Effect of downregulation of germline transcripts on immunoglobulin A isotype differentiation

Abstract

In this study we determined the role of immunoglobulin (Ig) germline transcripts in the isotype switch differentiation of the cloned lymphoma B cell line CH12.LX. In initial studies, we showed that addition of transforming growth factor β (TGF-β) and interleukin 4 (IL-4), either alone or in combination, augment switching from membrane (m)IgM+ to mIgA+ cells, and that increased switching is preceded and paralleled by an increase in the steady-state level of α germline transcripts (aGLT). Interestingly, TGF-β and IL-4 affect switching in different ways, as shown by the fact that IL-4 increases and TGF-β decreases the number of dual-positive (mIgM+/ mIgA+) cells; in addition, TGF-β and IL-4 have different effects on the time course of induction of αGLT. In subsequent studies, we established that we could downregulate αGLT levels in CH12.LX B cells by transfecting an expression vector that can be induced to produce transcripts antisense to the Iα exon. Using this approach we downregulated αGLT in CH12.LX B cells undergoing switching in the presence of TGF-β and IL-4 and showed that such downregulation led to decreased switching, as evidenced by decreased appearance of dual-positive B cells as well as decreased IgA synthesis relative to IgM synthesis. This result was corroborated by the fact that incubation of CH12.LX cells with phosphorothio-oligo antisense DNA to Iα sequence also led to a decrease in the number of dual-positive cells and in the IgA/IgM secretion ratio. In summary, IgA isotype differentiation in CH12.LX B cell, particularly the steps necessary for the elaboration of mIgM+/mlgA+ switch intermediate cells, is inhibited by downregulation of αGLT; it is therefore apparent that αGLT plays a key role in the initial stage of isotype switch differentiation.