Project Summary Allergic airway diseases (AAD) such as asthma require ongoing medical management and currently have no cure. IL-9 and IL-13 are cytokines previously reported to be essential to the pathology of AAD and are largely produced by CD4+ helper T cell subsets. IL-9 is primarily secreted by Th9 cells whereas IL-13 is classically associated with Th2 cells. There is additionally a subset of Th9 cells reported to produce Th2 cytokines such as IL-13. These multi-cytokine producing Th9 cells have demonstrated increased pathogenicity in an allergic disease context as compared to conventional Th9 cells. Our preliminary data shows that the cytokine TL1A is sufficient to increase IL-9 expression and induce robust IL-13 expression in Th9 cells in vitro. Also, we show that blockage of TL1A in a chronic model of AAD significantly decreases the multi-cytokine Th9 compartment. We hypothesize that TL1A is a critical factor in promoting IL-13 production by Th9 cells which increases their pathogenic potential in AAD. To test this hypothesis, we propose two aims. In the first aim, we will clarify how TL1A enhances production of IL-9 and IL-13 in Th9 cells through two sub-aims. The first sub-aim focuses on identifying TL1A-induced histone modifications associated with heterochromatin or euchromatin at the Il9 and Il13 loci using ChIP and discerning physical interactions between promoters and enhancers of the Il9 and Il13 loci through Hi-C (high-throughput chromatin conformation capture). In the second sub-aim, we will focus on assessing the importance of Nfkb2 and Stat5a in the TL1A-mediated increase in multi-cytokine Th9 cells. We will perform ChIP against Nfkb2 and Stat5a to assess for binding at the Il9 and Il13 loci and will also perform siRNA knockdown of Nfkb2 and Stat5a and assess whether TL1A is able to enhance development of the multi- cytokine Th9 compartment in the absence of either Nfkb2 or Stat5a. Completing this first aim will give us insight into how TL1A signaling impinges on the chromatin landscape of multi-cytokine Th9 cells and the relative contributions of Nfkb2 and Stat5a in their development. In the second aim of our proposal, we want to address the pathogenicity aspect of our hypothesis. To do this, we will assess how TL1A-primed Th9 cells affect disease severity relative to conventionally primed Th9 cells in an OVA-challenge model of murine AAD. Completion of this second aim will provide much needed information regarding the pathogenicity of TL1A- induced multi-cytokine Th9 cells. Together, the aims outlined in the following proposal will clarify the mechanism by which TL1A enhances development and pathogenicity of the multi-cytokine Th9 compartment. This will provide insight into novel therapeutic targets in the treatment of allergic airway diseases.