A disease-associated cellular immune response in type 1 diabetics to an immunodominant epitope of insulin

Abstract

The 9-23 amino acid region of the insulin B chain (B9-23) is a dominant epitope recognized by pathogenic T lymphocytes in nonobese diabetic mice, the animal model for type 1 diabetes. We describe herein similar (B9-23)-specific T-cell responses in peripheral lymphocytes obtained from patients with recent-onset type 1 diabetes and from prediabetic subjects at high risk for disease. Short-term T-cell lines generated from patient peripheral lymphocytes showed significant proliferative responses to (B9-23), whereas lymphocytes isolated from HLA and/or age-matched nondiabetic normal controls were unresponsive. Antibody-mediated blockade demonstrated that the response was HLA class II restricted. Use of the highly sensitive cytokine-detection ELISPOT assay revealed that these (B9-23)-specific cells were present in freshly isolated lymphocytes from only the type 1 diabetics and prediabetics and produced the proinflammatory cytokine IFN-gamma. This study is, to our knowledge, the first demonstration of a cellular response to the (B9-23) insulin epitope in human type 1 diabetes and suggests that the mouse and human diseases have strikingly similar autoantigenic targets, a feature that should facilitate development of antigen-based therapeutics.

Figure 1

Type 1 diabetic patient response to insulin B_(9–23) peptide. A total of 10⁵ T lymphocytes from short-term cell lines of B_(9–23)-treated PBMCs from type 1 diabetic patients (i.e., P1–P12) were seeded per well of a 96-well round-bottom plate with 7 × 10⁴ irradiated autologous PBMCs in the presence or absence of different concentrations of insulin B_(9–23) peptide. Cells were cultured for 5 days in which each well was pulsed with [³H]thymidine for the final 18–20 hours, and the amount of incorporated radioactivity was counted. Values in all panels are the mean cpm ± SEM of triplicate cultures from one experiment representative of at least two experiments for all patients.

Figure 2

Role of HLA-DQ8 in insulin B(9–23)-specific response in a type 1 diabetic patient. A total of 10⁵ T lymphocytes from short-term cell lines of B(9–23)-treated PBMCs from type 1 diabetic patient P2, homozygous for HLA-DR4 (DRB1*0402/0405) and DQ8 (DQB1*0302), were treated in the presence or absence of 20 μg/ml anti-DQ or anti-DR blocking antibody. These antibodies were added to stimulator autologous PBMCs 30 minutes before responder T cells were added. Cells were cultured for 5 days during which each well was pulsed with [³H]thymidine for the final 18–20 hours and the amount of incorporated radioactivity was counted. Isotype control antibody had no significant effect on proliferation. Values are the mean cpm ± SEM of triplicate cultures from one experiment representative of three experiments.

Figure 3

Example of cytokine responses to insulin B_(9–23) by type 1 diabetic patients and control subjects. 3 × 10⁵ freshly isolated PBMCs from two type 1 diabetic patients (P5 and P9) and two age-matched control subjects (C12 and C13) were seeded per well of a 96-well anti–IFN-γ mAb–coated ELISPOT assay plate in the presence or absence of insulin B_(9–23) and incubated at 37°C for 24 hours. Spots representing IFN-γ–producing cells (denoted by the number in parentheses) were developed using a biotinylated anti–IFN-γ secondary antibody and avidin-labeled peroxidase with AEC substrate and quantified using the Series-1 Immunospot Analyzer.