The overall goal of this project is to gain a cellular and molecular definition of the events governing the differentiation of IgA B cells. During this period we focused on the effects of various cytokines on IgA B cell switching, both in normal spleen b cells and in the B cell line CH12.LX. In addition, we examined the molecular basis of dual-bearing sIgM+/sIgA+CH12.LX cells. In initial studies, we determined the effect of TGF-beta on normal spleen B cell activation stimuli: 1) LPS; 2) anit-delta -dextran; 3) membrane components of an irradiated, activated T cell clone (D10); and 4) polyclonal cognate interaction. We showed that TGF-beta induces small increases in surface IgA expression (up to 2-3% of the cell present) following B cell activation by each of these stimuli, but caused increases in IgA secretion only when cells were activated by LPS. The small amount of switching induced by TGF-beta in these various activation systems provides strong evidence that TGF-beta is not a primary switch factor; instead, it is an enhancer of IgA switching induced by the activation stimulant itself. In the next series of studies, we determined the effects of TGF-beta and IL-4 on IgA switching in the B cell line, CH12.LX. Here, we showed first that CH12.LX cells are precommitted to switch to IgA since they produce IgA but not IgG subclasses following LPS stimulation, have demethylated I alpha and Calpha gene regions and produce germline alpha mRNA. Next, we showed that TGF-beta, IL-4 and particularly the combination of the two, induces 60% of CH12.LX to undergo switch to IgA. The massive switch induced by TGF-beta in CH12.LX B cells supports the contention that TGF-beta enhances IgA switching only after an initial commitment to IgA switching has been taken. In a final series of studies analyzing dual-bearing sIgM+/sIgA+ CH12.LX cells, we first obtained clones of CH12.LX cells representing the various cellular stages of isotype switching in this cell line; we then showed that: 1) mu mRNA and alpha MRNA transcripts in dual-bearing CH12.LX clones have identical VDJ regions; and 2) the VDJ gene segment in such clones are juxtaposed to a Cmu gene, not a Calpha gene. Thus, these studies suggest that isotype switching is accompanied by a discrete cellular stage in which an unrearranged CH region gives rise to both mu and alpha mRNA transcripts.
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