Regulation of IgG subclass switching in human B cells

Sammanfattning: Human IgG can be subdivided into four subclasses, each encoded by a separate heavy chain constant (CH) region gene and endowed with unique biological and functional properties. Deficiencies of single or multiple IgG subclasses have been suggested to be associated with increased susceptibility to recurrent infections in humans. Class, or isotype switching is the process whereby a B cell alters the heavy chain class by changing the CH region expressed. The switch in antibody class is brought about by a deletional DNA recombination event called switch recombination, which occurs between tandemly repeated sequences in the switch (S) regions located upstream of the CH genes. The mechanism of differential regulation of switching to human IgG subclasses is still largely unknown. Initially, we analyzed the structure of Sy regions. A crucial nuclear factor-[kappa]B (NF-[kappa]B) protein binding motif was found to be mutated in the (g) allotype-associated S[gamma]3, suggesting that polymorphisms within S[gamma]3 may contribute to allotype-associated differences in switching. We also found that there is no allotype-associated difference in the I[gamma]3 region. However, individual mutations in the I[gamma]3 region and differential splicing of GL l[gamma]3 transcripts were found and may affect the switching process. We subsequently found that the polymorphism of the [gamma]4 locus is due to differences in length of the S[gamma]4 region, and that the length of this region seems to play a role in IgG4 production. We furthermore characterized 44 S[my]-S[gamma]3 and 26 S[my]-S[gamma]4 recombination junctions from in vivo switched human B cells and a comparison was made with all previously published S[my]-S[gamma] sequences. Our analysis allowed us to clarify several issues related to the switching process. We also demonstrated that the region upstream of the initiation sites for [gamma]3 GL transcripts contains a functional promoter which is synergistically induced by IL-4, antibody to CD40 ([alpha]CD40) and phorbol dibutyrate (PDBu) in transient transfetion assays in a human cell line. Linker-scanning mutations identified multiple elements in the 3' half of the evolutionary conserved sequence (ECS) that were required for inducibility. Functional analysis of the promoter suggested that STAT6, NF-[kappa]B p50/p65 and C/EBP[gamma] synergistically activated this promoter and the recently described human 3' enhancer elements were found to provide a strong orientation-independent but copy numberdependent enhancer effect.