Tracking Regulatory T Cell Development in the Thymus Using Single-Cell RNA Sequencing/TCR Sequencing.

Recent studies have demonstrated that regulatory T cells (T) develop in the thymus via two pathways involving distinct T progenitors (TP): CD25FOXP3 (CD25 TP) and CD25FOXP3 (FOXP3 TP) T progenitors. To examine this process in more detail, we carried out single-cell RNA sequencing (scRNA-Seq) and TCR-Seq on sorted murine CD4CD8 double-positive (DP) thymocytes, CD4 single-positive (CD4SP) thymocytes, CD25FOXP3CD73 TP, CD25FOXP3CD73 TP, newly generated mature CD25FOXP3CD73 T, and FOXP3CD73 recirculating/long-term resident T (RT-T). Sorted populations were individually hashtagged and then combined into one scRNA-Seq/TCR-Seq library before sequencing and subsequent analysis. We found that both CD25 TP and FOXP3 TP arise via an initial agonist-activated state that gives rise to a second transitional stage before differentiating into mature T Using both scRNA-Seq and bulk RNA-Seq on sorted thymocyte subsets, we demonstrate that CD25 TP are significantly enriched for production, suggesting that they are the major source of IL-2 needed to convert TP into mature T Using TCR-Seq, we found that several TCRs were clearly biased in favor of the conventional or T lineages, but that a large fraction of TCRs were found in both these lineages. Finally, we found that RT-T in the thymus are not monomorphic but are composed of multiple distinct subsets and that these RT-T express the most diverse TCR repertoire of all CD4SP thymocytes. Thus, our studies define multiple stages of T differentiation within the murine thymus and serve as a resource for future studies on CD4 thymocyte development and T differentiation.