Proinsulin C-peptide is an autoantigen in people with type 1 diabetes

Abstract

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells, found within the islets of Langerhans in the pancreas, are destroyed by islet-infiltrating T cells. Identifying the antigenic targets of beta-cell reactive T cells is critical to gain insight into the pathogenesis of T1D and develop antigen-specific immunotherapies. Several lines of evidence indicate that insulin is an important target of T cells in T1D. Because many human islet-infiltrating CD4⁺ T cells recognize C-peptide-derived epitopes, we hypothesized that full-length C-peptide (PI_33-63), the peptide excised from proinsulin as it is converted to insulin, is a target of CD4⁺ T cells in people with T1D. CD4⁺ T cell responses to full-length C-peptide were detected in the blood of: 14 of 23 (>60%) people with recent-onset T1D, 2 of 15 (>13%) people with long-standing T1D, and 1 of 13 (<8%) HLA-matched people without T1D. C-peptide-specific CD4⁺ T cell clones, isolated from six people with T1D, recognized epitopes from the entire 31 amino acids of C-peptide. Eighty-six percent (19 of 22) of the C-peptide-specific clones were restricted by HLA-DQ8, HLA-DQ2, HLA-DQ8trans, or HLA-DQ2trans, HLA alleles strongly associated with risk of T1D. We also found that full-length C-peptide was a much more potent agonist of some CD4⁺ T cell clones than an 18mer peptide encompassing the cognate epitope. Collectively, our findings indicate that proinsulin C-peptide is a key target of autoreactive CD4⁺ T cells in T1D. Hence, full-length C-peptide is a promising candidate for antigen-specific immunotherapy in T1D.

Keywords: CD4+ T cells; HLA; epitope; proinsulin; type 1 diabetes.

Fig. 1.

CD4⁺ T cell responses to C-peptide in PBMC. A CDI ≥ 3.0, dotted line, is a positive response. The means of triplicate measurements for individuals with recent-onset T1D (n = 23), long-standing T1D (n = 15), and without T1D (n = 13) are plotted. Open red symbols indicate responses from which clones were isolated. Statistical significance was determined using unpaired Student’s t test, *P < 0.05 after log transformation of the CDIs.

Fig. 2.

Analysis of epitope specificity of C-peptide–specific CD4⁺ T cell clones. CD4⁺ T cell clone responses to antigen were measured by the secretion of IFN-γ measured by ELISA, mean ± SEM of triplicate IFN-γ measurements are shown. (A) Clone H11.5 was tested against 18mer peptides (10 μM) spanning C-peptide. (B) Peptides truncated by a single amino acid. (C) Clone K9.5 doesn’t have a detectable response to the overlapping 18-mer peptides (10 μM). Clone K9.5 epitope mapping using truncated peptides (D and E) and a series of substituted peptides (F). Experiments were performed at least twice with similar results. In all figures, the parallel lines delineate the sequence of the minimum epitope determined.

Fig. 3.

Comparison of the stimulatory capacity of full-length C-peptide and 18mer peptides. Titration of full-length C-peptide (closed circles) and 18mer peptides (open squares) incorporating the cognate epitope. KJ EBV or HLA-DR4 transfected BLS cells (both at 2 × 10⁴ cells/well), were used as APC. T cell responses were measured in triplicate by IFN-γ ELISA (pg/mL). Mean (±SEM) of triplicate IFN-γ measurements are shown. Dose–response curves for the following clones are shown: K9.6 (A), H11.5 (B), D1.1 (C), E2.3 (D), T17.1 (E), K9.5 (F), B3.3 (G), H3.3 (H). Amino acid substitution experiments (50 μM peptide), where each amino acid is changed to alanine or lysine, reveal that clones T17.1 (I) and H3.3 (J) recognize contiguous 7- to 10-aa-long epitopes. One representative of at least two experiments is shown.