Identification of disease-related T cell clones and arthritogenic antigens in ankylosing spondylitis

强直性脊柱炎（AS）是一种非常普遍的慢性炎症性疾病，可影响中轴骨骼和周围关节。其发病机制尚不清楚，但值得注意的是，90%以上的患者都有一个特定的HLA等位基因，即HLA I类等位基因HLA- b *27:05（白种人）。HLA分子向T细胞呈递抗原肽并启动免疫反应。第二强的遗传标记是“ERAP1”，它是一种酶，可以将抗原肽剪切到HLA分子的结合间隙中。在其他关于AS发病机制的假说中，有一种假说认为，“致关节炎肽”是由HLA-B27分子呈递给T细胞，启动并维持炎症过程。然而，到目前为止，还没有发现这样的肽。在这里，我们建议通过绕道疾病相关的T细胞来鉴定关节炎肽。为此，我们将首先研究T细胞受体（TCR）库，并鉴定被认为与疾病相关的克隆扩增CD8+ T细胞。然后在体外表达它们的TCRs，利用重组TCRs鉴定HLA-B*27分子递呈给T细胞的抗原肽抗原。在初步研究中，我们通过下一代TCR α链和β链测序，鉴定了从5例AS伴外周性关节炎患者的滑液中分离的抗原经历CD45RO+ CD8+记忆T细胞的寡克隆T细胞扩增。在所有患者中，扩增仅限于少数克隆，来自不同AS患者的几个克隆具有高度同源的TCR链。我们计划在更大的队列中完成这些库分析，并表达约20个重组tcr，用于体外抗原搜索。为此，我们将使用我们实验室最近开发的一项新技术，该技术基于质粒编码的组合肽库和极其敏感的检测系统。我们建议鉴定HLA-B*27向CD8+ T细胞呈递的肽抗原，并研究候选的人关节炎肽在共生微生物或病原体中是否有同源物。为了研究多肽的分子特性及其在体内的生成，我们将研究ERAP1在亲本蛋白加工中的作用。为了研究候选疾病相关肽是否可以在更大的AS患者队列中检测到，我们将生成HLA-B*27肽四聚体，并测定患者和对照组外周血和其他体液中抗原特异性T细胞的频率，并计划使用PCR和HLA-B*27肽四聚体在AS患者脊柱活检样本中鉴定疾病相关T细胞克隆。鉴定致关节炎肽可能对早期诊断有影响，也可能有治疗用途。

Ankylosing spondylitis (AS) is a highly prevalent chronic inflammatory disease, which affects both the axial skeleton and peripheral joints. Its pathogenesis is unclear, but it is remarkable that significantly more than 90 percent of all patients share a particular HLA allele, namely the HLA class I allele HLA-B*27:05 (in Caucasians). HLA molecules present antigenic peptides to T cells and initiate immune responses. The second strongest genetic marker is "ERAP1", an enzyme that trims antigenic peptides into the binding cleft of HLA molecules. Amongst other hypotheses for the pathogenesis of AS, it has therefore been assumed that "arthritogenic peptides" are presented by HLA-B27 molecules to T cells that would initiate and sustain the inflammatory process. However, so far none such peptides have been identified. Here we propose to identify arthritogenic peptides by a detour via disease-related T cells. To this end, we will first study the T cell receptor (TCR) repertoire and identify clonally expanded CD8+ T cells that are putatively disease-related. Then we will express their TCRs in vitro, and use the recombinant TCRs to identify antigenic peptide antigens presented by HLA-B*27 molecules to the T cells. In preliminary studies, we have identified oligoclonal T cell expansions of antigen experienced CD45RO+ CD8+ memory T cells isolated from synovial fluid of five AS patients with peripheral arthritis by next generation sequencing of TCR alpha- and beta-chains. In all patients the expansions were limited to only a few clones and several clones from different AS patients had highly homologous TCR chains. We are planning to complete these repertoire analyses in a larger cohort and to express about 20 recombinant TCRs which will be used for in vitro antigen searches. To this end, we will use a new technology recently developed in our labs that is based on plasmid-encoded combinatorial peptide libraries and an extremely sensitive detection system. We propose to identify peptide antigens presented by HLA-B*27 to CD8+ T cells and investigate whether candidate arthritogenic human peptides have homologues in commensal microbes or pathogens. To investigate molecular properties of the peptides and their generation in vivo, we will study the role of ERAP1 in processing of the parent proteins. To study whether or not the candidate disease-related peptides are detectable in larger cohorts of AS patients, we will generate HLA-B*27-peptide tetramers and determine frequencies of antigen-specific T cells in peripheral blood and other body fluids of patients and controls and are planning to identify disease-related T cell clones in spinal biopsy samples of AS patients using PCR and HLA-B*27-peptide tetramers. Identification of arthritogenic peptides may have impact in early diagnostics and may possibly also be of therapeutic use.