Mapping the two distinct proliferative bursts early in T‐cell development

Abstract

AbstractT‐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 β‐selection and one after, and these are responsible for the expansion. While the proliferation following β‐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 β‐selection. We subsequently employ fluorescence‐activated cell sorting–based analysis for DNA content to confirm these two proliferative bursts.