Airway epithelial cell and lymphocyte interactions in chronic lung allograft dysfunction pathogenesis

肺移植后长期存活的主要障碍是肺功能的进行性丧失， 称为慢性肺移植物功能障碍（CLAD），其中小气道中的收缩性纤维化是病理性的 标志CLAD在移植后4年影响超过一半的肺移植受者， 与移植相关的生活质量和功能改善。皮特、多伦多和加州大学旧金山分校肺部 移植程序已经完善了肺移植小气道刷的转录分析 受体作为一种新的技术，以了解基因表达的变化，在解剖部位，CLAD 病理学发展。我们已经发表了与CLAD相关的基因表达变化，这些变化在我们的研究中得到了验证。 中心.该提案利用这种创新方法来了解CLAD发病机制。 我们的初步数据显示，在气道刷拭中，低氧途径的早期上调，包括 在低氧条件下使用气道上皮细胞培养物招募和激活细胞毒性T淋巴细胞， 气道刷状转录组的通路分析。这种缺氧信号可能反映了 微血管系统，缺乏支气管循环和与肺移植相关的血管炎症。在 募集的T淋巴细胞，我们还观察到肿瘤坏死因子超家族（TNFSF）基因的上调， 它们是淋巴细胞靶细胞凋亡的主要驱动因素。我们的数据显示气道俱乐部中的优先凋亡 细胞，小气道的保护者和祖细胞，与上调的TNF相关凋亡相关， 诱导配体（TRAIL）表达。我们的单细胞研究在气道刷和支气管肺泡 灌洗液（BAL）显示这些途径在上皮细胞和淋巴细胞类型中的分离。基于 根据这些数据，我们推测气道缺氧先于TNFSF基因表达，T细胞介导的气道炎症可能是导致气道炎症的重要因素。 club细胞凋亡驱动CLAD发病机制。为了验证这一假设，我们将生成平行队列， 在三个中心研究CLAD与对照的大量和单细胞转录组， 基因表达特征的严格交叉验证。我们会用细胞培养来补充这些研究- 的方法来确定驱动这些基因表达变化的机制。目标1：量化 缺氧相关转录在气道刷细胞方面的CLAD，并确定缺氧如何可以促进 淋巴细胞性炎症在目的2中，我们将确定TNFSF共刺激因子的细胞来源和动力学。 使用气道刷和BAL液在CLAD中的分子表达。在目标3中，我们将研究TRAIL是否 优先诱导俱乐部细胞凋亡。三个大型肺移植转化研究的协同作用 具有世界一流的跨机构生物统计基础设施的计划提供了一个独特的机会， 严格地解决这个假设。CLAD时程中细胞特异性基因表达特征 通过这项研究产生的发展是迫切需要的替代生物标志物，以支持 靶向治疗的临床试验，并开创一种新的方法来诊断CLAD。

The major barrier to long term survival following lung transplantation is a progressive loss of lung function, termed chronic lung allograft dysfunction (CLAD), for which constrictive fibrosis in small airways is a pathologic hallmark. CLAD affects over half of lung transplant recipients by 4 years post-transplant and negates much of the quality of life and functional improvements associated with transplantation. Pitt, Toronto, and UCSF Lung Transplant programs have refined transcriptional analysis of small airway brushings from lung transplant recipients as a novel technique to understand the gene expression changes at the anatomical site where CLAD pathology develops. We have published gene expression changes associated with CLAD validated across our centers. This proposal leverages this innovative approach to understand mechanisms of CLAD pathogenesis. Our preliminary data show an early upregulation of hypoxia pathways in airway brushings including genes that recruit and activate cytotoxic T lymphocytes using both airway epithelial cell culture in hypoxic conditions and pathway analysis of airway brush transcriptomes. This hypoxia signaling may reflect disordered microvasculature, absent bronchial circulation, and vascular inflammation associated with lung transplant. In recruited T lymphocytes, we also observed upregulation of tumor necrosis factor superfamily (TNFSF) genes, which are major drivers of apoptosis in lymphocyte targets. Our data show preferential apoptosis in airway club cells, the protectors and progenitors of small airways, in association with upregulated TNF-related apoptosis- inducing ligand (TRAIL) expression. Our single cell investigations in airway brushings and bronchoalveolar lavage (BAL) fluid show the segregation of these pathways across epithelial and lymphoid cell types. Based on these data, we hypothesize that airway hypoxia precedes TNFSF gene expression and T cell-mediated airway club cell apoptosis that drive CLAD pathogenesis. To test this hypothesis, we will generate parallel cohorts investigating bulk and single cell transcriptomes of CLAD versus controls across three centers, allowing for rigorous cross-validation of gene expression signatures. We will complement these studies with cell culture- based methods to determine mechanisms driving these gene expression changes. In Aim 1, we will quantify hypoxia-related transcripts in airway brush cells with respect to CLAD and determine how hypoxia can promote lymphocytic inflammation. In Aim 2, we will determine the cellular sources and kinetics of TNFSF co-stimulatory molecule expression in CLAD using airway brushes and BAL fluid. In Aim 3, we will investigate whether TRAIL preferentially induces club cell apoptosis. The synergy of three large lung-transplant translational research programs with world-class cross-institutional biostatistical infrastructures provides a unique opportunity to address this hypothesis rigorously. The cell-specific gene expression signatures over the time course of CLAD development that will be generated through this study are critically needed as surrogate biomarkers to support clinical trials of targeted therapies and to pioneer a novel approach to CLAD diagnosis.