Project I: Recent in vitro studies have established that activated B cells express OX40 ligand (L) and become stimulated to proliferate and secrete immunoglobulin (Ig) after crosslinking of OX40L by its counterreceptor OX40, which is expressed on activated T cells. In the present study we investigated the in vivo role of this receptor-ligand pair for the interaction of T and B cells in the course of the T-dependent B cells response against 2,4,6-trinitrophenyl-keyhole limpet hemocyanin. We demonstrated that blocking of OX40-OX40L interaction with a polyclonal anti-OX40 antibody resulted in a profound decrease of the anti-hapten IgG responses, whereas the anti-hapten IgM responses were grossly unchanged. In addition, we showed that this antibody treatment leads to an inhibition of the development of PALS-associated B cell foci, whereas the formation of germinal centers remained intact. We conclude from these data that the OX40-OX40L interaction in vivo is necessary to the differentiation of activated B cells into highly Ig-producing cells. Project II: In these studies, we compared the activation requirements of sIgM+/sIgD+ B cells with those of isotype-switched sIgM-/sIgA+ B cells. We found that whereas sIgM+ B cells respond to TI and TD Ags with no significant bias toward one or the other stimulus, sIgA+ B cells were deficient in their ability to respond to Ag-receptor crosslinking but responded remarkably well to TD stimuli. Thus, anti-IgA-dextran, anti-kappa-dextran, or various immobilized anti-IgA Abs induced only low-level IgA B cell proliferation and no IgA secretion in the presence of various lymphokines; in marked contrast, sIgA+ B cells responded to cognate T cell stimulation as well as to stimulation by CD40L-bearing fibroblasts by secreting large amounts of IgA (up to 240,000 ng/ml/105 cells). In confirmation of these results, whole Peyer~s patch (PP) or lamina propria (LP) populations containing less than 15% sIgA+ B cells stimulated with a noncognate T cell stimulus of T cell membrane secreted mainly IgA (68%-94% of the total Ig secreted) and relatively little IgM. In further studies, we analyzed the role of Ig receptor crosslinking in T cell dependent stimulation of both sIgM+/sIgD+ B cells and sIgA+ B cells. We demonstrate that purified sIgA+ B cells pretreated with anti-IgA-dextran with higher doses (1 and 10 microgram/ml of antibody) led to a profound suppression of IgA secretion (greater than 90%). In contrast, sIgM+/sIgD+ B cells pretreated with anti-IgD-dextran or with anti-IgM-dextran did not show significant inhibition. Finally, we showed that anti-IgA-dextran-mediated suppression could be reversed by stimulation of sIgA+ B cells with fibroblasts expressing CD40L. These studies imply that Ig receptor crosslinking renders postswitch sIgA B cells unresponsive to subsequent stimulation via activated T cells but that this unresponsiveness is overcome by a persistent CD40Lhigh signal.
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