Developing a model of thymic regulatory T cell development with single-cell RNA sequencing

Abstract CD4SP thymocytes that receive moderate to strong TCR signals upregulate either FOXP3 or CD25, generating distinct FOXP3lo or CD25+ Treg progenitor (TRP) populations, respectively. Stimulation of these TRP populations with IL-2 induces co-expression of CD25 and FOXP3, completing thymic Treg differentiation. The alterations in gene expression that drive these respective Treg differentiation programs, as well as the identification of agonist selected cells within conventional CD4SP thymocytes remain unclear. To address these questions, we performed single-cell RNA-Seq with paired TCR sequencing of conventional and Treg lineage thymocytes, including recirculating Tregs. Interestingly, we found that IL-2 was exclusively produced within the most immature CD25+ TRP undergoing agonist selection. TCR analysis demonstrated that some TCRs were enriched in CD4SP receiving agonist stimulation that gave rise to Tregs, or were subjected to negative selection, while others were excluded from this pathway. Finally, we found several distinct subsets of recirculating Tregs, likely pointing to a variety of functions these cells play in thymic homeostasis and function. We are currently carrying out spatial transcriptome studies to identify the specific localization of distinct Treg intermediates within the thymus. We are using these combined single cell data sets to generate a comprehensive model of thymic Treg development.