Characterization of peripheral and lesional single B cell autoreactivity in patients with Sjögren’s syndrome and rheumatoid arthritis

Abstract

Sjögren’s syndrome (SS) and rheumatoid arthritis (RA) are characterised by breach of self-tolerance with high affinity circulating autoantibodies and peripheral B cell disturbances in the naïve and memory B cell compartments. In addition, both SS and RA develop functional ectopic B cell follicles in the respective target organs, i.e. the salivary glands and the joint synovium, whereby autoreactive B cell undergo antigen selection and affinity maturation. However, the exact stage at which errors in B cell tolerance checkpoints accumulate is unknown. In this PhD project, I amplified and sequenced Ig VH and VL gene transcripts from single B cells which were FACS sorted either from the peripheral blood of SS patients or from the RA synovium. Healthy donors (HD) were used as controls. Subsequently, I cloned and expressed recombinant monoclonal antibodies displaying identical antigenic specificity of the original B cells. Finally, I tested the poly- and autoreactivity profile of these antibodies against SS and RA-associated autoantigens. In SS, I analysed 353 VH and 293 VL sequences and obtained 114 recombinant antibodies from circulating naïve (n=66) and memory (n=48) B cells of 4 SS patients and compared their autoreactive and polyreactive profile to 45 naïve clones from 2 HD. Analysis of the VH and VL gene usage showed no significant differences between SS and HD. Conversely, I observed accumulation of circulating autoreactive naïve B cells in SS as demonstrated by Hep-2 cells, ENA, Ro/SSA and/or La/SSB reactivity. The elevated frequency of autoreactive naïve B cells in the circulation of SS patients supports the existence of early defects in B cell tolerance checkpoints in this condition In RA, I analysed the Ig gene repertoire and the VH gene somatic mutation rate of 139 VH and 175 VL sequences of synovial CD19+ B cells which demonstrated evidence of antigen selection and hypermutated alpha>gamma>mu VH chains with presence of intra-synovial clonal diversification. Recombinant antibodies from synovial B cell clones were then screened for reactivity towards citrullinated antigens with a plan for a wider analysis using autoantigen microarrays. Overall, these results highlighted the existence of B cell abnormalities and loss of tolerance for self-antigens both in the peripheral and/or lesional compartment of SS and RA. Further analysis of the fine specificity and pathogenicity of recombinant antibodies from autoreactive B cells will be invaluable in order to dissect the mechanisms and the antigens driving the development and the persistence of autoimmunity in RA and SS.