Most polyreactive and antinuclear antibodies are removed from the human antibody repertoire during B cell development. To elucidate how B cell receptor (BCR) signaling may regulate human B cell tolerance, we tested the specificity of recombinant antibodies from single peripheral B cells isolated from patients suffering from X-linked agammaglobulinemia (XLA). These patients carry mutations in the Bruton's tyrosine kinase (BTK) gene that encode an essential BCR signaling component. We find that in the absence of Btk, peripheral B cells show a distinct antibody repertoire consistent with extensive secondary V(D)J recombination. Nevertheless, XLA B cells are enriched in autoreactive clones. Our results demonstrate that Btk is essential in regulating thresholds for human B cell tolerance.

Impaired immune functions leading to primary immunodeficiencies often correlate with paradoxical autoimmune complications; patients with hyper-IgM syndromes who are deficient in activationinduced cytidine deaminase (AID), which is required for classswitch recombination and somatic hypermutation, are prone to develop autoimmune diseases. To investigate the impact of AIDdeficiency on early B-cell tolerance checkpoints in humans, we tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from AID-deficient patients. New emigrant/ transitional and mature naive B cells from AID-deficient patients express an abnormal Ig repertoire and high frequencies of autoreactive antibodies, demonstrating that AID is required for the establishment of both central and peripheral B-cell tolerance. In addition, B-cell tolerance was further breached in AID-deficient patients as illustrated by the detection of anti-nuclear IgM antibodies in the serum of all patients. Thus, we identified a major and previously unsuspected role for AID in the removal of developing autoreactive B cells in humans.

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Objective Treatment-naïve rheumatoid arthritis (RA) patients suffer from defective early B cell tolerance checkpoints and fail to remove developing autoreactive B cells. However, it is unclear if these B cell tolerance checkpoint defects are primary to the disease or the result of ongoing inflammatory processes in these patients. The aim of this study is to assess the impact of standard immunosuppressive treatments, methotrexate and anti-TNFα agents, on early B cell tolerance checkpoints in RA patients. Methods We tested the reactivity of recombinant antibodies cloned from single B cells from RA patients pre- and post-therapy with methotrexate and/or anti-TNFα agents. We were able thereby to determine the evolution of the frequency of autoreactive clones in the mature naïve B cell compartment of RA patients before and after treatment. Results We found that post-treatment frequencies of autoreactive mature naïve B cells in the blood of RA patients were elevated and remained similar to those of the same patients pre-treatment. Conclusion Despite patients’ improvement, methotrexate and anti-TNFα agents do not correct the accumulation of peripheral autoreactive mature naïve B cells in RA patients, suggesting that inflammation is not responsible for defective early B cell tolerance checkpoints in RA.

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