Immune destruction of beta (β) cells is a feature of type 1 diabetes (T1D), yet the pathophysiological role of autoantibodies (Abs) is poorly understood. Recently, proteomic interrogation of Abs indicates that restricted heavy (H) and light (L) chain gene usage underpins targeting of classic autoantigens in systemic autoimmunity. In the current study, we have taken a similar approach to characterise B cell clonotypes resulting in targeting of the major T1D autoantigen, IA-2. Firstly, we developed a novel ELISA assay for the major IA-2 epitopes using an MBP-fusion protein (FP) incorporating intracellular domain of IA-2. We next established ELISA-based affinity purification (AP) method using the IA-2-MBP FP to isolate enriched IA-2 specific Abs from 2 patients with verified (by RSR commercial ELISA) anti-IA-2 autoimmunity. The AP IA-2 Abs were then analysed for Ig H and L chain clonality, variable region gene usage and AA sequence using high-res electrophoretic fractionation and de-novo/database-driven analysis of AA peptides (TripleTOF 5600+ MS). We report the presence of 2 unique Ig Kappa-restricted clonotypes giving rise to IA-2 autoimmunity; namely a IGHV3.23 H chain paired with either a IGKV1.9 or IGKV3.20 L chain. Remarkably, both clones were present in AP Ig from 2 unrelated T1D patients, with shared AA replacement mutations. The Ig signatures were not present in healthy, age/sex matched controls, T1D patients negative for anti-IA-2, or Lupus. These extraordinary findings suggest the pathogenic mechanisms leading to IA-2 autoimmunity may be virtually identical across individuals, leading to the intriguing possibility of intervention in the processes underpinning the disease. Furthermore, the ability to AP and identify genuine human clonal IA-2 Abs will unlock functional studies aimed at establishing the role of these Abs in the pathophysiology of β cell loss.


T. Mendis: None. J. Wang: None. M. Pietropaolo: None. M.W. Jackson: None.

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