Research Summary Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system that results in neurological deficits from demyelination and neuron loss from chronic neuroinflammation. MS is more prevalent in women yet improvement in clinical symptoms occurs during pregnancy followed by disease exacerbations post-partum. It is well accepted that sex hormones have immunoregulatory properties and may protect individuals with MS during pregnancy. Our previous work has shown pretreatment with low dose estrogen (17β- estradiol, E2) protects mice from developing experimental autoimmune encephalomyelitis (EAE) and promotes regulatory B cells. We have demonstrated that E2 protects mice by inducing regulatory B cells that express PD- L1 and/or IL-10. Recently we showed E2 can protect IL-10 KO mice from developing EAE (indicating that IL-10 is not required for protection) and that these mice up regulate proteins in the PD-1/PD-L1/2 pathway as well as increasing different subsets of regulatory B cells. In this proposal we thus will examine in detail these two major pathways through which E2 appears to protect IL-10 KO mice during EAE in order to address our hypothesis that IL-10 knockout mice are protected from EAE by E2 through compensatory mechanisms. Aim 1, will define the role of the PD-1/PD-L pathway in protecting IL-10 knockout mice compared to wild type mice. PD-L1, PD-L2, and PD-1 will be inhibited with neutralizing antibodies to determine the extent of compensation provided by each protein in E2 pretreated IL-10 KO mice with EAE; Aim 2, will determine which subsets of regulatory B cells are compensating for the loss of IL-10 secreting regulatory B cells in E2 mediated protection of IL-10 knockout mice. Regulatory B cells from the spleen and lymph nodes will be examined in E2 pretreated wild type and IL-10 KO EAE mice to determine which regulatory B cells are produced in each strain of mouse; Aim 3, will identify which cytokine and chemokine pathways are up/down-regulated in E2 pretreated mice lacking IL-10 compared to wild type mice. Spinal cords will be evaluated using a cytokine array and RT-PCR analysis at 21 days post immunization in four groups of mice: wild type + E2, wild type + sham, IL-10 KO + E2, and IL-10 + sham. Overall this proposal will provide a more comprehensive understanding of the immunoregulatory properties of E2 and could lead to the identification of dominant pathways that could be activated or expanded for the treatment of MS.