Delineation of Dendritic Cell Haematopoiesis in Spleen

Dendritic cells (DC) are specialized antigen presenting cells critical for activating T cell immunity. In vitro studies using cytokine-driven culture systems have informed the function and precursor origin of these rare cells. Our lab has developed an in vitro long-term culture (LTC) system which continuously produces immature myeloid DC. Recently, we identified a novel DC subset in spleen with phenotypic and functional identity to LTC-DC. DC development in LTC is dependent on a monolayer of endothelial stromal cells and requires no additional cytokines. LTC continuously maintain 2 populations of large-sized DC and small-sized precursors/progenitors. We have now characterised precursors maintained in LTC, and subsequently the equivalent in vivo precursor population in spleen. Sorting of phenotypically distinct LTC cell subsets, followed by co-culture over splenic stromal cells (STX3), led to identification of Lin-ckitloSca1-CD34- small cells as DC precursors. Based on cell subset identity in LTC, we have also defined a Lin-ckitlo spleen cell subset which also produces this same, novel DC subset after co-culture above STX3 stroma. Since LTC derived from spleen maintain DC production for years, it has been hypothesized that LTC may also maintain self-renewing haematopoietic stem cells (HSC). Indeed, LTC also contains an HSC-like Lin-ckithiSca1+ population. The haematopoietic potential of LTC-derived cells was tested in a competitive reconstitution assay, by adoptive transfer of cells into CD45 allotype-distinct lethally irradiated mice. Ability to give long-term reconstitution typical of HSC was confirmed by phenotype analysis of progeny cells at ~20 weeks when 3 out of 8 chimeras showed evidence of LTC-derived progeny cells. Subsequent chimera studies have identified HSC in spleen as biased in their capacity to generate DC of this novel subset over other haematopoietic cell types. All of these studies point to a definitive role for spleen in supporting DC haematopoiesis.