B cell-adaptive Immune Profile in Emphysema-predominant COPD

项目摘要 最近的研究表明，并不是所有的COPD患者，但只有那些有肺气肿目前突出的肺 B细胞分子特征，而与气流限制的程度无关。我们的初步数据显示 和循环B细胞反应在COPD患者中上调， COPD严重程度相同。我们将检验中心假设，即肺气肿的特征是 B细胞转录组是导致肺结核的过度和脱靶B细胞应答的基础 杀伤性在目标1中，我们将检验以下假设：B细胞、淋巴滤泡（LF）和B细胞产物计数， 与肺气肿相比，肺气肿肺中特异性LF B细胞克隆和转录后变化增加 肺气肿程度较轻在目标1A中，我们将使用常规免疫染色和图像进行免疫染色， 质谱细胞术，COPD和对照肺，并关联B细胞，LF和其他细胞参与的数量。 B细胞反应，以及计算机断层扫描（CT）定义的肺叶气肿的程度。在目标1B中，我们将 使用激光捕获显微切割从COPD和对照肺切片中提取LF B细胞，然后：1）评估 LF的克隆性;和2）使用下一代免疫荧光技术研究B细胞受体（BCR）的重排。 测序（NGS）测定。在目标2中，我们将检验以下假设：夸大的B细胞活化， 具有转录组学变化，存在于肺气肿肺中。我们将从COPD中分离肺B细胞 患者和对照，以便：1）测试响应于以下的B细胞活化和免疫球蛋白（IG）产生： 使用体外培养物和ELISPOT测定来鉴定抗原; 2）使用流式细胞术来表征B细胞库（Aim 2A）; 3）使用单细胞RNA测序研究肺B细胞转录组和BCR重排 （scRNAseq）; 4）产生单克隆Ig并用质谱法鉴定其在肺中的结合靶标 (Aim 2B）。我们将把肺部B细胞活化的读数与CT扫描定义的肺叶范围相关联。 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的假设 与肺气肿程度相关的相关性研究中的细胞特征。在目标3A中，我们将：1）检测血液B细胞 使用体外培养物和ELISPOT测定，响应于抗原的活化和IG产生; 2）表征 使用流式细胞术检测血液B细胞库; 3）研究血液B细胞转录组和BCR重排 使用scRNAseq.在目的3B中，我们将使用COPD和对照血浆：1）定量血液IG同种型， 使用质谱法鉴定肺中的血液IG结合靶;和2）测量B细胞水平， 相关生物标志物和IgG对肺基质的反应性。我们将把B细胞活化的读数与 血液和肺来自同一受试者，具有CT扫描定义的肺气肿的程度。这些研究将定义 临床表型（可能是肺气肿）中B细胞应答的性质，可从治疗中获益 靶向B细胞或B细胞产物，导致COPD的更早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.