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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., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR-MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR-MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR-MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein-Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR-MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR-MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR-MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We have developed a B cell immortalization method for low B cell numbers per well using simultaneous B cell stimulation by CpG2006 and B cell infection by Epstein-Barr virus (EBV), followed by an additional CpG2006 and interleukin-2 (IL-2) stimulus. Using this method, immunoglobulin G (IgG)-producing immortalized B cell lines were generated from peripheral blood IgG(+)CD22(+) B cells with an efficiency of up to 83%. Antibody can already be obtained from the culture supernatant after 3-4 weeks. Moreover, clonality analysis demonstrated monoclonality in 87% of the resulting immortalized B cell lines. Given the high immortalization efficiency and monoclonality rate, evidence is provided that no further subcloning is necessary. An important application of this B cell immortalization method is the characterization of (autoreactive) antibodies from patients with autoimmune disease. This could eventually lead to the identification of new autoantigens, disease markers or targets for therapy., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The receptor-associated protein of the synapse (rapsyn) is required for anchoring and stabilizing the nicotinic acetylcholine receptor (AChR) in the postsynaptic membrane of the neuromuscular junction (NMJ) during development. Here we studied the role of rapsyn in the maintenance of the adult NMJ by reducing rapsyn expression levels with short hairpin RNA (shRNA). Silencing rapsyn led to the average reduction of the protein levels of rapsyn (31% loss) and AChR (36% loss) at the NMJ within 2 weeks, corresponding to previously reported half life of these proteins. On the other hand, the sodium channel protein expression was augmented (66%) in rapsyn-silenced muscles. Unexpectedly, at the ultrastructural level a significant increase in the amount of secondary folds of the postsynaptic membrane in silenced muscles was observed. The neuromuscular transmission in rapsyn-silenced muscles was mildly impaired. The results suggest that the adult NMJ can rapidly produce postsynaptic folds to compensate for AChR and rapsyn loss.

The BB rat is a well-established model for spontaneous thyroid autoimmune disease. Since antigen presentation in thyroid autoimmunity is still a matter of debate, we studied the presence of antigen-presenting dendritic cells in the thyroid of the BB/O rat during the development of the disease in relation to the presence of other leucocytes and the aberrant expression of class II MHC determinants by thyrocytes. Thyroid glands, as well as thyroid-draining lymph nodes, were investigated in enzyme histochemistry and immune histochemistry. The appearance of anti-colloid antibodies in the circulation at 6 weeks of age was accompanied by an increase in the weight of the thyroid-draining cervical lymph nodes, which contained many anti-thyroglobulin-producing plasma cells. The only noteworthy event in the thyroid gland in this early stage of the disease was an increase in the number of dendritic cells. T cells, B cells, and plasma cells were virtually absent from the thyroid, and thyrocytes were invariably negative for class II MHC determinants. Only after 18 weeks of age, when large accumulations of dendritic cells, B lymphocytes, and T lymphocytes were seen in 40% of the BB thyroids, could some class II MHC positive thyroicytes be observed. At this stage the thyroid also contained some anti-thyoglobulin-producing plasma cells. Our observations suggest that dendritic cells play a role in antigen presentation in the early stages of the thyroid autoimmune response.