The theme of this proposal is the analysis and comparison of the molecular mechanisms that control the synthesis of IgE and IgG4 in human B cells. IgE and IgG4 are expressed as a result of IL4-induced sequential switching and therefore share antigen specificity. Because IgE is critical for the pathogenesis of allergy and asthma, and antigen-specific IgG4 can block IgE-dependent responses in vivo, our long-term objective is to determine the mechanisms that lead to preferential production of antigen-specific IgG4. Our preliminary data show that IL-4-dependent antibody responses can be uncoupled in vivo, i.e., allergen-specific IgG4 can be detected in the absence of specific IgE. Furthermore, the IgE/IgG4 ratio for Alternaria is significantly higher in sensitized asthmatics than in sensitized patients without asthma. IL-10 suppresses IgE while enhancing IgG4 responses. Interestingly, in our population IL-10 promoter polymorphisms associated with lower IL-10 production are associated with increased IgE levels in early life. Thus our overall hypothesis is that (1) the ratio between allergen-specific IgE and IgG4, more than the absolute levels of either Ig class, is critical for the clinical outcome of allergic responses, and (2) IL-10 plays a critical role in regulating the IgE/IgG4 ratio. Therefore this proposal is aimed at elucidating the molecular mechanisms underlying the differential regulation of IgE and IgG4, and the role played by IL-10 in this process. Because the isotype targeted by switch recombination is determined through germline (GL) transcription, our analysis will focus on the mechanisms that control the activation of the epsilon, and gamma4 GL promoters. Here we propose:
Specific Aim 1. To analyze and compare chromatin remodeling at the IgG4 and IgE locus in IL-4-stimulated human B cells. We will use nuclease hypersensitivity (HS) assays to monitor and compare chromatin accessibility at the IgG4 and IgE locus in human B cells stimulated with IL-4 and/or CD40 crosslinking. Furthermore we will investigate the role of DNA methylation and histone acetylation in the control of IgG4 and IgE GL gene expression.
Specific Aim 2 : To analyze and compare the regulation of the proximal gamma4 and epsilon GL promoter, and to analyze the promoter-distal elements corresponding to nuclease HS sites for their role in the regulation of GL transcription. We will use reporter assays and transient transfections to define elements important in the transcriptional regulation of the proximal epsilon and gamma4 GL promoters in response to IL4 and CD40 crosslinking, and we will identify the nuclear proteins that bind the promoters. Furthermore we will define the function of the promoter-distal elements containing HS sites, and we will identify the transcription factors that bind those elements, establishing whether they modulate accessibility of the region.
Specific Aim 3 : To determine the mechanisms underlying the differential regulation of IgE and IgG4 expression by IL-10. We will determine whether IL-10 differentially affects IL-4-dependent transcriptional activity and DNA/protein interactions at the epsilon and gamma4 GL promoters. As a corollary, we will analyze how signals delivered through B7 molecules, a major target of IL-10, contribute to full activation of the IL-4-dependent GL promoters.
