Abstract
Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission.In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies.At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies are pathogenic.
Highlights
Various antibody-mediated autoimmune diseases are caused by the IgG1 and IgG3 subclasses [1e6], through cross-linking-induced endocytosis of cell surface antigens, and activation of complement that attacks the cell membrane resulting in cell damage and loss of antigen function [7e15]
MuSK IgG4 from each patient serum (n 1⁄4 32) bound to the MuSK-coated ELISA plates (Supplementary Fig. 1A, each data-point corresponding to a patient serum) correlated broadly with the MuSK RIA titer (r2 1⁄4 0.28; p 1⁄4 0.001) of note, two sera were negative in the MuSK IgG4 ELISA while they were strongly positive by RIA
To determine whether Fab-arm exchange of patient-derived autoantibodies could be induced in vitro, sera from muscle specific kinase myasthenia gravis (MuSK MG) patient or healthy controls were first incubated for 48 h at 37 C with an acetylcholine receptors (AChRs) monoclonal antibody of IgG4 subclass (AChR mAb IgG4-637 [25,83]), under either normal (PBS) or reducing conditions (0.5 mM GSH, Fig. 2A)
Summary
Various antibody-mediated autoimmune diseases are caused by the IgG1 and IgG3 subclasses [1e6], through cross-linking-induced endocytosis of cell surface antigens, and activation of complement that attacks the cell membrane resulting in cell damage and loss of antigen function [7e15]. This relies upon the ability of IgG1 and IgG3 subclasses to bind divalently (i.e. two antigen molecules crosslinked by the antibody‘s two identical antigen-binding fragments, Fab) to their cognate antigen on the cell surface and to activate the complement cascade via C1q
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