Single-cell RNA sequencing of thymic EC and TEC from control and RANKL treated aged mice
Age-related thymic involution, leading to reduced T cell production, is one of the major causes of immunosenescence. This results in an increased susceptibility to cancers, infections, and autoimmunity and in reduced vaccine efficacy. Here, we identified that the receptor activator of nuclear factor ?B (RANK)-RANK ligand (RANKL) axis in the thymus is altered during aging. Using a conditional transgenic mouse model, we demonstrated that endothelial cells depend on RANK signaling for their cellularity and functional maturation. Decreased RANKL availability during aging resulted in a decline in cellularity and function of both endothelial cells and thymic epithelial cells, contributing to thymic involution. We then found that, whereas RANKL neutralization in young mice mimicked thymic involution, exogenous RANKL treatment in aged mice restored thymic architecture as well as endothelial cell and epithelial cell abundance and functional properties. Consequently, RANKL improved T cell progenitor homing to the thymus and boosted T cell production. This cascade of events resulted in peripheral T cell renewal and effective antitumor and vaccine responses in aged mice. Furthermore, we conducted a proof-of-concept study that showed that RANKL stimulates endothelial cells and epithelial cells in human thymic organocultures. Overall, our findings suggest that targeting the RANK-RANKL axis through exogenous RANKL administration could represent a therapeutic strategy to rejuvenate thymic function and improve T cell immunity during aging. Overall design: Expression profiling by high throughput sequencing. CD31+ EC were sorted 3 weeks after GST and RANKL treatment in aged mice and mixed in equal ratios. Five hundred WT splenic B cells were added to each pool to ease batch correction if necessary. Cells were individually loaded onto a Chromium Next GEM chip K (10X Genomics), and cells were droplet-encapsulated with a Chromium Controller (10X Genomics). Libraries were prepared with the Chromium single cell 5' v2 kit (10X Genomics) according to the manufacturer's instructions with dual index. The concentration and quality of cDNA libraries were determined using the Qubit dsDNA HS assay kit (Thermo Fisher Scientific) for the Qbit fluorometer and the High Sensitivity DNA Analysis Kit (Agilent) for the bioanalyzer, respectively. Libraries were sequenced on an Illumina NextSeq 2000 platform with a 100-cycles P2 flow cell.