Higashide Takashi, Kawamura Tomoyuki, Nagata Masao, Kotani Reiko, Kimura Kayo, Hirose Masakazu, Inada Hiroshi, Niihira Shizuhiro, Yamano Tsunekazu
Department of Pediatrics, Osaka City University Graduate School of Medicien, Osaka, Japan.
Pediatr Res. 2006 Mar;59(3):445-50. doi: 10.1203/01.pdr.0000200803.72985.3c.
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-gamma-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.
1型糖尿病（T1D）是一种由T细胞介导的自身免疫性疾病。胰岛素似乎是自身反应性T细胞识别的关键抗原。在本研究中，我们使用串联重叠肽对日本T1D患者的胰岛素进行了T细胞表位作图。基于胰岛素序列制备了由23个肽组成的串联重叠胰岛素肽，每个肽长15个氨基酸。通过酶联免疫斑点（ELISPOT）分析研究了18例新发T1D患者和12例确诊T1D患者外周T细胞针对这些肽的细胞因子分泌情况，并与17名健康对照者进行了比较。在ELISPOT分析中，77.8%的新发T1D患者、50.0%的确诊T1D患者以及0%的健康对照者中观察到针对几种胰岛素肽的分泌γ干扰素的T细胞，而非分泌白细胞介素-4的T细胞。T细胞识别的所有表位均在胰岛素的B链中被鉴定。最常见的表位存在于B10 - 24区域（9/18），其次是B1 - 15和B11 - 25区域（各6/18），部分患者中还鉴定出了B4 - 18、B9 - 23和B12 - 26。这些数据与患者的胰岛素自身抗体或HLA - DRB1无关。这是首次在T1D中使用胰岛素的单氨基酸串联重叠肽进行T细胞表位作图的报告。ELISPOT分析显示新发和确诊T1D患者中频繁存在胰岛素肽特异性T细胞。在我们的队列中，胰岛素的T细胞表位相似但不完全相同，这可能解释了使用胰岛素预防人类T1D时遇到困难的原因。
