Session Type: Abstract Submissions (ACR)
Background/Purpose: Elevated titers of serum autoantibodies expressing the 9G4 idiotype are highly specific for SLE and correlate with disease activity and clinical manifestations. 9G4+ antibodies have been shown to be reactive to a wide variety of autoantigens, including B cells, dsDNA, and cardiolipin. In this study we analyze the molecular characteristics that contribute to this autoreactivity.
Methods: A panel of 9G4+ monoclonal antibodies was generated from single FACS-sorted naïve (CD19+IgD+CD27-CD24+CD38+) and isotype-switched memory (CD19+IgD-CD27+) B cells from SLE patients and healthy controls. Site-directed mutagenesis of the hydrophobic patch and light chain exchange were performed. Monoclonal antibodies were tested by ANA, dsDNA, and cardiolipin ELISA. Reactivity to B cells and apoptotic cells was determined by flow cytometry. The contribution of the hydrophobic patch, the HCDR3, and light chains to binding of the various self-antigens was analyzed.
Results: A significant percentage of 9G4+ monoclonal antibodies were reactive to autoantigen, including Hep-2 nuclear antigens (65%), B cells (48%), apoptotic cells (22%), dsDNA (9%), and cardiolipin (7%). Strong binding to apoptotic cells, dsDNA, and cardiolipin was more common among antibodies derived from SLE memory cells. Site-directed mutagenesis showed that B cell binding is mediated by the VH4-34-encoded FR1 hydrophobic patch (which also determines the expression of the 9G4 idiotype). By contrast, autoreactivity to the other SLE-related antigens is patch-independent and is actually enhanced by elimination of the 9G4 idiotype. In addition, apoptotic cell and ANA-dsDNA reactivity correlate with the charge of the HCDR3, while B cell binding does not. Finally, light chains can substantially change VH4-34-associated autoreactivity.
Conclusion: Our findings show that 9G4+ antibodies are reactive to several antigens important in SLE pathogenesis, and bind antigen in two fundamentally different ways. We also show that the intrinsic autoreactivity imprinted by the expression of 9G4+ VH4-34 heavy chains can be substantially modulated by somatic hypermutation and light chain replacement.
A. S. Chida,
D. G. Adlowitz,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/molecular-analysis-of-9g4-antibodies-in-systemic-lupus-erythematosus/