B Cell Tolerance and BCR Signaling Dysregulation in NF155-Mediated Autoimmune Nodopathies.

Objective: Autoimmune nodopathies (AINs) are a group of rare, acquired autoimmune neuropathies with distinct clinical features and the presence of circulating autoantibodies - often of the immunoglobulin G4 (IgG4) subclass - targeting proteins at the node of Ranvier. Defects in B cell tolerance checkpoints have been implicated in several autoimmune diseases. Prior work identified defective B cell tolerance-reflected by a high frequency of self-reactive naïve B cells-in patients with MuSK-positive myasthenia gravis (MG), mediated by IgG4 autoantibodies. Here, we investigated whether tolerance defects exist in neurofascin-155-mediated AIN (NF155-AIN), similar to MuSK+ MG. Additionally, we analyzed B and T cell transcriptomics and interactions at the single-cell level to explore the underlying pathomechanism. Methods: Using a well-established assay, we assessed B cell tolerance fidelity by generating recombinant antibodies from new emigrant (NE) and mature naïve (MN) B cell populations- directly downstream of key tolerance checkpoints-from three NF155-AIN patients, and testing these antibodies for polyreactivity and autoreactivity, thereby determining the frequency of polyreactive and autoreactive B cells. The transcriptome of peripheral blood mononuclear cells (PBMC) was studied, with a special focus on naïve B cells and CD4+ T cells at the single-cell level, along with characterization of cell-cell interactions. Results: NF155-AIN patients have an elevated frequency of polyreactive B cells in the NE (37.4% compared to 9.7% in healthy controls (HCs), p = 0.03) and MN (31.5% compared to 10.5% in HCs, p = 0.03) compartments with increased B cell clones expressing autoreactive antibodies, consistent with a breach in early tolerance checkpoints. We observed abnormal B cell receptor (BCR) signaling characterized by low CD79B, CSK, BLNK, and BTK expression, which may contribute to a breach in B cell tolerance. We also observed evidence of impaired follicular helper T cells (Tfh) and regulatory T cells (Treg), which may limit the normal development and suppression of autoreactive B cells. Moreover, comparative gene expression analysis of B cells and CD4+ T cells from three patients with chronic inflammatory demyelinating polyneuropathy (CIDP) -a related autoimmune neuropathy- confirmed that these differences are largely specific to NF155-AIN, supporting a distinct pathophysiology in this subset. Conclusion: These findings demonstrated a breach in early B cell tolerance checkpoints, defective BCR signaling, and disrupted T cell-B cell interactions in NF155-AIN, all of which may contribute to the development of pathogenic autoreactivity. These immunologic abnormalities appear distinct from those seen in CIDP, supporting NF155-AIN as a unique immunopathologic entity.