PB0667 - Specific defects of immature B cells in secondary recombination drive autoantibody production in immune thrombocytopenia

Background: Immune thrombocytopenia (ITP) is characterized by over-production of antiplatelet autoantibodies with unknown mechanisms. Receptor editing is the predominant mechanism underlying central B-cell tolerance.

Aims: To elucidate the link between receptor editing and autoantibody production using ITP-patient samples and original murine models.

Methods: We performed next-generation sequencing on B cells of ITP patients and healthy controls. We used single-cell RT-PCR to clone Ig heavy and Ig light chains genes, and we produced the recombinant monoclonal antibodies (rmAbs) derived from single naïve B cells of patients and healthy controls, respectively. The reactivity of these rmAbs to platelet antigens was tested using ELISA, immunofluorescence, and flow cytometry. And we developed a novel murine model by suppressing secondary rearrangements using Elvitegravir, a potent inhibitor of RAG functions and Ig rearrangements.

Results: Our results showed that in peripheral B cells of ITP patients, the clone types and diversities of Ig heavy chains are diminished, the usage of downstream IGK variable and upstream IGK joining genes is increased, and the V-J distance in immunoglobulin kappa-chain genes is reduced.

Employing single-cell antibody cloning technique, we found high-frequency platelet-reactive naïve B cells, most of which were polyreactive, in ITP patients. IgH CDR3s with more positively charged AAs and Igκ CDR3s with longer sequences were preferentially associated with platelet+ rmAbs. And RAG expression was reduced in ITP immature B cells.

By sequential transfusion of immature or transitional B cells (isolated from the bone marrow of B6-G/R mice) in RAG-inhibited μMT mice and CD61-knockout mice, we found that impairment of RAG function in immature B rather than in later-stage increased antiplatelet antibody production.

Conclusion(s): In conclusion, we demonstrated that receptor editing is impaired in immature B cells, leading to a disruption in central B cell tolerance and resulting in the accumulation of antiplatelet and polyreactive naïve B cells in patients with ITP.