B cell-adaptive Immune Profile in Emphysema-predominant COPD

项目总结 最近的研究表明，并不是所有的慢性阻塞性肺疾病患者，只有那些患有肺气肿的患者才会出现肺突出。 B细胞分子特征与气流受限程度无关。我们的初步数据显示，肺部 肺气肿较大与较轻的COPD患者循环B细胞反应上调 COPD严重程度分级相同。我们将检验这一中心假设，即肺气肿的特征是 B细胞转录组，它是导致肺的B细胞反应被夸大和偏离靶点的基础 毁灭。在目标1中，我们将测试B细胞、淋巴滤泡(LFS)和B细胞产物计数的假设， 肺气肿患者肺组织中特异性LfB细胞克隆和转录后变化增加 肺气肿较轻。在目标1A中，我们将使用常规免疫染色和图像进行免疫染色 质量细胞术、COPD和对照肺，并将B细胞、LFS和其他参与 B细胞反应，以CT确定的肺气肿程度为标准。在目标1B中，我们将 用激光捕获显微切割法提取慢性阻塞性肺疾病患者的LfB细胞，对照肺切片：1)评估 LFS的克隆性；以及2)使用下一代B细胞受体(BCR)重排 测序(NGS)分析。在目标2中，我们将检验这样一种假设，即夸大的B细胞激活与 随着转录的改变，出现在肺气肿的肺。我们将从COPD患者中分离出肺B细胞 患者和对照组：1)测试B细胞的活化和免疫球蛋白(Ig)的产生 用体外培养和ELISPOT试验检测抗原；2)用流式细胞术(AIM)鉴定B细胞池 2)；3)用单细胞RNA测序研究肺B细胞转录和bcr重排 (ScRNAseq)；4)制备单抗免疫球蛋白，并用质谱仪鉴定其在肺中的结合靶点 (目标2B)。我们将把肺中B细胞激活的读数与CT扫描确定的肺叶的范围相关联 肺气肿。在目标3中，我们将检验循环B细胞功能和表型反映肺B细胞的假设 与肺气肿程度相关研究中的细胞特征。在目标3A中，我们将：1)检测血液B细胞 用体外培养和ELISPOT试验检测抗原的激活和免疫球蛋白产生；2)表征 用流式细胞术检测血B细胞池；3)研究血B细胞转录和bcr重排 使用scRNAseq。在目标3B中，我们将使用COPD和对照血浆来：1)定量血液Ig亚型和 用质谱仪鉴定肺中的血液免疫球蛋白结合靶点；2)测量B细胞水平- 相关生物标志物和免疫球蛋白对肺基质的反应。我们将把B细胞激活的读数与 血液和肺来自同一受试者，具有CT扫描确定的肺气肿程度。这些研究将界定 临床表型(可能是肺气肿)中B细胞反应的性质，可从治疗中受益 针对B细胞或B细胞产品，导致对COPD的早期和更个性化的治疗干预。

PROJECT SUMMARY Recent studies show that not all the COPD patients, but only those having emphysema present a prominent lung B cell molecular signature irrespectively of the degree of airflow limitation. Our preliminary data show that lung and circulating B cell responses are upregulated in COPD patients with greater vs. lesser emphysema within the same COPD severity stage. We will test the central hypothesis that emphysema is characterized by changes in B cell transcriptome which underlie exaggerated and off-targeted B cell responses contributing to lung destruction. In Aim 1, we will test the hypothesis that B cell, lymphoid follicles (LFs), and B cell product counts, and specific LF B cell clonal and post-transcriptional changes are increased in the emphysematous lung vs. lung with lesser emphysema. In Aim 1A, we will immunostain, using both conventional immunostaining and image mass cytometry, COPD and control lungs and correlate the numbers of B cells, LFs, and other cells involved in B cells responses, with the extent of computed tomography (CT)-defined lobar emphysema. In Aim 1B, we will use laser capture microdissection to extract LF B cells from COPD and control lung sections to then: 1) assess the clonality of the LFs; and 2) study the rearrangement of the B cell receptor (BCR) using a next-generation sequencing (NGS) assay. In Aim 2, we will test the hypothesis that an exaggerated B cell activation, associated with transcriptomic changes, is present in the emphysematous lung. We will isolate lung B cells from COPD patients and controls in order to: 1) test B cell activation and immunoglobulin (Ig) production in response to antigens using in vitro cultures and ELISPOT assays; 2) characterize the B cell pool using flow cytometry (Aim 2A); 3) study lung B cell transcriptomes and BCR rearrangements using single-cell RNA sequencing (scRNAseq); 4) generate monoclonal Igs and identify their binding targets in the lung with mass spectrometry (Aim 2B). We will correlate the readouts of B cell activation in the lung with the extent of CT scan-defined lobar emphysema. In Aim 3, we will test the hypothesis that circulating B cell function and phenotypes reflect lung B cell features in correlation studies relating to the extent of emphysema. In Aim 3A, we will: 1) test blood B cell activation and Ig production in response to antigens using in vitro cultures and ELISPOT assays; 2) characterize the blood B cell pool using flow cytometry; and 3) study blood B cell transcriptomes and BCR rearrangements using scRNAseq. In Aim 3B, we will use COPD and control plasma to: 1) quantify the blood Ig isotypes and identify the blood Ig binding targets in the lung using mass spectrometry; and 2) measure the levels of B cell- related biomarkers and IgG reactivity against lung matrix. We will correlate the readouts of B cell activation in blood and lung from the same subject, with the extent of CT scan-defined emphysema. These studies will define the nature of the B cell responses in a clinical phenotype (likely emphysema) which could benefit from therapies targeting B cells or B cell products, leading to earlier and more personalized therapeutic intervention for COPD.