Myelopoiesis in spleen-producing distinct dendritic-like cells

Abstract 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)−ckitlo and Lin−ckithi 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−ckithi 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−ckithi subset. A model is advanced for development of L-DC directly from haematopoietic progenitors in spleen and dependent on the spleen microenvironment.


Introduction
The mouse is a well-recognized model for studying haematopoiesis. At birth, HSC migrate from foetal liver to bone where they remain for the life of the animal. HSC also appear in mouse spleen soon after birth [1] and are maintained there for life. They also exist in extramedullary sites and small numbers mobilize through blood and lymph into tissues like spleen, liver, lung, brain and intestine in the steady-state [2,3]. The number of haematopoietic stem/ progenitor cells (HSPC) in blood, spleen and liver also expands noticeably following acute inflammation or drug treatment [4,5]. The small number of progenitors in extramedullary tissues in the steady-state should not discount their importance or potential contribution to the immune response. Findings from this laboratory indicate that spleen in the steady-state does contain HSC detectable by their long-term reconstitution ability upon adoptive transfer to irradiated host mice [6]. The importance of bone marrow (BM) in haematopoiesis is clear however since neonatally splenectomized mice can maintain normal BM haematopoiesis [1,6]. The relative haematopoietic contribution of HSC from spleen and BM of adults is however not known, and it has long been assumed that spleen fills the role of an emergency or backup site in times of stress or disease.
Spleen is also a central organ for development of DC that take up and present antigen to lymphocytes. Multiple subsets have been identified including the CD8␣ Ϫ and CD8␣ ϩ conventional (c)DC and plasmacytoid (p)DC [7], and the less well-defined regulatory DC [8][9][10]. Monocyte-derived DC (moDC) are also found in spleen but only under conditions of inflammation [11]. An essential element of DC biology is the definition of progenitors and precursors since this underpins the formation of lineages of cells with distinct function. Initially, cDC and pDC progenitors were defined as a Flt3 ϩ subset amongst common myeloid and lymphoid progenitors (CMP/CLP) [12]. Current evidence points to a common monocyte/dendritic progenitor (MDP) in BM which gives rise to all monocyte/macrophage and dendritic-type cells [13,14], and a more committed common dendritic progenitor (CDP) for cDC and pDC in BM [15,16]. While CDP and MDP are not present in spleen or blood [17], spleen does harbour cDC precursors, which have a high turnover and are replaced by blood-borne precursors [18]. There is also evidence from parabiotic mouse studies to suggest that some splenic DC might arise from endogenous progenitors [19], although others have questioned that result [20].
In this laboratory, a novel dendritic-like antigen presenting cell was discovered in spleen on the basis of similarity with dendriticlike cells developing in splenic long-term cultures (LTC) [21,22]. 'LTC-DC' have a characteristic immature phenotype as CD11c lo CD11b hi MHC-II Ϫ CD8␣ Ϫ cells, distinguishing them from cDC, pDC and monocytes [22,23]. They are also distinguishable as large, endocytic cells specialized in cross-presentation of antigen to CD8 ϩ T cells [24], a function usually attributed to CD8␣ ϩ cDC [25]. They are also distinct in their very weak ability to activate CD4 ϩ T cells, consistent with their MHC-II Ϫ phenotype. Since they can be derived from GM-CSF Ϫ/Ϫ mice [26], they are distinct from moDC [11] or 'Tip-DC' [27] that develop in response to inflammatory factors like GM-CSF/TNF-␣. The in vivo equivalent 'L-DC' subset is readily distinguishable from cDC, pDC and monocytes on the basis of CD11b and CD11c expression, as well as many other markers including CD8␣, MHC-II, CD205 and myeloid markers like Ly6G and Mac3 [24]. L-DC show similar antigen cross presenting function as LTC-DC [24] and are functionally distinct from described subsets of regulatory DC which inhibit T cell proliferation [8][9][10]. The ontogeny and lineage origin of this subset appears to be distinct from other known DC and myeloid subsets in spleen.
It is hypothesized that spleen maintains a lineage of dendriticlike cells, which arise from endogenous haematopoietic progenitors maintained in spleen. Such tissue-specific production of DC has been previously reported for Langerhans cells in skin which are continuously renewed from radio-resistant, skin-derived progenitors [28], only being replaced by blood-borne progenitors under inflammatory conditions [29]. Splenic stromal cells which support haematopoiesis of L-DC in vitro have been shown to have an endothelial origin [30,31], and L-DC have been shown to arise in co-cultures of BM progenitors or spleen subsets over a splenic stromal cell line [32]. Both neonatal and adult splenocytes contain progenitors that produce L-DC when co-cultured over STX3 spleen stroma [33]. This study identifies and characterizes L-DC progenitors in adult spleen in terms of capacity to produce L-DC in stromal co-cultures and to undergo haematopoiesis for L-DC production upon transplantation into irradiation chimeras.

Enrichment for spleen precursors
Whole splenocytes were enriched for precursors by negative depletion of T cell and B cell populations using antibody-coated magnetic beads as described previously [33], which are specific for CD19 (eBio1D3), Thy1.2 (30-H12) and TER-119, (eBioscience). Recovered cells were washed and then stained with antibody for subsequent isolation of subsets by sorting.

Co-culture assays to assess DC development
Spleen stromal line STX3 is a spleen stromal cell line derived from a long-term culture which ceased production of DC over time with passage [34]. STX3 grows as a confluent monolayer and is passaged by scraping and cell transferral. When spleen or BM cells are co-cultured over STX3 (1-5 ϫ 10 5 cells/ml), haematopoiesis is established and myeloid dendritic-like cells are produced. For co-culture maintenance, half medium is exchanged every 3-4 days and non-adherent cells are collected for flow cytometric analysis.

Production of murine chimeras
Chimeras were generated using CD45-allotype distinct donor and host mice as described previously [24]. Haematopoietic cells (CD45.1) were transferred intravenously into lethally irradiated hosts (9.5 Gy) to assess in vivo reconstitution potential of blood cell lineages. Recipients were also given host (CD45.2 ϩ ) BM (10 5 cells) to ensure survival.

Statistical analysis
Data are presented as mean Ϯ S.E., n ϭ 3. With only small sample sizes, a normal distribution cannot be assumed. The Wilcoxon Rank Sum Test was therefore used to assess significance (P Յ 0.05).

Characterization of L-DC progenitors in adult spleen
Candidate progenitor subsets were identified in adult spleen following staining with markers for HSPC ( Fig. 1). A significant fraction of cells (10%) was found to be ckit lo CD11c hi cells, a population reflective of cDC [35]. CD11c ϩ cells expressing ckit were thus excluded from further analysis. The remaining Lin Ϫ ckit ϩ cells could be divided into subsets of Lin Ϫ ckit lo and Lin Ϫ ckit hi cells, representing ~3% and 1.7% of cells, respectively (Fig. 1A). These were analysed for expression of other known haematopoietic markers [36]. Sca1 was expressed on only ~3% of Lin Ϫ ckit hi cells, and the HSPC markers CD34 and Flt3 (not shown) were not expressed on Lin Ϫ ckit ϩ cells. The absence of HSPC in spleen expressing CD34 or Flt3 has been confirmed by others [17,20]. IL-7R, a marker of lymphoid progenitors in BM [37], was also absent, while a large population of cells were found to be Lin Ϫ ckit Ϫ CD11b ϩ (61%), reflecting myeloid precursors.

Differentiation of splenic progenitors in co-cultures
In line with marker analysis shown in Figure 1A, splenocytes depleted of T and B cells (Thy1.2 Ϫ CD19 Ϫ ) were gated by flow cytometry to exclude CD11c ϩ CD11b ϩ cells and sorted to give

Absence of immediate L-DC precursors amongst spleen Lin ؊ ckit ؉ progenitors
The presence of immediate L-DC precursors amongst the adult spleen Lin Ϫ ckit ϩ subsets was further investigated by adoptive transfer of 5 ϫ 10 4 sorted Lin

The spleen Lin ؊ ckit hi subset contains LT-HSC and L-DC progenitors
A unique characteristic of spleen LTC compared with other in vitro DC culture systems is that productivity is sustained for years. One explanation is that self-renewing stem cells maintained in culture differentiate to give L-DC perhaps via formation of an L-DC progenitor [32,38,39]. Only the spleen Lin Ϫ ckit hi subset produced progeny cells with multilineage long-term reconstitution in 7/7 mice consistent with the presence of LT-HSC (Fig. 4). Multilineage reconstitution by HSC within the spleen Lin Ϫ ckit hi subset was indicated by the detection of donor-derived DC, myeloid cells, T cells and B cells (Fig. S1). No progeny cell reconstitution was achieved with the spleen Lin Ϫ ckit lo subset (0/3 mice), or with the Lin  (Table 1). Three out of four chimeras given donor Lin Ϫ ckit hi spleen cells showed complete reconstitution with donor myeloid cells, while a fourth chimera showed partial reconstitution. Control chimeras given donor-derived BM alone also gave complete long-term multilineage reconstitution with donor cells in three out of four mice, and also showed new HSC production.

B cells and CD8
ϩ T cells were detected in lymph node. ‡ HSC were identified in spleen as a Lin ckit hi Sca1 ϩ subset.
The latter population was also identified in terms of granulocyte and macrophage subsets using specific antibody (data not shown). The population distribution of DC/myeloid subsets in spleens of chimeras was similar to control mice, indicating full haematopoietic reconstitution (Fig. 5B). Each DC or myeloid compartment in spleen was restored to homeostatic levels, with myeloid cells representing the largest population, followed by CD8␣ Ϫ cDC, CD8␣ ϩ cDC and L-DC (Fig. 5B). However, only L-DC in spleen showed full reconstitution with donor-derived (CD45.1 ϩ ) HSC present in spleen Lin Ϫ ckit hi cells or in BM (Fig. 5C). The CD8␣ Ϫ cDC, CD8␣ ϩ cDC and myeloid cell compartments were only partially replaced by donor-type cells. In chimeras of this type, donor-derived HSC do colonize BM in low number (data not shown). However, there is no clear evidence yet for L-DC development in BM, although a similar but distinct subset of cells is under further investigation. A comparison of relative numbers of donor-versus hostderived cells confirmed differing levels of chimerism for each cell subset. A distinct trend in relative donor:host levels for different DC subsets was evident across chimeras analysed at 15 to 18.5 weeks, despite variance in overall donor cell reconstitution levels between individual mice. When the fold-increase in donor versus host cell numbers was calculated for each APC subset and standardized to CD8␣ Ϫ cDC (donor:host ratio ϭ 1.0), L-DC consistently exceeded myeloid cells and cDC in terms of relative donor to host cell contribution (Fig. 6). By this analysis, donor-derived progenitors reconstituted CD8␣ Ϫ cDC and CD8␣ ϩ cDC subsets to equal levels, but gave ~2-fold more myeloid cells. These results are developmentally consistent with a common progenitor (CDP) for cDC subsets [16], and a common upstream progenitor (MDP) for cDC and macrophages [13]. In contrast, donor-derived progenitors gave rise to significantly higher numbers of L-DC (4-12 fold increase; mean ϭ 8.7) compared with CD8␣ Ϫ cDC (Fig. 6), perhaps indicative of a separate developmental origin for L-DC compared with cDC and macrophages. Since this same trend was also observed for control mice given only donor BM intravenously (Figs 4 and 5), we concluded that the source of HSC was not important. The lodgement of donor-type progenitors into empty niches in spleen would appear to determine the development of L-DC in higher relative numbers than other myeloid/dendritic cell types in spleen.

Discussion
This study addresses myelopoiesis in spleen leading to the development of a novel dendritic-like cell type, namely L-DC, which is unique in terms of its phenotype and immune functional potential [24]. Adult murine spleen is clearly an extramedullary haematopoietic site containing low numbers of multipotent HSC [1]. The hypothesis that extramedullary haematopoiesis mediates the production of tissue-specific APC like L-DC with sitespecific functions, is consistent with compartmentalism of the immune response to meet the needs of distinct tissue sites and their respective pathogens. While multiple extramedullary sites clearly contribute to haematopoiesis, the nature of cells produced and the conditions under which this occurs are not yet well defined. In order to identify an APC subset as distinct it is necessary to show that the lineage origin and progenitor of those cells differs from that of other common dendritic and myeloid subsets. Marker analysis of adult spleen has led to the identification of minor subsets of Lin Ϫ ckit lo and Lin Ϫ ckit hi cells which lack markers like Flt3, CD34 and IL-7R associated with myeloid and lymphoid haematopoietic progenitor subsets in BM (Fig. 1) [17,20]. There is already evidence that HSC in different tissue sites are different since the marker profile of foetal liver HSC differs from that of BM HSC [40]. Here we have localized the progenitor of L-DC within the Lin Ϫ ckit hi subset of spleen, which also contains LT-HSC. The presence of LT-HSC in this subset was evident since this subset gave long-term multilineage reconstitution of chimeras out to 54 weeks (Fig. 6). This study therefore identifies a close, if not linked, relationship between LT-HSC and the L-DC progenitor in murine spleen. Further work is under way to obtain the full marker expression profile of splenic HSC, and to determine whether this cell type differs from that of BM HSC with the same differentiative capacity. Both adult Lin Ϫ ckit lo and Lin Ϫ ckit hi adult spleen subsets were found to contain progenitors which differentiate to give L-DC in co-culture over the STX3 splenic stroma. However, only the Lin Ϫ ckit hi subset and not the Lin Ϫ ckit lo subset contains cells, which reflect self-renewing LT-HSC as demonstrated by their long-term multilineage reconstitution potential in irradiated mice. The differential function of these two subsets in vivo did not mirror their common in vitro differentiative capacity. A first explanation is that they contain a common progenitor, not yet identifiable with available antibodies. The second explanation is that the Lin Ϫ ckit lo subset contains L-DC progenitors that derive directly from progenitors within the Lin Ϫ ckit hi subset of spleen, and that this differentiation occurs when Lin Ϫ ckit hi cells are co-cultured over STX3 stroma. The transition of Lin Ϫ ckit hi cells into Lin Ϫ ckit lo cells within co-cultures has proven very difficult to test because of the small size of these subsets in spleen, and the difficulty of recovering enough cells from co-cultures to perform an analysis of phenotypic change. Indeed, the L-DC progenitor in spleen is phenotypically distinct from other described myeloid and dendritic progenitors present in BM. The CDP and MDP are phenotypically Flt3 ϩ cells [13], with no counterpart subset in spleen. The CDP has distinct differentiative capacity for cDC, and the MDP differentiates to give macrophages and DC [17]. Furthermore, neither the splenic Lin Ϫ ckit lo nor Lin Ϫ ckit hi subsets described here resemble the immediate cDC precursor or pre-cDC subset previously identified as ckit ϩ CD11c lo cells, which respond to Flt3L to produce mature cDC and pDC [15,18]. This pre-cDC subset would have been excluded by our sorting protocol, which specifically gated out Lin ϩ cells including CD11c ϩ DC. L-DC are a distinct CD11c lo CD11b hi MHC-II Ϫ dendritic-like subset in spleen with strong cross presentation capacity for CD8 ϩ T cell activation [24]. While these cells are phenotypically distinct from monocytes, which are CD11c Ϫ and are also unable to cross present antigen [24], it is not yet known whether L-DC share a common lineage relationship with monocytes. Since monocytes, macrophages and cDC /pDC all originate from BM progenitors like MDP and CDP, one expectation is that these cell types might all be reconstituted to similar levels following HSC transfer. This prediction was in fact verified in radiation chimeras shown here, and multiple chimeras demonstrated equal long-term reconstitution of donor-derived splenic monocytes/macrophages, and the CD8␣ Ϫ and CD8␣ ϩ cDC populations (Fig. 6). However, these same chimeras showed an average 9-fold increase in donor over host reconstitution of L-DC compared with CD8␣ Ϫ cDC, and a 7-fold Fig. 6 Lin Ϫ ckit hi adult spleen cells give preferential reconstitution of L-DC in irradiation chimeras. The long-term reconstituting potential of Lin Ϫ ckit hi spleen cells for L-DC, CD8␣ ϩ cDC, CD8␣ Ϫ cDC and myeloid cells was assessed in irradiation chimeras described in Figure 4. These mice were given a mix of donor-type Lin Ϫ ckit hi spleen cells and host-type bone marrow. Controls included two irradiation chimeras reconstituted with donor-type bone marrow. The individual myeloid subsets of donor (CD45.1 ϩ ) versus host (CD45.2 ϩ ) type were identified in spleen using flow cytometry as shown in Figure 5. The relative prevalence of donor:host cells for individual subsets was calculated for each animal, based on a relative value of 1.0 for CD8␣ Ϫ cDC. Mean Ϯ SE is shown for SPL chimeras (n ϭ 3). Cell subsets having significantly higher representation of donor:host-type cells compared with CD8 Ϫ cDC are indicated (*) [P Յ 0.05 (Wilcoxon Rank Sum Test)].