IGE PRODUCTION REGULATION VIA CD23 STALK ENGAGEMENT AND CELL CYCLE STIMULATION

CD23, the low affinity receptor for IgE, is expressed mainly on B cells and has been shown to regulate IgE production. Previously, recombinant mouse and human CD23 were constructed with a trimerizing isoleucine zipper motif attached in frame to the N-terminus of the entire extracellular CD23 (lz-ECCD23). The goal was to examine the role of the necessity of the CD23 stalk for binding IgE. Using PCR-based mutagenesis to delete the majority of the stalk, binding to IgE was lost. Further studies examined the effect of lz-ECCD23 in preventing IgE from binding FcεRI and therefore acting as a therapeutic agent. It was determined that the lz-ECCD23 construct was capable of doing this, albeit most effectively at 4°C rather than at physiological temperature. In addition, antibodies to the stalk region of CD23 were developed and assessed for their capacity to modulate IgE. Rabbit anti-CD23stalk (RAS) antibodies were found to inhibit IgE production in IL-4/antiCD40 stimulated B cells. The inhibition observed was dependent on the Fc portion of the antibody, implicating a role for FcγRIIb, an inhibitory receptor, in the IgE reduction. It was also shown that the addition of anti-stalk antibodies caused significant release of soluble CD23 (sCD23). Finally, I show that optimal IgE production was cell density dependent and was achieved through the addition of IL-21 and/or IL-10 to IL-4/antiCD40 stimulated B cells. While IgE production is inversely proportional to plated cell densities, it is directly correlated to increased cellular division, as determined by CFSE staining, and to increased cellular differentiation, as determined by FACS analysis for differentiation markers. This work is the first demonstration that IgE production in humans is dependent on cell density, that IL-21 affects all isotypes tested, and that maximal Ig production is found at lower cell densities, correlating with increased cell division. I also show for the first time that the increase in IgE observed after IL-10 addition to IL-4 and anti-CD40 stimulated cells correlates with increased cellular division. When IL-10 and IL-21 were added together, there was a synergistic increase in IgE, but interestingly, no further cell division was seen, suggesting an increase in differentiation