Aim
We planned this study to identify diabetogenic glutamic acid decarboxylase (GAD65) peptides possibly responsible for human leucocyte antigen (HLA)-DR3/DQ2-mediated activation of GAD65-specific CD4 T cells in type 1 diabetes (T1D).
Conclusion
GAD65 peptides, particularly those belonging to PP2, induced CD4 T cells to express IFN-γ and IL-17 cytokines in T1D patients, suggesting that group 2 peptides possibly presented by HLA-DR3 molecule to CD4 T cells shift immune balance toward inflammatory phenotype in patients. 目的:我们进行这项研究以确定与1型糖尿病(T1D)中人类白细胞抗原(HLA)-DR3/DQ2介导的谷氨酸脱羧酶(GAD65)特异性CD4 T细胞激活可能相关的致糖尿病性GAD65肽。 方法:选取与HLA-DR3/DQ2分子在体外结合强烈的前30个GAD65肽, 分为4个池组。使用这些肽来刺激研究对象的CD4 T细胞, 在16小时PBMC培养中分析CD4 T细胞对干扰素(IFN-γ), 白介素(IL)-17, 肿瘤坏死因子-α(TNF-α)和IL-10的激活表达, 使用流式细胞术进行分析。 结果:所有四个GAD65肽池(PP1-4)都导致CD4 T细胞的IFN-γ表达明显增加(p=0.003, p<0.0001, p=0.026 和 p=0.002, 分别), 只有池2在T1D患者与健康对照组之间显示出显著的IL-17表达增加(p<0.0001)。肽间组间比较显示, 在患者中, PP2组与其他组相比, IFN-γ和IL-17的表达显著增加, IL-10的表达显著降低(p<0.0001, p=0.02 和 p=0.04), 但在对照组中没有显示出明显差异。此外, 2号组肽对于HLA-DRB1 * 03-DQA1 * 05-DQB1 * 02 + 患者而言, 导致CD4 T细胞的IFN-γ和IL-17表达显著增加(p=0.002), 而IL-10表达显著降低(p=0.04)。在HLA-DRB1 * 03-DQA1 * 05-DQB1 * 02 +的最近诊断的T1D患者中, CD4 T细胞的IL-17表达显著增加(p=0.03)。 结论:GAD65肽, 尤其是属于PP2的肽段, 在T1D患者中能够诱导CD4 T细胞表达IFN-γ和IL-17细胞因子, 这表明在患者中2号组肽可能通过HLA-DR3分子递呈给CD4 T细胞, 使免疫平衡朝向炎症表型转变。.
Methods
Top 30 GAD65 peptides, found to strongly bind in silico with HLA-DR3/DQ2 molecules, were selected and grouped into four pools. The peptides were used to stimulate CD4 T cells of study subjects in 16-h peripheral blood mononuclear cell culture. CD4 T cells' stimulation in terms of interferon-gamma (IFN-γ), interleukin (IL)-17, tumor necrosis factor-alpha (TNF-α), and IL-10 expression was analyzed using flow cytometry.
Results
Although all four GAD65 peptide pools (PP1-4) resulted in significantly higher expression of IFN-γ by CD4 T cells (p = .003, p < .0001, p = .026, and p = .002, respectively), only pool 2 showed significant increase in IL-17 expression (p < .0001) in T1D patients vs healthy controls. Interpeptide group comparison for immunogenicity revealed significantly higher IFN-γ and IL-17 expressions and significantly lower IL-10 expression for PP2 compared to other groups (p < .0001, p = .02, and p = .04, respectively) in patients but not in controls. Further, group 2 peptides resulted in significant increase in CD4 T cells' expression of IFN-γ and IL-17 (p = .002 for both) and significant decrease in IL-10 (p = .04) in HLA-DRB1*03-DQA1*05-DQB1*02+ patients vs HLA-DRB1*03-DQA1*05-DQB1*02+ controls. The CD4 T cells' expression of IL-17 was significantly higher (p = .03) in recently diagnosed vs long-standing HLA-DRB1*03-DQA1*05-DQB1*02+ T1D patients.