Summary Multiple Sclerosis is a progressive multifocal demyelinating disease which affects approximately 2.5 million people worldwide. It is the leading cause of neurologic disability in young adults. The contribution of the immune system to this disease is well established. Multiple treatments targeting B and T lymphocytes have been developed against the most prevalent Recurring Remitting form of the disease (RRMS). However, etiology of the disease remains unclear. Studies from human MS and rodent Experimental Autoimmune Encephalomyelitis (EAE) models suggest that nervous system-derived myelin antigens are important in the development of the disease. However, the format of these antigenic signals and how they are transported to the peripheral lymphoid tissues remain poorly understood. Recent evidence suggest that exosomes secreted by mature oligodendrocytes (OLs) are enriched in a variety of myelin antigens. Exosomes are the secreted vesicles that are increasingly appreciated to play important roles in cell-cell communications. Importantly, myelin-containing exosomes have been reported peripherally in MS patients. The specific roles of OL-derived exosomes either in human MS or rodent EAE has not been investigated. We hypothesize that exosomes produced by myelinating cells, especially OLs, may serve as early sources for central and peripheral myelin antigen presentation, ultimately facilitating pathogenic accumulation of autoreactive lymphocytes in the CNS parenchyma. Specifically, we propose to investigate this hypothesis by first analyzing in situ dynamics of the myelinating cell-derived exosomes in CNS and peripheral immune tissues in mouse EAE in Aim 1 using a cell-type specific exosome reporter mouse line, recently developed by the MPI lab. For these experiments we will use a modified EAE model (Cup-EAE) which includes both disruption of central myelin using short treatment with cuprizone followed by the induction of the peripheral immune response. We will then examine both the ability of exosomes to reach various peripheral tissues as well as their uptake by relevant cell types within the CNS under control versus Cup-EAE conditions. We also plan to expand on our promising preliminary data to further characterize the protection from EAE by the exosome release inhibitor. In Aim 2, we will focus on understanding the functional properties of exosomes produced in vitro by control and MS-associated factors (TNFa and glutamate)-treated OLs. Various properties of these vesicles will be assessed including size, cargo, uptake by antigen presenting cells (APCs), and ability of these exosomes to support myelin antigen presentation by APCs to myelin-specific T cells in vitro. Overall, we believe that our proposal utilizing an innovative exosome reporter mouse will provide important and informative data to begin the understanding of the roles of exosomes produced by myelinating cells in MS with specific emphasis on OL-derived myelin-containing exosomes.