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

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.

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.

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.

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.

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.