Currently separate areas of investigation are being performed in our laboratory aimed at understanding the mucosal immune response through identifying factors which regulate this process at the cellular level and by studying the molecular regulatory elements involved in the immunoglobulin class switch. Several key studies have shown that TGF-Beta plays a role in IgA class switching and, more specifically, that its effects are at the level of switch differentiation rather than terminal differentiation. At the cellular level, TGF-Beta was shown to act on surface IgA- (sIgA-) B cells by increasing the frequency of B cell clones secreting IgA rather than increasing the number of IgA B cells per clone. This effect was later shown by Ehrhardt et al. to be specific for sIgA- B cells since TGF-Beta inhibits IgA production by already switched sIgA+ B cells. Furthermore, TGF-Beta has been shown to have no selective effect on B cell viability. At the molecular level, in LPS-activated B cells, TGF-Beta induces production of C-alpha germline transcripts - an earlier molecular event in the immunoglobulin isotype switch process. Collectively, these data have established beyond question that TGF-Beta does act as an IgA switch factor, however, the magnitude of this effect was minimal in earlier in vitro systems - in these studies, TGF-Beta induced only a small fraction of LPS-stimulated sIgM+ B cells to switch to sIgA+ B cells. Thus, by these in vitro studies, it appears that the action of TGF-Beta on LPS-activated B cells only induces IgA switching at a minor non-physiologic rate that is in no way comparable to the rate observed at in vivo IgA induction sites, namely the Peyer's patches. Later, McIntyre et al. developed an in vitro system in which B cells were co-stimulated with either LPS plus anti-IgD linked to dextran (anti-IgD- dextran) or CD40L plus anti-IgD-dextran in the presence of IL-4, IL-5, and TGF-Beta -- the latter being added at a 10-fold higher concentration compared to that used in previous studies. In this system, B cells demonstrated a 10-fold enhancement in IgA switching and a 500-fold enhancement, at minimum, in IgA secretion compared to cells cultured in the absence of the dual activation stimulus. Costimulation by LPS/anti-IgD-dextran or CD40L/anti-IgD-dextran also provided an enhanced proliferative signal to B cells in the face of higher, typically immunosuppressive, levels of TGF-Beta. These studies have laid the groundwork for our present studies which are aimed at more specifically identifying the contribution of anti-IgD-dextran to this in vitro IgA switching system as a means to more completely understand the IgA switching process as it occurs in vivo. Based on apoptosis studies, our recent data suggests that anti- IgD-dextran may be providing a viability signal which extends the life of B cells in culture and allows them to be responsive to higher concentrations of TGF-Beta needed to drive high level IgA secretion. We are extending this observation with studies to more accurately assess the temporal aspects of this viability signal (with respect to germline C-alpha transcription and expression of IgA on the B cell surface) and to identify the specific intracellular molecules, such as anti-apoptotic factors, which may be regulated by anti-IgD-dextran. Related to, yet separate from the above studies, we are working in collaboration with Dr. Charles Chu at North Shore University Hospital, NY School of Medicine to isolate and characterize genes expressed specifically in murine B cells that are undergoing IgA and IgG1 class switching using Representational Difference Analysis (RDA). Using the modified RDA method devised by Chu and Paul, genes for IL-5 induced IgG1 class switching will be obtained by subtracting cDNA obtained from splenic B cells cultured in the presence of anti-IgD-dextran + IL-4 + IL-5 from cells cultured with anti-IgD-dextran + IL-4. Similarly, genes for TGF-Beta, CD40L, and IL-5-induced IgA class switching will be obtained by subtracting cDNA obtained from splenic B cells cultured in the presence of anti-IgD-dextran + CD40L + IL-4 + IL-5 + TGF-Beta from cells cultured with anti-IgD- dextran + CD40L + IL-4 +IL-5, anti-IgD-dextran + IL-4 + IL-5 + TGF-Beta, or anti-IgD-dextran + CD40L + IL-4 + TGF-Beta, respectively. Specific genes that are identified will be sequenced and further characterized and their particular functions will be analyzed. Total RNA has been purified from B cells cultured for IgG1 switching in the above manner and has been provided to Dr. Chu for analysis.
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