Single-Cell Analysis of Thymocyte Differentiation: Identification of Transcription Factor Interactions and a Major Stochastic Component in αβ-Lineage Commitment

T cell commitment and αβ/γδ lineage specification in the thymus involves interactions between many different genes. Characterization of these interactions thus requires a multiparameter analysis of individual thymocytes. We developed two efficient single-cell methods: (i) the quantitative evaluation of the co-expression levels of nine different genes, with a plating efficiency of 99–100% and a detection limit of 2 mRNA molecules/cell; and (ii) single-cell differentiation cultures, in the presence of OP9 cells transfected with the thymus Notch1 ligand DeltaL4. We show that during T cell commitment, Gata3 has a fundamental, dose-dependent role in maintaining Notch1 expression, with thymocytes becoming T-cell-committed when they co-express Notch1, Gata3 and Bc11b. Of the transcription factor expression patterns studied here, only that of Bcl11b was suggestive of a role in Pu1 down-regulation. Individual thymocytes became αβ/γδ lineage-committed at very different stages (from the TN2a stage onwards). However, 20% of TN3 cells are not αβ/γδ-lineage committed and TN4 cells comprise two main subpopulations with different degrees of maturity. The existence of a correlation between differentiation potential and expression of the pre-TCR showed that 83% of αβ-committed cells do not express the pre-TCR and revealed a major stochastic component in αβ-lineage specification.胸腺中的T细胞定向分化（T cell commitment）与αβ/γδ谱系特化（αβ/γδ lineage specification）过程涉及众多不同基因间的相互作用。因此，解析此类基因相互作用需对单个胸腺细胞开展多参数分析。本研究开发了两种高效的单细胞分析方法：(i) 9种不同基因共表达水平的定量检测技术，其铺板效率达99%~100%，检测限为2个mRNA分子/细胞；(ii) 单细胞分化培养体系，该体系依托转染了胸腺Notch1配体DeltaL4的OP9细胞构建。研究结果显示，在T细胞定向分化阶段，Gata3在维持Notch1表达过程中发挥关键的剂量依赖性作用；当胸腺细胞共表达Notch1、Gata3与Bc11b时，即完成T细胞定向分化。在本研究检测的转录因子表达模式中，仅Bcl11b的表达模式提示其可能参与Pu1的下调调控。单个胸腺细胞的αβ/γδ谱系定向分化起始于多个不同阶段（自TN2a阶段起）。然而，仍有20%的TN3细胞未完成αβ/γδ谱系定向分化；而TN4细胞可分为两个成熟度不同的主要亚群。分化潜能与前T细胞受体（pre-TCR）表达水平的相关性分析表明，83%的αβ谱系定向细胞并不表达前T细胞受体，同时揭示了αβ谱系特化过程中存在显著的随机调控组分。