CD4+ T-cell Repertoires of the Lung in Rheumatoid Arthritis

项目摘要 类风湿性关节炎（RA）是一种自身免疫性的炎症性关节炎，可导致侵蚀性关节损伤.而 瓜氨酸蛋白抗原（ACPAs）抗体是RA中B细胞参与的良好特征， 已知T细胞在RA病因和发病机制中的反应性和功能性。研究都集中 主要是关节和外周血的T细胞。然而，联合国反映了长期存在的 自身免疫，其由于表位扩散而在适应性免疫应答中引入偏差。ACPA存在 全身和粘膜部位的关节炎症发作之前，这表明起源部位 RA的自身免疫性是关节外的。肺是被认为是可能的位置之一， 引发RA相关的自身免疫值得注意的是，在超过33%的RA风险患者中可检测到痰ACPA 超过66%的类风湿性关节炎患者此外，在超过70%的RA患者中观察到肺部异常， 肺部疾病是RA常见的关节外表现。该提案的目标是描述T- 细胞库，以更好地了解疾病的发病机制。这涉及到调查双方 通过单细胞RNA-seq的RA相关肺T细胞的表型和功能以及抗原性 当与滑液抗原一起呈递时，通过IL-2生物测定这些T细胞的反应性。学习T- 由于取样的限制，肺的细胞反应一直是一个挑战。诱导痰是一种便宜， 非侵入性样本，但它通常含有1%或更少的淋巴细胞。我们已经克服了 通过使用新的微流体技术回收痰用于研究肺的T细胞应答的痰 淋巴细胞在一个完全触摸自由和公正的方式。这使我们能够研究肺中的T细胞， RA和比较他们的RA滑膜，因为我们假设将有共享的效应状态和抗原 RA中肺源性和滑膜源性CD 4 + T细胞之间的反应性。我们的总体假设是T细胞 来源于肺的T细胞对RA的T细胞库具有共同的效应子状态和抗原反应性 滑膜我们将评估RA患者痰液淋巴细胞中Vβ17+外周辅助性T细胞（Tph）的存在， RA痰液中Vβ14+细胞毒性CD 4 + T细胞及其T细胞受体模体和抗原反应性的比较 到RA滑膜。从单细胞RNA-seq数据，我们还将获得RA痰CD 4 + T细胞的TCR序列。 T细胞我们将使用先前发表的包含TCR α和β链序列的数据集， RA滑膜和肺之间的相关TCR和基序。从我们的提案中得出的结果将允许 提高了对T细胞参与肺和关节RA的认识。

PROJECT SUMMARY Rheumatoid arthritis (RA) is an inflammatory arthritis of autoimmune origin leading to erosive joint damage. While B-cell involvement in RA is well characterized by antibodies to citrullinated protein antigens (ACPAs), much less is known about T-cell reactivity and functionality in the etiology and pathogenesis of RA. Studies have focused largely on the T-cells of the joint and peripheral blood. However, the joint is reflective of long-standing autoimmunity which introduces a bias in adaptive immune responses due to epitope spreading. ACPAs exist systemically and in mucosal sites preceding the onset of joint inflammation, which suggests the originating site of autoimmunity in RA is extra-articular. The lung is one of these sites considered to be a possible location for initiating RA-related autoimmunity. Notably, sputum ACPAs are detectable in over 33% of those at risk for RA and over 66% of those with RA. Furthermore, lung abnormalities are seen in over 70% of those with RA, and lung disease is a common extra-articular manifestation of RA. The goal of this proposal is to characterize the T- cell repertoire of the lung in RA to better understand disease pathogenesis. This involves investigating both phenotype and functionality of RA-related lung T-cells through single cell RNA-seq as well as the antigenic reactivity of these T-cells through IL-2 bioassays when presented with synovial fluid antigens. Studying the T- cell responses of the lung has been a challenge due to sampling limitations. Induced sputum is an inexpensive, non-invasive sample but it typically contains 1% or fewer lymphocytes. We have overcome the limitations of sputum for studying T-cell responses of the lung by the use of a novel microfluidic technique to recover sputum lymphocytes in a completely touch-free and unbiased manner. This allows us to study the T-cells of the lung in RA and compare them to the RA synovium as we hypothesize there will be shared effector states and antigenic reactivity between lung-derived and synovium-derived CD4+ T-cells in RA. Our overall hypothesis is T-cells derived from the lung have common effector states and antigenic reactivity to the T-cell repertoire of the RA synovium. We will assess sputum lymphocytes in RA for the presence of Vβ17+ peripheral helper T (Tph) and Vβ14+ cytotoxic CD4+ T-cells and compare T-cell receptor motifs and antigenic reactivity of T-cells in RA sputum to RA synovium. From the single-cell RNA-seq data, we will also obtain TCR sequences of the RA sputum CD4+ T-cells. We will use previously published datasets containing TCR α and β chain sequences to determine closely related TCRs and motifs between the RA synovium and lung. The findings from our proposal will allow for an improved understanding of T-cell involvement in RA in both the lung as well as the joint.