Abstract
BackgroundSplenic stroma overlaid with hematopoietic progenitors supports in vitro hematopoiesis with production of dendritic-like cells. Co-cultures of murine lineage-depleted bone marrow over the 5G3 stromal line produce two populations of cells, characterised as CD11b+CD11c+MHC-II− dendritic-like ‘L-DC’, and CD11b+CD11c+MHC-II+ cells, resembling conventional dendritic cells (cDC). To date, the functional capacity of these two subsets has not been clearly distinguished.ResultsHere we show both the L-DC and cDC-like subsets can be activated and induce proliferation of OT-I CD8+ T cells, being strong inducers of IL-2 and IFN-γ production. Both subsets lack ability to induce proliferation of OT-II CD4+ T cells. The cDC-like population is shown here to resemble regulatory DC in that they induce FoxP3 expression and IL-10 production in OT-II CD4+ T cells, in line with their function as regulatory DC. L-DC did not activate or induce the proliferation of CD4+ T cells and did not induce FoxP3 expression in CD4+ T cells. L-DC can be distinguished from cDC-like cells through their superior endocytic capacity and expression of 4-1BBL, F4/80 and Sirp-α. A comparison of gene expression by the two subsets was consistent with L-DC having an activated or immunostimulatory DC phenotype, while cDC-like cells reflect myeloid dendritic cells with inflammatory and suppressive properties, also consistent with functional characteristics as regulatory DC. When a Transwell membrane was used to prevent hematopoietic cell contact with stroma, only cDC-like cells and not L-DC were produced, and cell production was dependent on M-CSF production by stroma.ConclusionCo-cultures of hematopoietic progenitors over splenic stroma produce two distinct subsets of dendritic-like cells. These are here distinguished phenotypically and through gene expression differences. While both resemble DC, there are functionally distinct. L-DC activate CD8+ but not CD4+ T cells, while the cDC-like population induce regulatory T cells, so reflecting regulatory DC. The latter can be enriched through Transwell co-cultures with cell production dependent on M-CSF.
Highlights
Splenic stroma overlaid with hematopoietic progenitors supports in vitro hematopoiesis with production of dendritic-like cells
The CD11b+CD11c+ subset of dendritic-like cells was further divided on the basis of MHC-II, CD8α and B220 expression for delineation of CD11b+CD11c+MHC-II−CD8α−B220− ‘L-Dendritic cells (DC)’ and CD11b+CD11c+MHC-II+CD8α−B220− ‘conventional dendritic cells (cDC)-like cells’ as described previously [23, 24]
At the end of incubation, cells were stained for markers, and L-DC and cDC-like subsets gated on the basis of phenotype as CD11b+CD11c+MHC-II− and CD11b+CD11c+MHC-II+ cells, respectively. These were assessed flow cytometrically for endocytosis in terms of % cells taking up FITC-ovalbumin (OVA) compared with controls cells activated with L-DC, and with T cells activated by the control antigen presenting cells (APC) subsets of CD8+ cDC and CD8− cDC sorted from normal adult spleen (Fig. 4). In these experiments we showed that CD8− cDC were stronger activators of OT-I T cells than CD8+ cDC in terms of IL-2 and IFN-γ produced (Fig. 4)
Summary
Splenic stroma overlaid with hematopoietic progenitors supports in vitro hematopoiesis with production of dendritic-like cells. Stromal lines isolated from spleens of mouse [20,21,22] have since been shown to support restricted myelopoiesis from overlaid bone marrow progenitors with highly reproducible production of two distinct dendritic-like subsets [22,23,24,25]. These two distinct subsets are not developmentally linked and arise through differentiation of distinct progenitors/precursors [23, 24]. They are here investigated in terms of their similarity with L-DC and with reported subsets of DCregs produced in vitro
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