Basement Membrane Matrix Encapsulated Cell Aggregation for Investigating Murine Spleen Tissue Formation.

The spleen is an immune organ that plays a key role in blood-borne immune responses. The anatomical or functional loss of this tissue increases susceptibility to severe blood infections and sepsis. Auto-transplantation of spleen slices has been used clinically to replace lost tissue and restore immune function. However, the mechanism driving robust and immunologically functional spleen tissue regeneration has not been fully elucidated. Here, we aim to develop a method for aggregating and encapsulating spleen cells within a semi-solid matrix in order to investigate the cellular requirements for spleen tissue formation. Basement membrane matrix encapsulated cell constructs are amenable to both in vitro tissue culture of three-dimensional organoids as well as transplantation under the kidney capsule to directly assess in vivo tissue formation. By manipulating the input cells for aggregation and encapsulation, we demonstrate that graft-derived PDGFRß+MAdCAM-1- neonatal stromal cells are required for spleen tissue regeneration under animal transplantation models.

Transplanted spleen stromal cells with osteogenic potential support ectopic myelopoiesis.

Spleen stromal lines which support in vitro hematopoiesis are investigated for their lineage origin and hematopoietic support function in vivo. Marker expression and gene profiling identify a lineage relationship with mesenchymal stem cells and perivascular reticular cells described recently in bone marrow. Stromal lines commonly express Cxcl12, Pdgfra and Pdgfr typical of bone marrow derived perivascular reticular cells but reflect a unique cell type in terms of other gene and marker expression. Their classification as osteoprogenitors is confirmed through ability to undergo osteogenic, but not adipogenic or chondrogenic differentiation. Some stromal lines were shown to form ectopic niches for HSCs following engraftment under the kidney capsule of NOD/SCID mice. The presence of myeloid cells and a higher representation of a specific dendritic-like cell type over other myeloid cells within grafts was consistent with previous in vitro evidence of hematopoietic support capacity. These studies reinforce the role of perivascular/perisinusoidal reticular cells in hematopoiesis and implicate such cells as niches for hematopoiesis in spleen.

Determinants of postnatal spleen tissue regeneration and organogenesis.

The spleen is an organ that filters the blood and is responsible for generating blood-borne immune responses. It is also an organ with a remarkable capacity to regenerate. Techniques for splenic auto-transplantation have emerged to take advantage of this characteristic and rebuild spleen tissue in individuals undergoing splenectomy. While this procedure has been performed for decades, the underlying mechanisms controlling spleen regeneration have remained elusive. Insights into secondary lymphoid organogenesis and the roles of stromal organiser cells and lymphotoxin signalling in lymph node development have helped reveal similar requirements for spleen regeneration. These factors are now considered in the regulation of embryonic and postnatal spleen formation, and in the establishment of mature white pulp and marginal zone compartments which are essential for spleen-mediated immunity. A greater understanding of the cellular and molecular mechanisms which control spleen development will assist in the design of more precise and efficient tissue grafting methods for spleen regeneration on demand. Regeneration of organs which harbour functional white pulp tissue will also offer novel opportunities for effective immunotherapy against cancer as well as infectious diseases.

Stromal Cell Subsets Directing Neonatal Spleen Regeneration.

Development of lymphoid tissue is determined by interactions between stromal lymphoid tissue organiser (LTo) and hematopoietic lymphoid tissue inducer (LTi) cells. A failure for LTo to receive appropriate activating signals during embryogenesis through lymphotoxin engagement leads to a complete cessation of lymph node (LN) and Peyer's patch development, identifying LTo as a key stromal population for lymphoid tissue organogenesis. However, little is known about the equivalent stromal cells that induce spleen development. Here, by dissociating neonatal murine spleen stromal tissue for re-aggregation and transplant into adult mouse recipients, we have identified a MAdCAM-1+CD31+CD201+ spleen stromal organizer cell-type critical for new tissue formation. This finding provides an insight into the regulation of post-natal spleen tissue organogenesis, and could be exploited in the development of spleen regenerative therapies.

Redefining Myeloid Cell Subsets in Murine Spleen.

Spleen is known to contain multiple dendritic and myeloid cell subsets, distinguishable on the basis of phenotype, function and anatomical location. As a result of recent intensive flow cytometric analyses, splenic dendritic cell (DC) subsets are now better characterized than other myeloid subsets. In order to identify and fully characterize a novel splenic subset termed "L-DC" in relation to other myeloid cells, it was necessary to investigate myeloid subsets in more detail. In terms of cell surface phenotype, L-DC were initially characterized as a CD11b(hi)CD11c(lo)MHCII(-)Ly6C(-)Ly6G(-) subset in murine spleen. Their expression of CD43, lack of MHCII, and a low level of CD11c was shown to best differentiate L-DC by phenotype from conventional DC subsets. A complete analysis of all subsets in spleen led to the classification of CD11b(hi)CD11c(lo)MHCII(-)Ly6C(lo)Ly6G(-) cells as monocytes expressing CX3CR1, CD43 and CD115. Siglec-F expression was used to identify a specific eosinophil population, distinguishable from both Ly6C(lo) and Ly6C(hi) monocytes, and other DC subsets. L-DC were characterized as a clear subset of CD11b(hi)CD11c(lo)MHCII(-)Ly6C(-)Ly6G(-) cells, which are CD43(+), Siglec-F(-) and CD115(-). Changes in the prevalence of L-DC compared to other subsets in spleens of mutant mice confirmed the phenotypic distinction between L-DC, cDC and monocyte subsets. L-DC development in vivo was shown to occur independently of the BATF3 transcription factor that regulates cDC development, and also independently of the FLT3L and GM-CSF growth factors which drive cDC and monocyte development, so distinguishing L-DC from these commonly defined cell types.

Murine spleen tissue regeneration from neonatal spleen capsule requires lymphotoxin priming of stromal cells.

Spleen is a tissue with regenerative capacity, which allows autotransplantation of human spleen fragments to counteract the effects of splenectomy. We now reveal in a murine model that transplant of neonatal spleen capsule alone leads to the regeneration of full spleen tissue. This finding indicates that graft-derived spleen stromal cells, but not lymphocytes, are essential components of tissue neogenesis, a finding verified by transplant and regeneration of Rag1KO spleen capsules. We further demonstrate that lymphotoxin and lymphoid tissue inducer cells participate in two key elements of spleen neogenesis, bulk tissue regeneration and white pulp organization, identifying a lymphotoxin-dependent pathway for neonatal spleen regeneration that contrasts with previously defined lymphotoxin-independent embryonic spleen organogenesis.

Characterization of the effect of LPS on dendritic cell subset discrimination in spleen.

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is a potent inflammatory mediator and a leading cause of bacterial sepsis. While LPS is known to activate antigen-presenting cells, here we find that LPS down-regulates expression of CD11c and CD11b on splenic dendritic cell subsets, thus confounding the ability to identify these subsets following treatment. This has implications with regard to tracking the response to LPS in terms of the cell subsets involved, and should be considered whenever such studies are undertaken.

Spleen stroma maintains progenitors and supports long-term hematopoiesis.

Hematopoietic stem/progenitor cells (HSPC) differentiate in the context of stromal niches producing cells of multiple lineages. Limited success has been achieved in the past with induction of hematopoiesis in vitro. Previously, spleen long-term stromal cultures (LTC) were shown to continuously support restricted hematopoiesis for production of novel dendritic-like cells (LTC-DC). An in vivo equivalent dendritic cell type was then described which is specific for spleen. The in vivo counterpart cell was termed 'L-DC' and represents a dendritic-like CD11c(lo)CD11b(hi)CD8α-MHC-II- cell which differs phenotypically and functionally from monocytes/macrophages and conventional and plasmacytoid DC. Splenic stroma is now shown to maintain HSPC and to support their restricted in vitro differentiation to give this 'L-DC' subset. In order to characterise progenitors of this distinct cell type, LTC were analysed for cell subsets produced, and these subsets sorted and assessed for hematopoietic potential in subsequent co-cultures over STX3 stroma. Progenitors were defined as a lineage (Lin)(-)ckit(lo) subset reflecting HSPC. Furthermore, when Lin(-)ckit(hi)Sca1(+)Flt3(-) HSPC were sorted from bone marrow, they colonised splenic stroma with long-term production of L-DC. The maintenance of HSPC by splenic stroma was confirmed when non-adherent cells collected from LTC showed oligopotent reconstitution of the hematopoietic compartment of lethally irradiated mice. All data support a model whereby spleen houses a niche for HSPC in the resting state, with production of progenitors, and their differentiation to give tissue-specific antigen presenting cells.

Novel splenic antigen-presenting cells derive from a Lin(-) c-kit(lo) progenitor.

The main DC subsets in murine spleen arise from BM-derived precursors. Recently, a novel APC type was described in spleen. To determine if spleen contains the progenitors of this subset, a stromal coculture system was used to assess candidate progenitors for their hematopoietic potential. Here, the progenitor of that subset is identified as a spleen endogenous Lin(-)c-kit(lo) hematopoietic progenitor and is most highly enriched among the Lin(-)c-kit(lo)CD34(+) subset. Dendritic-like cells produced in vitro functionally resemble the previously described in vivo equivalent subset with high endocytic capacity and capability for antigen-specific activation of CD8(+) T cells but not CD4(+) T cells.

Myelopoiesis in spleen-producing distinct dendritic-like cells.

Dendritic cells (DC) represent a heterogeneous class of antigen presenting cells (APC). Previously we reported a distinct myeloid dendritic-like cell present in spleen, as an in vivo counterpart to cells produced in murine spleen long-term cultures (LTC-DC). These cells, named 'L-DC', were found to be functionally and phenotypically distinct from conventional (c)DC, plasmacytoid (p)DC and monocytes. These results suggested that spleen may represent a niche for development of L-DC from endogenous progenitors. Adult murine spleen has now been investigated for the presence of L-DC progenitors. Lineage-negative (Lin)(-) ckit(lo) and Lin(-) ckit(hi) progenitor subsets were identified as candidate populations, and tested for ability to produce L-DC; in vitro upon co-culture with the spleen stromal line STX3, and in vivo after adoptive therapy into mice. Both subsets colonized STX3 stroma in vitro for L-DC production, indicating that they contained either a common or two distinct progenitors for L-DC. However, only the Lin(-) ckit(hi) subset gave progeny cells after adoptive transfer into lethally irradiated mice. In vivo development was however multilineage and not restricted to L-DC development. Multilineage reconstitution reflects long-term reconstituting haematopoietic stem cells (LT-HSC), suggesting a close relationship between L-DC progenitors and LT-HSC. L-DC were however produced in vivo in much higher number than monocytes/macrophages and cDC, indicating the presence of a specific L-DC progenitor within the Lin(-) ckit(hi) subset. A model is advanced for development of L-DC directly from haematopoietic progenitors in spleen and dependent on the spleen microenvironment.

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.

Identification of a novel antigen cross-presenting cell type in spleen.

Antigen-presenting cells (APC), like dendritic cells (DC), are essential for T-cell activation, leading to immunity or tolerance. Multiple DC subsets each play a unique role in the immune response. Here, a novel splenic dendritic-like APC has been characterized in mice that has immune function and cell surface phenotype distinct from other, described DC subsets. These were identified as a cell type continuously produced in spleen long-term cultures (LTC) and have an in vivo equivalent cell type in mice, namely 'L-DC'. This study characterizes LTC-DC in terms of marker phenotype and function, and compares them with L-DC and other known splenic DC and myeloid subsets. L-DC display a myeloid dendritic-like phenotype equivalent to LTC-DC as CD11c(lo) CD11b(hi) MHC-II(-) CD8α(-) cells, distinct by high accessibility and endocytic capacity for blood-borne antigen. Both LTC-DC and L-DC have strong antigen cross-presentation ability leading to strong activation of CD8(+) T cells, particularly after exposure to lipopolysaccharide. However, they have weak ability to stimulate CD4(+) T cells in antigen-specific responses. Evidence is presented here for a novel DC type produced by in vitro haematopoiesis which has distinct antigen-presenting potential and reflects a DC subset present also in vivo in spleen.

In vitro haematopoiesis of 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 from progenitors in bone marrow and to seed lymphoid and tissue sites during development. This study advances in vitro methodology for haematopoiesis 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 haematopoiesis is initiated in spleen, and the type of progenitor involved.

Artificial engineering of secondary lymphoid organs.

Secondary lymphoid organs such as spleen and lymph nodes are highly organized immune structures essential for the initiation of immune responses. They display distinct B cell and T cell compartments associated with specific stromal follicular dendritic cells and fibroblastic reticular cells, respectively. Interweaved through the parenchyma is a conduit system that distributes small antigens and chemokines directly to B and T cell zones. While most structural aspects between lymph nodes and spleen are common, the entry of lymphocytes, antigen-presenting cells, and antigen into lymphoid tissues is regulated differently, reflecting the specialized functions of each organ in filtering either lymph or blood. The overall organization of lymphoid tissue is vital for effective antigen screening and recognition, and is a feature which artificially constructed lymphoid organoids endeavor to replicate. Synthesis of artificial lymphoid tissues is an emerging field that aims to provide therapeutic application for the treatment of severe infection, cancer, and age-related involution of secondary lymphoid tissues. The development of murine artificial lymphoid tissues has benefited greatly from an understanding of organogenesis of lymphoid organs, which has delineated cellular and molecular elements essential for the recruitment and organization of lymphocytes into lymphoid structures. Here, the field of artificial lymphoid tissue engineering is considered including elements of lymphoid structure and development relevant to organoid synthesis.

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 CD11b(hi)CD11c(lo)CD8(-)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 CD11c(lo) 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.

Investigation of murine spleen as a niche for hematopoiesis.

BACKGROUND: Spleen as a lymphoid tissue is specialized for monitoring blood and mounting immunity against blood-borne antigens. Antigen-presenting cells present in spleen commonly develop from bone marrow-derived precursors that enter blood circulation. However, a distinct splenic myeloid antigen-presenting cell subset described in this laboratory, namely "dendritic-like cells" (L-DC), has been hypothesized not to share a bone marrow origin. METHODS: In this study, the presence of endogenous hematopoietic progenitors in spleen was investigated by transplanting intact spleen into allotype-distinct recipients and monitoring development of progeny cells in grafted tissues. RESULTS: Successful engraftment of donor spleens was achieved for up to 4 weeks. After 2 weeks, donor-type myeloid cells, dendritic cells (DC) and few B cells were observed in spleen grafts indicative of spleen-endogenous hematopoiesis. An influx of host-type hematopoietic stem cell, as well as host-type lymphoid and myeloid cells, was also observed. CONCLUSION: Evidence for the maintenance of donor-type myeloid cells after 2 to 4 weeks reflected development from hematopoietic progenitors endogenous to spleen. L-DC were also detected in spleen grafts; however, within the graft microenvironment they were one of the several DC and myeloid subsets, including activated DC, resembling L-DC. Overall, this study adds further evidence that spleen can support endogenous myelopoiesis.

Haematopoietic stem cells in spleen have distinct differentiative potential for antigen presenting cells.

Dendritic cells (DC) are known to develop from macrophage dendritic progenitors (MDP) in bone marrow (BM), which give rise to conventional (c)DC and monocytes, both dominant antigen presenting cell (APC) subsets in spleen. This laboratory has however defined a distinct dendritic-like cell subset in spleen (L-DC), which can also be derived in long-term cultures of spleen. In line with the restricted in vitro development of only L-DC in these stromal cultures, we questioned whether self-renewing HSC or progenitors exist in spleen with restricted differentiative capacity for only L-DC. Neonatal spleen and BM were compared for their ability to reconstitute mice and to give rise to L-DC, as well as other splenic APC. Neonatal spleen cells were transplanted into allotype-distinct lethally irradiated hosts along with host-type competitor BM cells, and assayed over 8 to 51 weeks for haematopoietic reconstitution of L-DC and cDC subsets, along with other lymphoid and myeloid cells. In this study, neonatal spleen showed multilineage haematopoietic reconstitution in mouse chimeras, rather than specific or restricted ability to differentiate into L-DC. However, the representation of individual APC subsets was found to be unequal in chimeras partially reconstituted with donor cells, such that more donor-derived progeny were seen for L-DC than for myeloid and cDC subsets. The ability of HSC in spleen to develop into L-DC was indicated by a strong bias in the subset size of these cells over other splenic APC subsets. This type of evidence supports a model whereby spleen represents an important site for haematopoiesis of this distinct DC subset. The conditions under which haematopoiesis of L-DC occurs in spleen, or the progenitors involved, will require further investigation.

Splenic stromal niches support hematopoiesis of dendritic-like cells from precursors in bone marrow and spleen.

OBJECTIVE: The aims of this study are to test the ability of stromal cells from murine spleen to support hematopoiesis, to define the tissue source of precursors that seed these hematopoietic niches, and to determine the type of cells produced. MATERIALS AND METHODS: Cloned isolates of murine spleen stroma have been developed that support hematopoiesis. Analysis has been investigated in terms of tissue source of progenitors. Type and number of cells produced were analyzed by flow cytometry. RESULTS: Hematopoietic precursors that seed cocultures exist in spleen and bone marrow (BM), but not thymus. Cell production is highest if overlay cells are enriched for hematopoietic precursors. BM contains more precursors than spleen, but the cell types produced are different. Cocultures established from spleen maintain a high proportion of a distinct class of dendritic-like cells produced in only low numbers in BM cocultures. These reflect the immature myeloid dendritic cell (DC) produced continuously in long-term spleen cultures established previously in this laboratory. Stroma-conditioned medium alone does not support DC development, but does support early outgrowth of myelomonocytic cells from precursors in both spleen and BM. CONCLUSION: The outcome has been development of a coculture system that supports hematopoiesis of immature myeloid dendritic-like cells in vitro. Although production of monocytes can occur in the presence of stroma-conditioned medium alone, production of DC is dependent on stromal cell interaction. Results presented here raise questions about the role of spleen as a site for DC hematopoiesis from endogenous precursors.

Hematopoiesis of immature myeloid dendritic cells in stroma-dependent spleen long-term cultures occurs independently of NF-KB/RelB function.

OBJECTIVE: The nuclear factor-kappaB (NF-KB)/RelB transcription factor plays an essential role in development of some dendritic cell (DC) subsets in mice. In this laboratory, immature myeloid DC are produced in vitro in a stroma-dependent murine spleen long-term culture (LTC) system. In LTC, DC differentiate from hematopoietic progenitors maintained within the stromal cell matrix. Expression and function of RelB in development of LTC-DC has been investigated, with a view to assessing the relationship between DC produced in this system and other known subsets of DC. MATERIALS AND METHODS: RelB expression by LTC-DC was confirmed by detection of protein by Western blotting, RNA by reverse transcription polymerase chain reaction, and nuclear protein with DNA-binding function in electrophoretic mobility shift assays. The role of RelB in cell development was assessed by addition of antisense RelB oligonucleotides into LTC and colony assays established above STX3 stromal cells. RelB(-/-) mice were also examined for ability to produce LTC, and for presence of DC progenitors in spleen and bone marrow that can generate DC when overlaid on STX3 in cocultures. RESULTS: Functional RelB was detected in both LTC-DC and in STX3 stromal cells. A critical role for RelB in DC differentiation from spleen progenitors was confirmed, because antisense RelB oligonucleotides specifically and completely inhibited production of large differentiated myeloid DC in LTC. Further investigation using RelB(-/-) mice revealed that RelB expression by stromal cells rather than hematopoietic cells was required for production of LTC-DC. This was evidenced by a combination of factors, including 1) inability to generate productive LTC from RelB(-/-) mice; 2) presence of DC precursors in RelB(-/-) bone marrow and spleen, which could produce DC in stromal cocultures; and 3) increased myeloid precursor frequency among RelB(-/-) spleen cells over RelB(+/+) control cell populations. CONCLUSION: Specific development of fully differentiated, but immature myeloid CD11c(+)CD11b(+)MHC-CII(-)CD8alpha(-)CD40(-) DC in spleen LTC is dependent on expression of activated NF-kappaB/Rel-B. However, this appears to relate to stromal cell function rather than to the function of hematopoietic cells. Altogether these data confirm the importance of splenic stromal cells in myelopoiesis leading to development of immature DC as produced in LTC.

Concise review: Dendritic cell development in the context of the spleen microenvironment.

The dendritic cell (DC) population in spleen comprises a mixture of cells including endogenous DC progenitors, DC precursors migrating in from blood and bone marrow, and DC in different states of differentiation and activation. A role for different microenvironments in supporting the dynamic development of murine DC of different types or lineages is considered here. Recent evidence for production of DC dependent on splenic stromal cells is reviewed in the light of evidence that cell production is dependent on cells comprising an endothelial niche in spleen. The possibility that self-renewing progenitors in spleen give rise to DC with tolerogenic or regulatory rather than immunostimulatory function is considered.