PROJECT SUMMARY / ABSTRACT Myelin oligodendrocyte glycoprotein (MOG) has been recognized as an autoantigen (autoAg) in EAE and as a putative T cell and antibody (Ab) target in MS. Recently, MOG has been identified as the target in several CNS autoimmune conditions, including acute disseminated encephalomyelitis (ADEM), optic neuritis (ON), and transverse myelitis (TM). Collectively, this spectrum is known as MOG Ab-associated disease (MOGAD). Currently, there are no FDA-approved treatments for MOGAD. Key aspects regarding MOGAD pathogenesis have not been elucidated. MOG-specific Abs in MOGAD are T cell-dependent and are not pathogenic in the absence of T cell-mediated CNS inflammation. Therefore, we hypothesize that MOG-specific T cells have a central role in MOGAD and cooperate with B cells, and possibly MOG-specific Abs, to promote CNS injury. MOG-induced EAE is an invaluable model to evaluate how MOG-specific T cells cooperate with MOG- specific B cells and Abs in MOGAD. Like MOGAD, MOG-induced EAE manifests as ON, TM and encephalomyelitis. Besides serving as a source of MOG-specific Abs, B cells are Ag presenting cells (APC). EAE induction by MOG protein is B cell MHC II-dependent. Whether B cell Ag presentation promotes development of a distinct pathogenic T cell repertoire is unknown. We hypothesize that B cell Ag presentation expands a unique TCRa/b repertoire of MOG-specific pathogenic T cells. Susceptibility to MOG protein-induced EAE is sensitive to certain human-specific amino acid (aa) sequence differences. Novel MOG T cell epitopes identified from studying MOGAD patients are located within the region where many aa sequence differences are clustered. To understand cellular and humoral responses to human MOG, and to better translate our findings, we created humanized MOG knock-in mice by replacing mouse MOG genomic sequence with human genomic MOG. In this program, we propose: (1) To identify and characterize MOG-specific T cells in patients with distinct MOGAD phenotypes. Peripheral blood mononuclear cells will be collected from patients enrolled at three collaborating institutions. MOG specificity will be determined by stimulation with overlapping MOG peptides and TCRa/b repertoire will be examined by single cell RNA sequencing (scRNA-Seq). (2) In subaim 2a we will determine how B cell Ag presentation in vivo may shape development of the pathogenic MOG-specific T cell repertoire. T cells from wild-type, B cell-deficient and MOG-specific B cell receptor (BCR) mice immunized with MOG protein (B cell-dependent) or MOG peptide (B cell-independent) will be subjected to TCRa/b repertoire analysis by scRNA-Seq. In subaim 2b, our humanized MOG mice will be characterized for MOG-specific T cell and humoral responses, and requirement for B cell-T cell cooperation in EAE. Serum MOG-specific Abs from MOGAD patients will also be tested for pathogenic potential in recipient humanized MOG mice. Our results should provide invaluable knowledge reg...