CLONAL ETIOLOGY OF EMERGENCY DENDRITIC CELL DEVELOPMENT

Dendritic cells (DCs) are critical mediators of both innate and adaptive immune responses. Increasing DC numbers by administration of Flt3 ligand (FL) provides therapeutic benefit for infection and cancer immunotherapy. However, how FL signals regulate emergency DC development at the clonal level is largely unknown. To interrogate the early cellular and molecular events within hematopoietic stem and progenitor cells (HSPCs) after FL stimulation, we develop a single cell multi-omics profiling approach. This allows simultaneous measurement of surface marker phenotype, division history and the transcriptome of single HSPCs treated with PBS or FL after transfer into non-irradiated recipients. We observe hyperproliferation of most HSPCs after FL treatment. In particular, we identify a unique group of hyper-proliferative cells that co-expresses gene signatures of both ‘multipotent progenitors’ and ‘DC progenitors’, indicative of the earliest molecular and cellular events during emergency DC development. To understand how FL affects HSPC fate towards other lineages at the clonal level, we tag individual HSPCs with unique DNA barcodes and compare their development, with or without FL stimulation in vivo. We observe no evidence of lineage diversion of HSPCs away from alternative fates and instead determine that pre-existing multi-/oligo-potent retain their potency and selectively expand a DC-producing sub-branch, leading to preferential production of DCs. Collectively, our results suggest that lineage programs within single HSPCs are established and stable, and that selective clonal expansion, not fate ‘plasticity’ of HSPCs via extrinsic regulation, explains FL-mediated emergency DC development. These findings provide new insight into the control and regulation of fate during hematopoiesis.