Distinct subpopulations of DN1 thymocytes exhibit preferential γδ T lineage potential

The αβ and γδ T cell lineages are both are thought to differentiate in the thymus from common uncommitted progenitors. The earliest stage of T cell development is known as known as CD4 - CD8 - double negative 1 (DN1). These thymocytes have previously been revealed to be a heterogenous mixture of cells, which of only the CD117 + fraction have been proposed to be true T cell progenitors that progress to the DN2 and DN3 thymocyte stages, at which point the development of the αβ and γδ T cell lineages diverge. However, recently, it has been shown that at least some γδ T cells are actually derived from a subset of CD117 - DN thymocytes. Along with other ambiguities, this suggests that T cell development may not be as straightforward as previously thought. To better understand early T cell development, particularly the heterogeneity of DN1 thymocytes, we performed single cell RNA sequence (scRNAseq) of mouse DN and γδ thymocytes and show that the various DN stages are indeed comprised of transcriptionally diverse subpopulations of cells. We also show that multiple subpopulations of DN1 thymocytes exhibit preferential development towards the γδ lineage. Furthermore, specific γδ-primed DN1 subpopulations preferentially develop into IL-17 or IFNγ-producing γδ T cells. We show that DN1 subpopulations that only give rise to IL-17-producing γδ T cells already express many of the transcription factors associated with type 17 immune cell differentiation, while the DN1 subpopulations that can give rise to IFNγ-producing γδ T cell already express transcription factors associated with type 1 immune cell differentiation.