An Integrated Epigenomic and Transcriptomic Map of Mouse and Human αβ T Cell Development

αβ lineage T cells, most of which are CD4⁺ or CD8⁺ and recognize MHC I- or MHC II-presented antigens, are essential for immune responses and develop from CD4⁺CD8⁺ thymocytes. The absence of in vitro models and the heterogeneity of αβ thymocytes have hampered analyses of their intrathymic differentiation. Here, combining single-cell RNA and ATAC (chromatin accessibility) sequencing, we identified mouse and human αβ thymocyte developmental trajectories. We demonstrated asymmetric emergence of CD4⁺ and CD8⁺ lineages, matched differentiation programs of agonist-signaled cells to their MHC specificity, and identified correspondences between mouse and human transcriptomic and epigenomic patterns. Through computational analysis of single-cell data and binding sites for the CD4⁺-lineage transcription factor Thpok, we inferred transcriptional networks associated with CD4⁺- or CD8⁺-lineage differentiation, and with expression of Thpok or of the CD8⁺-lineage factor Runx3. Our findings provide insight into the mechanisms of CD4⁺ and CD8⁺ T cell differentiation and a foundation for mechanistic investigations of αβ T cell development.