Medicine (St Vincent's) - Theses
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ItemSingle-cell analysis of ab versus gd T cell development( 2022)T cells are divided into the alphabeta and gammadelta lineages. It is currently thought both lineages develop in the thymus from common uncommitted progenitors that are seeded from the bone marrow. Over the years, studies at a population level have identified many of the specific genes that must be activated or silenced as T cell progenitors differentiate towards either lineage. Despite much effort over the years, it is still unknown whether this combination of activation and silencing occurs in each cell as they differentiate. In addition, the exact branch point at which the alphabeta and gammadelta lineages diverge remains contentious. Although the prevailing T cell developmental model assumes that the gammadelta lineage branches from the main developmental pathway around the DN2b and DN3a stages, which is when definitive gammadelta lineage markers become obvious, it is still unclear whether this is actually the case. To better understand the development of the gammadelta lineage, the kinetics of alphabeta versus gammadelta T cell development were tracked in OP9-DL1 cultures. This revealed the two lineages may develop along different pathways. Moreover, cellular barcoding analysis of CD4-CD8- double negative 1 (DN1) thymocytes, which suggested these thymocytes may in fact be a heterogenous mixture consisting of distinct subpopulations that develop into either alphabeta cells or gammadelta cells. To investigate the early alphabeta versus gammadelta T cell development more precisely, single-cell RNA-sequencing (scRNA-seq) was employed to determine the transcriptional profiles of individual DN and gammadelta thymocytes, which was then used to re-assemble de novo a model of the early stages in T cell development. >20,000 DN and gammadelta 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 early T cell development. Computational trajectory analyses suggested that the alphabeta versus gammadelta decision may actually be established at the DN1 stage, which is consistent with the previous cellular barcoding experiments. Eight transcriptionally distinct DN1 subpopulations were identified. One or more of these may be the bipotent precursor(s) of both alphabeta and gammadelta lineages. Alternatively, it may be that specific subpopulations are precursors of the alphabeta lineages, while others are precursors of the gammadelta lineage. To investigate this, these subpopulations were sorted and analysed for the lineage outcomes in OP9-DL1 co-cultures. I show that distinct DN1 subpopulations are in fact restricted to the alphabeta lineage, while others are restricted to the gammadelta lineage. Moreover, specific gammadelta-primed DN1 subpopulations preferentially develop into IL-17 or IFNg-producing gammadelta T cells, well before the expression of lineage-defining T cell receptors. Thus, T cell lineage decisions are already hardwired from the earliest stages of T cell development. These new insights into hardwiring of lineage commitment represents a paradigm-change in our understanding of early T cell development. This has the potential to form the basis for more comprehensive research to elucidate underlying molecular mechanisms and framework to address how the lineage decision is made. Ultimately, this will have significant implications on designing novel therapeutics targets for treating diseases.