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

Recent studies have demonstrated that regulatory T cells (T regs ) develop in the thymus via two pathways involving distinct T reg progenitors (T reg P): CD25 + FOXP3 - (CD25 + T reg P) and CD25 - FOXP3 lo (FOXP3 lo T reg P) T reg progenitors. To examine this process in more detail, we carried out single-cell RNA sequencing (scRNA-Seq) and TCR-Seq on sorted murine CD4 + CD8 + double-positive (DP) thymocytes, CD4 + single-positive (CD4SP) thymocytes, CD25 + FOXP3 - CD73 - T reg P, CD25 - FOXP3 lo CD73 - T reg P, newly generated mature CD25 + FOXP3 + CD73 - T regs , and FOXP3 + CD73 + recirculating/long-term resident T regs (RT-T regs ). 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 + T reg P and FOXP3 lo T reg P arise via an initial agonist-activated state that gives rise to a second transitional stage before differentiating into mature T regs Using both scRNA-Seq and bulk RNA-Seq on sorted thymocyte subsets, we demonstrate that CD25 + T reg P are significantly enriched for Il2 production, suggesting that they are the major source of IL-2 needed to convert T reg P into mature T regs Using TCR-Seq, we found that several TCRs were clearly biased in favor of the conventional or T reg lineages, but that a large fraction of TCRs were found in both these lineages. Finally, we found that RT-T regs in the thymus are not monomorphic but are composed of multiple distinct subsets and that these RT-T regs express the most diverse TCR repertoire of all CD4SP thymocytes. Thus, our studies define multiple stages of T reg differentiation within the murine thymus and serve as a resource for future studies on CD4 + thymocyte development and T reg differentiation.