulated. DCPCR was also used to show that when splenocytes are cultured with IL-4 along with anti-delta-dextran (as a stimulus for proliferation), addition of IL-5 is required for significant DNA switch recombination. To explore the mechanism of the switch event we have initiated studies on the role of the B cell-specific protein BSAP, encoded by the Pax-5 gene, which binds to several cultured murine splenocytes with a BSAP antisense oligonucleotide that reduces the detectable BSAP protein causes a decrease in cell proliferation and in IgG1 surface expression, suggesting an important role of BSAP in these cells. With our UCLA collaborators we have been studying human immunoglobulin epsilon mRNA isoforms resulting from alternative splicing. We now have evidence for nine different isoforms, encoding six distinct predicted epsilon proteins. We are exploring which of these proteins exist in significant amounts and how they may differ in parameters of binding to the Fc-epsilon receptor.
