Identification of Unique Antigenic Determinants in the Amino Terminus of IA-2 (ICA512) in Childhood and Adult Autoimmune Diabetes: New Biomarker Development

Abstract

Objective: The characterization of diverse subtypes of diabetes is a dynamic field of clinical research and an active area of discussion. The objective of this study was to identify new antigenic determinants in the neuroendocrine autoantigen IA-2 (ICA512) and assess whether circulating autoantibodies directed to new IA-2 epitopes identify autoimmune diabetes in young and adult populations with diabetes.

Research design and methods: Clinically diagnosed patients with type 2 diabetes (n = 258; diabetes duration: 0.01-31 years) were evaluated using a new biomarker detecting autoantibodies directed to the extracellular domain of the neuroendocrine autoantigen IA-2 (IA-2ec). The proportion of IA-2ec autoantibodies was also evaluated in newly diagnosed patients with type 1 diabetes (n = 150; diabetes duration: 0.04-0.49 years). In addition, IA-2 (intracellular domain), GAD65, and zinc transporter 8 autoantibodies were assayed.

Results: IA-2ec autoantibodies were detected in patients with type 1 diabetes and, surprisingly, in 5% of patients with type 2 diabetes without serologic responses to other IA-2 antigenic epitopes or other islet autoantigens. We also assessed the ability of IA-2ec-derived peptides to elicit CD4⁺ T-cell responses by stimulating peripheral blood mononuclear cells from patients with type 1 diabetes (n = 18) and HLA-matched healthy subjects (n = 13) with peptides and staining with the peptide/DQ8-specific tetramers, observing disease-associated responses to previously unreported epitopes within IA-2ec.

Conclusions: We developed a new antibody biomarker identifying novel antigenic determinants within the N terminus of IA-2. IA-2ec autoantibodies can be detected in patients with type 1 diabetes and in a subgroup of adult autoimmune patients with type 2 diabetes phenotype negative for conventional islet autoantibody testing. These observations suggest that islet autoimmunity may be more common in clinically diagnosed type 2 diabetes than previously observed.

Figure 1

A: Autoantibodies directed to IA-2ec (amino acids 26–577) can be detected in patients with type 1 and 2 diabetes. Dashed line represents the cutoff point for the assays. Eight percent of patients with type 1 diabetes and 5% of patients with type 2 diabetes exhibited antibody responses to IA-2ec. *P = 0.0371; **P = 0.0023. Two-dimensional SDS-PAGE fractionation of IA-2ec immunoprecipitates followed by autoradiography illustrates that serum from one patient with type 1 diabetes and from another patient with type 2 diabetes (blot, lanes 1 and 2, respectively) strongly reacted with a 60-kDa band (IA-2ec), unlike serum from a healthy volunteer (blot, lane 3). B: The majority of patients with clinically diagnosed type 2 diabetes carrying IA-2ec autoantibodies tested negative for IA-2ic, GAD65, ZnT8, and mIAA. *Positive for both IA-2ec and GAD65 autoantibodies.

Figure 2

Venn diagrams of autoantibody combinations in autoantibody-positive patients with type 1 and 2 diabetes. The frequency of autoantibodies in patients with type 1 (A) and type 2 (B) diabetes (IA-2ec, IA-2ic, GAD65, and ZnT8 autoantibodies). Intersecting regions (lightly shaded) indicate the number of patients positive for different combinations of islet autoantibodies. Insulin autoantibodies were not included in this analysis because many patients were on insulin therapy. *Both patients with type 2 diabetes were negative for IA-2ec antibody (Ab) autoantibodies. **All patients with type 2 diabetes were negative for IA-2ic autoantibodies. †One out of 16 patients with type 1 diabetes was IA-2ec autoantibody positive. ‡One out of two patients with type 1 diabetes was IA-2ic autoantibody positive.

Figure 3

Specific inhibition of autoantibody binding to IA-2ec (circles) and to IA-2ic (squares) with sera from patients with type 1 and 2 diabetes. A: Binding to IA-2ec of serum of a patient with type 2 diabetes positive for IA-2ec antibodies was not inhibited by preincubation with unlabeled IA-2ic, whereas it was specifically inhibited by preincubation with unlabeled IA-2ec (left panel). Binding to labeled IA-2ic of serum of a patient with type 2 diabetes positive for IA-2ic antibodies was not inhibited by preincubation with unlabeled IA-2ec, but it was specifically inhibited by preincubation with unlabeled IA-2ic (right panel). B and C: Specific inhibition of autoantibody binding to IA-2ec and IA-2ic in patients with type 1 diabetes with different combinations of these autoantibodies. Ab, antibody.

Figure 4

CD4⁺ T-cell responses to IA-2ec peptides are elevated in subjects with type 1 diabetes. A: Representative tetramer staining of T cells using tetramers loaded with a negative control peptide (chromogranin A 198 [ChgA₁₉₈]), DQ8-restricted IA-2 peptides (as indicated on each panel), or an influenza peptide (MP-97) after 2 weeks of in vitro expansion. B: Comparison of IA-2–specific T-cell responses measured in 18 subjects with type 1 diabetes (T1D) and 13 HLA-matched control subjects (all with DQ8⁺ haplotypes) by tetramer staining (combined percentages for all four epitopes). Responses >1.1% (1 SD above the mean response for controls, indicated by the dashed line) were above the positive threshold. The magnitude of responses to IA-2ec peptides were significantly higher in subjects with type 1 diabetes (P = 0.0017, two-tailed t test with Welch’s correction).