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 CD11bhiCD11cloMHCII−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 CD11bhiCD11cloMHCII−Ly6CloLy6G− cells as monocytes expressing CX3CR1, CD43 and CD115. Siglec-F expression was used to identify a specific eosinophil population, distinguishable from both Ly6Clo and Ly6Chi monocytes, and other DC subsets. L-DC were characterized as a clear subset of CD11bhiCD11cloMHCII−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.

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.
Keywords: dendritic cells, monocytes, spleen, myeloid cells inTrODUcTiOn Characterization of dendritic cell (DC) subsets in spleen has progressed rapidly in terms of phenotype and function; however, other splenic myeloid subsets are less clearly defined. The term "myeloid" has been used as an umbrella term describing DC, granulocytes, and macrophages/monocytes, which descend from a common myeloid progenitor (CMP) in bone marrow (1). Granulocytes comprise neutrophils, eosinophils, basophils, and mast cells (2), while monocytes have been classified into distinct resident and inflammatory populations, in line with monocyte subsets described in peripheral blood (3). Recent definition of a common dendritic progenitor (CDP) now separates the development of conventional (c) and plasmacytoid (p) DC from other myeloid cell types (4,5). The current classification of splenic myeloid subsets is based on cell surface phenotype, although full phenotypic profiles are not yet available. To date, studies focusing on specific subsets do not compare one subset against another to ensure pure populations and do not achieve a comprehensive picture of the relationship between subsets. Accurate identification of splenic myeloid cell types is essential for distinguishing specific subsets and their function, and for making studies in the field comparable.
Monocytes develop in bone marrow from CMP and traverse into blood as mature cells (1). Resident monocytes were described as having a longer half-life in blood than inflammatory monocytes (6) and were originally thought to act as precursors of tissue resident macrophages in the steady-state, hence their name "resident" monocytes (7). They were identified further as CC-chemokine receptor 2 (CCR2) − and CX3-chemokine receptor 1 (CX3CR1) hi Ly6C lo Ly6G − cells, which play an important role in the detection of vascular integrity (3,8). Inflammatory monocytes were described as distinct as CCR2 + CX3CR1 lo Ly6C hi Ly6G − cells (3,7,(9)(10)(11). Several studies have now highlighted how tissue-specific macrophages including microglial cells, alveolar macrophages, Langerhans cells and splenic macrophages have a yolk sac-derived origin rather than a bone marrow origin (12)(13)(14)(15).
Most studies on monocytes have involved blood as a source of cells, but more recently the relationship between splenic monocytes and their blood counterpart has been considered. The studies of Swirski and colleagues demonstrated that splenic monocytes resemble their blood counterpart on the basis of phenotype, morphology and gene expression (16). Spleen was identified as a reservoir for monocytes, where Ly6C hi inflammatory monocytes were efficiently deployed to sites of inflammation during cardiac arrest. Monocytes in blood and spleen are now identified as cells with a CD11b hi CD11c −/lo phenotype, with further distinction as Ly6C hi and Ly6C lo cells resembling inflammatory and residential monocytes, respectively (17). Neutrophils have not been extensively characterized in spleen and were previously delineated on the basis of phenotype as a blood equivalent CD11b + Ly6C + Ly6G + subset (18). Similarly, eosinophils have been described in blood as CD11b + Siglec-F + CCR3 + F4/80 + cells and in spleen as Ly6C int Ly6G − SSC hi Siglec-F + cells (17,19).
The lineage of DC is equally diverse with multiple subsets defined in mice and humans (20,21). All subsets are capable of antigen uptake, processing and presentation for T cell activation. Conventional DC are predominant in spleen, comprising CD8 − and CD8 + populations, phenotypically distinct as CD11c hi CD11b + CD8α − MHCII + and CD11c hi CD11b − CD8α + MHCII + cells, respectively (22). These two dominant DC subsets are distinct in function, cytokine production and ability to cross-present antigen (23). Spleen also contains a smaller population of pDC existing as a plasmacytoid preDC (p-preDC) in the steady-state. These are, however, long-lived, circulating cells, which produce high levels of Type I interferon important in anti-viral immunity (24). Spleen also contains a variable population of poorly defined "myeloid DC. " As an example, under inflammatory conditions, circulating CD115 + Ly6C hi CCR2 + monocytes in blood may be recruited into spleen where they differentiate to become "monocyte-derived (mo)-DC" (25)(26)(27). Different subsets of mo-DC can subsequently be observed in different disease states.
Novel dendritic-like cells termed L-DC, were recently discovered in murine and human spleen on the basis of their resemblance with dendritic-like cells produced in vitro in splenic long-term cultures (LTC-DC) (28)(29)(30), or in co-cultures of bone marrow precursors over selected splenic stroma (31,32). L-DC have a distinct CD11b hi CD11c lo MHCII − CD8 − phenotype and dendritic-like appearance (33). Splenic stroma maintains continuous but restricted in vitro development of only this cell type, without addition of cytokines like GM-CSF, M-CSF, or Flt3L used by others to induce DC development in vitro from bone marrow precursors (34). An in vivo equivalent L-DC subset has been partially characterized in spleens of mice and human (35,36). While L-DC resemble myeloid DC on the basis of CD11c and CD11b expression, lack of MHCII expression has raised criticism that L-DC may more resemble splenic monocytes/ macrophages than DC. Low expression of CD11c on L-DC is, however, consistent with some myeloid DC and pDC described as CD11c lo cells (37)(38)(39).
Analysis of cell surface phenotype using specific antibodies and flow cytometry is widely accepted as a means to identify and distinguish ex vivo cell subsets. A staining protocol and gating strategy were therefore developed here to more accurately delineate and identify DC, monocytes, and other myeloid subsets in spleen. This study now identifies the L-DC subset in vivo as distinct from monocytes, granulocytes and cDC. The development of L-DC has also been investigated in relation to cDC and myeloid subsets in the spleens of Batf-3 −/− , GM-CSF −/− , and Flt3L −/− mutant mice. The GM-CSF and FLT3 ligand (FLT3L) growth factors are known to be important in the differentiation of dendritic and other myeloid cells (34), and the BATF3 transcription factor was recently described as essential for CD8 + cDC development (40,41).

MaTerials anD MeThODs
animals MACS-labeling buffer (2 mM EDTA/0.5% BSA in PBS) on ice for 25 min. Cells were then washed twice with MACS-labeling buffer, resuspended in buffer (10 8 cells/mL) followed by the addition of 20 μL of anti-biotin microbeads/10 8 cells (Miltenyi), with incubation for 25 min on ice. The washing step was repeated, and cells resuspended in MACS-labeling buffer. T, B, and red blood cells were depleted by running cells through LS columns (Miltenyi) in a SuperMACS II Separation Unit (Miltenyi), washing thrice with 3 mL of buffer and collection of unbound cells as flow-through. antibody staining Antibody staining followed by flow cytometry was performed to analyze cell surface marker expression as described previously (32). Non-specific antibody binding via

cell sorting
Cell populations were isolated by sorting following flow cytometry with fluorochrome-conjugated antibodies. Cells were prepared as described above and all incubation and washing steps performed in 1% fetal calf serum in Dulbecco's Modified Eagle Medium (DMEM). After a final wash prior to sorting, cells were filtered through a 70-μm nylon cell strainer (Becton Dickinson) for removal of cell clumps. Cell sorting was performed using a FACSAria cell sorter (Becton Dickinson). Sorted populations were collected in complete medium (10% fetal calf serum in DMEM) as described previously (32).

May-grünwald-giemsa staining
Cell staining with Giemsa was employed to morphologically differentiate cells within sorted populations. Sorted cells (10 3 -10 6 ) were pelleted on to a glass slide using a cytospin centrifuge. Cells were fixed in methanol, then stained in a two-step procedure with CliniPure staining solution 1 (0.25% Eosin YO/Soresen buffer, pH 6.8) followed by staining solution 2 (0.25% methylene blue polychrone/Soresen buffer, pH 6.8) (HD Scientific: Sydney, NSW, Australia) for 5 s at each step. Excess solution was rinsed off and slides dried before mounting. Non-aqueous Depex mounting agent (Fluka Analytical: Buchs, Switzerland) was used to prevent leaching of dye from stained cells. Photographs were taken with a LEICA DFC digital camera connected to a LEICA brightfield inverted microscope (LEICA Microsystems: Wetzlar, Germany).

statistical analysis
Data have been presented as mean ± SE for sample size n. For sample size n ≤ 5, the Wilcoxon Rank-Sum test was used to test significance (p ≤ 0.05). Where a normal distribution could be assumed (n > 5), Students' t-test was used to determine significance (p ≤ 0.05).

resUlTs identification of Myeloid subsets in spleen
In blood, both the described populations of Ly6C hi inflammatory and Ly6C lo -resident monocytes do not express Ly6G (3). Ly6C hi monocytes are distinct from neutrophils which express Ly6G as well as Ly6C. In order to identify myeloid cells in spleen, CD11b hi CD11c − MHCII − cells were initially gated, then further delineated by Ly6C and Ly6G expression to yield subsets with a Ly6C hi Ly6G − , Ly6C lo Ly6G − , and Ly6C + Ly6G − phenotype as previously described in blood ( Figure 1A). These myeloid subsets were then distinguishable on the basis of SSC, with the Ly6C hi subset as SSC lo cells, Ly6C lo cells as SSC hi and neutrophils as SSC mid ( Figure 1A). Staining with antibodies specific for CD11b, CD11c, MHCII, Ly6C and Ly6G, and analysis of SSC can therefore be used to distinguish three main myeloid subsets in spleen, equivalent to those described previously in blood (7,25).
The novel L-DC subset resembles myeloid DC in spleen on the basis of high CD11b expression. In order to further explore the relationship between L-DC and the three major myeloid subsets described above, T, B, and red blood cell-depleted splenocytes were gated as CD11b hi CD11c lo MHCII − cells, and further tested for Ly6C and Ly6G expression. Since three distinct populations of cells were observed, Ly6C + Ly6G + (19.9%), Ly6C + Ly6G − (27.1%), and Ly6C − Ly6G − (47.1%) ( Figure 1B), these were selected for further study and termed "L-DC candidates. " FSC and SSC profiles distinguished Ly6C + Ly6G + and Ly6C + Ly6G − candidates as FSC hi SSC mid , while the Ly6C − Ly6G − subset was predominantly FSC lo SSC lo ( Figure 1B). To fully delineate the L-DC subset and to distinguish it from myeloid cells, it was necessary to identify further markers and to characterize all myeloid subsets concurrently.
Ly6C lo -resident monocytes in blood were previously distinguished from Ly6C hi -inflammatory monocytes on the basis of higher CD43 expression (10). In addition, eosinophils present in steady-state spleen were distinguishable from other myeloid cells through expression of Siglec-F (17,19). Myeloid cells identified in Figure 1 were therefore stained for these markers. All CD11b hi CD11c − cells delineated by Ly6C and Ly6G expression expressed CD43 (Figure 2A). The majority of Ly6C hi Ly6G − monocytes (~99%) and Ly6C + Ly6G + neutrophils (~97%) did not express Siglec-F ( Figure 2B). The Ly6C lo Ly6G − population contained a distinct CD43 hi subset (83.7%), which corresponded to SSC hi Siglec-F + cells and therefore resembled  (Figures 2A,B). All L-DC candidates, irrespective of Ly6C/Ly6G delineation, expressed CD43 (Figure 2A). The Ly6C lo Ly6G − candidates displayed the largest CD43 hi population (40.6%), also a reflection of resident monocytes. While the majority of cells in all subsets were Siglec-F − , each Ly6C − Ly6G − , Ly6C + Ly6G − , and Ly6C + Ly6G + subset showed some low level contamination with Siglec-F + eosinophils ( Figure 2B). The presence of Siglec-F + cells among splenic myeloid cells therefore necessitated mapping out the eosinophil phenotype in relation to myeloid cells. As shown in Figure 2B, Ly6C lo Ly6G − myeloid cells contained a large subset of Siglec-F + cells (79.3%), indicating a majority of eosinophils. The presence of eosinophils was also confirmed by transcriptome analysis, which revealed specific expression of CCR3 by this subset (unpublished data). In order to confirm the specificity of Siglec-F staining, a backgating strategy was applied to Siglec-F + cells ( Figure S1 in Supplementary Material). Siglec-F + eosinophils were found to be mainly CD11b hi CD11c − Ly6C lo Ly6G − CD43 + cells. Since this phenotype also reflects Ly6C lo monocytes in blood, future stainings therefore involved Siglec-F gating to distinguish or exclude eosinophils from splenic subsets of interest.

redefining splenic Myeloid subsets
Myeloid subsets in spleen were investigated further in terms of the expression of specific markers which are known to Frontiers in Immunology | www.frontiersin.org distinguish myeloid cells. CX3CR1 expression has been associated with the differentiation of macrophages and DC from a macrophage/dendritic progenitor (MDP) (46)(47)(48), and Ly6C loresident monocytes and Ly6C hi -inflammatory monocytes in blood have been described as CX3CR1 hi and CX3CR1 lo cells, respectively (3,7). CX3CR1 transgenic mice tagged with green fluorescence protein (GFP) were therefore used to confirm that the subset of CD11b hi CD11c − Ly6C lo Ly6G − Siglec-F + cells did not express CX3CR1 in line with the designation of eosinophils. When splenic SSC hi Ly6G − cells corresponding to CD11b hi CD1 1c − Ly6C lo Ly6G − Siglec-F + cells were gated, very few cells were found to express CX3CR1 (6.78%; Figure 2D), consistent with their classification as eosinophils rather than monocytes. The gated CD11b hi CD11c − Ly6C hi Ly6G − cells were found to be CX3CR1 lo , so confirming their identity as Ly6C hi -inflammatory monocytes described by Geissmann et al. (7) (Figure 2D). Finally, Ly6G + Ly6C + cells were found to be CX3CR1 − and so were likely to be neutrophils. L-DC candidates were then analyzed for CX3CR1 expression. For this experiment, they were divided into three populations based on SSC and Ly6G staining, and Ly6C expression on candidate subsets gated on the basis of SSC and Ly6G was also verified by independent staining (data not shown). Of the three L-DC candidate subsets, SSC hi Ly6C lo Ly6G − cells were largely CX3CR1 hi (63%), and along with their CD115 expression, reflected a phenotype consistent with Ly6C lo monocytes ( Figure 2D). The Ly6C + Ly6G + subset classified as neutrophils appeared heterogeneous with 43% CX3CR1 hi cells. The SSC lo Ly6C − Ly6G − L-DC candidate, negative for CD115 expression, was also CX3CR1 + . Low CX3CR1 expression was also observed on 48% of gated CD8 − cDC and 23% of gated CD8 + cDC (Figure 2D), consistent with previous reports (49,50). In terms of L-DC, the expression of CX3CR1 on SSC lo Ly6C − Ly6G − cells is consistent with a dendritic/ monocyte cell type.

l-Dc are a Distinct lineage from cDc
While L-DC are phenotypically distinct from cDC, they show similarity in their endocytic and cross-presentation ability (36).
To determine if L-DC and cDC derive from a common lineage progenitor, their prevalence was investigated in Batf3 −/− mice. The expression Batf3 has been described for pre-cDC, with peak expression in differentiated CD8 + and CD8 − cDC (51)(52)(53).
Batf3 −/− mice contain decreased numbers of CD8 + cDC, but not CD8 − cDC, suggesting that Batf3 is essential in the development of CD8 + cDC from pre-cDC (51). Batf3 may co-operate with another factor to induce the final differentiation of CD8 + cDC, while CD8 − cDC differentiation may occur independently of Batf3 (52).
The proportional representation of dendritic and other myeloid subsets in spleen relative to total splenic myeloid subsets of CD11b + and/or CD11c + cells was compared in mutant and wild-type mice. Subsets were delineated as described in Figure 3 and Table 1. A significant drop in the number of CD8 + cDC was observed in Batf3 −/− mice compared with wild-type mice consistent with previous reports (Figure 5). However, the number of CD8 − cDC in Batf3 −/− varied with 4 of 10 Batf3 −/− mice showing an increase in CD8 − cDC, while another four showed lower numbers of CD8 − cDC compared with controls. Variability in CD8 − cDC numbers could suggest either multiple interactive effects leading to highly variable numbers, or heterogeneity among the CD8 − cDC subset delineated here. The percentage of Ly6C hi -inflammatory monocytes also dropped significantly in Batf3 −/− mice (Figure 4). L-DC, Ly6C lo -resident monocytes, and neutrophils displayed no change in percentage due to the Batf3 mutation. Eosinophils were the only cells that displayed an increase in percentage in Batf3 −/− mice, but this could be consistent with increased inflammation in these mice.

l-Dc Develop independently of FlT3l and gM-csF
Fms-like tyrosine kinase 3 (FLT3) signaling has been described as essential for cDC development in steady-state spleen (54). Administration of FLT3L during in vitro and in vivo cell development leads to an increase in the number of splenic cDC and pDC (55)(56)(57). Similarly, knockdown of Flt3L leads to a two-to threefold drop in the number of splenic cDC and pDC in adult mice (54). In order to investigate whether L-DC development is also dependent on FLT3L, the percentage representation of cDC, L-DC and other myeloid subsets was analyzed relative to the total dendritic and myeloid cell population in adult Flt3L −/− and wild-type mice. A sixfold reduction in proportion of CD11c hi cells was observed in Flt3L −/− mice compared with wild-type mice, consistent with the literature (58, 59) (data not shown). A significant ~7-fold drop in percentage CD8 − cDC among total dendritic and myeloid cells, and a significant drop in percentage of CD8 + cDC were observed in Flt3L −/− mice compared with wild-type mice ( Figure 5A). There was also a significant drop in the percentage of Ly6C hi inflammatory and Ly6C lo -resident monocytes in Flt3L −/− mice ( Figure 5A). In contrast, neither L-DC nor eosinophils were affected by absence of Flt3L expression during development. Last, the percentage of neutrophils increased in Flt3L −/− mice ( Figure 5A). These results predict a role for FLT3L in the development of cDC, and all monocytes, but not L-DC and eosinophils. The proportional increase in neutrophils could reflect inflammation in a mouse model which has reduced immune capacity.
During inflammation, Ly6C hi monocytes can give rise to mo-DC that produce TNF-α and inducible NO synthase. These have been called "Tip-DC" (7,42,60). Similarly, granulocytemacrophage colony-stimulating factor (GM-CSF) can induce formation of inflammatory type mo-DC after addition to cultures of bone marrow progenitors (34,61,62). In order to investigate a role for GM-CSF in the development of splenic dendritic and other myeloid subsets, changes in the populational representation of cDC, L-DC and myeloid subsets were investigated in GM-CSF −/− mice ( Figure 5B). Previous studies have reported no change in the proportion of pDC and cDC in GM-CSF −/− mice (63,64). In this study, however, percentages of both CD8 + cDC and CD8 − cDC were higher in GM-CSF −/− mice (Figure 5B), while percentages of both Ly6C hi and Ly6C lo were significantly lower ( Figure 5B). The percentage of eosinophils in GM-CSF −/− mice was significantly increased, while the percentage of neutrophils remained unaffected (Figure 5B). Proportional representation of L-DC was not significantly different, although more variable in GM-CSF −/− compared with wild type mice.
L-DC development therefore occurs independently of both FLT3L and GM-CSF, while the development of monocyte subsets is clearly dependent on both factors (Figure 5). While cDC subsets are dependent on FLT3L for development, they develop independently of GM-CSF. In fact, in the absence of GM-CSF, there appears to be a compensatory increase in the development of cDC as well as eosinophils (Figure 5). These results distinguish L-DC from both cDC and other myeloid subsets, since L-DC develop independently of BATF3, FLT3L and GM-CSF.

DiscUssiOn
In addressing the issue of myeloid cell subset classification in spleen, a recent paper suggested a unified nomenclature for DC, monocytes, and macrophages based primarily on ontogeny, and secondly by location, function, and phenotype (65). The delineation of splenic Ly6C lo monocytes was made on the basis of the described phenotype and function of previously described resident monocytes in blood (7,25). Now, it is shown here that the spleen contains distinct populations of Ly6C hi CD11c − and Ly6C lo CD11c lo monocytes, as well Ly6C lo CD11c − eosinophils.
The latter population appears to be present in higher number in spleen and so more likely to obscure the Ly6C lo CD11c lo monocyte population in the absence of staining for CD11c and the SiglecF marker specific for eosinophils.
In order to better define an in vivo equivalent L-DC population in relation to splenic myeloid subsets, it was necessary to analyze dendritic and other myeloid subsets concurrently. Since blood monocytes are the most widely characterized monocytes, their phenotype was used as the starting point for analysis of splenic monocytes (19,47). Ly6C lo and Ly6C hi monocytes in blood were previously described as CD11b hi CD11c − MHCII − Ly6G − cells (3,6,7,10,11). Siglec-F, an inhibitory receptor expressed by murine eosinophils (66,67) proved definitive for analysis of eosinophils in this subset (Figure 2). The CD11b hi CD11c − MHCII − Ly6C + Ly6G − subset of cells in spleen was found to comprise a majority (87%) of Siglec-F + eosinophils (Figure 2), with Ly6C hi monocytes clearly Siglec-F − (Figure 2). The results of this study now confirm the definition of splenic Ly6C lo monocytes as CD11c lo cells (9,10,68) lying within the Ly6C + Ly6G − population.
To further test these initial predictions, the myeloid markers CX3CR1 and CD115 were employed. CX3CR1 is a known marker of Ly6C lo -resident monocytes (7,25), and CD115 is a marker of bone marrow-derived myeloid cells (43,44). High levels of CX3CR1 were shown to be expressed by CD11c lo cells that were also Ly6C lo Ly6G − , so confirming their similarity with resident monocytes (Figure 2D). In addition, these cells also expressed CD115 (Figure 2C). Since CD115 was not expressed by the putative L-DC subset, this suggests different lineage origins for L-DC and Ly6C lo monocytes. In line with the literature, Ly6C hi monocytes are CD115 + , while both neutrophils and eosinophils lacked CD115 expression ( Figure 2C).
Phenotypic studies were informative although not definitive in the identification of L-DC in relation to other myeloid subsets. Morphological characterization by May-Grünwald-Giemsa staining was used to confirm predicted subsets. Blood monocytes have a bi-lobate nucleus and minimal cytoplasm devoid of granules (Figure 4) (7,10). Ly6C hi monocytes in spleen displayed this morphology in line with their blood cell counterparts (Figure 4) (16,19). Giemsa staining also confirmed that the splenic subset defined here as eosinophils had a characteristic multi-lobate nucleus with orange granules in the cytoplasm, consistent with their phenotypic classification as Siglec-F + eosinophils (Figure 4) (69). The identified Ly6C lo monocyte subset identified as CD11c lo cells displayed morphology consistent with blood monocytes and not DC. These cells had a bi-lobate nucleus with minimal cytoplasm which lacked granules (Figure 3). Therefore morphological studies supported phenotypic studies, showing that spleen resident Ly6C lo monocytes lie within the CD11b hi CD11c lo Ly6C + Ly6G − population. Morphological analysis also showed that all Ly6G + subsets comprised a majority of neutrophils with characteristic multi-lobate nucleus (Figure 4). This was also confirmed by their 7/4 staining (data not shown).
The morphology of splenic DC is known to be to be quite distinct from the first described DC subset of Langerhans cells, which have long membrane projections (70,71). Here, CD8 + cDC were shown to reflect a majority of mononuclear cells with vacuoles evident in the cytoplasm. Similarly, CD8 − cDC and up to 60% of the Ly6C − Ly6G − L-DC subset also demonstrated similar cDC morphology with cytoplasmic vacuoles (Figure 3). These cells also showed veiled membranes with a few small dendrities, although this cannot be clearly be distinguished on cytospun cells. Combined phenotypic and morphological studies therefore identify L-DC as a subset of CD11b hi CD11c lo Ly6C − Ly6G − cells, with the majority demonstrating the morphology of a DC. A combination of studies shown here, therefore identify L-DC as a distinct cell type, reflecting the in vivo equivalent of cells produced in longterm cultures of spleen, LTC-DC. While it has been possible to phenotypically and morphologically identify an in vivo counterpart of the in vitro-produced L-DC, classification of this cell type as dendritic will be dependent on demonstration of their ability to activate naïve T cells and to cross present antigen as shown previously for in vitro produced cells (31,33).
Knockout mice studies have played a definitive role in identifying genes essential for development. Since none of the mutant mice studied here are embryonically lethal, and none of the genes mutated are crucial for survival, it was possible to obtain essential information about the development of L-DC in relation to other splenic myeloid subsets. CD11b hi CD11c lo Ly6C − Ly6G − L-DC development occurs independently of factors that regulate cDC and monocyte development, including BATF3, FLT3L, and GM-CSF. These findings distinguish L-DC from cDC as a separate lineage of cells, and identify them as distinct from splenic monocyte and granulocyte subsets.
Previously, this lab published evidence that L-DC can arise in vitro from self-renewing bone marrow-derived hematopoietic stem cells (HSC) and multipotential progenitors (MPP) following co-culture over splenic stromal lines (72,73). These findings confirm that L-DC arise from a progenitor distinct from the CDP also present in bone marrow (74). The production of L-DC was unaffected in Batf-3 −/− mice, suggesting that L-DC develop as a distinct lineage separated from cDC. In Batf-3 −/− mice, a significant drop in the percentage of inflammatory monocytes was also observed. Recent studies in Batf-3 −/− mice also showed a defect in the differentiation of CD4 T helper cells into T helper 17 (Th17) cells (75,76) essential for supporting inflammatory responses involving pathogens or autoimmunity (77,78). It is interesting to speculate that Ly6C hi monocytes may be important mediators of Th17 cell development from CD4 + T helper cells.
The binding of FLT3L to FLT3 triggers the development of cDC from pre-cDC (34,63,79), and the knockout of either Flt3 or Flt3L adversely affects cDC development (63,79). In this study, a significant drop in the production of splenic CD8 + cDC and CD8 − cDC was observed in Flt3L −/− mice (34,63). FLT3 is also a marker of common lymphoid progenitors (CLP) and CMP (59), such that the delivery of additional FLT3L to mice can lead to an expansion of the monocyte pool along with cDC (58,59). Expression of the FLT3 receptor on myeloid precursors, and the development of monocytes following FLT3L stimulation suggests that FLT3 receptor tyrosine kinase signaling plays a role in monocyte development. In agreement with this, Waskow et al. (79) showed an increase in the numbers of MDP which give rise to CDP and monocytes following FLT3L stimulation in vivo and in vitro. A reduction in the number of Ly6C lo resident and Ly6C hiinflammatory monocytes in Flt3L −/− mice shown here also supports a role for FLT3 signaling in monocyte development. Since L-DC development is not compromised in Flt3L −/− mice, L-DC are distinguishable in terms of development and lineage origin from both cDC subsets, and from the major monocyte subsets in spleen.
Both FLT3L and GM-CSF have been used for in vitro expansion of DC. Previously, it was shown that the culture of bone marrow precursors with FLT3L-generated cDC and pDC, while GM-CSF in the same system, generated inflammatory mo-DC (61,63). GM-CSF −/− mice were employed here to determine any role for GM-CSF in the in vivo development of L-DC. Deficiency in GM-CSF production did not affect the development of L-DC, suggesting that L-DC are not likely to be an inflammatory type cell dependent on GM-CSF signaling for development from myeloid precursors. By comparison, in GM-CSF −/− mice, the percentage of the two main monocyte subsets was reduced, consistent with their development in response to GM-CSF and probably inflammation. The percentage of Ly6C hi -inflammatory monocytes was fourfold lower in GM-CSF −/− mice, while Ly6C loresident monocytes showed a smaller but significant 1.4-fold reduction. Previous studies in GM-CSF −/− mice showed no significant change in peripheral blood myeloid subsets, although alveolar macrophages were found to be defective (80). However, that study did not investigate splenic myeloid subsets to the level of phenotypic detail used here. Under steady-state conditions, the development of cDC occurs independently of GM-CSF (63). In this study, the percentage of CD8 + cDC and CD8 − cDC was found to be significantly increased in GM-CSF −/− mice, although an increase in cDC numbers could occur as a compensatory change in spleen due to reduction in the number of monocytes. This study also showed that eosinophils increased in number in spleens of GM-CSF −/− mice, suggesting that the development of eosinophils occurs independently of GM-CSF. An increase in eosinophil number could also reflect an inflammatory or allergic state in GM-CSF −/− mice.
In conclusion, a novel dendritic-like cell type termed L-DC has been defined among multiple spleen myeloid subsets as CD11b hi CD11c lo MHCII − CD43 lo CD115 − Siglec-F − CX3CR1 lo Ly6 C − Ly6G − cells, resembling in vitro grown LTC-DC (32,33). In defining L-DC, populations of monocytes and granulocytes were also closely considered. Another outcome has therefore been the definition of eosinophils in spleen as CD11b hi CD11c − Ly6C + Ly6G − Siglec-F + cells. Ly6C lo -resident monocytes in spleen have now been identified more completely as CD11b hi CD11c lo Ly6C + Ly6G − CD43 + CX3CR1 hi CD115 + Siglec-F − cells. A study of subset prevalence in knockout mouse models confirmed the definition of L-DC in terms of their distinct lineage origin, establishing that L-DC develop independently of Batf3 expression essential for cDC development, and also independent of the FLT3L/GM-CSF growth factors necessary for cDC and monocyte development in spleen.

aUThOr cOnTriBUTiOns
YH: performance of experiments, analysis and assembly of data, manuscript writing. JT: analysis and interpretation of data, manuscript review. HO: project design and management, planning experiment, analysis and assembly of data, manuscript writing.