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

Hematopoiesis leading to a diversity of dendritic antigen-presenting cell types

Hematopoietic stem cells (HSCs) undergo expansion and differentiation, giving rise to all terminally differentiated blood cells throughout life. HSCs are found in distinct anatomical sites during development, and in adults, hematopoiesis occurs predominantly on the luminal side of the bone cavity in bone marrow. Millions of newly formed blood cells are generated per second to accommodate the short half-life of hematopoietic cells. For this to happen, HSCs must sustain their self-renewal capacity as well as their capability to commit and differentiate toward multiple cell lineages. Development of the hematopoietic system is finely regulated as the animal ages, so that it does not become exhausted or misdirected. This review covers aspects of hematopoietic development from the embryonic period through adult life in relation to development of dendritic cells. It also considers a role for HSCs in extramedullary sites and their possible role in myelopoiesis, with formation of tissue-specific antigen-presenting cells.NHMRC (National Health and Medical Research Council of Australia

Distinct progenitor origin distinguishes a lineage of dendritic-like cells in spleen

The dendritic cell (DC) compartment comprises subsets of cells with distinct phenotypes. Previously this lab reported methodology for hematopoiesis of dendritic-like cells in vitro dependent on a murine splenic stromal cell line (5G3). Co-cultures of lineage-depleted bone marrow (Lin(-) BM) over 5G3 continuously produced a distinct population of dendritic-like "L-DC" for up to 35 days. Here the progenitor of L-DC is investigated in relation to known BM-derived hematopoietic progenitors. It is shown here that L-DC-like cells also derive from the CD150(+)Flt3(-) long-term reconstituting-hematopoietic stem cells (HSC), and also from the Flt3(+) multipotential progenitor subset in BM. Lin(-) BM co-cultures also produce a transient population of cells resembling conventional (c) DC. Production of cDC-like cells is shown here to be transient and M-CSF dependent, and also appears following co-culture of described common dendritic progenitors or monocyte dendritic progenitors over 5G3. BM cells from C57BL/6-flt3L(tm1lmx) and C57BL/6-Csf2(tm1Ard) mice which lack cDC precursors and monocytes, are shown here to contain L-DC progenitors which can seed 5G3 co-cultures. L-DC are functionally distinct cells, in that they arise independently of M-CSF, and by direct differentiation from HSC.This work was supported by project grant #585443 to Helen Christine O’Neill from the National Health and Medical Research Council of Australia. Sawang Petvises was supported by a graduate scholarship from the Royal Thai Government

Characterisation of dendritic cells arising from progenitors endogenous to murine spleen

Heterogeneity amongst dendritic cell (DC) subsets leads to a spectrum of immune response capacity against pathogens. Several DC subsets in spleen have been described which differ in terms of phenotype and function. We have previously reported a distinct population of CD11c(lo)CD11b(hi)MHC-II(-)CD8(-) dendritic-like "L-DC" in murine spleen, which can also be generated in splenic stromal longterm cultures. Here, the ontogeny of L-DC development in perinatal mice has been compared with other known splenic DC subsets. Flow cytometric analysis has revealed the presence of L-DC at embryonic age (E)18.5 spleen, while plasmacytoid (p)DC and conventional (c)DC appear at 2 and 4 days following birth. Co-cultures of E18.5 spleen above splenic stroma also showed production of only L-DC, while spleen cells from D0 through D5 neonates showed production of both L-DC and cDC-like cells. Addition of an M-CSFR inhibitor to co-cultures revealed that while the development of cDC-like cells depended on M-CSF, many L-DC developed independently of M-CSF. Furthermore, purified hematopoietic stem cells (HSC) and multipotential progenitors (MPP) isolated from neonatal D1 spleen are capable of developing into L-DC in co-cultures. These studies reveal a lineage of dendritic-like cells developing in the spleen microenvironment, and which appear to arise from endogenous progenitors laid down in spleen during embryogenesis.This work was supported by project grant #585443 to H.C.O. from the National Health and Medical Research Council of Australia. S.P. was supported by a graduate scholarship from the Royal Thai Government.

Impact of the c-MybE308G mutation on mouse myelopoiesis and dendritic cell development

Booreana mice carrying the c-Myb308G point mutation were analyzed to determine changes in early hematopoiesis in the bone marrow and among mature cells in the periphery. This point mutation led to increased numbers of early hematopoietic stem and progenitor cells (HSPCs), with a subsequent reduction in the development of B cells, erythroid cells, and neutrophils, and increased numbers of myeloid cells and granulocytes. Myelopoiesis was further investigated by way of particular subsets affected. A specific question addressed whether booreana mice contained increased numbers of dendritic-like cells (L-DC subset) recently identified in the spleen, since L-DCs arise in vitro by direct differentiation from HSPCs co-cultured over splenic stroma. The non-lethal c-Myb mutation in booreana mice was associated with significantly lower representation of splenic CD8- conventional dendritic cells (cDCs), inflammatory monocytes, and neutrophils compared to wild-type mice. This result confirmed the bone marrow origin of progenitors for these subsets since c-Myb is essential for their development. Production of L-DCs and resident monocytes was not affected by the c-MybE308G mutation. These subsets may derive from different progenitors than those in bone marrow, and are potentially established in the spleen during embryogenesis. An alternative explanation may be needed for why there was no change in CD8+ cDCs in booreana spleen since these cells are known to derive from common dendritic progenitors in bone marrow.This work was supported by a project grant #585443 to HO from the National Health and Medical Research Council (NHMRC) of Australia. PP was supported by an NHMRC CJ Martin Fellowship. YH was supported by a postgraduate scholarship from the Australian National Universit

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

Hematopoiesis in the context of the spleen microenvironment

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

Distinct progenitor origin distinguishes a lineage of dendritic-like cells in spleen

The dendritic cell (DC) compartment comprises subsets of cells with distinct phenotypes. Previously this lab reported methodology for hematopoiesis of dendritic-like cells in vitro dependent on a murine splenic stromal cell line (5G3). Co-cultures of lineage-depleted bone marrow (Lin− BM) over 5G3 continuously produced a distinct population of dendritic-like “L-DC” for up to 35 days. Here the progenitor of L-DC is investigated in relation to known BM-derived hematopoietic progenitors. It is shown here that L-DC-like cells also derive from the CD150+Flt3− long-term reconstituting-hematopoietic stem cells (HSC), and also from the Flt3+ multipotential progenitor subset in BM. Lin− BM co-cultures also produce a transient population of cells resembling conventional (c) DC. Production of cDC-like cells is shown here to be transient and M-CSF dependent, and also appears following co-culture of described common dendritic progenitors or monocyte dendritic progenitors over 5G3. BM cells from C57BL/6-flt3Ltm1lmx and C57BL/6-Csf2tm1Ard mice which lack cDC precursors and monocytes, are shown here to contain L-DC progenitors which can seed 5G3 co-cultures. L-DC are functionally distinct cells, in that they arise independently of M-CSF, and by direct differentiation from HSC

Myelopoiesis related to perinatal spleen

Adult murine spleen is known to have a major role in the development of dendritic cell (DC) subsets, including conventional DC and plasmacytoid DC. In this lab, long-term cultures (LTCs) established from murine spleen support continuous production of novel dendritic-like cells, termed LTC-DC. An in vivo equivalent subset also exists in spleen, namely L-DC. As co-cultures using LTC-derived splenic stroma support the outgrowth of L-DC from spleen and bone marrow sources, it is likely that spleen represents an important niche for DC development. To investigate the appearance of L-DC during ontogeny, spleen was isolated from embryonic and neonatal mice of different ages for analysis of myeloid and DC subsets. Perinatal spleen was also used to establish co-cultures for identification of progenitors, and LTCs were established from spleens for assessment of stromal competence. Although spleen from 16-day embryos (E16.5) contained myeloid cells, DC subsets did not appear until day 4 after birth (D4). However, murine spleen at D0 contained progenitors, which could seed co-cultures for L-DC production. LTC could not be established from spleen until D4. The appearance of L-DC after D4 in spleen is dependent on the formation of the appropriate stromal microenvironment which occurs in the early postnatal period