PROJECT SUMMARY The aim of this proposal is to discover the cellular and molecular pathways regulated by IL-13 that promote B cell production of high affinity IgE to allergens. Despite the importance of IgE in allergic responses, the signals that instruct B cells to produce high affinity, sialylated and long-lived IgE are not well understood. Recently we discovered in mice and humans a rare subset of T follicular helper (Tfh) cells, known as Tfh13 cells, that produce IL-13 and are required for high affinity IgE production. Ablation of of IL-13 specifically from Tfh13 cells causes a loss of high affinity IgE and prevents anaphylaxis. Additionally, we have demonstrated that the receptor for IL- 13, IL-13 receptor alpha 1 (IL-13Rα1), is highly expressed on IgG1 GC B cells, IgE GC B cells, and IgE plasma cells as compared to naïve B cells. These findings suggest that Tfh13-derived IL-13 acts directly on B cells to promote high affinity IgE responses. However, where in the lymph node Tfh13 cells interact with B cells and how Tfh13-produced IL-13 promotes high affinity IgE production are unknown. Our hypothesis is that IL-13 acts on IgE+ B cells to promote their survival. In Aim 1, we will identify the stage of mouse and human IgE+ B cell differentiation and/or functions that are regulated by IL-13. We will use specialized in vitro culture systems to generate both mouse and human germinal center-like B cells to test whether IL-13 promotes IgE switching, IgE+ plasma cell differentiation, or IgE+ plasma cell survival. We will also use mass spectrometry to investigate whether Tfh13 cells and/or IL-13 play a role in the regulation of IgE glycosylation in vitro and in vivo. Additionally, we will determine whether the expression of IL13Rα1 differs on various B cell subsets in allergic versus healthy donors and will test how this impacts their response to IL-13. In Aim 2, we will define the role of IL-13Rα1 on mouse IgE+ B cells in vivo. To achieve this, we will use high-resolution immunofluorescence imaging and spatial transcriptomics/proteomics to characterize the sub-anatomic regions in the lymph node where IL-13-expressing Tfh13 cells and B cells interact. Additionally, using a novel conditional Il13ra1 knockout mouse we generated we will determine which stages of the IgE+ B cell response are disrupted by the loss of IL-13 stimulation in vivo. Lastly, we will investigate whether B cell intrinsic IL-13Rα1 is necessary for the production of sialylated and high affinity IgE to allergens and downstream anaphylaxis in vivo. In Aim 3, we will identify the IL13Rα1-specific signaling pathways that promote IgE production by mouse and human B cells. To accomplish this, we will use a combination of phospho flow, ChIP-Seq, and RNA-Seq to characterize STAT6-dependent and -independent signaling pathways in human and mouse B cells downstream of IL-4 versus IL-13 stimulation. Subsequently, we will use knockout cells and inhibitors to alter the balance of type I and type ...