Spatiotemporal multiomics reveal a default CD4 fate and a stem-like CD8 T cell subset in the thymus

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BACKGROUND: T cell development in the thymus proceeds through coordinated transcriptional and spatial transitions. While key stages have been described, the precise timing and regulatory logic of lineage bifurcation-such as the separation between αβ and γδ T cells, and the commitment to CD4 or CD8 fate remains incompletely understood. A high-resolution, integrated view is needed to clarify how distinct T cell identities are established. OBJECTIVES: The goal is to construct a comprehensive, multimodal map of thymocyte development that defines early lineage decisions, intermediate states, and spatial checkpoints underlying T cell maturation. METHODS: We generated an integrated single-cell atlas by combining single-cell transcriptomics, protein profiling (CITE-seq), chromatin accessibility (scATAC-seq), and spatial transcriptomics of murine thymus. Data integration was used to resolve developmental trajectories, identify transcription factor programs, and map the anatomical localization of thymocyte subpopulations. RESULTS: Our analysis revealed that αβ and γδ lineages diverge as early as the DN1 stage. Within the αβ lineage, we identified a continuum of double-positive thymocyte substages distinguished by shifts in cell surface markers, cell-cycle activity, and recombination gene expression. CD4 lineage commitment emerged as a transcriptional default program initiated by Gata3. It was maintained independently of class II MHC signaling, whereas CD8 specification required Runx3 activation and class I MHC signaling, consistent with a sequential reprogramming model. We also identified a stem-like CD8-positive population enriched for self-renewal and developmental potential genes. Spatial mapping showed medullary enrichment of CD4 cells and localization of thymocyte exit cues to the cortico-medullary junction. CONCLUSIONS: This multimodal atlas provides a detailed framework for the molecular and spatial logic of T cell development. The findings uncover early lineage bifurcation events, refine and mechanistically characterize intermediate developmental states, and highlight anatomical checkpoints critical for thymic output and immune competence.