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. Overall design: 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.