Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden.
Amsterdam Rheumatology and Immunology Center, and Academic Medical Center, Amsterdam, The Netherlands.
Arthritis Rheumatol. 2016 May;68(5):1124-35. doi: 10.1002/art.39553.
Antibodies against citrullinated type II collagen (Cit-CII) are common in the sera and synovial fluid of patients with rheumatoid arthritis (RA); however, the known T cell epitope of CII is not dependent on citrullination. The aim of this study was to identify and functionally characterize the Cit-CII-restricted T cell epitopes that are relevant to RA.
Peripheral blood mononuclear cells (PBMCs) from HLA-DRB110:01-positive patients with RA and healthy donors were stimulated in vitro with candidate CII peptides. CD154 up-regulation was measured as a marker of antigen-specific activation, and anti-HLA-DR-blocking experiments confirmed HLA restriction. Cytokine production was measured using a Luminex technique. Direct peptide-binding assays using HLA-DRB110:01 and HLA-DRB1*04:01 monomeric proteins were performed. The T cell receptor (TCR) β-chain of CD154-enriched antigen-specific T cells was analyzed using high-throughput sequencing.
A novel Cit-CII peptide was identified based on its ability to activate CD4+ T cells from HLA-DRB110:01-positive individuals. When stimulated in vitro, Cit-CII autoreactive T cells produced proinflammatory cytokines. Cit-CII(311-325) bound (with low affinity) to HLA-DRB110:01 but not to HLA-DRB1*04:01, while the native form was unable to bind either protein. In addition, highly expanded clones were identified in the TCRβ repertoire of Cit-CII(311-325) -stimulated PBMCs.
These results illustrate the ability of the citrullination process to create T cell epitopes from CII, a cartilage-restricted protein that is relevant to RA pathogenesis. The exclusive binding of Cit-CII(311-325) to HLA-DRB1*10:01 suggests that recognition of citrullinated epitopes might vary between individuals carrying different RA-associated HLA-DR molecules.
抗瓜氨酸化 II 型胶原（Cit-CII）抗体常见于类风湿关节炎（RA）患者的血清和滑液中；然而，已知的 CII 表位并不依赖瓜氨酸化。本研究旨在鉴定和功能表征与 RA 相关的 Cit-CII 限制性 T 细胞表位。
来自 HLA-DRB110:01 阳性 RA 患者和健康供体的外周血单核细胞（PBMC）在体外用候选 CII 肽刺激。CD154 的上调被作为抗原特异性激活的标志物进行测量，抗 HLA-DR 阻断实验证实了 HLA 限制。使用 Luminex 技术测量细胞因子产生。使用 HLA-DRB110:01 和 HLA-DRB1*04:01 单体蛋白进行直接肽结合测定。使用高通量测序分析 CD154 富集的抗原特异性 T 细胞的 TCRβ 链。
基于其激活来自 HLA-DRB110:01 阳性个体的 CD4+T 细胞的能力，鉴定了一种新型 Cit-CII 肽。体外刺激时，Cit-CII 自身反应性 T 细胞产生促炎细胞因子。Cit-CII(311-325) （亲和力低）与 HLA-DRB110:01 结合，但不与 HLA-DRB1*04:01 结合，而天然形式无法与任何一种蛋白结合。此外，在 Cit-CII(311-325)刺激的 PBMC 中 TCRβ 库中鉴定出高度扩增的克隆。
这些结果说明了瓜氨酸化过程从软骨受限蛋白 CII 中产生 T 细胞表位的能力，该表位与 RA 发病机制相关。Cit-CII(311-325) 对 HLA-DRB1*10:01 的特异性结合表明，对瓜氨酸化表位的识别可能因携带不同 RA 相关 HLA-DR 分子的个体而异。
