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Transmission electron microscopy showing characteristic features of cells produced in spleen culture (15 d) under various magnifications. Clockwise from top left: ×5800, ×700, ×3000, ×2900, ×2900, ×6500.
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A stromal cell-dependent long-term culture (LTC) system has been developed from spleen which continuously generates non-adherent cells with dendritic cell (DC) characteristics. Bioassays using factor-dependent cell lines have revealed that both spleen and thymic stromal cultures secrete interleukin (IL)-3 and IL-6-like growth factors. Conditioned m...
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... Inhibitors of known signaling pathways including Notch, Wnt, and the tyrosine kinases c-KIT and PDGFR have been tested in coculture assays for impact on cell production, maintenance of progenitors, and production of myeloid cells including L-DC. [13,14]. These have been shown to comprise a stromal cell monolayer which continuously supports the production of distinct myeloid cell types. ...
There are very few model systems which demonstrate hematopoiesis in vitro . Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro . Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic cells and to resemble perivascular reticular cells in the bone marrow through gene expression. In particular, 5G3 resembles CXCL12-abundant reticular cells or perivascular reticular cells, which are important niche elements for hematopoiesis in the bone marrow. To analyse the hematopoietic support function of 5G3, specific signaling pathway inhibitors were tested for the ability to regulate cell production in vitro in cocultures of stroma overlaid with bone marrow-derived hematopoietic stem/progenitor cells. These studies identified an important role for Wnt and Notch pathways as well as tyrosine kinase receptors like c-KIT and PDGFR. Cell production in stromal cocultures constitutes hematopoiesis, since signaling pathways provided by splenic stroma reflect those which support hematopoiesis in the bone marrow.
... FRCs were isolated from single-cell suspensions of naive BALB/c splenocytes using a modification of previously published methods (12). First, the splenocytes were depleted of CD45 + cells using anti-CD45 beads (Miltenyi Biotec, Cologne, Germany) and magnetic separation. ...
... We hypothesized that coculture of splenocytes with FRCs would facilitate in vitro modeling of the in vivo CD4 T cell priming environment and potentially enable high-throughput screening of adjuvants to predict their impact on CD4 T cell priming during vaccination. As a first step toward developing such a predictive in vitro priming system, we established cultures of normal FRCs from BALB/c spleen using previously published methods (12). The cultivation of CD45-depleted splenocytes under the conditions used generated homogeneous populations of viable fibroblastic cells that appeared capable of long-term survival and growth in a contact-inhibited manner. ...
Effective subunit vaccines require the incorporation of adjuvants that stimulate cells of the innate immune system to generate protective adaptive immune responses. Pattern recognition receptor agonists are a growing class of potential adjuvants that can shape the character of the immune response to subunit vaccines by directing the polarization of CD4 T cell differentiation to various functional subsets. In the current study, we applied a high-throughput in vitro screen to assess murine CD4 T cell polarization by a panel of pattern recognition receptor agonists. This identified lipopeptides with TLR2 agonist activity as exceptional Th1-polarizing adjuvants. In vivo, we demonstrated that i.v. administration of TLR2 agonists with Ag in mice replicated the findings from in vitro screening by promoting strong Th1 polarization. In contrast, TLR2 agonists inhibited priming of Th1 responses when administered cutaneously in mice. This route-specific suppression was associated with infiltrating CCR2⁺ cells in the skin-draining lymph nodes and was not uniquely dependent on any of the well characterized subsets of dendritic cells known to reside in the skin. We further demonstrated that priming of CD4 T cells to generate Th1 effectors following immunization with the Mycobacterium bovis bacillus Calmette-Guérin (BCG) strain, a lipoprotein-rich bacterium recognized by TLR2, was dependent on the immunization route, with significantly greater Th1 responses with i.v. compared with intradermal administration of BCG. A more complete understanding of route-dependent TLR2 responses may be critical for informed design of novel subunit vaccines and for improvement of BCG and other vaccines based on live-attenuated organisms. The Journ Al of Immunology, 2018, 201: 3604-3616.
... The continuous stromal cell lines 5G3 and 3B5 have been previously described [21,22]. They were cloned from an original STX3 splenic stroma [13,23,24], established from a longterm culture (LTC) of B10.A(2R) mouse spleen which had ceased production of DC over time due to loss of hematopoietic cells. Stroma was maintained by scraping cells for passage into a new flask and culture at 37˚C in 5% CO 2 in air in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 5 x 10 -4 M 2-mercaptoethanol, 10mM HEPES, 100U/ml penicillin, 100ug/ml streptomycin, 4mg/L glucose, 6mg/L folic acid, 36mg/L L-asparagine, 116mg/L L-asparagine hydrochloric acid (sDMEM). ...
... Double stranded cDNA was synthesized from RNA in a two-step process. The first strand of cDNA was synthesis using T7-(dT) 24 primers and Superscript II reverse transcriptase (Invitrogen Life Technologies: Mount Waverley, VIC, Australia). This was followed by second strand cDNA synthesis. ...
Cultured splenic stroma has been shown to support in vitro hematopoiesis in overlaid bone marrow and spleen progenitors. These co-cultures support longterm production of a novel dendritic-like cell type along with transient production of myeloid cells. They also maintain a progenitor cell population. The splenic stromal lines 5G3 and 3B5 have been identified as a supporter and a non-supporter of hematopoiesis. Based on their gene expression profile, both 5G3 and 3B5 express genes related to hematopoiesis, while 5G3 cells express several unique genes, and show upregulation of some genes over 3B5. Based on gene expression studies, specific inhibitors were tested for capacity to inhibit hematopoiesis in co-cultures. Addition of specific antibodies and small molecule inhibitors identified VCAM1, CXCL12, CSF1 and SPP1 as potential regulators of hematopoiesis, although both are expressed by 5G3 and 3B5. Through inhibition of function, SVEP1 and ALDH1 are also shown here to be deterministic of 5G3 hematopoietic support capacity, since these are uniquely expressed by 5G3 and not 3B5. The achievement of inhibition is notable given the dynamic, longterm nature of co-cultures which involve only small numbers of cells. The alternate plan, to add recombinant soluble factors produced by 5G3 back into 3B5 co-cultures in order to recover in vitro hematopoiesis, proved ineffective. Out of 6 different factors added to 3B5, only IGF2 showed any effect on cell production. The identification of differentially expressed or upregulated genes in 5G3 has provided an insight into potential pathways involved in in vitro hematopoiesis leading to production of dendritic-like cells.
... As with all previous 5G3 co-culture studies involving overlaid hematopoietic progenitors, only myelopoiesis or DC development was detected in either boo/boo or WT type co-cultures, with no production of lymphoid cells or DC subsets expressing markers like CD8 + or B220 + [42][43][44]. L-DC production was maintained by co-cultures established with both boo/boo and WT bone marrow for up to 6 months (data not shown), which was consistent with all previous studies on in vitro hematopoiesis with L-DC production [26,34,42]. ...
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.
... The 5G3 splenic stromal line supports hematopoiesis in cocultured bone marrow and splenocytes. [27][28][29] The 5G3 stromal cells were maintained by scraping up adherent cells to passage every 3-4 days. Stromal cells were cultured at 37uC in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4 mg/ml glucose, 6 mg/ml folic acid, 36 mg/ml L-asparagine, 116 mg/ml L-asparagine hydrochloric acid, to which was added 10% fetal calf serum, 5310 24 M 2-mercaptoethanol, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 100 U/ml penicillin and 100 mg/ml streptomycin (sDMEM). ...
Dendritic cells (DCs) and monocyte subpopulations present in the human spleen were analyzed by flow cytometry in an attempt to identify the presence of a novel dendritic-like cell subset described previously in mice and named L-DCs. In this study, an equivalent of this novel murine subset was characterized in the human spleen, thus increasing our knowledge of the antigen-presenting cell types present in the human spleen. Human L-DCs were identified as a hCD11c(+)hCD11b(+)HLA-DR(-)hCD86(+) subset in the spleen, along with the previously described subsets of hCD1c(+) DCs, hCD123(+) plasmacytoid DCs (pDCs), hCD16(+) DCs and hCD141(+) DCs. Three subsets of monocytes were also characterized. DC and monocyte subsets in human spleen had phenotypes similar to those of subsets in human blood. In line with murine studies, the presence of L-DC progenitors within the spleen was also investigated. When human splenocytes depleted of T and B cells were cocultured with the murine stromal line 5G3, hematopoiesis ensued and hCD11c(+)HLA-DR(+) and hCD11c(+)HLA-DR(-) cells were produced. The latter resemble L-DCs, which are also produced in murine spleen cocultures. Both subsets expressed hCD80 and hCD86, which identifies them as antigen-presenting cells, particularly DCs, and were highly endocytic. It is noteworthy that murine splenic stroma can serve as a support matrix for human hematopoiesis and DC production. These results support the hypothesis that 5G3 must express both cell-associated and soluble factors that can signal hematopoiesis in human and murine progenitors.Cellular & Molecular Immunology advance online publication, 20 April 2015; doi:10.1038/cmi.2015.16.
... 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 selfrenewing 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 2 ) BM or purified HSC [8,17,18]. ...
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.
... The heterogeneous stromal layer which forms within early splenic LTC comprises a network of fibroblast-like and endothelial-like cells [38]. The STX3 stromal line was cloned by single cell sorting to determine its constituent cell components, and 102 cloned lines were derived [39]. ...
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 from progenitors in bone marrow and to seed lymphoid and tissue sites during development. This study advances in vitro methodology for hematopoiesis of dendritic-like cells from progenitors in spleen. Since spleen progenitors undergo differentiation in vitro to produce these cells, the possibility exists that spleen represents a specific niche for differentiation of this subset. The fact that an equivalent cell subset has been shown to exist in spleen also supports that hypothesis. Studies have been directed at investigating the specific functional role of this novel subset as an APC accessible to blood-borne antigen, as well as the conditions under which hematopoiesis is initiated in spleen, and the type of progenitor involved.
... STX3 was isolated from a LTC that no longer produces DCs as a result of loss of progenitors [8]. The small cell population containing DC progenitors requires stromal cell contact for growth and proliferation [6,9], and substitution of stroma with growth factors does not allow DC development [10,11]. Phenotypic characterization of cells produced in LTCs revealed a heterogeneous population of hematopoietic cells among the "small" cells, with a homogeneous population of large dendritic-like cells (LTC-DC) [6]. ...
... Cell culture Cells were cultured at 37 C in 5% CO 2 in air in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FCS, 5 Â 10 À4 mol/L 2-mercaptoethanol, 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 U/mL penicillin, 100 mg/ mL streptomycin, 4 g/L glucose, 6 mg/L folic acid, 36 mg/L L-asparagine, 116 mg/L L-arganine HCl (sDMEM). The establishment of LTC from neonatal spleens has been described previously [11,14,15]. LTCs show continuous production of dendritic-like cells for more than 1 year [5]. ...
Novel APC have been described in murine spleen. Cells have a distinct CD11c(lo)CD11b(hi)MHC-II(-)CD8α(-) phenotype as highly endocytic dendritic-like cells which cross-present antigen to CD8(+) T cells but fail to activate CD4(+) T cells. They are named 'L-DC' since they reflect cells produced in longterm spleen cultures (LTC-DC). Similar cells were produced when bone marrow progenitors were co-cultured over the splenic stromal line 5G3. Co-cultures continuously produced a majority of L-DC, and a transient population of conventional (c) DC. 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-DC 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-DC that are continuously produced presumably from self-renewing progenitors. These subsets are not developmentally linked via a precursor/progeny relationship. L-DC and cDC-like cells are also distinct in terms of cytokine expression, with 65 out of 84 tested genes displaying >2-fold difference by qRT-PCR. Splenic stroma supports production of two APC subsets reflecting different lineage origins.
... Spleen long-term cultures (LTC) that exclusively support hematopoiesis of dendritic-like cells were first reported more than a decade ago [7] [8]. Cells produced were termed 'LTC- DC' and found to be large cells expressing the common DC surface markers CD11c and CD11b [8]. ...
... While L-DC progenitors exist within the embryo, the splenic microenvironment required for development of L-DC may not form or become competent until the neonatal period. This is consistent with evidence that productive LTC of spleen can only be generated from neonatal mice after day 6 [7]. The presence of L-DC progenitors within spleen during the perinatal period has since been confirmed by establishment of cocultures over STX3 [45]. ...
The concept of extramedullary hematopoiesis for production of organ-specific antigen presenting cells has importance in immunity in terms of the compartmentalisation of the immune response in different tissue sites. A new and distinct dendritic-like antigen presenting cell subtype is described which is dependent on the spleen microenvironment for development. Cells arise by a unique developmental pathway distinct from other dendritic cells (DC). In particular, a self-renewing progenitor of these cells has been identified in spleen upstream of the earliest DC progenitor currently identified in bone marrow. This progenitor depends on the splenic microenvironment for maintenance and proliferation, adding further support for spleen as a site for hematopoiesis.
