S region transcription per se promotes basal IgE class switch recombination but additional factors regulate the efficiency of the process

Abstract

Stimulation of B lymphocytes with a combination of lipopolysaccharide (LPS) and interleukin-4 (IL-4) induces germline transcription of and subsequent switching to the epsilon heavy chain constant region (C epsilon) gene. Mature germline C epsilon transcripts contain a non-coding exon (I epsilon exon) spliced to the C epsilon exons. To distinguish between the potential roles of germline transcription and those of germline transcripts in regulating the class switch process, we replaced the LPS- and IL-4-inducible I epsilon promoter and exon in ES cells with an LPS-inducible E mu enhancer/VH promoter expression cassette. Wildtype, heterozygous or homozygous mutant ES cells were injected into RAG-2 deficient blastocysts to generate somatic chimeras in which all B cells derived from ES cells. In contrast to normal B cells, heterozygous and homozygous mutant B cells had substantial transcription through the epsilon switch recombination region (S epsilon) following treatment with LPS alone and, under these conditions, both underwent low level switching (10- to 100-fold less than wildtype cells stimulated with LPS + IL-4) to IgE production. Heterozygous mutant cells underwent switching to IgE at essentially wildtype levels when stimulated with LPS and IL-4. However, homozygous mutant cells still showed extremely low levels of switching to IgE upon LPS and IL-4 stimulation. Analyses of hybridomas from heterozygous mutants indicated that the mutation is cis-acting and normal switching to other isotypes indicated that it is specific for IgE. Thus transcription per se generates low levels of class switch recombination in the absence of I region sequences. However, we demonstrate for the first time that, for optimal efficiency, the process requires the presence of the intact I region and/or I region promoter in cis, implicating factors beyond transcription through the S region in the regulation of class switching.