Key Points Proinsulin contains several T cell epitopes recognized by human CD4+ T cells. HLA-DQ2–restricted proinsulin epitopes B18-26 and C22-33 are naturally processed. Proinsulin-reactive TCR sequences are enriched in pancreatic lymph node samples. Visual Abstract Type 1 diabetes (T1D) is a T cell–mediated autoimmune disease in which the insulin-producing β cells within the pancreas are destroyed. Identification of target Ags and epitopes of the β cell–reactive T cells is important both for understanding T1D pathogenesis and for the rational development of Ag-specific immunotherapies for the disease. Several studies suggest that proinsulin is an early and integral target autoantigen in T1D. However, proinsulin epitopes recognized by human CD4+ T cells have not been comprehensively characterized. Using a dye dilution–based T cell cloning method, we generated and characterized 24 unique proinsulin-specific CD4+ T cell clones from the peripheral blood of 17 individuals who carry the high-risk DR3-DQ2 and/or DR4-DQ8 HLA class II haplotypes. Some of the clones recognized previously reported DR4-restricted epitopes within the C-peptide (C25-35) or A-chain (A1-15) of proinsulin. However, we also characterized DR3-restricted epitopes within both the B-chain (B16-27 and B22-C3) and C-peptide (C25-35). Moreover, we identified DQ2-restricted epitopes within the B-chain and several DQ2- or DQ8-restricted epitopes within the C-terminal region of C-peptide that partially overlap with previously reported DQ-restricted epitopes. Two of the DQ2-restricted epitopes, B18-26 and C22-33, were shown to be naturally processed from whole human proinsulin. Finally, we observed a higher frequency of CDR3 sequences matching the TCR sequences of the proinsulin-specific T cell clones in pancreatic lymph node samples compared with spleen samples. In conclusion, we confirmed several previously reported epitopes but also identified novel (to our knowledge) epitopes within proinsulin, which are presented by HLA class II molecules associated with T1D risk.

中文翻译：

---

1 型糖尿病相关 HLA II 类分子限制的胰岛素原 T 细胞表位的表征

要点 胰岛素原含有几个被人类 CD4+ T 细胞识别的 T 细胞表位。HLA-DQ2 限制性胰岛素原表位 B18-26 和 C22-33 是自然加工的。胰腺淋巴结样本中富含胰岛素原反应性 TCR 序列。视觉摘要 1 型糖尿病 (T1D) 是一种 T 细胞介导的自身免疫性疾病，其中胰腺内产生胰岛素的 β 细胞被破坏。鉴定 β 细胞反应性 T 细胞的靶抗原和表位对于了解 T1D 发病机制和合理开发针对该疾病的抗原特异性免疫疗法都很重要。几项研究表明，胰岛素原是 T1D 早期和不可或缺的靶自身抗原。然而，人类 CD4+ T 细胞识别的胰岛素原表位尚未得到全面表征。使用基于染料稀释的 T 细胞克隆方法，我们从携带高危 DR3-DQ2 和/或 DR4-DQ8 HLA II 类单倍型的 17 名个体的外周血中生成并表征了 24 个独特的胰岛素原特异性 CD4+ T 细胞克隆。一些克隆识别先前报道的胰岛素原 C 肽 (C25-35) 或 A 链 (A1-15) 内的 DR4 限制性表位。然而，我们还对 B 链（B16-27 和 B22-C3）和 C 肽（C25-35）内的 DR3 限制性表位进行了表征。此外，我们鉴定了 B 链内的 DQ2 限制性表位和 C 肽 C 末端区域内的几个 DQ2 或 DQ8 限制性表位，这些表位与先前报道的 DQ 限制性表位部分重叠。DQ2 限制性表位中的两个，B18-26 和 C22-33，被证明是由全人胰岛素原自然加工而成。最后，我们观察到与脾脏样本相比，胰腺淋巴结样本中与胰岛素原特异性 T 细胞克隆的 TCR 序列匹配的 CDR3 序列频率更高。总之，我们确认了几个先前报道的表位，但也确定了胰岛素原内的新表位（据我们所知），这些表位由与 T1D 风险相关的 HLA II 类分子呈现。