Defective Central B Cell Tolerance In Primary Immune Thrombocytopenia

Background:Primary immune thrombocytopenia (ITP) is a common bleeding disorder characterized by low platelet counts. One hallmark of ITP is the production of platelet-associated immunoglobulin (PAIg), resulting in accelerated platelet consumption and impaired platelet production. Nevertheless, the underlying mechanism that accounts for autoantibody production is not clear.Both central and peripheral self-tolerance for B cells are needed to avoid production of auto-reactive antibodies. Mechanisms that influence the periperal tolerance and facilitate autoreactive B cell survival in peripheral are well studied. These mechanisms include skewed cytokine profile, elevated BAFF, suppressed regulatory T cells, antigen-presenting cell dysfunction, and infections. In contrast, the status of the early B cell tolerance is unknown. We expect that the central tolerance for B cells in ITP patients is defective,which has great significance in improving the mechanism of autoimmunity in ITP.Objective:To gain an insight of the mechanism of defective central B cell tolerance in ITP, we study the immunoglobulin repertoire in naive B cell compartment and test the anti-platelet reactivity of recombinant antibodies by ELISA in primary ITP patients.Methods:Peripheral blood was taken from health adults and ITP patients. Peripheral blood mononuclear cells(PBMCs) of health adults and ITP patients were isolated from peripheral blood by Ficoll Hypaque method, and naive B cells were isolated from the PBMCs by Magnetiel Labelled Bead Cell Separation(MACS) method. we performed single cell RT-PCR analysis to study the immunoglobulin repertoire in naive B cell compartment in primary ITP patients. The anti-platelet reactivity of recombinant antibodies is tested by ELISA. The sequences were analyzed by programs including IMGT, IgBLAST, and VH Replacement Analyzer(VHRFA-1).Result:The analysis of testing the anti-platelet reactivity of recombinant antibodies by ELISA shows that the ratio of platelet-associated immunoglobulin (PAIg) in recombinant antibodies derived from ITP is significantly higher than those of health adults. The analysis of the sequences of Ig genes shows that the Ig light chains preferentially use downstream Vk and upstream Jk. The frequencies of VH replacement products are increased in ITP. Furthermore, the IgH CDR3 regions are enriched with positively charged amino acids.Conclusion:1.B cells against platelet antigens are accumulated in the naive B cell compartment in ITP patients, indicating the defective early B cell tolerance in ITP patients.2.The usages of downstream Jk genes are decreased in ITP naive B cells, indicating insufficient receptor editing during early B cell development in ITP patients.3.The frequencies of VH replacement products are elevated in ITP naive B cells, indicating increased occurrence of VH replacement in ITP bone marrow. Most of the VH replacement products have extra positively charged amino acids,which can facilitate the reactivity with platelet glycoproteins that have more negative charged amino acids.4.Based on these data, we conclude that the early B cell tolerance is defective in patients with primary ITP and this may contribute to the generation of PAIg.