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.

αβ 谱系 T 细胞，其中大部分是 CD4 ⁺或 CD8 ⁺并识别 MHC I 或 MHC II 呈递的抗原，对于免疫反应至关重要，并由 CD4 ⁺ CD8 ⁺胸腺细胞发育而来。体外模型的缺乏和 αβ 胸腺细胞的异质性阻碍了对其胸腺内分化的分析。在这里，结合单细胞 RNA 和 ATAC（染色质可及性）测序，我们确定了小鼠和人类 αβ 胸腺细胞的发育轨迹。我们证明了 CD4 ⁺和 CD8 ^{+ 的}不对称出现谱系，将激动剂信号细胞的分化程序与其 MHC 特异性相匹配，并确定了小鼠和人类转录组学和表观基因组模式之间的对应关系。通过对 CD4 ^{+ -}谱系转录因子 Thpok的单细胞数据和结合位点的计算分析，我们推断出与 CD4 ⁺ - 或 CD8 ^{+ -}谱系分化以及 Thpok 或 CD8 ^{+ -}谱系因子的表达相关的转录网络运行x3。我们的发现提供了对 CD4 ⁺和 CD8 ⁺ T 细胞分化机制的深入了解，并为 αβ T 细胞发育的机制研究奠定了基础。