Population and functional diversity of aged lymph node stromal cells during viral infection

Aging correlates with delayed and blunted adaptive immune responses following infection and vaccination leading to increased susceptibility to infectious diseases. Lymph node stromal cells (LNSC) are a diverse and rare population of fibroblast and endothelial cell subsets that assist in LN structure and immunological function. The negative impact of aging on the function of hematopoietic immune cells has been described; however, the contribution of aging to LNSC function has not been elucidated. Here, we utilized single cell RNA sequencing (scRNA-seq) to investigate adult and aged LNSC transcriptional dynamics during an acute West Nile virus infection to gain in vivo insight into the active immune response in these rare and diverse cellular subsets. We identified three major LNSC subsets as well as 15 individual stromal cell types present in adult and old LNs. Overall, the adult and aged LNSC response to WNV is characterized by the robust activation of antiviral signaling pathways, including expression of interferon stimulated genes. Population dynamics of LNSCs from adult and old LNs were largely similar, although aged LNs were found to be enriched in fibroblast subsets, including TRC and MedRC. Infection induced metabolic reprograming of the LNSC subsets in the adult setting; however, this reprogramming was dampened in the aged setting. Collectively, these studies demonstrate that aged LNSCs do not display age-related impairments in antiviral signatures; however, age-related changes influence the aged LN immune response. The data presented here is the first to examine age-dependent LNSC dynamics during viral infection on the single cell level. Lymph nodes were harvested and digested from adult and old mice. These mice were either naïve or 2 days post West Nile viral (WNV-Kunjin) infection. Lymph node stromal cells (LNSCs) were then isolated from lymph node suspensions using a magnetic microbead-based separations, based on the absence of CD45 expression and presence of CD31 and/or gp38 expression. The resulting LNSCs were then analyzed using single cell RNA sequencing (scRNA-seq). Adult LNSC scRNA-seq experiments were performed in duplicate, whereas the old LNSC scRNA-seq experiment contains a single replicate.

衰老与感染及疫苗接种后适应性免疫应答的延迟与钝化相关，进而导致传染病易感性升高。
淋巴结基质细胞（Lymph node stromal cells, LNSC）是一类异质性且稀缺的成纤维细胞与内皮细胞亚群，对淋巴结（LN）结构及免疫功能起到维持作用。
衰老对造血免疫细胞功能的负面影响已有报道，但衰老对LNSC功能的贡献尚未阐明。
本研究利用单细胞RNA测序（single cell RNA sequencing, scRNA-seq）技术，探究急性西尼罗河病毒（West Nile Virus, WNV）感染过程中成体与衰老小鼠LNSC的转录组动态变化，以在体揭示这类稀缺异质性细胞亚群的活跃免疫应答机制。
我们在成体与衰老小鼠的淋巴结中鉴定出3大类LNSC亚群，以及15种独立的基质细胞类型。
总体而言，成体与衰老小鼠LNSC对WNV的应答以抗病毒信号通路的显著激活为特征，包括干扰素刺激基因的表达。
成体与衰老小鼠淋巴结LNSC的群体动态大体相似，但衰老小鼠淋巴结的成纤维细胞亚群（包括TRC与MedRC）丰度更高。
感染可诱导成体小鼠LNSC亚群发生代谢重编程，但该重编程过程在衰老小鼠体内受到抑制。
综上，本研究证实衰老小鼠LNSC并未在抗病毒应答特征上出现衰老相关的功能损伤，但衰老相关的改变会影响衰老小鼠淋巴结的免疫应答。
本研究提供的数据是首个在单细胞层面探究病毒感染过程中年龄依赖性LNSC动态变化的研究。
研究对象为成体与衰老小鼠，采集其淋巴结并进行消化处理；小鼠分为未感染组与WNV-Kunjin株感染后2天组。
随后基于CD45阴性、CD31和/或gp38阳性的表达特征，利用磁珠分选法从淋巴结悬液中分离得到LNSC。
最终获得的LNSC通过单细胞RNA测序（scRNA-seq）进行分析。成体小鼠LNSC的scRNA-seq实验设置了两次生物学重复，而衰老小鼠LNSC的scRNA-seq实验仅设置了一次生物学重复。