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Waters, P.

Publications and source records attributed to Waters, P..

2 recordsLinked to original sources

Permissive central tolerance plus defective peripheral checkpoints licence pathogenic memory B cells in CASPR2-antibody encephalitis

Autoimmunity affects 10% of the population. Within this umbrella, autoantibody-mediated diseases targeting one autoantigen provide a unique opportunity to comprehensively understand the developmental pathway of disease-causing B cells and autoantibodies. While such autoreactivities are believed to be generated during germinal centre reactions, the roles of earlier immune checkpoints in autoantigen-specific B cell tolerance are poorly understood. We address this concept in patients with CASPR2-autoantibody encephalitis and healthy controls. In both groups, comparable and high ([~]0.5%) frequencies of unmutated CASPR2-reactive naive B cells were identified. By contrast, CASPR2-reactive memory B cells were exclusive to patients, and their B cell receptors demonstrated affinity-enhancing somatic mutations with heterogenous binding kinetics. These effector molecules possessed epitope-dependent pathogenic effects in vitro neuronal cultures and in vivo. The unmutated common ancestors of these memory B cells showed a distinctive balance between strong CASPR2 reactivity and very limited binding across the remaining human proteome. Our results are the first to propose mechanisms underlying autoantigen-specific tolerance in humans. We identify permissive central tolerance, defective peripheral tolerance and heterogenous autoantibody binding properties as sequential pathogenic steps which licence CASPR2-directed pathology. By leveraging the basic immunobiology, we rationally direct tolerance-restoring approaches in CASPR2-antibody diseases. This paradigm is applicable across autoimmune conditions.

immunology↗

Isolation and characterization of rare circulating autoantibody-producing cells from patients with muscle-specific kinase myasthenia gravis

Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by autoantibodies that bind to functional membrane proteins at the neuromuscular junction. Most patients have autoantibodies to the acetylcholine receptor (AChR), but a subset of patients instead have autoantibodies to muscle specific tyrosine kinase (MuSK). MuSK is an essential component of the Agrin/Lipoprotein receptor-related protein 4(LRP4)/MuSK/downstream of tyrosine kinase 7 (DOK7) pathway that is responsible for synaptic differentiation, including clustering of AChRs at the neuromuscular junction, both during development and in adult muscle. Nerve-released Agrin binds to LRP4 which then binds to MuSK, stimulating autophosphorylation and recruitment of DOK7 to complete the membrane component of the pathway. Serum-derived IgG4 subclass MuSK autoantibodies prevent the binding of LRP4 to MuSK, subsequently impairing autophosphorylation, resulting in the loss of Agrin-induced AChR clustering in the mouse myotube-forming C2C12 line. Although this autoimmune mechanism appears well understood, MuSK autoantibodies from patients are polyclonal. Most are IgG4 but IgG1, 2 and 3 are also present and can achieve similar results on AChR clustering. In addition, most bind the first Ig-like domain in MuSK, however some patients harbor serum autoantibodies that recognize other domains in MuSK. We sought to establish individual MuSK IgG clones so that the disease mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from MuSK MG patients with a fluorescent-tagged MuSK antigen multimer and generated a panel of human monoclonal autoantibodies (mAbs) from these cells. We produced 77 mAbs from single B cells collected from six MuSK MG patients. Here we focused on three highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells and at mouse neuromuscular junctions where they co-localized with AChRs. These three IgG isotype mAbs (two IgG4 and one IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The three MuSK mAbs inhibited Agrin induced-AChR clustering in C2C12 myotubes, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation. This approach for ex vivo isolation of MuSK MG autoantibody-producing cells and production of human recombinant mAbs has identified distinct autoantibody specificities and likely divergent effector mechanisms. Collectively, these findings will enable a better understanding of MuSK autoantibody-mediated pathology.

immunology↗