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The Journal of Immunology, 2004, 173: 2362-2372.
Copyright © 2004 by The American Association of Immunologists

IL-10-Producing B220+CD11c– APC in Mouse Spleen1

Fiona Burke, Andrew J. Stagg, Penelope A. Bedford, Nicholas English and Stella C. Knight2

Antigen Presentation Research Group, Faculty of Medicine. Imperial College London, Harrow, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
APC acting at the early stages of an immune response can shape the nature of that response. Such APC will include dendritic cells (DCs) but may also include populations of B cells such as marginal zone B cells in the spleen. In this study, we analyze APC populations in mouse spleen and compare the phenotype and function of B220+CD11c– populations with those of CD11c+ spleen DC subsets. Low-density B220+ cells had morphology similar to DCs and, like DCs, they could stimulate naive T cells, and expressed high levels of MHC and costimulatory molecules. However, the majority of the B220+ cells appeared to be of B cell lineage as demonstrated by coexpression of CD19 and surface Ig, and by their absence from RAG-2–/– mice. The phenotype of these DC-like B cells was consistent with that of B cells in the marginal zone of the spleen. On bacterial stimulation, they preferentially produced IL-10 in contrast to the DCs, which produced IL-12. Conventional B cells did not produce IL-10. The DC-like B cells could be induced to express low levels of the DC marker CD11c with maturational stimuli. A minority of the B220+CD11c– low-density cells did not express CD19 and surface Ig and may be a DC subset; this population also produced IL-10 on bacterial stimulation. B220+ APC in mouse spleen that stimulate naive T cells and preferentially produce IL-10 may be involved in activating regulatory immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigen-presenting cells that act at early stages of an immune response can form a bridge between innate and acquired immunity and are able to shape the developing Ag-specific response. The APC influences whether an active response or nonresponsiveness is the outcome of a particular encounter with a naive lymphocyte. It may also determine, in part by the production of cytokines such as IL-12, IL-10, and IL-4, whether a type 1 or type 2 response predominates or whether regulatory cells are generated (1). The APC can also control the homing characteristics of lymphocytes it activates so that they can be targeted to the appropriate tissues (2, 3, 4).

Dendritic cells (DCs) are one of the few types of APC able to activate naive T cells in vitro (5) and play a central role in the initiation of primary immune responses in vivo (6). "Immature" DCs, highly efficient at Ag uptake but expressing low levels of costimulatory molecules, constitutively migrate from most tissues into draining lymph nodes and are probably involved in the maintenance of nonresponsiveness to self-Ags (7, 8). In response to microbial products or the production of cytokines that indicate local tissue damage, DCs undergo a process of maturation and become fully stimulatory cells.

There is considerable heterogeneity within the DC pool. The nature of the activating DC as well as its maturation status can determine the type of response generated. In the human immune system, CD11c+ ("myeloid") and CD11c– ("plasmacytoid") DC subsets have been described previously (9, 10) and the plasmacytoid DC is the major IFN-{alpha}-producing cell in the blood (11). In the mouse, all recognized DC subsets express the CD11c integrin (12) but again myeloid and IFN-{alpha}-producing plasmacytoid DCs have again been identified (13, 14, 15). A third major murine CD11c+ DC subset has been described, delineated by the presence of the CD8{alpha}{alpha} homodimer (16), but there is currently no human equivalent for this subpopulation and its developmental relationship to other mouse DC subsets remains controversial.

Recent evidence suggests that in addition to DCs, other cell types could function as APC during the early stages of an immune response in vivo. Like DCs, B cells are heterogeneous. Mature B cells can be divided into B1 cells located in the peritoneal and pleural cavities, and B2 cells comprise follicular and marginal zone (MZ) B cells. MZ B cells comprise 5–10% of adult mouse B cells and are located at the red pulp/white pulp junction (17). They rapidly capture blood-borne Ags and differentiate into Ab-secreting plasma cells (18, 19), suggesting that they can play a critical role in host defense against bacterial pathogens (20). In response to Ag, some MZ B cells migrate to the T cell zone of the spleen and subsequently to the B cell follicles (21). In a process reminiscent of DC maturation, MZ B cells can up-regulate MHC Ags and costimulatory molecules in response to bacterial Ags such as LPS or signals from activated T cells (22). The rapidity of this process suggests that MZ B cells may participate in the earliest stages of in vivo T cell activation in the spleen. Ag-capturing myeloid DCs are also resident in the MZ and migrate in response to Ag exposure to the T cell zones of the spleen. Thus, DCs and MZ B cells may act in a coordinated and cooperative fashion to initiate early T cell responses in the spleen.

Other links between DC and B cells are suggested. The differentiation of DCs from early B cells has established a link between DC and B cell ontogeny (23, 24). The murine CD11c+ plasmacytoid DC subset coexpresses B220 (13, 14, 15), an Ag associated with the B cell lineage and an APC population with the properties of DC has been isolated from the mouse liver that has the phenotype CD11c–B220+ (25). These liver-derived APC produce IL-10 and have a regulatory role in the direction of the immune response.

In light of these findings, we extended our investigation of DC subsets in the mouse to study B220+CD11c– cells present in the low-density (LD) fraction of spleen cells. Analysis of the B220+CD11c– LD cells revealed two populations, both of which produced IL-10 in response to bacterial stimulation. The majority of B220+CD11c– LD cells were a specialized subset of B cells, distinct from conventional B cells in their ability to stimulate naive T cells and produce IL-10. They had a phenotype similar to B cells found in the MZ of the spleen. The second population, smaller in number, was not related to mature B cells and instead may be a B220+ DC subset. The ability of both of these B220+ APC populations to stimulate naive T cells and preferentially produce IL-10 suggests they may be involved in activating a regulatory immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female BALB/c, CBA, and C3H mice were bred under specific pathogen-free conditions at Northwick Park Institute for Medical Research or were purchased from Harlan U.K. (Bicester, U.K.) and housed at Northwick Park Institute for Medical Research. Mice were between 6 and 12 wk old at the time of use. Female BALB/c RAG-2–/– mice were kindly provided by Dr. J. Langhorne (Division of Parasitology, National Institute for Medical Research, Mill Hill, London, U.K.).

Chlamydia trachomatis

Buffalo green monkey kidney cells grown to confluence (DMEM/10% FCS/2 mM L-glutamine/25 µg/ml gentamicin; Sigma-Aldrich Cell Culture, Dorset, U.K.) were infected with a crude preparation of Chlamydia trachomatis elementary bodies (EBs)/serovar L1/440/LN (1/1000) and harvested 48 h after infection. The EBs were purified by centrifugation through a 25% (v/v) Isopaque gradient (Nycomed, Oslo Norway) at 29,000 x g for 60 min at 4°C, washed by centrifugation at 14,000 x g for 30 min at 4°C, and stored at 4°C in PBS for use in experiments.

Isolation of spleen LD cells

Single-cell suspensions of murine spleens were prepared by enzymatic digestion. Spleens were digested in 1 mg/ml collagenase D and 20 µg/ml DNase 1 (Boehringer Manheim, Lewes, U.K.) in RPMI 1640 HEPES medium containing 2% FCS for 2 h at 37°C with gentle agitation and passed through a metal cell strainer. Cells were washed twice by centrifugation at 350 x g for 5 min, resuspended in complete medium (RPMI 1640 Dutch modification medium with 10% FCS/2 mM L-glutamine/100 U/ml penicillin/streptomycin; Sigma-Aldrich Cell Culture, and 5 x 10–5 M 2-ME; Life Technologies, Grand Island, NY) and transferred to 25-cm2 Falcon tissue culture flasks (one spleen per flask) for overnight culture in a humidified incubator at 37°C in 5% CO2. Nonadherent cells were overlaid onto a metrizamide gradient (Nycomed; analytical grade, 13.7% w/v in HEPES-buffered RPMI 1640 medium containing 10% FCS) and centrifuged at 600 x g for 10 min. LD cells were collected at the interface, washed twice by centrifugation at 350 x g for 5 min, resuspended, and replicates pooled together (minimum of two spleens per experiment). High-density (HD) cells remaining in the pellet were carefully resuspended; the RBC were lysed with water and used as a source of conventional B cells.

Purification of B220+CD11c– LD cells by immunomagnetic separation

B220+ cells were separated from the DCs in the LD fraction by positive selection using biotinylated B220 Ab. The LD fraction was washed in miniMACS buffer (PBS/5% BSA/5 mM EDTA; Sigma-Aldrich) at 4°C, labeled with the Ab for 30 min at 4°C, and washed twice by centrifugation at 350 x g for 5 min. Streptavidin microbeads (Miltenyi Biotec, Bisley, U.K.) were then added to the cells for 15 min at 4°C, and again the cells were washed by centrifugation twice in cold buffer. The cells were gently resuspended and passed over the miniMACS column according to the protocol provided by the manufacturer (Miltenyi Biotec). The positively selected B220+ LD cells were then washed back into complete medium for subsequent experiments or into FACS buffer for analysis by flow cytometry. Subdivision of B220+ LD cells to obtain B220+CD19+IgM+CD11c– and B220+CD19–IgM–CD11c– LD cell populations was conducted by positive selection using FITC-conjugated IgM, followed by anti-FITC microbeads as described above. A second positive selection using biotinylated B220 Ab was performed to select for the B220+ LD cells remaining in the IgM-negative fraction and the negative fraction of the two sequential immunomagnetic separations was used as a source of purified DCs. Conventional B cells were isolated from the HD cells by positive selection using biotinylated B220 Ab as described above.

Mixed Leukocyte Reactions

Lymph node cell suspensions were obtained by passing lymph nodes from CBA mice through a cell strainer. LD cells and separated B220+ LD populations were used to stimulate proliferation of allogeneic lymph node responder cells in 20-µl hanging drop cultures (26). Stimulator cells were added to lymph node responder cells at varying concentrations in triplicate to 60-well Terasaki plates. The Terasaki plates were inverted and hanging drop cultures were incubated over saline, in a humidified incubator, at 37°C in 5% CO2. On day 3 of culture, the cells were pulsed with 1 µg/ml [3H]thymidine with a specific activity of 2 Ci/mmol (Amersham International, Amersham, U.K.) for 2 h and the hanging drop cultures were blotted onto filter paper. Filters were washed with saline, 5% TCA, and dried with alcohol. Uptake of thymidine was assessed by liquid scintillation counting or by measurement on a phosphor imager (Amersham Biosciences, Amersham, U.K.). Thymidine was used at nonlimiting concentrations to reflect DNA synthesis.

Cytokine measurement

Purified cell populations were stimulated for 2 h with EBs then cultured for 24 h or cultured overnight with EBs. Cytokine production in the cell supernatants was measured by capture ELISA using OptEIA mouse IL-10 and IL-12 ELISA sets (BD Pharmingen, San Diego, CA) according to the manufacturer’s instructions. Standards and samples were assayed in duplicate. A standard curve was plotted and cytokine production by test samples was expressed in picograms per milliliter (group mean with SEs).

Flow cytometry

mAb to the following Ags were purchased from BD Pharmingen for use in flow cytometry experiments: CD11b (Mac-1), anti-I-Ad (AMS-32.1), anti-IgD (11--26c.2a), and CD21/35 (CR2/CR1) (7G6) conjugated to FITC; CD23 (B3B4), CD24 (heat-stable Ag) (M1/69), CD44 (PgP-1, Ly-24), CD5 (Ly-1) (53-7.3), and Gr-1 (Ly-6G and Ly-6C) (RB6-8C5) conjugated to PE; CD1d (1B1), CD9 (KMC8), and CD43 (Ly-48) conjugated to biotin; anti-CD19 (ID3), anti-CD4 (RM4-5), anti-CD40 (HM40-3), anti-CD80 (16-10-A1), anti-CD86 (GL1), and anti-IgM (R6-60.2) conjugated to FITC or PE; anti-CD11c (HL3), anti-CD45R (B220) (RA3-6B2), and anti-CD8{alpha} (53-6.7) conjugated to FITC, PE, or biotin; anti-DEC-205 conjugated to FITC was a gift from Dr. A. Lane (Serotec, Oxford, U.K.). Corresponding isotypes to the above mAbs, conjugated to the appropriate fluorochromes, were used as controls for nonspecific binding of Ab. Second layer reagents used to bind biotinylated Abs were as follows: streptavidin conjugated to PerCP (BD Pharmingen), R-PE-Cy5 (CytomationDako, Cambridgeshire, U.K.), or ECD (Beckman Coulter, Fullerton, CA). Cells were washed in FACS buffer comprising 5% FCS, 0.02% sodium azide, and 1 mM EDTA in PBS. FCS (15 µl) and 2 µl of purified CD16/32 (Fc{gamma}III/II receptor) (2.4G2; BD Pharmingen) were added to each tube to inhibit nonspecific binding. mAbs were added to the appropriate tubes for 30 min at 4°C. The cells were washed twice by centrifugation in cold FACS buffer at 350 x g for 5 min and the second layer reagent was added for 30 min at 4°C. Finally, the cells were washed twice by centrifugation in FACS buffer and fixed in 1% paraformaldehyde. Cells were acquired on a FACSCalibur (BD Biosciences U.K., Oxford, U.K.) or a Coulter EPICS XL-MCL flow cytometer (Beckman Coulter). To quantitate cell numbers, absolute count beads (Flow Count; Beckman Coulter) were added to each sample before acquisition. Flow cytometry data were analyzed using the CellQuest software on the Apple Macintosh and WinList (Verity, Topsham, MA) version 4 on the PC.

Electron microscopy

LD cells were labeled with biotinylated Ab, followed by 10-nm anti-biotin gold particles for identification by electron microscopy. Cells were fixed in 3% glutaraldehyde in 0.1 M sodium phosphate buffer and washed, and the pellet was embedded in low-gelling temperature agarose. Cells were postfixed in 1% osmium tetroxide in 0.1% (pH 7.4) sodium phosphate, washed with water, and block stained in 2% uranyl acetate. The cells were dehydrated using an acetone gradient and gradually infiltrated with araldite resin. Blocks of cells were sectioned (100-nm thick) using a Reichert-Jung Ultracut E microtome, stained with Reynolds lead citrate, and carbon coated. Sections were viewed using a JEOL JEM-1200 EX electron microscope (Osaka, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enrichment of DCs from murine spleen

Analysis of freshly isolated spleen cells by flow cytometry demonstrated DCs identified by their expression of CD11c and MHC class II (Fig. 1A). After overnight culture, a metrizamide gradient was used to select for DCs from nonadherent spleen cells on the basis of their LD and differential shrinkage. Two DC subsets were present in the LD fraction of the spleen, delineated by the presence or absence of the CD8{alpha}{alpha} homodimer as previously described (27). A population of B220+CD11c– LD cells copurified with the DCs was demonstrated by flow cytometry. A small number of the B220+ LD cells expressed a low level of CD11c. The expression of B220 enabled the cells to be separated from the DCs by immunomagnetic separation with >90% purity (Fig. 1A).



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  		FIGURE 1. Isolation of B220+CD11c– LD cells from the spleen that display properties of DCs. A, Whole spleen cells labeled with Ab to CD11c and MHC class II after RBC lysis and analyzed by flow cytometry (upper left dot plot). LD cells isolated over metrizamide after overnight culture and labeled with Ab to CD11c and B220 (upper right dot plot). LD cells labeled with Ab to CD8{alpha} to identify subsets of DCs (lower left dot plot). B220+ LD cells positively selected from the LD fraction of the spleen using immunomagnetic separation and analyzed for purity by flow cytometry, >90% purity (lower right dot plot). B, A MLR stimulated with B220+ LD cells in parallel with whole LD cells. Two thousand stimulator cells were added to varying concentrations of allogeneic lymph node responders in a hanging drop microculture system and proliferation was measured by incorporation of titrated thymidine in cpm. Similar results were obtained with 500 and 1000 stimulator cells. C, B220+ LD cells, identified by absence of CD11c, analyzed for expression of Ag-presenting molecules and costimulatory molecules by flow cytometry; overlays represent isotype-matched controls.

 
Splenic B220+CD11c– LD cells display properties of DCs

The unique ability of DCs to stimulate naive T cells in the MLR provides a functional assay for their identification. Surprisingly, the separated B220+CD11c– LD cells stimulated a MLR with comparable potency to that of the whole LD fraction containing the DCs (Fig. 1B). Consistent with the ability to stimulate a MLR, the B220+CD11c– LD cells expressed high levels of Ag-presenting molecules and costimulatory molecules on the cell surface. Analysis by flow cytometry demonstrated that the B220+CD11c– LD cells expressed comparable levels of MHC class II, CD40, and CD86 to the DCs, with more heterogeneous expression of CD80 (Fig. 1C).

Morphology of splenic B220+CD11c– LD cells

The morphology of the LD cells was observed by electron microscopy; the B220+ LD cells were identified by immunogold labeling for B220 Ag. Electron microscopy revealed that the B220+CD11c– LD cells ranged in size and appearance (Fig. 2), from cells with a more uniform appearance, round or oval (plasmacytoid), with heterochromatic nuclei, rare to numerous short cytoplasmic processes and a darker cytoplasm (Fig. 2, A and D), to larger cells with a strikingly similar appearance to that of DCs (identified as B220–), with more irregular nuclei becoming euchromatic and a paler cytoplasm (Fig. 2, A–C).



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  		FIGURE 2. Transmission electron microscopy of LD cells. LD cells were isolated as described in Materials and Methods, labeled with biotinylated B220 followed by goat anti-biotin gold particles (10 nm) and observed under the electron microscope. The electron micrographs demonstrated the B220+ LD cells that range in size and appearance (A). B220+ LD cells with similar morphology to DC (A) and at higher magnification (B) and revealed the immunogold labeling indicating the presence of B220 on the cell surface (C). B220+ LD cells with numerous short cytoplasmic processes (D), B220 expression confirmed by the presence of immunogold particles (E). Bars, 1 µm (A, B, and D); 100 nm (C and E).

 
Cytokine profile of splenic LD cells

To ascertain the potential role of the B220+CD11c– LD cells in directing the subsequent immune response, cytokine production by these cells in response to stimulation with C. trachomatis was analyzed by ELISA. The B220+CD11c– LD cells produced large amounts of IL-10 on stimulation, with little or no IL-12 present in the supernatant (Fig. 3A). In contrast, the DCs produced negligible IL-10 and large amounts of IL-12 with bacterial stimulation, as previously observed (28, 29). Although IL-10 production by the B220+CD11c– LD cells was only evident on stimulation, the DCs demonstrated spontaneous IL-12 production in the absence of stimulation. The LD fraction containing the B220+CD11c– LD cells and the DCs produced both immunomodulatory cytokines upon bacterial stimulation. The production of IL-10 by the B220+CD11c– LD cells on bacterial stimulation suggested that these APC may potentially exert an immunomodulatory effect over the subsequent direction of the immune response. Production of IL-10 by the LD cells on stimulation with C. trachomatis was independent of the TLR-4 signaling pathway as the cytokine was also produced in LPS nonresponsive C3H/HeJ mice (Fig. 3B). Material prepared from uninfected Buffalo green monkey kidney cells (sham EB) stimulated neither IL-10 (Fig. 3B) nor IL-12 production (data not included).



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  		FIGURE 3. Cytokine profile of LD cells. A, B220+ LD cells positively selected from the LD fraction of the spleen by immunomagnetic separation and cultured in parallel with DCs (B220– fraction) and unseparated LD cells for 24 h in the presence or absence of C. trachomatis. Supernatants were analyzed for production IL-10 (A, left histogram) and IL-12 (A, right histogram) by ELISA (nd, no detectable cytokine). B, LD cells isolated from C3H/HeN and C3H/HeJ (lpsd) mice and cultured overnight with EBs (1.8 x 104–1.8 x 106/ml) and analyzed for IL-10 production by ELISA. LD cells cultured with sham EB (uninfected buffalo green monkey kidney cells) were used as a control for IL-10 production.

 
Phenotype of splenic B220+CD11c– LD cells

The functional and morphological analysis of the B220+CD11c– LD cells had demonstrated that these cells display some of the classic hallmarks of DC. However, phenotypic analysis revealed that the major fraction of the B220+CD11c– LD cells coexpressed CD19, a surface Ag more restricted to the B cell lineage than B220. This suggested that the majority of the cells were indeed a subset of the B cell lineage. A small number of B220+CD11c– LD cells did not express CD19 (Fig. 4).



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  		FIGURE 4. Expression of CD19 by B220+CD11c– LD cells. LD cells were isolated as described in Materials and Methods, labeled with Ab to B220 and CD19, and analyzed by flow cytometry (left dot plot). The isotype control for CD19 was included to detect nonspecific binding by the B220+CD11c– LD cells (right dot plot).

 
To clarify further the phenotype of the B220+CD11c– LD cells, the expression of a number of surface Ags used to discriminate between B cell subpopulations was examined using four-color flow cytometry. Expression of CD19 was used to gate the B220+CD11c– LD cells, and cell surface Ag expressed was compared with that of conventional HD B cells in the spleen and to the DCs present in the LD fraction identified by the absence of CD19 (Fig. 5). Compared with the conventional HD B cell population, the B220+CD11c– LD cells were enriched for CD1d, expressed high levels of CD24 (heat-stable Ag), and were CD21/35 (CR2/CR1) and IgM+ and IgDlow. The cells expressed CD43, a surface Ag not present on conventional resting peripheral B cells, in addition to high levels of CD44, a surface Ag associated with activated cells. A low level of CD5 was also expressed, an Ag associated with B-1 cells (Fig. 5, middle and right-hand panels). Notably, a number of the surface Ags used to discriminate between B cell subsets, with the exception of the Igs, were also present at comparable levels on the DCs, namely, CD1d, CD43, and CD5 and were not exclusive to cells of a B cell lineage (Fig. 5, left-hand panel). The phenotype of the B220+CD11c– LD cells was distinct from conventional B cells present in the HD fraction of the spleen. Expression of high levels of CD1d, IgM, and CD21/35 was suggestive of the B cells found in the MZ of the spleen (30). To investigate this possibility, the B220+CD11c– LD cells were analyzed for other cell surface Ags associated with B cells in the MZ. The phenotype of the B220+CD11c– LD cells was then compared with that of the HD B cells (Fig. 6A) and freshly isolated spleen B cells (data not shown). As demonstrated in Fig. 6, the majority of the B220+CD11c– LD cells expressed high levels of both CD1d and CD9, were CD21/35highCD23low, and were IgMhighIgDlow, a phenotype consistent with B cells described in the MZ (31, 32). In contrast, HD B cells expressed high levels of IgD, low levels of IgM and CD21/35, and did not express CD1d or CD9. In comparison to fresh spleen cells, HD cells were depleted of the MZ-like population found in cells of LD (data not shown).



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  		FIGURE 5. Phenotype of B220+CD19+CD11c– LD cells. LD cells were isolated as described in Materials and Methods, labeled with a panel of Abs used to identify subsets of B cells, and analyzed by flow cytometry. Expression of surface markers by the B220+CD19+CD11c– LD cells (middle panel) was compared with that of conventional splenic B lymphocytes in the HD fraction of the spleen (right panel) and to the DCs present in the LD fraction identified by the absence of CD19 (left panel).

 


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  		FIGURE 6. B220+CD11c– LD cells have the phenotype of B cells in the MZ of the spleen. A, LD spleen cells and HD spleen cells were isolated as described in Materials and Methods, labeled with a panel of Abs used to identify B cells in the MZ of the spleen, and analyzed by flow cytometry. Expression of surface markers by the B220+CD11c– LD cells (left panels) was compared with that of conventional splenic B lymphocytes in the HD fraction of the spleen (right panels). B, LD spleen cells were prepared from mice of the BALB/c and C3H strains reported to have low and high numbers of MZ B cells, respectively, and stained with anti-B220 and anti-CD11c. Percentages represent the proportion of LD cells that were B220+.

 
Consistent with their phenotypic similarity to MZ B cells, B220+CD11c– cells were increased in the LD fraction isolated from C3H mice (Fig. 6B), a strain known to have high numbers of MZ B cells.

Separation of B220+CD11c– LD cell on the basis of surface IgM

Although the majority of the B220+CD11c– LD cells were CD19+, there was heterogeneous expression of many of the surface markers analyzed; a discrete population of the B220+CD11c– LD cells was negative for CD19 and surface Ig (Figs. 4 and 5E). These B220+CD19– IgM– LD cells may be related to the B220+ DC subset recently reported in the spleen (13, 14, 15). To confirm that the properties of DCs and the production of IL-10 were attributable to the specialized subset of B cells we had described and not to a contaminating B220+ DC subset, the B220+CD19+IgM+CD11c– LD cells were separated from the B220+CD19–IgM–CD11c– LD cells on the basis of surface Ig. The functional properties of these separated populations were examined and compared with those of the DCs and HD B cells.

The B220+CD19+IgM+CD11c– LD cells stimulated naive T cells in a MLR, confirming that the ability of these cells to stimulate naive T cells was not due to a contaminating B220+ DC subset (Fig. 7A). The purified DCs were the most potent APC as demonstrated by the dosimetry of the stimulator populations observed in Fig. 7A, which showed that 500 DCs was sufficient for maximal stimulation of lymph node responders, while T cell proliferation on stimulation with B220+CD19+IgM+CD11c– LD cells was still increasing at 1000 stimulator cells. The B220+CD19–IgM– LD cells also stimulated T cell proliferation; however, it is notable that they did contain some contaminating DCs that may account for this stimulation.



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  		FIGURE 7. Functional analysis of separated B220+ LD cells. A, B220+ LD cells, separated based on their differential expression of IgM, were used to stimulate a MLR in parallel with DCs (B220–IgM–). Two hundred fifty (left panel), 500 (middle panel), and 1000 (right panel) stimulator cells were added to varying concentrations of allogeneic lymph node responders in a hanging drop microculture system and proliferation was measured by incorporation of titrated thymidine in cpm. B, Separated B220+ LD cells were cultured overnight with EBs in parallel with DCs (B220–IgM–), unseparated LD cells, and HD B cells; cytokine production was measured in the supernatants by ELISA. C, LD cells were isolated from RAG-2–/– mice using a method identical to that used in wild-type mice; LD cells were cultured overnight with or without EBs and cytokine was measured in the supernatants by ELISA.

 
Next, we examined the cytokine profile of the purified LD cell populations on stimulation with C. trachomatis (Fig. 7B). As before, the B220+CD19+IgM+CD11c– LD cells produced large amounts of IL-10 and negligible IL-12, confirming that theywere a source of this immunomodulatory cytokine. TheB220+CD19–IgM–CD11c– LD cells also produced IL-10 and although this population did contain some contaminating DCs, it was unlikely that IL-10 production was due to the DCs, as we had demonstrated that they produce negligible IL-10 on stimulation. Production of IL-12, however, by this population may have been due to DC contamination within the B220+CD19–IgM– LD cells.

The cytokine profiles indicated that we had identified both a major and minor population of IL-10-producing B220+ cells present in the LD fraction of the spleen; the former appeared to be a specialized subset of B cells that displayed properties of DCs, DC-like B cells, while the latter did not express mature B cell markers and may represent the recently described B220+ DC subset in the mouse. However, the lack of CD11c and the ability to produce IL-10 was more analogous with the regulatory liver-derived DC described by Lu et al. (25).

Absence of B220+CD11c– LD cells in BALB/c RAG-2–/– mice

The presence of the two B220+CD11c– LD cell populations was analyzed in the LD fraction of BALB/c RAG-2–/– mice devoid of circulating B cells (33). Spleen cells from RAG-2–/– mice were cultured overnight after enzymatic digestion, and LD cells were isolated over metrizamide as described in the previous experiments. As expected, the majority of the B220+CD11c– LD cells were absent in these mice, confirming their identity as a specialized subset of the B cell lineage. A discrete population of B220+ cells, however, was present in the LD fraction of RAG-2–/– mice. This supported the presence of a B220+ DC in the LD fraction of wild-type mice, unrelated to mature B cells. The B220+ LD cells in RAG-2–/– mice, expressed a low level of CD11c, a low and intermediate level of MHC class II, and contained both CD8{alpha}+ and CD8{alpha}– and Gr-1+ and Gr-1–B220+ LD cells (Fig. 8).



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  		FIGURE 8. Analysis of LD cells in BALB/c RAG-2–/– mice. LD cells were isolated from the spleens of BALB/c RAG-2–/– mice as described in Materials and Methods and analyzed for the presence of B220+ LD cells by flow cytometry. The B220+ LD cells present were labeled with a panel of Abs to determine their phenotype. Percentage positive for CD11c and MHC class II was calculated by enhanced negative subtraction of the isotype using WinList software (version 4). Positive expression is represented by the shaded areas.

 
LD cells in RAG-2–/– mice produced both IL-10 and IL-12 upon bacterial stimulation. Since no IL-10 production by the DCs had been demonstrated in earlier experiments, this indicated that the source of the IL-10 may be the B220+ LD cells present in RAG-2–/– mice, supporting our previous findings of IL-10 production by B220+CD19–IgM–CD11c– LD cells in the wild-type mice.

Induction of CD11c expression by B220+CD11c– LD cells

The analysis of LD cells in RAG-2–/– mice had highlighted a population of B220+ LD cells that coexpressed a low level of CD11c. We had observed a small number of B220+ LD cells in wild-type mice that did express a low level of CD11c, and we investigated whether the levels of CD11c expression could be up-regulated with maturational stimuli on a subpopulation of our B220+ LD cells. B220+ LD cells were isolated as before and cultured for an additional 24 h with or without stimulation C. trachomatis and expression of CD11c was analyzed by flow cytometry. As shown in Fig. 9, the level of expression of CD11c on the B220+ LD cells was not up-regulated and remained low. The number of B220+ LD cells expressing a low level of CD11c, however, increased from 12.7% of the B220+ LD cells to >37.1% on culture; no further increase was evident with bacterial stimulation. The observed increase in CD11c expression was not due to preferential survival of the B220+ LD cells coexpressing a low level of CD11c, as absolute cell numbers of B220+ LD cells expressing a low level of CD11c increased (data not shown). The low level of CD11c surprisingly appeared to be on the B220+CD19+IgM+ LD cells as the proportion of cells expressing CD19 and surface Ig remained unchanged with maturational stimuli, despite the induction of CD11c on a substantial proportion of the cells. This was concurrent with a decrease in the level of expression of both CD19 and surface Ig on these cells (data not shown). The expression of CD11c demonstrated another property of DCs shared by the DC-like B cells we had identified.



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  		FIGURE 9. Induction of CD11c by B220+CD11c– LD cells. LD cells were isolated over a metrizamide gradient following overnight incubation as described and either labeled with Ab to CD11c and B220 on day 1 (left histogram) or cultured for an additional 24 h and labeled on day 2 (right histogram). Expression of CD11c by the B220+ LD cells was analyzed by flow cytometry. B220+ LD cells were gated on and percentage positive for CD11c was calculated by enhanced negative subtraction using WinList software (version 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the investigation of murine spleen DC subsets, in addition to the identification of the CD8{alpha}-positive and -negative DC subsets previously described (16), we describe a heterogeneous population of B220+CD11c– cells present in the LD fraction of the spleen that displayed properties of DCs. Despite recent reports of a B220+ DC subset in the mouse (13, 14, 15), the B220+ cells that we describe lacked CD11c and the majority appeared to be of B cell lineage. Nevertheless, the B220+CD11c– LD cells shared properties with DCs, in particular, the ability to stimulate naive T cells. The ability of B cells to stimulate naive T cells has previously been demonstrated using B cells activated first in vitro (34, 35). However, the cells that we describe did not require prior activation in vitro to stimulate naive T cells and expressed high levels of MHC class II, costimulatory molecules, CD43, and CD44 when freshly isolated. It is widely accepted that resting B cells are inefficient at stimulating naive T cells (5, 34, 36, 37) and weak proliferative responses that do occur have been explained by the presence of small numbers of in vivo-activated LD B cells (34). These were thought to be present in naive mice under resting conditions as a result of exposure to environmental Ag and as such their significance in the stimulation of primary immune responses has not been fully explored. Our results, in which we have consistently isolated a functionally unique population of B220+CD11c– LD cells from the spleen of naive mice, clearly indicate a discrete subset of B cells that is present in mice under resting conditions. Like DC, these B cells can stimulate naive T cells and secrete immunomodulatory cytokines and therefore have the potential to play a specialized role in the immune response. Evidence of B cells displaying properties of DCs has been documented previously. The formation of dendrites on mature B cells has been described on B cells that have been activated in vitro (38, 39, 40). In addition, DC-like B cells have recently been isolated from human peripheral blood (41); despite their B cell phenotype, these cells expressed DC-associated markers, stimulated a MLR, and displayed morphology akin to the cells we describe with a uniform appearance and short processes. Parallels between our work and the work by Zhong et al. (41) gives rise to the possibility that the DC-like B cells we describe in the mouse are also present in humans and that their presence may not be restricted to the spleen. A number of recent studies have suggested closer links between B cells and cells of the myeloid lineage than previously recognized. Some B cells retain the potential to differentiate into DC (23, 24) and B cell/macrophage lineage switching by B-1 cells has been reported in both healthy animals and human patients with B cell malignancy (42)

The phenotype of the DC-like B cells we describe was consistent with B cells previously described in the MZ of the spleen as demonstrated by high levels of expression of CD1d, CD21/35 (CR2/CR1), and IgM, a low level of IgD, positive expression of CD9, and absence of CD23 (30, 32, 32). MZ B cells, like B-1 cells, may play a role in the innate immune response. These innate B cells express germline-encoded receptors with limited diversity that recognize conserved structures, many of which are of self-origin rather than the diverse repertoire of receptors expressed by conventional B cells involved in the adaptive immune response (43). Such innate B cells are described as having a natural memory rather than a naive phenotype and are thought to be early effector cells in the immune response. The cells we describe may indeed correspond to this specialized subset of the B cell lineage and our studies extend their unique properties to both the production of the immunomodulatory cytokine IL-10 and properties more commonly associated with DCs, primary APC, so supporting their role as early effector cells in the immune response.

A small number of the B220+CD11c– LD cells were CD19– surface Ig– and appeared to share similarities to the B220+CD11c–CD19–DCs described by Lu et al. (25), particularly as they produced IL-10 on stimulation. Using the methods here, there was no evidence of a B220+ DC subset in the spleen that coexpressed CD11c, as reported by other groups (13, 15), although they were present in the lymph node (data not shown); their absence may be a result of the overnight incubation step used here in the isolation of splenic LD cells over metrizamide.

The expression of CD11c by the DC-like B cells on further culture demonstrated another property that these cells shared with DCs; expression of CD11c on the B220+CD19+ LD cells highlighted the need for caution in the exclusive use of CD11c as a marker for the identification of murine DCs. In humans, CD11c is expressed at low levels on a number of other cell populations (44, 45, 46, 47, 48, 49). However, in mouse CD11c, expression has long been thought to be restricted to DCs and not present on other cells (12). A low level of CD11c expression has been demonstrated on a subset of intestinal intraepithelial lymphocytes, only in the presence of intestinal flora (50), and on a discrete population of thymic eosinophils with an "activated" phenotype (51). In support of our observation of CD11c expression by the DC-like B cells, a study of DCs from Peyer’s patches and spleen found that a significant proportion of cells selected with anti-CD11c from both tissues expressed B220, some of which were CD19+. In light of our own study, this observation highlights the presence of heterogeneous populations of B220+ cells in lymphoid tissue that coexpress a low level of CD11c, some of which are also CD19+ (52).

Both the DC-like B cells and the smaller population of B220+CD19–CD11c– LD cells preferentially produced IL-10 on bacterial stimulation. B cells can produce an array of cytokines on activation; in particular, IL-10 production by B cells has previously been demonstrated (53). Despite this, the influence of cytokine production by B cells has not yet been fully explored and has mainly been thought to play an auxiliary role, amplifying the ongoing immune response. Recently, polarized cytokine production by splenic B cells has been demonstrated upon coculture with the respective Th1 and Th2 cells (54); however, the particular population of B cells responsible for producing cytokine was not investigated. We demonstrate that the DC-like B cells we have isolated, which may correspond to a population of MZ B cells, preferentially produced IL-10 on stimulation, while conventional B cells did not. This indicates that cytokine production by B cells may be attributable to particular subsets of B cells with distinct roles in the immune response. Interestingly, IL-10 production by peritoneal B cells but not unseparated splenic B cells has been demonstrated (55). Peritoneal B cells are enriched in B-1 cells, which as stated earlier share many properties with the B cells described in the MZ of the spleen and may indicate along with the work described here that innate B cells are specialized in the preferential production of IL-10.

It is notable that IL-10 production by the subset of B cells we describe occurred in the absence of T cell help and taken together with the ability to stimulate naive T cells indicates that these APC may be involved in initiating the polarization of a primary immune response. In particular, the preferential production of IL-10 implicates these APC in the activation of regulatory immune responses. The role of regulatory T cells in the maintenance of tolerance both at mucosal sites and in transplantation tolerance has been demonstrated (56, 57). IL-10 is critical in mediating the tolerance conferred by CD4+CD45RBlow regulatory T cells and tolerance can be abrogated by IL-10-neutralizing Abs (56). CD4+CD45RBlow regulatory T cells from IL-10–/– mice do not demonstrate regulatory properties, indicating that IL-10 is required for their effector function (57); however, it may also be required for the generation of regulatory T cells. The APC involved in activating IL-10-mediated regulatory immune responses has not yet been fully explored; however, it has been reported that CD11c+CD8{alpha}– DCs in the lung produce IL-10 and are crucial in conferring tolerance to respiratory allergen (58), and that mucosal DCs in the Peyer’s patches preferentially produce IL-10 (52). In a model of inflammatory bowel disease, a population of regulatory B cells has been described; suppression of the pathogenic T cells by the regulatory B cells in this model was dependant on CD40 and CD86 and not CD80 and furthermore was independent of Ig. Subsequently, it has been shown that these cells were enriched for CD1d and produced IL-10 (59, 60) similar to the DC-like B cells we describe. Ligation of CD1d has been reported to result in production of IL-10 (61). The similarity between the regulatory B cells in the gut and the cells we describe in the spleen may indicate that a subset of B cells exists in vivo located in lymphoid organs and at mucosal sites that are specialized in the generation of regulatory immune responses. Regulatory B cells that produce IL-10 have also been described in experimental autoimmune encephalitis (62) and in a mouse model of arthritis (63); however, unlike the cells described here, regulatory B cells in these models are dependent on T cell help for production of IL-10 and do not appear to have an innate B cell phenotype.

The minor population of IL-10-producing B220+CD11c–CD19– LD cells that we describe shared similarities with the liver-derived DCs described by Lu et al. (25), which were shown to regulate T cell responses (25) and may support a regulatory role for the B220+CD11c–CD19– LD cells we describe.

In conclusion, both populations of IL-10-producing B220+ APC we have identified in mouse spleen may be involved in activating regulatory immune responses. The effector cells stimulated by these APC require further investigation.


   Acknowledgments
 
We thank Dr. Jean Langhorne (Division of Parasitology, National Institute for Medical Research, Mill Hill, London, U.K.) for providing the BALB/c RAG-2–/– mice used in our experiments.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by the Medical Research Council and Dunhill Medical Trust. Back

2 Address correspondence and reprint requests to Prof. Stella C. Knight, Antigen Presentation Research Group, Faculty of Medicine, Imperial College London, Northwick Park Campus, Level 7W, Watford Road, Harrow, HA1 3UJ, U.K. E-mail address: s.knight{at}ic.ac.uk Back

3 Abbreviations used in this paper: DC, dendritic cell; LD, low density; HD, high density; MZ, marginal zone; EB, elementary body. Back

Received for publication October 18, 2002. Accepted for publication May 13, 2004.


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