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

项目总结 类风湿关节炎(RA)是一种自身免疫性炎性关节炎，可导致侵蚀性关节损害。而当 B细胞在RA中的参与很好地表征了抗瓜氨酸蛋白抗原(ACPA)的抗体，更少 已知T细胞反应性和功能在类风湿关节炎的病因和发病机制中的作用。研究集中在 主要分布在关节和外周血液的T细胞上。然而，这个关节反映了历史悠久的 自身免疫由于抗原表位的扩散而导致适应性免疫反应的偏差。ACPA的存在 在关节炎症开始之前的全身和粘膜部位，这表明起源部位 类风湿性关节炎的自身免疫性是关节外的。肺是这些被认为可能的位置之一 启动类风湿性关节炎相关自身免疫。值得注意的是，在33%以上的RA高危人群中，痰中可检测到ACPA 超过66%的类风湿性关节炎患者。此外，超过70%的RA患者出现肺部异常，以及 肺部疾病是类风湿关节炎常见的关节外表现。这项提案的目标是描述T- 为更好地了解类风湿关节炎的发病机制，研究类风湿关节炎的肺细胞谱。这涉及到调查两个人 单细胞RNA-SEQ检测RA相关肺T细胞表型和功能及其抗原性 这些T细胞在滑液抗原存在下，通过IL-2生物检测的反应性。研究T-- 由于采样的限制，肺的细胞反应一直是一个挑战。诱导痰是一种廉价的， 非侵入性样本，但通常含有1%或更少的淋巴细胞。我们已经克服了 用新型微流控技术回收痰研究肺T细胞反应 以一种完全无接触和无偏见的方式处理淋巴细胞。这使我们能够研究肺中的T细胞 RA并将其与RA滑膜进行比较，因为我们假设将存在共享的效应器状态和抗原性 RA患者肺和滑膜来源的CD4+T细胞之间的反应性。我们的总体假设是T细胞 来自肺的具有共同的效应状态和对RA的T细胞谱系的抗原反应性 滑膜。我们将评估RA患者的痰淋巴细胞是否存在Vβ17+外周血辅助性T细胞(TPh)和 Vβ14+细胞毒T细胞及类风湿关节炎患者外周血中T细胞受体模式和抗原性的比较 至RA滑膜。从单细胞RNA-seq数据中，我们还将获得RA痰中CD4+的TCR序列 T细胞。我们将使用之前发表的包含TCRα和β链序列的数据集来密切确定 RA滑膜和肺之间的相关TCR和模体。我们提案的调查结果将允许 提高了对T细胞参与类风湿关节炎肺部和关节的了解。

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.