Poster Presentation Lorne Infection and Immunity 2022

Single-cell analysis of αβ versus γδ T cell development (#194)

Seungyoul SO Oh 1 2 , Xin XL Liu 1 , Daniel DG Gray 3 , Mark MC Chong 1 2
  1. Genomics and Immunology, St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia
  2. Department of Medicine, University of Melbourne, Parkville, Victoria, Australia
  3. The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

T cells develop in the thymus, where they acquire a range of distinct functional identities. T cells are divided into the αβ or γδ lineages. Progenitors that enter the thymus remain multipotent, with the potential to differentiate into a range of cell types. T lineage identity is then fixed early in T cell development. Later, γδ T cells branch off, while αβ development continues onto additional stages. Studies over the years have defined, at a population level, many of the specific genes that have to be activated or silenced as T cell progenitors differentiate toward either lineage. However, it is still unknown whether this combination of activation/silencing actually occurs in each and every cell as they differentiate. To investigate this, single-cell RNA-sequencing (scRNA-seq) was employed to re-assemble de novo a model of the early stages in T cell development based on the transcriptional profiles of individual cells. >20,000 CD4-CD8- double negative (DN) and γδ thymocytes were analysed using Chromium 10x scRNA-seq over three runs. Hierarchical clustering revealed that early thymocyte populations are much more complex than the standard view of T cell development. Assembly of these thymocyte populations into developmental trajectories based on gene expression suggested that the decision to differentiate into αβ versus γδ T cells occurs at a much earlier stage than the current model and that distinct DN1 and DN2 sub-populations are specific to either the αβ or γδ developmental pathways. To confirm that specific sub-populations are restricted to either αβ or γδ lineages, they were sorted and analysed for lineage outcomes in OP9-DL1 cultures. We show that lineage is already restricted in DN1 thymocytes, which is considered the earliest stages of T cell development. Thus, γδ developmental appears to occur in a parallel with αβ development and not a branch as previously thought.