We are studying the immunoglobulin isotype switch induced by interleukins -4, -10 and -13 in human B lymphocytes, focussing on the events at the DNA level. To estimate the extent of switch recombination induced by these interleukins we have developed strategies for amplifying the reciprocal recombination products, which exist in the cells as transient circular DNA molecules. From the sequences flanking the germline gamma switch regions (which we recently completed) we developed a method to estimate the contribution of each of the four human gamma genes to reciprocal products containing gamma-epsilon composite switch regions. We are exploring the relationship between these data and our recent data on the contribution of the individual gamma genes to composite switch regions of the structure S(mu)-S(gamma)-S(epsilon) that are retained on the chromosomal DNA. Using a similar strategy we have devised a technique for assessing the role of IL-10 in promoting switch recombination to various human gamma genes. We are also completing an analysis of the effect of the cytokine TGF-b on the isotype switch to epsilon expression; this cytokine appears to affect a novel step in the process after the synthesis of germline epsilon transcripts. In collaboration with Dr. Burd we have analyzed the ability of activated mast cells to secrete a cytokine which appears identical to IL-13 in its ability to induce human B lymphocytes to switch to epsilon expression. An additional study of the contribution of EBV infection to epsilon expression and allergy in humans has been initiated. To further explore the mechanism of the switch event we are constructing an artificial switch substrate that will be transfected into cells and used to characterize sequence dependencies for switch recombination. Our studies are continuing on the function of the B cell-specific protein BSAP, encoded by the Pax-5 gene; this protein binds to several sites in the immunoglobulin heavy chain locus and appears to play a role in switch recombination. In collaboration with Drs. Neurath and Strober we have used anti-sense and triple-helix-forming oligonucleotides to probe BSAP function, and have accumulated suggestive evidence that at one site of BSAP binding in the immunoglobulin 3' alpha enhancer, this protein acts by displacing a novel protein we have designated NF-alphaP, which is related to the Ets family of transcription factors. Finally, with our UCLA collaborators we have been studying human immunoglobulin epsilon mRNA isoforms resulting from alternative splicing. To complete the characterization we are trying to determine the 3' end of the mRNAs that include """"""""membrane"""""""" exons.
