Abstract Females are more to susceptible many autoimmune diseases. In multiple sclerosis (MS), not only is there a 3-4 fold increase in disease incidence, but there are sex-determined differences in the average age of onset and clinical course. Yet the cellular and molecular underpinnings of this sex-dimorphism have remained undefined. The SJL mouse model of MS recapitulates these differences in that female mice exhibit higher incidence, more severe disease, and a more consistent relapsing-remitting pattern than their male counterparts. This difference in disease susceptibility corresponds to qualitatively distinct anti-myelin Th cell cytokine responses. Whereas females generate pro-inflammatory Th1/Th17-dominant responses, the response in males is Th2-skewed and non-pathogenic. In this application we provide evidence that type 2 innate lymphoid cells (ILC2s) exert a male-specific protective influence. Best studied in allergic airway models, ILC2s are c-kit+ and are essential for inducing Th2 immunity through production of IL-13. We propose that mast cell activation in immunized in male mice elicits production of ILC2 activating factors such as IL-33 that promote ILC2 functionality. The inability to generate a robust IL-33 response in females leads to a functional deficit in ILC2 activity. Mast cells (c-kit+ FcεR1+) are one important source of IL-33 in vivo and testosterone directly induces Il33 exclusively in male-derived cells, despite equivalent androgen receptor expression by female-derived mast cells. These data suggest a cellular and molecular target of testosterone and identify a potential mechanism of action for testosterone-mediated protection in CNS autoimmune disease. Specific Aim 1: Determine the IL-33 expression kinetics, cellular source (s) and its requirement for protection in immunized male mice. Using IL-33- reporter mice (Il33Cit/+) or IL-33-deficient mice (Il33Cit/Cit (Il33-deficient) these experiments will define the IL-33-expressing cells, when and where it is produced. Specific Aim 2: Determine how testosterone influences the expression of IL-33 and other factors that regulate sex-dimorphic EAE protection. IL-33 gene expression will be evaluated in mice treated with testosterone or androgen receptor (AR) antagonists. We will ask if testosterone induces epigenetic changes at the Il33 locus conferring changes in chromatin accessibility. Other AR target genes will also be examined. Specific Aim 3: Explore the contributions of sex determining genes on ILC2 and mast cell function in EAE. The potential effect of sex chromosomes independent of hormonal influences on ILC2 and mast cell gene expression and function will be examined using four core genotype mice.