To advance our long-term goal of understanding the regulation of immunoglobulin isotype switching in human B lymphocytes, we have been studying three enhancer regions lying downstream of the immunoglobulin human C-alpha genes to determine how these regions integrate signals from the B cell milieu to regulate Ig expression in conjunction with Ig gene promoters. These three enhancers correspond to DNase I hypersensitivity (HS) sites designated HS3, HS12 and HS4. In transient transfection assays using luciferase reporter constructs, we have found that CD40 ligand (CD40L) -- which upregulates Ig gene expression in some B cells -- inhibits the function of a minimal HS12 enhancer in a human B cell line designated CL01 with germinal center phenotype. In addition, a larger fragment of the HS12 enhancer appears to confer both CD40L- and IL-4 dependent inhibition of enhancer function in the same cell line, while HS4 demonstrates significant inhibition by IL-4 but a lesser inhibition by CD40L. Both CD40L and IL-4 inhibit the function of a minimal Vk or VH promoter, while neither significantly alters the activity of an I-gamma3 promoter or a control non-Ig promoter. These effects on Ig gene regulatory regions may reflect the mechanism of a recently reported physiological decrease in Ig secretion mediated by CD40L in germinal center cells. This decrease has been hypothesized to prevent secreted Ig from interfering with selection of high affinity B cell receptors after somatic mutation. Experiments currently under way are aimed at identifying the DNA motifs responsible for the down-regulation of Ig enhancer and promoter regions by IL-4 and CD40L. In addition, we have created constructs designed to test the activity of these regions as Locus Control Regions, and are attempting to derive a panel of stable B cell transfectants for analysis of LCR function and cytokine response elements. In a collaborative project with Drs. Matthias Wabl and Rolf Jessberger, we are attempting to evaluate the role of SWAP70, a candidate component of the switch recombinase machinery discovered by these scientists. This protein was identified as a B cell-specific component of a protein complex that could participate in an in vitro reaction bearing similarities to the immunoglobulin isotype switch recombination. Because initial experiments using antisense phosphorothioate oligonucleotides were unsuccessful in reducing SWAP70 expression, and stable constructs constitutively expressing antisense SWAP70 mRNA appeared to block cell proliferation, thereby preventing isolation of stable antisense-expressing lines, we have been developing a system of stable cell lines expressing antisense mRNA under the control of a tetracycline-inducable promoter. Using these cells, we will be assessing whether antisense-induced reduction in SWAP70 levels affects the ability of this cell line to undergo cytokine-induced isotype switching.
