ABSTRACT
T-cell development occurs in the thymus and is tightly regulated to produce a diverse enough repertoire of mature T cells that can recognize any potential pathogen. The development of T cells is dependent on small numbers of uncommitted precursors that continually seed the thymus from the bone marrow. As they progress along the developmental pathway, there is a massive expansion in cell number to generate the necessary diversity in T-cell receptor chain usage. It is recognized that there are two proliferative bursts that occur early in T-cell development, one prior to ß-selection and one after, and these are responsible for the expansion. While the proliferation following ß-selection is well-characterized, the earlier proliferative burst has yet to be precisely defined. In this study, we employ single-cell RNA sequencing coupled to trajectory inference methods to pinpoint when in T-cell development thymocytes are induced into cell cycle. We show that the first proliferative burst is initiated in the double-negative (DN) 2a stage before T lineage commitment occurs, with cell cycling downregulated by the DN3a stage. A second burst is then initiated at the DN3b stage, immediately after ß-selection. We subsequently employ fluorescence-activated cell sorting-based analysis for DNA content to confirm these two proliferative bursts.