Project I: In ongoing molecular studies of B cell differentiation, we further defined the regulation of the Ig heavy chain 3'alpha enhancer. Earlier, we showed that the Pax5 transcription factor, BSAP, binds to and represses the activity of this enhancer. We now show that such repression is mediated via regulation of binding of a newly identified Ets family protein, NF alpha-P, to a positive regulatory element lying approximately 50bp downstream of the BSAP binding site. In in vivo footprint analyses, we first showed that the alpha-P site is occupied only in plasma cells and not in B cells, whereas the reverse is true for the BSAP site. We then showed that when BSAP binding is blocked in vivo by transfection of a triple helix-forming oligonucleotide that binds to the BSAP site,an alpha-P footprint appears and Ig heavy chain transcription is increased. Finally, we showed that the triple helix-forming oligo-nucleotide also increases enhancer activity of a transfected 3'alpha enhancer construct in B cells, but only when the alpha-P site is intact. Thus, BSAP negatively regulates the 3'alpha enhancer by blocking the binding of NF- alphaP to a positive regulatory element, alphaP. Project II:In these studies we defined the role of the T cell/B cell interaction mediated by OX40L-OX40 on B cell differentiation. In initial studies we showed that crosslinking of OX40L on CD40L (or anti-ID- dextran) stimulated B cells, or both, results in enhanced B cell proliferation and Ig secretion, independent of added cytokines. We then showed that OX40L crossbinding results in the downregulation of the above-mentioned BSAP which, in turn, leads to occupation of the alpha-P binding site in the 3'alpha enhancer. Thus, in this novel pathway of T cell dependent B cell differentiation, the cell surface interaction acts through 3'alpha enhancer activation. Project III: In a broad study of T cell differentiation and regulation in Peyer's patches (PP) we have continued our examination of PP dendritic cells (DC). Using immunoperoxidase staining of frozen sections of murine PP, we demonstrated the presence of a dense layer of cells with DC morphology, just beneath the PP dome epithelium, that stained with antimurine CD11c (mAb IV418) an antibody associated with DCs. Such cells were distinct from a second population of DCs that were also stained with the NLDC-145 and M342 mAb, intracellular DC markers associated with more differentiated DCs. In functional studies we showed that PP dendritic cells can be loaded with oral administered antigen to present to T cells bearing receptors for the fed antigen. Finally, we showed that PP DCs, when stimulated with activated T cells, produce significantly more IL-12 than spleen DCs, thus accounting for a previous finding that PP DCs induce T cells producing 5-10 fold more IFN-gamma than spleen DCs. Overall, these studies show that PP DCs take up antigen that enters the PP (probably via M cells) and then present the antigen to T cells to induce Th1-type T cell responses.
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