BACKGROUND HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterisation of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described. RESULTS Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-β, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n = 7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n = 14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n = 5) and early RA patients (n = 5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n = 10) and found that the group of patients achieving minimum disease activity (DAS28 < 2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells. CONCLUSIONS Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterisation of antigen-specific T cells in ex vivo patient samples including RA 'at risk' subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.

中文翻译：

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瓜氨酸反应性T细胞的多HLA II类四聚体分析和类风湿关节炎的早期治疗反应。

背景技术HLA II类四聚体可用于抗原特异性CD4 + T细胞的离体计数和表型表征。它们越来越多地应用于变态反应，疫苗接种和自身免疫性疾病等环境中。类风湿关节炎（RA）是一种慢性自身免疫性疾病，已针对其描述了许多自身抗原。结果使用多参数流式细胞仪，我们开发了一种多HLA II类四聚体方法，以同时研究RA患者样品中的几种抗原特异性。我们专注于先前描述的α-烯醇酶，纤维蛋白原-β，波形蛋白以及软骨中间层蛋白（CILP）的瓜氨酸化HLA-DRB1 * 04：01限制性T细胞表位。首先，我们检查了测定间的变异性和健康捐献者外周血中测定的敏感性（n = 7）。下一个，我们证实了一组重复抽血的RA患者的鲁棒性和敏感性（n = 14）。然后，我们在两个不同的设置中应用了我们的方法。我们评估了血清阳性的关节痛（n = 5）和早期RA患者（n = 5）的淋巴组织，并可以在有发展RA风险的个体中证明自身反应性T细胞。最后，我们研究了早期RA患者（n = 10）的外周血，发现在6个月的随访中达到最小疾病活动（DAS28 <2.6）的患者组显示瓜氨酸特异性T细胞的频率降低。结论我们的研究证明了灵敏的四聚体研究小组的发展，该研究可以同时表征离体患者样品中的抗原特异性T细胞，包括RA高危人群。