Upregulation of CD90 correlates with T lineage commitment at the DN2a stage of T cell development

Early thymic progenitors (ETPs) retain the potential for alternative fates but commit to the T-cell lineage as they progress through the CD4^-CD8^- double-negative (DN) thymocyte stages early in T-cell development. Current evidence suggests that T-lineage commitment occurs during the transition from the DN2a to DN2b stage, a process characterized by downregulation of c-KIT and CD44 expression. Moreover, Bcl11b activation in DN2a thymocytes closely coincides with T-lineage commitment. Despite this heterogeneity in Bcl11b-YFP expression within DN2a thymocytes, these cells have predominantly been studied as a single population due to the absence of cell-surface markers capable of resolving the corresponding substages. To define the T-lineage commitment transition process at higher resolution, we profiled c-KIT-expressing DN thymocytes in the mouse thymus using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). This revealed heterogeneity within DN2a thymocytes. We identified two subpopulations, which we termed DN2a-1 and DN2a-2, based on differences in CD34, CD90, and Bcl11b expression. DN2a-1 thymocytes co-expressed multipotency and T-lineage-associated markers, whereas DN2a-2 cells mainly displayed a transcriptional profile consistent with T-lineage commitment. Functional assays demonstrated that DN2a-1 thymocytes were more developmentally advanced than ETPs but remained multipotent, whereas DN2a-2 thymocytes were more mature and largely restricted to the T lineage. Furthermore, in vivo clonal lineage tracing indicated that these subsets were on a developmental continuum. Together, these findings reveal that the heterogeneous DN2a stage can be resolved further by CD90 expression into pre- and post-T lineage-restricted subsets.