Division of Rheumatology, Department of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden.
Translational Research Program, BRI at Virginia Mason, Seattle, (WA), USA.
BMC Immunol. 2020 May 18;21(1):27. doi: 10.1186/s12865-020-00357-w.
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.
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.
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.
HLA Ⅱ类四聚体可用于体外计数和表型分析抗原特异性 CD4+T 细胞。它们在过敏、疫苗接种和自身免疫性疾病等领域的应用越来越广泛。类风湿关节炎（RA）是一种慢性自身免疫性疾病，已经描述了许多自身抗原。
我们使用多参数流式细胞术开发了一种多 HLA Ⅱ类四聚体方法，以同时研究 RA 患者样本中的几种抗原特异性。我们专注于先前描述的来自α-烯醇化酶、纤维蛋白原-β、波形蛋白以及软骨中间层蛋白（CILP）的 HLA-DRB1*04:01 限制性 T 细胞表位。首先，我们在健康供体（n=7）的外周血中检查了实验内变异性和检测方法的灵敏度。然后，我们在重复采血的 RA 患者队列（n=14）中验证了其稳健性和灵敏度。接下来，我们在两种不同的情况下应用了我们的方法。我们评估了血清阳性关节痛（n=5）和早期 RA 患者（n=5）的淋巴组织，并在有发生 RA 风险的个体中证明了自身反应性 T 细胞的存在。最后，我们研究了早期 RA 患者（n=10）的外周血，发现 6 个月随访时达到最低疾病活动度（DAS28<2.6）的患者组中，瓜氨酸特异性 T 细胞的频率降低。
我们的研究表明，开发了一种敏感的四聚体面板，允许同时在包括 RA“风险”患者在内的体外患者样本中对抗原特异性 T 细胞进行特征分析。这种多四聚体方法可用于具有已知 HLA 限制元件和多个候选抗原的任何疾病的纵向免疫监测。
