Defining the tuberculosis lung landscape during disease and latency using single cell technologies

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), infects approximately one-fourth of the world's population. The majority of infected persons are asymptomatic, but latent TB infection (LTBI) can progress to active clinical disease in 5-10% of infected individuals. The immune mechanisms that govern progression from latent to active pulmonary TB (PTB) remain poorly defined. An in-depth understanding of immune factors correlating with TB disease, as well as protection during TB, is necessary for developing new immunotherapies to promote immune control of Mtb. Experimentally Mtb-infected non-human primates (NHP) mirror the disease progression and pathology observed in humans and can recapitulate both PTB and LTBI. In the present study, we have characterized the lung immune landscape in NHPs with LTBI and PTB using high-throughput technologies including single-cell RNA sequencing (scRNA-seq) and Time of flight cytometry (CyTOF). We show that the three defining features of PTB in macaque lungs are the influx of plasmacytoid DCs (pDCs), an Interferon (IFN)-exhibiting alveolar macrophage population and predominant activated T cell responses. These features contribute to uncontrolled inflammation and disease without mediating Mtb control. In contrast, a CD27+ Natural killer (NK) cell subset accumulated in the lungs of LTBI macaques and in circulation in individuals with LTBI, thus providing novel insights into the protective lung landscape that functions during TB latency. A comprehensive understanding of the lung immune landscape as described here will improve our overall understanding of TB disease immunopathogenesis and provide novel targets for design of new therapies and vaccines for TB control. Overall design: To delineate the lung landscape at a high resolution at the single cell level, we performed 10X scRNA-seq on dematricized single cells that underwent CD45+ enrichment from the lungs of uninfected (control, n=3), PTB (n=5) and LTBI (n=3) macaques.

结核病（Tuberculosis, TB）由结核分枝杆菌（Mycobacterium tuberculosis, Mtb）引起，全球约四分之一的人口受到感染。大多数感染者无临床症状，但潜伏性结核感染（Latent TB Infection, LTBI）患者中，有5%~10%会进展为活动性临床疾病。目前，调控潜伏感染向活动性肺结核（Pulmonary TB, PTB）进展的免疫机制仍未明确。深入解析与结核病发病相关的免疫因素以及结核感染中的保护性免疫机制，是开发新型免疫疗法以实现对Mtb免疫控制的必要前提。

实验性感染Mtb的非人灵长类动物（non-human primates, NHP）可模拟人类的疾病进展与病理特征，且能复现活动性肺结核与潜伏性结核感染两种疾病状态。本研究利用高通量技术，包括单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与飞行时间流式细胞术（Time of flight cytometry, CyTOF），对潜伏性结核感染与活动性肺结核非人灵长类动物的肺部免疫微环境进行了系统特征分析。

研究结果显示，猕猴肺部活动性肺结核的三大标志性特征为：浆细胞样树突状细胞（plasmacytoid DCs, pDCs）的浸润、表达干扰素（Interferon, IFN）的肺泡巨噬细胞亚群的存在，以及以活化T细胞应答为主的免疫反应。上述特征会介导失控的炎症反应与疾病进展，却无法实现对Mtb的免疫控制。与之相反，CD27+自然杀伤（Natural killer, NK）细胞亚群会在潜伏性结核感染猕猴的肺部以及潜伏性结核感染个体的循环系统中富集，这为结核潜伏感染期间发挥保护作用的肺部免疫微环境提供了全新的研究视角。

本研究所揭示的肺部免疫微环境全貌，将有助于我们更全面地理解结核病的免疫发病机制，并为开发新型结核病防治疗法与疫苗提供全新的靶点。

整体实验设计：为在单细胞水平实现肺部免疫微环境的高分辨率解析，本研究对未感染（对照组，n=3）、活动性肺结核（n=5）与潜伏性结核感染（n=3）猕猴肺部经CD45+富集处理的解离单细胞样本，开展了10X单细胞RNA测序。