Visualizing thymic regulatory T cell development with single-cell RNA sequencing

Abstract

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 IL2 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 CD4SP thymocytes. This data set contained cells of the entire range of maturity, from recent DP-CD4SP converts to mature CD4 T cells competent for thymic export. In conventional CD4SPs we were able to identify a subset of cells undergoing agonist selection that differentiates into TRP cells along with cells undergoing negative selection. The transcriptomes of the subsequent CD25+ and FOXP3lo TRP subsets were clearly distinct. For example, we found that the source of IL2 required for conversion of both TRP subsets into mature Tregs was uniquely found within the most immature CD25+ TRP undergoing agonist selection. Finally, TCR analysis demonstrated that some TCRs were enriched in CD4SP receiving agonist stimulation that gave rise to Tregs, or undergoing negative selection, while others were excluded from this pathway. We are currently carrying out spatial transcriptome studies to identify the specific localization of distinct Treg intermediates within the thymus. Interrogation of these single cell and spatial transcriptomic data sets will generate a comprehensive model of thymic Treg development.