PROJECT ABSTRACT MS is an immune-mediated demyelinating disease of the CNS, and despite first line drugs that limit symptoms, disease progresses over time and is incurable. Given its early onset and rise in prevalence for nearly 1 million Americans, MS presents immense health and economic burdens on the United States. Given the success of B cell depletion therapy (BCDT), understanding the pathogenic roles of B cells during disease is of high interest. Because of the CD20-targeted nature of BCDT, it is understood that B cells contribute to MS pathology through mechanisms beyond antibody production, likely through support of autoreactive CD4 T cells. Most animal models of MS (experimental autoimmune encephalomyelitis or EAE) are B cell-independent, which limits impactful insights into pathogenic B:T interactions in vivo. To circumvent this, we have recently developed a B cell-dependent, antibody-independent animal model of MS featuring CD4 T cell immunoreactivity to the extracellular domain sequences of the highly abundant and 100% conserved myelin proteolipid protein (PLPECD). Through rigorous preliminary studies, we have identified B cell-mediated antigen presentation to CD4s through MHC class II as the required pathogenic B cell mechanism in PLPECD-induced EAE, where B cells engage PLPECD through the B cell receptor and are superior vs. non-B cell antigen presenting cells at processing and presenting immunodominant residues from within PLPECD to PLP178-191-reactive CD4 T cells. Further mimicking the sustained pathogenic B cell involvement seen in MS and unlike B cell- independent EAE driven by PLP178-191, BCDT robustly ameliorates established PLPECD disease. B cells’ role in supporting CD4s during neuroinflammation and shaping the diverse T helper cell (Th) profiles observed in MS remains unclear. Understanding these dynamics in MS may prove significant in identifying which patients will respond to BCDT and thus modeling pathogenic B cell involvement in vivo is becoming increasingly important. Our objective is to use this powerful B cell-dependent EAE model to understand how B cells shape the CD4 response and to investigate B cell-mediated support of CD4s in the CNS. These are key aspects of B cell-mediated pathology that are not currently understood and difficult to model appropriately in vivo. Our model put us in a unique position to answer these questions. We hypothesize that B cell-provided cytokines shape a biased Th profile and that CNS B cell:CD4 interactions are critical in promoting demyelination in PLPECD EAE. Specific Aim 1 will determine how B cells shape pathogenic CD4 T cells by testing B cell-provided cytokines’ impact on Th1/17 bias. Specific Aim 2 will determine CNS B cells’ role in PLPECD-induced EAE and visualize B cell-supported neuroinflammation.

PROJECT NARRATIVE Multiple sclerosis (MS) is an incurable immune-mediated demyelinating disease of the central nervous system that affects nearly 1 million Americans. Successful B cell depletion therapy in MS patients has invigorated intense interest in how pathogenic B cells contribute to disease. We will use our powerful novel B cell-dependent, antibody-independent EAE model to dissect mechanisms by which B cells shape the CD4 T cell response and determine the role of CNS B cells in autoimmune demyelinating disease.