Abstract
BackgroundT 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.MethodsWe 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.ResultsWe 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.ConclusionsOur 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.
Highlights
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
We divided the cells into 7 developmental stages of thymocytes according to the expression of marker genes: DN progenitors (Il2ra+), immature SP thymocytes (ISPs, Cd4− Cd8+ Mki67+), DP blasts (DPblas; Cd4+ Cd8+ Mki67+), DP thymocytes undergoing rearrangement (DPres; Cd4+ Cd8+ Rag1high), DP cells under selection (DPsels; Cd4+ Cd8+ Itm2a+), and CD4/ CD8 SP thymocytes (CD4sps and CD8sps)
Cells in different stages were aggregated in a two-dimensional t-distributed stochastic neighbor embedding (t-SNE) plot that outlined the developmental trajectory of αβ-T cells (Fig. 1b)
Summary
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. Most CD4/ CD8 double-negative (DN) precursors develop into the αβ-T cell lineage and go through CD4/CD8 double-positive (DP) and CD4/CD8 single-positive (SP) stages to achieve maturation [2]. While developing into DP cells, thymocytes undergo rapid and extensive proliferation, which generates a burst of DP cells and supplies a large pool of T cell clones [3, 4]. When DP cells become quiescent from expansion, rearrangement of the TCRα chain is initiated to form mature TCRs. DP thymocytes are subjected to positive and negative selection, which depend on TCR interaction with the peptide/major histocompatibility complex (MHC) complex (pMHC). The TCRα chain is continuously rearranged until a MHC-restricted TCR αβ heterodimer is achieved [5, 6]. Thymocytes that survive the thymopoiesis process become naïve T cells and migrate out of the thymus
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