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Rheumatoid arthritis (RA) is a chronic autoimmune disease particularly affecting synovial joints. Anti-citrullinated protein autoantibodies (ACPA) are detected in the serum of about 2/3 of RA patients and are being used to classify the disease. These autoantibodies may occur years before any signs of arthritis which implies that they are a cause rather than a consequence of disease. By using modified autoantigens to detect ACPAs in large patient cohorts and by purifying polyclonal autoantibodies from patients, the hunt for disease promoting autoantigens has been going on for decades. However, the relationship between ACPA specificity and any functional effects remains unclear. In order to understand the evolution, specificity, and function of autoreactive B cells in RA, the focus of this thesis is the generation of monoclonal antibodies (mAbs) from paired variable heavy- and light-chain immunoglobulin (Ig) sequences from identified single B cells from various RA tissue compartments. We have identified citrulline-reactive autoantibody producing plasma cells in the synovium of RA patients with established disease. By generating single plasma cell derived mAbs, we learned that such autoantibodies may be directly involved in the pathogenesis of RA by promoting bone degrading osteoclasts. The mAbs were all multireactive to citrulline-peptides and citrullinated proteins, but with unique distinct binding patterns. We recognized glycine in +1 position to the citrulline and a fraction of the citrulline-reactive mAbs cross-reacted with carbamyl-peptides. In addition, the identified plasma cells displayed features of high somatic mutations and fragment antigen binding (Fab) variable N-glycosylation sites introduced by affinity maturation. By analyzing a selection of RA patient B cell derived mAbs for reactivity against apoptotic cells and activated neutrophils, we learned that a subset of the citrulline reactive mAbs bound nuclear antigens. Interestingly, a fraction of these m

Objective: Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross-reactivity. Methods: IgG-secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full-length mAb (n = 93) and also as germline-reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated. Results: Four unrelated anti-citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell-derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay. Conclusion: These findings suggest that the high level of cross-reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints.

QC 20190822

Rheumatoid Arthritis (RA) is a prevalent autoimmune disease characterized by inflammation of peripheral joints. Patients can be subdivided by the presence or absence of Rheumatoid Factor and anti-citrullinated protein antibodies (ACPA) in their circulation. Inflammation of the joint tissue is associated with infiltration of leukocytes from the blood, which can result in generation of lymphoid structures composed of B and T cells. Previous studies have shown that both memory B cells and antibody-secreting plasma cells populate the rheumatic joint tissue when captured from established and often long-standing disease. However, it has remained unclear, whether these cells are autoreactive and whether the associated lymphoid structures are present at the site of inflammation already at the time of diagnosis. Here, we used an integrated single cell and spatial transcriptomic approach to study B and plasma cells in synovial tissue of ACPA- and ACPA+ RA patients at this early time point. We found evidence for T cell help to B cells and presence of memory B and plasma cell pools in ACPA- as well as in ACPA+ RA. Our results demonstrated common supportive microenvironments in both patient subgroups, clonal relationships between the memory B and plasma cell pools and autoreactivity within the plasma cell compartment. These findings challenge our understanding of the dynamics of local adaptive immune responses in the RA joint of ACPA- and ACPA+ patients at the time of diagnosis, with direct implications for B and T cell targeting therapies for both patient subgroups., QC 20210322