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_regP): CD25⁺FOXP3² (CD25⁺ T_regP) and CD25²FOXP3^lo (FOXP3^lo T_regP) 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⁺FOXP3²CD73² T_regP, CD25²FOXP3^loCD73² T_regP, newly generated mature CD25⁺FOXP3⁺CD73² 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_regP and FOXP3^lo T_regP 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_regP are significantly enriched for Il2 production, suggesting that they are the major source of IL-2 needed to convert T_regP 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.