Hematopoiesis in the context of the spleen microenvironment

Abstract

Hematopoiesis involves the generation of fully differentiated blood cells from hematopoietic stem cells (HSC), commonly maintained within the bone marrow (BM) microenvironment. This lab has identified a role for the spleen microenvironment in supporting hematopoiesis and the development of dendritic-like cells. Here, the relationship between dendritic-like cells developing in vitro over splenic stroma and other DC subsets has been investigated through assessment of their progenitors, and ontogeny in spleen. Two populations of dendritic-like cells produced in splenic stromal co-cultures established with lineage-depleted (Lin) BM have been characterised: CD11b+CD11c+MHC-II- L-DC and CD11b+CD11c+MHC-II+ cDC-like cells. L-DC were further distinguished by their expression of 4-1BBL, F4/80 and Sirp-alpha. L-DC showed high capacity for activation of CD8+ T cells, but not CD4+ T cells, confirmed by release of IL-2 and IFN-gamma by effector T cells. cDC-like cells were unable to activate CD4+ T cells, but induced expression of Foxp3 and regulatory T cell formation, suggesting properties of regulatory DC. Isolated BM hematopoietic progenitors were tested for capacity to undergo hematopoiesis when overlaid on 5G3 stroma. (LT)-HSC gave rise to only L-DC in co-cultures, while multipotent progenitors (MPP) gave a majority of L-DC, with fewer cDC-like cells. Monocyte/dendritic cell progenitors and common dendritic cell progenitors produced only cDC-like cells in co-cultures. These findings distinguished L-DC as a novel antigen presenting cell (APC) subset arising from self-renewing HSC. Analysis of cytokine dependency for DC production in co-cultures showed that L-DC development occurs independently of Flt3L and GM-CSF. Further studies in booreana mutants indicate that the c-MybE308G mutation has no effect on L-DC development, and serves to distinguish L-DC from the monocyte/macrophage lineage. An in vivo equivalent of L-DC appeared early in E18.5 spleen, while pDC and cDC appeared at 2 and 4 days following birth. E18.5 spleen co-cultured produced only L-DC, while cDC-like cells were produced only in postnatal spleen co-cultures along with L-DC. These findings distinguish L-DC as a lineage distinct from common DC subsets in spleen. Indeed, LT-HSC frequency was increased in one-day old spleen, and produced only L-DC in 5G3 co-cultures. These results indicate that HSC endogenous to spleen may serve as progenitors for L-DC. Human L-DC were identified as a hCD11c+HLA-DR-hCD86+hCD11b+ subset in spleen, along with known subsets of hCD1c+ DC, hCD123+ pDC, hCD16+ DC and hCD141+ DC. Three subsets of monocytes were also characterised in human spleen. Indeed, analysis of DC and monocytic subsets in human spleens showed similarity with subsets in human blood. Stromal cells were established from cultured human spleen and tested for their ability to support in vitro hematopoiesis and DC development. The cloned hu7B2 stromal line supported development of dendritic-like cells from overlaid human BM, Lin- cord blood and splenocytes. In addition, the hu7B2 stroma supported murine L-DC and cDC-like cell development from overlaid Lin- BM. These studies demonstrated that the human splenic microenvironment can support DC hematopoiesis. This study has revealed extramedullary hematopoiesis in both murine and human spleen and predicts an important role for the splenic stromal microenvironment in the development of tissue-specific APC. Show more