Single Cell Analysis of Peripheral TB-Associated Granulomatous Lymphadenitis

We employed a single cell RNA sequencing (scRNA-seq) approach to describe the cells and communication networks characterizing granulomatous lymph nodes of TB patients. When mapping cells from individual patient samples, we uniformly identify several cell types that characterize granulomas. Whether high or low Mtb burden, overall we find the T cell cluster to be the most abundant. Other cell clusters that are uniformly detected, but that vary dramatically in abundance amongst the individual patient samples, are the B cell, plasma cell and macrophage/dendrocyte and NK cell clusters. When we combine all our scRNA-seq data from our current 23 patients (to gain greater n), we distinguish T, macrophage, dendrocyte and plasma cell subclusters. The sizes of these subclusters also varies dramatically amongst the individual patients. In comparing FNA composition we noted trends in which T cell populations and macrophage/dendrocyte populations were negatively or positively correlated with NK cell populations. In addition, we discovered that the scRNA-seq pipeline detects Mtb RNA transcripts and associates them with their host cell's transcriptome, thus identifying individual infected cells. The number of infected cells also varies dramatically in abundance amongst the patient samples. CellChat analysis identified predominating signaling pathways amongst the cells comprising the various granulomas. Overall design: Fine needle aspirates from periferal lymphadenopathies were collected from concenting patients arriving at the Port Moresby General Hospital (POMGH) Tuberculosis Clinic, Papua New Guinea, processed to collect the single cells, and fixed and sterilized with MeOH before returning to the University of Utah for processing for single-cell RNA sequencing using the 10X Genomics system.本研究采用单细胞RNA测序（single cell RNA sequencing, scRNA-seq）技术，对结核患者的肉芽肿性淋巴结的细胞组成与细胞通讯网络特征进行解析。在对单例患者样本的细胞进行注释时，我们统一鉴定出数种肉芽肿特征性细胞类型。无论结核分枝杆菌（Mycobacterium tuberculosis, Mtb）负荷高低，整体分析结果均显示T细胞簇为丰度最高的细胞群。其余可统一检测到的细胞簇包括B细胞、浆细胞、巨噬细胞/树突状细胞以及自然杀伤（natural killer, NK）细胞簇，但其在各患者样本中的丰度差异显著。当我们整合当前23例患者的全部scRNA-seq数据以提升样本量时，我们进一步区分出T细胞、巨噬细胞、树突状细胞与浆细胞的亚簇。这些亚簇的细胞数量在不同患者间同样存在显著差异。在比较细针抽吸活检（Fine Needle Aspiration, FNA）的细胞组成时，我们观察到T细胞群、巨噬细胞/树突状细胞群与NK细胞群分别呈现负相关或正相关的趋势。此外，本研究发现scRNA-seq流程可检测到Mtb的RNA转录本，并将其与宿主细胞的转录组进行关联，从而实现单个感染细胞的鉴定。各患者样本中的感染细胞数量同样存在显著差异。细胞通讯分析工具CellChat鉴定出构成各类肉芽肿的细胞间的主要信号通路。研究整体设计：从巴布亚新几内亚莫尔兹比港总医院（Port Moresby General Hospital, POMGH）结核门诊招募的知情同意患者中，采集外周淋巴结病变的细针抽吸活检样本，分离获取单细胞后采用甲醇（methanol, MeOH）固定并灭活，随后转运至犹他大学，采用10X Genomics系统完成单细胞RNA测序建库与测序。