| Abstract
| - We have developed an efficient in vitro class switching system using a subclone (CH12F3) of the lgM+ CH12.LX lymphoma cell line. CH12F3 cells switched from surface lgM+ cells to surface lgA+ cells at a high frequency (∼50%) after 72 h stimulation with IL-4, transforming growth factor(TGF)-β and CD40L. No other class isotype-producing cells were detected, indicating that the CH12F3 clone is exclusively committed to IgA isotype switching. To understand the molecular basis of the isotype commitment, we studied the methylation profiles of I region promoters and I region transcription of CH12F3 cells. No germline transcripts other than those from the lα region were detected and only the lα promoter was demethylated in uninduced CH12F3 cells. TGF-β, CD40L and IL-4 synergistically induced efficient switch recombination in CH12F3 cells, suggesting that the three stimulations up-regulate different steps of switch recombination in isotype-committed B cells such as CH12F3 cells. Stimulation of CH12F3 cells by IL-4 or TGF-β, but not by CD40L, induced transient but complete methylation of the lα region. TGF-β and CD40L, but not IL-4, increased the amounts of germline a transcripts. We found that the extents of methylation and the amounts of germline α transcripts do not necessarily correlate with the efficiency of recombination in induced CH12F3 cells. These results led to the proposal that switch recombination can be separated into at least two phases, i.e. commitment and recombination. The roles of IL-4, TGF-β and CD40L in the two phases are discussed.
|
