Title: Delineating Human Dendritic Cell Development
Description: The origin of human dendritic cells (DCs) has long been debated. DCs are a subset of innate immune cells that are essential for initiating adaptive immune responses. Determining their ontogeny is critical for vaccine development and for unveiling the molecular mechanism of DC insufficiency, which underlies many primary immune deficiency disorders and leukemia. Like all blood cells, human DCs develop from hematopoietic stem cells through a sequence of increasingly restricted progenitors. Initially it was assumed that DCs should derive exclusively from myeloid progenitors. However, during the past few decades, a number of myeloid and lymphoid progenitors have been described and shown to have DC potential, instigating the debate of the myeloid vs. lymphoid origin of DCs. Hindering the resolution of this debate, human DC-restricted progenitors had not been identified. Further, the potential of known progenitors could not be interrogated due to the lack of a culture system that supports simultaneous differentiation of all human DCs subsets, in conjunction with other myeloid and lymphoid cells.

In this work, we establish a culture system that supports the development of the three major subsets of DCs (plasmacytoid DCs or pDCs, and the two classical DCs, cDCs) as well as granulocytes, monocytes and lymphocytes. This system combines mitomycin C-treated murine stromal cell lines, MS5 and OP9, together with human recombinant cytokines, FLT3L, SCF and GM-CSF, and supports the differentiation of progenitor cells at a population and single cell level. Using this culture in combination with a phenotypic characterization of CD34+ progenitor cells, we identify four consecutive DC progenitors with increasing degree of commitment to DCs and describe their anatomical location of development. We show that DCs develop from a granulocyte-monocyte-DC progenitor (GMDP), which produces granulocytes and a monocyte-DC progenitor (MDP), which then generates monocytes and a common DC progenitor (CDP), which produces pDCs and a cDC precursor (pre-cDC), which finally produces cDCs only. Lastly, we establish a staining panel that allows the phenotypic identification and separation of newly found DC progenitors as well as all previously described myeloid and lymphoid progenitors.

We investigate their inter-developmental relationship and DC potential at the single cell level. We show that each progenitor population with homogeneous phenotype is heterogeneous in developmental potential and prove that cell surface marker expression cannot be directly equated to a specific developmental potential. In order to resolve the unreliability of phenotype to draw developmental pathways, we propose to use the quantitative clonal output in order to delineate the DC developmental pathway. In summary, our studies provide a new tool to determine DC potential in vitro, identify new stages of DC development, and propose a new method for tracing the developmental pathway for DC lineage. This will generate a new model of dendritic cell hematopoiesis that can explain and reconcile the conflicts of data on DC origin and development.