Abstract
Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease. Insulin seems to be a critical antigen recognized by autoreactive T cells. In this study, we performed T cell epitope mapping of insulin using serial overlapping peptides in Japanese patients with T1D. Serial overlapping insulin peptides comprising 23 peptides, which were each 15-amino acid long, were prepared based on insulin sequence. Cytokine secretion from peripheral T cells against these peptides was studied by enzyme-linked immunospot (ELISPOT) assay in 18 patients with recent-onset T1D and 12 patients with established T1D, and compared with 17 healthy control subjects. In ELISPOT assay, IFN-γ-secreting T cells, but not IL-4, against several insulin peptides were observed in 77.8% of patients with recent-onset T1D, 50.0% of patients with established T1D, and 0% of healthy control subjects. All epitopes recognized by T cells were identified in the B-chain of insulin. The most frequent epitope existed at the B10–24 region (9/18), followed by B1–15 and B11–25 regions (6/18, each), with B4–18, B9–23, and B12–26 identified in some patients. These data did not correlate with insulin autoantibodies or HLA-DRB1 of the patients. This is the first report of T cell epitope mapping using one amino acid serial overlapping peptides of insulin in T1D. ELISPOT assay revealed the frequent existence of insulin peptide-specific T cells in patients with recent-onset and established T1D. The T cell epitopes of insulin were similar but not identical in our cohort, which probably explains the difficulty encountered in prevention of human T1D by using insulin.
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Abbreviations
- ELISPOT:
-
enzyme-linked immunospot
- IA:
-
insulin antibody
- IAA:
-
insulin autoantibody
- IA-2:
-
insulinoma-associated tyrosine phosphatase-like protein-2
- NOD:
-
nonobese diabetic
- PBMCs:
-
peripheral blood mononuclear cells
- PHA:
-
phytohemagglutinin
- PPD:
-
purified protein derivative
- SI:
-
stimulation index
- T1D:
-
type 1 diabetes
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Acknowledgements
The authors thank Mr. H. Murayama and coworkers at Diagnostics Department of Yamasa Corporation for assaying IAA and IA.
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This work was supported in part by Grants-in-Aid 13670828 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Higashide, T., Kawamura, T., Nagata, M. et al. T Cell Epitope Mapping Study With Insulin Overlapping Peptides Using ELISPOT Assay in Japanese Children and Adolescents With Type 1 Diabetes. Pediatr Res 59, 445–450 (2006). https://doi.org/10.1203/01.pdr.0000200803.72985.3c
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DOI: https://doi.org/10.1203/01.pdr.0000200803.72985.3c
