MCSF drives regulatory DC development in stromal co-cultures supporting hematopoiesis

Background: Splenic 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. Results: Here 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 IL10 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 cDClike 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. Conclusion: Co-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. Keywords: Hematopoiesis, Dendritic cell, Regulatory dendritic cells, Regulatory T cellsThis work was supported by project grant #585443 to HO from the National Health and Medical Research Council of Australia. SP was supported by a graduate scholarship from the Royal Thai Government. PP was supported by an Australian National University Graduate Scholarship

Spleen as a site for hematopoiesis of a distinct antigen presenting cell type

While spleen and other secondary tissue sites contribute to hematopoiesis, the nature of cells produced and the environment under which this happens are not fully defined. Evidence is reviewed here for hematopoiesis occurring in the spleen microenvironment leading to the production of tissue-specific antigen presenting cells. The novel dendritic-like cell identified in spleen is phenotypically and functionally distinct from other described antigen presenting cells. In order to identify these cells as distinct, it has been necessary to show that their lineage origin and progenitors differ from that of other known dendritic and myeloid cell types. The spleen therefore represents a distinct microenvironment for hematopoiesis of a novel myeloid cell arising from self-renewing hematopoietic stem cells (HSC) or progenitors endogenous to spleen

Spleen as a Site for Hematopoiesis of a Distinct Antigen Presenting Cell Type

While spleen and other secondary tissue sites contribute to hematopoiesis, the nature of cells produced and the environment under which this happens are not fully defined. Evidence is reviewed here for hematopoiesis occurring in the spleen microenvironment leading to the production of tissue-specific antigen presenting cells. The novel dendritic-like cell identified in spleen is phenotypically and functionally distinct from other described antigen presenting cells. In order to identify these cells as distinct, it has been necessary to show that their lineage origin and progenitors differ from that of other known dendritic and myeloid cell types. The spleen therefore represents a distinct microenvironment for hematopoiesis of a novel myeloid cell arising from self-renewing hematopoietic stem cells (HSC) or progenitors endogenous to spleen

Studies on B Cells in the Fruit-Eating Black Flying Fox (Pteropus alecto)

The ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat's immunity. Here, we report a panel of cross-reactive antibodies against MHC-II, NK1.1, CD3, CD21, CD27, and immunoglobulin (Ig), that allows flow cytometry analysis of B, T and NK cell populations in two different fruit-eating bat species namely, Pteropus alecto and E. spelaea. Results confirmed predominance of T cells in the spleen and blood of bats, as previously reported by us. However, the percentages of B cells in bone marrow and NK cells in spleen varied greatly between wild caught P. alecto bats and E. spelaea colony bats, which may reflect inherent differences of their immune system or different immune status. Other features of bat B cells were investigated. A significant increase in sIg+ B cell population was observed in the spleen and blood from LPS-injected bats but not from poly I:C-injected bats, supporting T-independent polyclonal B cell activation by LPS. Furthermore, using an in vitro calcium release assay, P. alecto B cells exhibited significant calcium release upon cross-linking of their B cell receptor. Together, this work contributes to improve our knowledge of bat adaptive immunity in particular B cells

Stromal cells which direct dendritic cell development

Development of the immune system is depicted as a hierarchical process of differentiation from hematopoietic stem cells (HSC) to lineage-committed precursors, which further develop to give mature immune cells. In the case of dendritic cell (DC) development, this linear precursor-progeny approach has led to a complex picture of relationships between various subsets of DC identified in vivo. A possible reconciliation of the diversity of DC precursors and their progeny in vivo can be achieved by assessing the role of the microenvironment in hematopoiesis. One model is that the splenic microenvironment comprises one or more populations of stromal cells, each with a unique role in DC hematopoiesis. Stromal cells derived from spleen have been studied as a model microenvironment for DC development. Cloned splenic stromal cells lines derived from a spleen long-term culture (LTC) which formerly supported production of dendritic-like cells (LTC-DC), were tested for capacity to support DC hematopoiesis in co-cultures seeded with bone marrow (BM) as a source of progenitors. In particular, the 5G3 stroma, a good supporter of DC hematopoiesis was analysed using Affymetrix microarrays for genes specifically expressed or upregulated in 5G3 over the non-supporting stroma 3B5. This resulted in identification of many genes of interest. These included genes involved in hematopoiesis, like Clcal, Sfrp2, Rspo2, Abcgl, ApoD, Aldhlal and Serpinasn, and novel genes such as Ms4a4d, Svep1, Plxdc2 and Ctla2a. The expression of these genes by 5G3 stroma was further confirmed by Realtime PCR. Cells produced in 5G3 co-cultures seeded with lineage negative (Lin') BM were characterized in terms of phenotype and function. Two populations of dendritic-like cells were characterized as CDllc.cnub+MHC-Ir L-DC and CD11c+CD11b+MHC-11+ cells resembling conventional (c)DC. The L-DC subset was shown to be phenotypically and functionally similar to LTC-DC, in terms of high endocytic capacity, ability to crosspresent antigen to CDS+ T cells and inability to activate CD4+ T cells. These features identify L-DC as distinct in comparison with other DC subsets described in spleen. The cDC-like cells showed ability to crosspresent antigen to CD8+ T cells, and were weakly endocytic. In addition, they could activate CD4+ T cells, but did not induce proliferation of CD4+ T cells, suggesting a role in tolerance or suppression. Investigation of cytokine gene 0. expression by Realtime PCR revealed many differences between the two subsets. A 10-fold increase in ILlO expression in cDC-like cells over L-DC, was also consistent with ability of cDC-like cells to be immunosuppressive. In an attempt to elucidate the stroma-dependent progenitor responsible for development of L-DC, HSC with both long-term (LT) and short-term (ST) reconstituting potential, were sorted and co-cultured over 5G3 stroma. LT-HSC required stromal contact to produce progeny CD11c+CD11b+MHC-II L-DC. In contrast, ST-HSC produced mature, myeloid CD11c"CD11b+MHC-11+ cDC-like cells in a contact-independent manner. These results identified the L-DC progenitor amongst HSC subsets, and also distinguished the lineage origin of L-DC from that of cDC-like cells. In spleen, the L-DC progenitor was identified within the LinClrl lc' c_kit10 subset which also contains HSC. These results delineate a distinct DC type in terms of phenotype and function. It can be derived by in vitro differentiation from HSC in the context of a splenic stromal niche that has a unique ability to support hematopoiesis

Stroma-dependent development of two dendritic-like cell types with distinct antigen presenting capability

Novel antigen presenting cells (APCs) have been described in the murine spleen. Cells have a distinct CD11c(lo)CD11b(hi)MHC-II(-)CD8α(-) phenotype as highly endocytic dendritic-like cells that cross-present antigen to CD8(+) T cells but fail to activate CD4(+) T cells. These cells are named "L-DCs" because they reflect dendritic cells (DCs) produced in long-term spleen cultures (LTC). Similar cells were produced when bone marrow progenitors were cocultured over the splenic stromal line 5G3. Cocultures continuously produced a majority of L-DCs and a transient population of cells reflecting conventional dendritic cells (cDCs). Both the L-DC and cDC-like subsets cross-present antigen to CD8(+) T cells, inducing their activation and proliferation. However, as MHC-II(-) cells, L-DCs are unable to activate CD4(+) T cells, while MHC-II(+) cDC-like cells present antigen for CD4(+) T cell activation. These results distinguish two APC subsets produced in vitro: a transient population of cDC-like cells and L-DCs that are continuously produced, presumably from self-renewing progenitors. These subsets are not developmentally linked via a precursor or progeny relationship. L-DCs and cDC-like cells are also distinct in terms of cytokine expression, with 65 of 84 tested genes displaying greater than a twofold difference by quantitative reverse-transcriptase polymerase chain reaction. Splenic stroma supports production of two APC subsets reflecting different lineage origins.NHMRC (National Health and Medical Research Council of Australia

In vitro hematopoiesis reveals a novel dendritic-like cell present in murine spleen

Dendritic cells (DC) are important antigen presenting cells (APC) which induce and control the adaptive immune response. In spleen alone, multiple DC subsets can be distinguished by cell surface marker phenotype. Most of these have been shown to develop

Delineation of precursors in murine spleen that develop in contact with splenic endothelium to give novel dendritic-like cells

Hematopoietic cell lineages are best described in terms of distinct progenitors with limited differentiative capacity. To distinguish cell lineages, it is necessary to define progenitors and induce their differentiation in vitro. We previously reported in vitro development of immature dendritic-like cells (DCs) in long-term cultures (LTCs) of murine spleen, and in cocultures of spleen or bone marrow (BM) over splenic endothelial cell lines derived from LTCs. Cells produced are phenotypically distinct CD11bhiCD11c loCD8-MHC-II- cells, tentatively named L-DCs. Here we delineate L-DC progenitors as different from known DC progenitors in BM and DC precursors in spleen. The progenitor is contained within the lineage-negative (Lin)-c-kit+ subset in neonatal and adult spleen. This subset has multipotential reconstituting ability in mice. In neonatal spleen, the progenitor is further enriched within the c-kit lo and CD34+ subsets of Lin-c-kit+ cells. These cells seed cocultures of splenic endothelial cells, differentiating to give L-DCs that can activate T cells. L-DC progenitors are distinguishable from described splenic CD11clo DC precursors and from Fms-like tyrosine kinase 3+ DC progenitors in BM. Overall, this study confirms that LTCs are a physiologically relevant culture system for in vitro development of a novel DC type from spleen progenitors

Development of two distinct dendritic-like APCs in the context of splenic stroma

Murine splenic stroma has been found to provide an in vitro niche for hematopoiesis of dendritic-like APC. Two distinct cell types have been characterized. The novel "L-DC" subset has cross-presenting capacity, leading to activation of CD8+ T cells, but nFunding information: NHMRC (National Health and Medical Research Council of Australia