Steady-state and emergency dendritic cell development at a clonal level

Abstract

Recent clonal fate and single cell RNA-sequencing studies demonstrate that significant lineage imprinting is already in place within individual haematopoietic stem and progenitor cells (HSPCs). Dendritic cells (DCs) represent one such branch of haematopoiesis and are responsible for pathogen-sensing and activation of the adaptive immune response. At the population level, all three major DC subtypes including type 1 conventional DCs (cDC1s), type 2 conventional DCs (cDC2s) and plasmacytoid DCs (pDCs), can be generated from a restricted common DC progenitor (CDP) population downstream of HSPCs. However, recent clonal evidence has suggested earlier subtype-specific imprinting within the CDP and even early HSPC populations. Therefore, the current hierarchical model of haematopoiesis is insufficient to explain the complexity and dynamics of DC development. The aim of this thesis was to investigate the development of DCs at the single cell level.

One caveat of most prior single cell lineage tracing studies was that clonal fate was only measured at a single time point. Therefore, questions remain as to whether the fate bias observed at a snapshot in time is consistent with earlier or later times. Here, using cellular barcoding, I develop an experimental and computational framework to allow robust periodical examination of lineage outputs of thousands of transient clones during DC development in vitro. I reveal that single HSPC clones are largely programmed regarding the types of DCs to make (fate), the number of DCs to produce (size), and when DC generation occurs (timing). Together, I define these unique properties as a clone’s cellular trajectory. Importantly, I demonstrate that a large proportion of early HSPCs are already committed towards either cDC or pDC generation, even when clonal output is measured over time. This finding is consistent with and further complements the most recent evidence of DC subtype imprinting during early haematopoiesis.

Exogenous administration of Flt3 ligand (FL) is known to preferentially induce ‘emergency’ DC development, and is shown to provide promising therapeutic benefits in various conditions such as infection and cancer. However, how FL signals regulate cell proliferation and differentiation during early DC development is largely unknown. In this thesis, I investigate the clonal aetiology of this process. Using cellular barcoding, I demonstrate that emergency DC generation is predominantly driven by increased expansion of pre-existing HSPC clones that are already primed with DC potential. Consistently, enhanced cell cycle activity is found to be prominent within most early HSPCs after short exposure to FL. In particular, using a single cell multi-omics profiling approach, I identify key cellular and molecular events within a unique group of early HSPCs that are most responsive to FL stimulation, which include increased cell division, maintenance of hyper-proliferative potential and establishment of a DC lineage program.

Collectively, the findings presented in this thesis provide new insights into the control and regulation of DC fate within individual HSPCs during steady-state and emergency haematopoiesis, with important implications regarding the maintenance or manipulation of DC generation in health and disease.