Project Summary The increasing prevalence of asthma over the past few decades signals an urgent need to understand disease pathogenesis and to develop more effective therapeutics. High affinity IgE plays a central role in the disease by mediating mast cell and basophil degranulation, which releases chemical mediators responsible for asthma exacerbation. T follicular helper (Tfh) cells promote the relevant high affinity antibodies for a given immune response through the cytokines they secrete. Tfh cell-derived IL-4 is required to induce high affinity IgE in response to allergens. However, IL-4 has also been considered a canonical Tfh cell cytokine, produced even during microbial immunizations that do not elicit IgE. Such studies largely rely on the use of an IL-4 cytokine reporter that indicates activation of Il4 transcription but not necessarily transcript stability or translation. Our lab has established murine models of Alternaria-induced allergic airway inflammation (AAI), which involves high affinity IgE production, as well as lipopolysaccharide (LPS)-induced lung inflammation, which does not. Using these two models, I observed Il4 mRNA expression in Tfh cells from both conditions; in contrast, I found that only Tfh cells in AAI produce IL-4 protein. My preliminary data suggest that Il4 mRNA in Tfh cells from AAI is more stable than that from LPS-induced inflammation, uncovering a post-transcriptional regulatory mechanism that governs IL-4 production in Tfh cells. My overall hypothesis is that post-transcriptional regulation of Il4 is a checkpoint dictating the high affinity IgE-promoting capacity of Tfh cells. My proposal therefore suggests a new paradigm for the regulation of IL-4 protein production in Tfh cells and the molecular basis of IgE-mediated AAI. My first aim is to identify the mRNA regulatory sequences responsible for Il4 post-transcriptional regulation in Tfh cells during AAI versus LPS-induced lung inflammation. To accomplish this, I will clone Il4 expression constructs mutated in regulatory sequences to determine the effect on Il4 mRNA stability, IL-4 protein production, and high affinity IgE responses. My second aim is to evaluate the role of an RNA-binding protein (RBP) on IL-4 production in Tfh cells during AAI. To do this, I will map RBP-Il4 binding sites, mutate these binding sites in Il4 expression constructs, and evaluate the effect of such mutations on Il4 mRNA stability, IL-4 protein production, and high affinity IgE responses. If successful, this project will define a previously undescribed mechanism of IgE regulation in asthma while also elucidating the immunologic rules that prevent the inappropriate induction of IgE in non-allergic responses. Such findings will offer valuable insight into new strategies for blocking IgE development in asthma and other allergic diseases.