In vitro hematopoiesis produces a distinct class of immature dendritic cells from spleen progenitors with limeted T cell stimulation

School of Biochemistry and Molecular Biology, Australian National University, Canberra, ACT 0200, Australia.
International Immunology (Impact Factor: 2.54). 05/2004; 16(4):567-77.
Source: PubMed


The study of dendritic cells (DC) has been hampered by the difficulty of isolating rare cells for analysis of their phenotype and function. Interpretation of the DC lineage has been largely influenced by studies on cell populations which can be readily isolated and amplified in the presence of cytokines. Long term cultures (LTC) from murine spleen have been shown to support continuous in vitro hematopoiesis of DC dependent on interaction with a stromal cell monolayer. LTC-DC represent a single, stable class of DC derived by constant turnover of spleen DC progenitors maintained within stroma. They represent a resident DC population in spleen. The functional characteristics of LTC-DC have been studied in terms of capacity to stimulate T cells and response to activation by environmental stimuli. LTC-DC have many morphological, phenotypic and functional properties reflecting an immature or partially mature, marginal zone-like CD4(-)CD8(-) splenic DC subset. They are highly endocytic and can process and present protein antigen to naive hen egg lysozyme (HEL)-specific MHC-II-restricted TCR-Tg CD4(+) T cells. They do not, however, induce T cell proliferation in a mixed lymphocyte reaction. LTC-DC do not respond in a typical fashion to common DC activators like LPS and CD40L. They upregulate MHC-I and CD80/CD86 but not MHC-II and CD40. They reflect an endogenous, immature DC subset in spleen with properties distinct from immature DC located in peripheral tissues.

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    • "These cells are distinct in that they induce CD8+ T cell responses, but do not activate CD4+ T cells. Previous studies had shown that long-term cultures (LTC) of neonatal spleen maintained production of similar dendritic-like cells called “LTC-DC” over years, suggesting that they may be derived from self-renewing progenitors [14], [15], [16]. Cloned splenic stroma derived from LTC have since been shown to support development of equivalent cells called “L-DC” from overlaid lineage-depleted (Lin−) BM or purified HSC [8], [17], [18]. "
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    ABSTRACT: 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.
    PLoS ONE 02/2014; 9(2):e88311. DOI:10.1371/journal.pone.0088311 · 3.23 Impact Factor
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    • "L-DC express CD11b and CD11c molecules on their cell surface, but not other DC markers such as MHC-II, CD8α, or B220. Cells have high capacity to endocytose antigen and to induce CD8 T cell activation (5, 8, 31). L-DC produced in vitro or isolated from spleen have been shown to have powerful capacity to cross-present antigen to CD8+ T cells (7, 9). "
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    ABSTRACT: 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.
    Frontiers in Immunology 01/2014; 4:501. DOI:10.3389/fimmu.2013.00501
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    • "Consistent with this finding is evidence that the in vivo equivalent of this novel cell type can also be identified amongst resting splenic myeloid and DC subsets in normal mice, and it is not present in other organs (Tan et al., 2011). This novel in vivo subset parallels in terms of phenotype and function, with cells generated in vitro both in LT spleen cultures (Quah et al., 2004) and in co-cultures of HSC over stroma as shown here. "
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    ABSTRACT: 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" sub-set has cross-presenting capacity, leading to activation of CD8 + T cells, but not activating CD4 + T cells, which is consistent with their CD11c lo CD11b hi MHC-II − phenotype. For L-DC, an equivalent tissue-specific APC has been found only in spleen. A second population of CD11c hi CD11b lo MHC-II + cells resembling conventional dendritic cells (cDC) can activate both CD4 and CD8 T cells. Production of L-DC but not cDC-like cells is now shown to be dependent on contact between the L-DC progenitor and stroma such that the presence of a Transwell membrane can prevent L-DC development. Since L-DC can be produced contin-uously in vitro in stromal co-cultures overlaid with bone marrow (BM) progenitors, it was hypothesized that L-DC progenitors are self-renewing. The L-DC progenitor is shown here to be defined by the Flt3 − c-kit + Lin − Sca-1 + (F − KLS) subset of adult BM which contains primitive HSC. Since the less primitive F + KLS HSC subset also contains L-DC progenitors, Flt3 does not appear to be a defining marker for this progenitor. Precursors of the cDC-like subset are found only within the F + KLS subset and seed production of a transient popu-lation of APC. All data identify differentiation of L-DC from HSC, and of cDC-like cells from DC precursors, which occurs independently of inflammatory signals and is dependent on a splenic stromal microenvironment.
    Frontiers in Immunology 03/2013; 4(Article 73):1-11. DOI:10.3389/fimmu.2013.00073
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