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

强直性脊柱炎(AS)是一种高度流行的慢性炎症性疾病，可累及轴骨和周围关节。其发病机制尚不清楚，但值得注意的是，显著超过90%的患者共享一种特定的HLA等位基因，即HL A-I类等位基因HLA-B*27：05(在高加索人中)。人类白细胞抗原分子向T细胞递呈抗原肽，启动免疫反应。第二强的遗传标记是“ERAP1”，这是一种将抗原肽修剪成与人类白细胞抗原分子结合的裂隙的酶。在AS发病机制的其他假说中，有一种假设认为“致关节炎的多肽”是由人类白细胞抗原B27分子呈递给启动和维持炎症过程的T细胞。然而，到目前为止，还没有鉴定出这样的多肽。在这里，我们建议通过疾病相关的T细胞绕道鉴定致关节炎的多肽。为此，我们将首先研究T细胞受体(TCR)谱系，并鉴定可能与疾病相关的克隆性扩增的CD8+T细胞。然后，我们在体外表达它们的TCRs，并用重组TCRs来鉴定人类白细胞抗原-B*27分子呈递给T细胞的抗原肽。在初步研究中，我们通过下一代TCRα和β链测序，从5例AS患者的滑液中分离出经历了CD45RO+CD8+记忆T细胞的抗原寡克隆T细胞扩增。在所有患者中，扩增仅限于少数克隆和来自不同患者的几个克隆，因为患者具有高度同源的TCR链。我们计划在更大的队列中完成这些谱系分析，并表达约20个重组TCR，这些重组TCR将用于体外抗原搜索。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灵敏的检测系统。我们建议鉴定由人类白细胞抗原B*27呈递给CD8+T细胞的多肽抗原，并研究候选的致关节炎人源多肽在共生微生物或病原体中是否有同源物。为了研究这些多肽的分子性质及其在体内的生成，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多肽是否能在更大的AS患者队列中检测到，我们将产生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，并计划利用PCR和HLA-B*27-肽四聚体在AS患者脊柱活检样本中鉴定与疾病相关的T细胞克隆。对致关节炎多肽的鉴定可能对早期诊断有影响，也可能用于治疗。

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.