Department of oncology, The First Affiliated Hospital of USTC, Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medicine, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230021, Anhui, China.
The CAS Key Laboratory of Innate Immunity and Chronic Disease, CAS Center for Excellence in Molecular Cell Sciences, University of Science and Technology of China, Hefei, 230021, Anhui, China.
Genome Med. 2021 Mar 26;13(1):49. doi: 10.1186/s13073-021-00861-7.
T cells generated from thymopoiesis are essential for the immune system, and recent single-cell studies have contributed to our understanding of the development of thymocytes at the genetic and epigenetic levels. However, the development of double-positive (DP) T cells, which comprise the majority of thymocytes, has not been well investigated.
We applied single-cell sequencing to mouse thymocytes and analyzed the transcriptome data using Seurat. By applying unsupervised clustering, we defined thymocyte subtypes and validated DP cell subtypes by flow cytometry. We classified the cell cycle phases of each cell according to expression of cell cycle phase-specific genes. For immune synapse detection, we used immunofluorescent staining and ImageStream-based flow cytometry. We studied and integrated human thymocyte data to verify the conservation of our findings and also performed cross-species comparisons to examine species-specific gene regulation.
We classified blast, rearrangement, and selection subtypes of DP thymocytes and used the surface markers CD2 and Ly6d to identify these subtypes by flow cytometry. Based on this new classification, we found that the proliferation of blast DP cells is quite different from that of double-positive cells and other cell types, which tend to exit the cell cycle after a single round. At the DP cell selection stage, we observed that CD8-associated immune synapses formed between thymocytes, indicating that CD8sp selection occurred among thymocytes themselves. Moreover, cross-species comparison revealed species-specific transcription factors (TFs) that contribute to the transcriptional differences of thymocytes from humans and mice.
Our study classified DP thymocyte subtypes of different developmental stages and provided new insight into the development of DP thymocytes at single-cell resolution, furthering our knowledge of the fundamental immunological process of thymopoiesis.
从胸腺生成的 T 细胞对于免疫系统至关重要，最近的单细胞研究有助于我们在遗传和表观遗传水平上了解胸腺细胞的发育。然而，双阳性 (DP) T 细胞的发育，占胸腺细胞的大多数，尚未得到很好的研究。
我们应用单细胞测序对小鼠胸腺细胞进行测序，并使用 Seurat 分析转录组数据。通过应用无监督聚类，我们定义了胸腺细胞亚型，并通过流式细胞术验证 DP 细胞亚型。我们根据细胞周期特异性基因的表达对每个细胞的细胞周期阶段进行分类。为了检测免疫突触，我们使用免疫荧光染色和基于 ImageStream 的流式细胞术。我们研究和整合人类胸腺细胞数据来验证我们的发现的保守性，并且还进行了跨物种比较以检查物种特异性基因调控。
我们对 DP 胸腺细胞的原始、重排和选择亚型进行了分类，并通过流式细胞术使用表面标志物 CD2 和 Ly6d 来识别这些亚型。基于这个新的分类，我们发现原始 DP 细胞的增殖与双阳性细胞和其他细胞类型的增殖有很大不同，后者在单个循环后往往会退出细胞周期。在 DP 细胞选择阶段，我们观察到胸腺细胞之间形成了与 CD8 相关的免疫突触，表明 CD8sp 选择发生在胸腺细胞本身之间。此外，跨物种比较揭示了与人类和小鼠胸腺细胞转录差异相关的物种特异性转录因子 (TFs)。
我们的研究对不同发育阶段的 DP 胸腺细胞亚型进行了分类，并在单细胞分辨率上提供了 DP 胸腺细胞发育的新见解，进一步加深了我们对胸腺生成基本免疫学过程的认识。
