PROJECT SUMMARY FOXP3 is a key transcription factor for the development and function of regulatory T cells (Tregs) and amorphic mutations at this locus in humans and mice lead to systemic autoimmune diseases and allergic symptoms. Human FOXP3 encodes two major isoforms, a full length (FOXP3 FL isoform) and an alternatively spliced isoform lacking exon 2 (FOXP3 ∆E2 isoform), while mouse Foxp3 gene only produces the FOXP3 FL isoform. The roles of these two isoforms in health and disease have not been clearly defined. We generated mice with Foxp3 exon 2 deletion (Foxp3dE2) to study the functionality of FOXP3 ΔE2 isoform in vivo and demonstrated that expression of FOXP3 ΔE2 isoform resulted in significantly more severe allergic airway inflammation after repeated intranasal allergen (both OVA and HDM) challenge compared with wild type mice expressing only the FOXP3 FL isoform. Despite having comparable suppressive function to FOXP3 FL Tregs, FOXP3 ΔE2 Tregs are less stable and have greater propensity to transdifferentiate into T helper cells. Furthermore, FOXP3 ΔE2 Tregs express higher levels of Il4 and Il9 than FOXP3 FL Tregs. We hypothesize that dysregulated FOXP3 isoform expression in airway Tregs (i.e. elevated FOXP3 ΔE2 expression) is one of the risk factors for asthma persistence and severity. We will test our hypothesis in the following three specific aims using unique mouse strains developed in our lab: WT (Foxp3FL) mice expressing only the FOXP3 FL isoform, Foxp3dE2 mice expressing only the FOXP3 ΔE2 isoform, and humanized Foxp3huE2 mice co- expressing both isoforms through alternative splicing as in humans. (1) Define the role of the FOXP3 isoforms in severity and persistence of allergic airway inflammation. We will determine the severity of airway inflammation, Treg phenotypes, and FOXP3 isoform expression in Foxp3FL, Foxp3dE2 and Foxp3huE2 mice after chronic exposure to allergens. (2) Elucidate the mechanism of FOXP3 isoform regulation of Th9 differentiation. We will determine, at the molecular and cellular level, how FOXP3 affects Th9 differentiation and Treg to Th9 transdifferentiation. (3) Define the association of FOXP3 isoform expression with human asthma severity. We will examine the expression of human FOXP3 isoforms in patients with asthma and test whether FOXP3 isoform expression correlates with disease severity and impacts Th9 and Th17 differentiation. The information learned from these studies will provide novel insights into the physiological functions of these two naturally existing human FOXP3 isoforms and would pave the way to develop efficient clinical protocols aimed at prevention/treatment of allergic diseases.