Spatial Single-cell Transcriptome Atlas of Mouse Thymus Reveals the T Lymphocyte Dynamics During Development

The thymus provides a crucial microenvironment for lymphocyte development, undergoes rapid growth by progressive atrophy, yet the underlying molecular dynamics remain largely unclear. Here, we present a comprehensive single-cell transcriptomic data resource of mouse thymus across four developmental stages (3, 9, 15, and 33 weeks). We identified eleven major immune cell types, including γδ​cells, B cells, NKT cells, Dendritic cells, immature single-positive-T cells, DN3a cells, double positive (DP)-T cells-further subdivided into DP-1, DP-2, and DP-3-as well as CD4+ and CD8+ T cells. Our analysis revealed dynamic remodeling of thymic cell composition and transcriptional profiles over time. Notably, the proportion of CD8+ T cells and the expression of Smad4 and Smad7, key regulators of lymphocyte development, declined during thymic involution. The spatial single-cell atlas revealed that DP-2 and DP-3 formed distinct clusters, with dendritic cells (DCs) located in closer proximity to DP-2. Notably, DC-DP-2 interactions were enriched via the MHC-II pathway, potentially promoting the differentiation of DP-T cells. Further spatial trajectory analysis delineated the developmental landscape of DP-2 cells. Finally, autoimmune disease-associated risk genes were preferentially enriched in CD4+ and NKT cells, displaying distinct spatiotemporal expression patterns. Collectively, this study provides a valuable resource for dissecting thymic architecture and immune system development across different ages.