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Blood, 1 February 2007, Vol. 109, No. 3, pp. 1165-1173. Prepublished online as a Blood First Edition Paper on October 12, 2006; DOI 10.1182/blood-2006-05-015354.
IMMUNOBIOLOGY Production of interferons by dendritic cells, plasmacytoid cells, natural killer cells, and interferon-producing killer dendritic cells1 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia; and 2 the Bavarian Nordic GmbH, Munich, Germany
The capacity of mouse spleen conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) to produce interferon-  (IFN-) or IFN- was assessed, and compared with that of natural killer (NK) cells and the recently identified interferon-producing killer dendritic cells (IKDCs), both of which are frequent contaminants in DC preparations. Fully developed cDCs or pDCs, if free of NK cells or IKDCs, showed little capacity for IFN- production. However, an early developmental form of the CD4–8+ cDC subtype, and the Ly6C– Ly49Q– pDC subtype, both were able to produce moderate amounts of IFN-, although less than IKDCs. In response to toll-like receptor 9 stimuli, both the Ly6C+ Ly49Q+ and the Ly6C– Ly49Q– pDC subtypes were effective producers of IFN-. However, IKDCs, which efficiently produced IFN- and showed immediate cytotoxicity on NK target cells, did not produce IFN- un-der these conditions.
Interferons (IFNs) are key effector cytokines of the innate and adaptive immune systems. Type 1 interferons, such as IFN- , are produced in large quantities by activated plasmacytoid dendritic cells (pDCs), and are particularly important in resistance to virus infections.1–5 The inflammatory cytokine IFN- is produced in large quantities by Th1 effector CD4 T cells, by CD8 T cells, and by natural killer (NK) cells.6–8 Interferons also play crucial roles in the initiation of adaptive immune responses. Type 1 interferons help activate the conventional dendritic cells (cDCs) which are needed to initiate primary T-cell responses. IFN- itself is needed to initiate the differentiation of activated T cells toward the IFN-–producing Th1 state. Thus, the production of even relatively small amounts of interferons by even minor cell populations could be important in the early steps of immune responses.
It has been reported by several laboratories, including our own,9–12 that DCs are able to produce IFN-
A further complication to the assessment of IFN production by DC subtypes is the recent recognition of a type of cell with properties overlapping those of DC and NK cells. The existence of DC with NK-like killing properties had been recognized previously in rodents.13–16 Human NK clones have been shown to be effective at antigen presentation,17 and human pDC malignancies display NK cell markers.18–21 Recent studies by 2 groups have provided sufficient information to consider these as a distinct cell type in mice, now termed interferon-producing killer dendritic cells (IKDCs).22–24 Since IKDCs are potent producers of IFN-
To clarify these issues we isolated spleen cDC and pDC subtypes in such a way as to segregate them from NK cells and IKDCs, then we tested them for IFN-
Mice Mice were produced at the Walter and Eliza Hall Institute (WEHI) animal facility under specific pathogen-free conditions. Female 5- to 7-week-old C57Bl/6J Wehi mice were used in most experiments. cDC isolation and enrichment The standard procedure for isolation and enrichment of conventional DC (cDCs) was based on our earlier procedures25,26 and is summarized in Figure 1. All media were at pH 7.2 and iso-osmotic with mouse serum (308 mOs). Spleens (n=8) were cut into small fragments and digested, with frequent mixing, for 20 minutes at room temperature (22°C) in 7 mL modified RPMI-1640 medium, 2% fetal bovine serum (FCS) containing collagenase (1 mg/mL; Worthington Biochemical, Freehold, NJ; verified free of trypsinlike protease activity) and DNAase I (0.02 mg/mL; Boehringer Mannheim, Mannheim, Germany). EDTA solution (600 µL, 0.1 M, pH 7.2) was then added and mixing continued for 5 minutes. Undigested material was removed by filtration through a coarse sieve. All subsequent steps were at 0°C to 4°C, using a divalent metal–free buffered balanced salt solution containing EDTA (EDTA-BSS). Cells from the digest were centrifuged to a pellet and the pellet was resuspended in 10 mL of a 1.077 g/cm3 medium, made by dissolving Nycodenz powder (Nycomed, Oslo, Norway) in water as a dense stock (0.37 M) then diluting in EDTA-BSS to give the required density at 308 mOs. Further Nycodenz was layered below the Nycodenz cell suspension, EDTA-FCS layered above, then the tube centrifuged at 1700g for 10 minutes. The light density fraction (< 1.077 g/cm3) was collected, diluted in EDTA-BSS, recovered by centrifugation, then incubated for 30 minutes with the following monoclonal antibodies (mAbs): anti-CD3 (KT3-1.1); anti-Thy1 (T24/31.7); anti-B220 (RA3-6B2); anti-Gr1 (RB68C5); antierythrocyte (TER-119). All mAbs were titrated and used at near saturation conditions except anti-Thy1, which was used at 25% saturation. The cells were washed free of excess mAb, then antibody-coated cells were removed using anti–rat immunoglobulin (Ig)–coupled Biomag beads (Qiagen, Clifton Hill, Australia). The beads and cells at a 10:1 ratio were mixed as a concentrated slurry by slow rotation for 20 minutes, diluted with EDTA-BSS, then the beads and bound cells were removed with a magnet, using 2 removal steps. In the modified procedure to remove cells bearing NK markers, an additional depletion step was used prior to staining and sorting (Figure 1). The cells recovered after the anti–rat Ig depletion step were incubated with saturation levels of biotinylated mAb against CD49b (DX5) and against CD161c (NK1.1; clone PK136), washed, then incubated (in BSS containing 0.5% FCS) with antibiotin MACS magnetic particles (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions, then passed over a Miltenyi column (Miltenyi Biotec), collecting the unbound cells. The enriched spleen cDCs obtained by both protocols were around 80% pure.
Staining, sorting, and analysis of cDC subsets
To effect the final purification of cDCs and segregate them into subtypes, the enriched cDC preparation was normally stained with mAbs against CD11c (N418-FITC), CD4 (GK1.5-Alexa 594), and CD8 pDC isolation and enrichment The procedure was based on our previous study,27 and is summarized in Figure 2A. The production of a spleen cell suspension and isolation of the light density fraction was identical with the procedure for cDCs. The immunomagnetic bead depletion step was also similar, except that the cells were coated with a different cocktail of mAbs, which spared pDCs but allowed some depletion of cDCs. These were mAbs against: CD3 (KT3-1.1); CD90 (T24/31.7); CD19 (1D3); Ly6G (1A8); erythrocytes (TER-119); F4/80 antigen (F4/80); CD11b (M1/70); and CD205 (NLDC145). Note that the mAb against CD205 allowed only partial depletion of the CD205+ cDC subtypes. No additional depletion for cells bearing NK markers was used, although the procedure used for cDCs was effective if predepletion of NK cells and IKDCs was required.
pDC purification and analysis To effect final purification and analysis of pDC subsets (Figure 2A), the pDC-enriched preparation was normally stained for CD11c (N418-Alexa 594), CD45RA (14.8-APC), Ly6C (5075-3.6-FITC), and CD49b (DX5-biotin with PE-streptavidin second stage). When the cells were stained for Ly49Q (2E6-FITC), the mAb against Ly6C was a biotin conjugate with PE-streptavidin second stage, and CD49b staining was omitted. Dead cells were stained with propidium iodide as for cDCs. The sorting and analysis was as specified for cDCs. NK cell isolation Splenic cell suspensions were generated using collagenase digestion as in the DC isolation procedure. Red cells and dead cells were removed using a 1.091 g/cm3 Nycodenz density cut. The viable light density cells were incubated with anti-CD4 (GK1.5), anti-CD8 (53.6-7), anti-CD24 (M1/69), and anti-MHC II (M5/114) mAbs and coated cells were removed using anti–rat Ig magnetic beads. NK cells were then isolated by sorting for CD49b+(DX5+) CD3– cells. Cytokine production culture conditions
Sorted cells (105) were cultured in 200 µL modified RPMI 1640 medium that contained 10% FCS, in 96-well round-bottom plates at 37°C under 10% CO2-in-air. After culture, supernatants were collected and stored for a short time at –20°C prior to analysis. To determine IFN- Analysis of cytokine production by ELISA
Culture supernatants were assayed for the presence of cytokine by 2-site enzyme-linked immunosorbent assay (ELISA). Purified capture monoclonal antibodies to IFN- Cytotoxicity assays
The YAC-1 target cells were harvested in exponential growth phase, washed, and labeled by incubating 106 cells with 100 µL (51Cr) sodium chromate in 100 mL medium for 1.5 hours at 37°C then washed 4 times. Target cells (104) and various levels of effector cells were plated in 0.2 mL modified RPMI-1640 10% FCS medium in 96-well round-bottom plates, centrifuged for 1 minute at 335g, then incubated 4 hours at 37°C. After centrifugation (5 minutes, 400g) 100 µL supernatant was removed and the chromium release (E) was measured by Antigen cross-presentation assays For the in vitro presentation assay the cDC subsets were freshly isolated from unstimulated mice. For the in vivo presentation assay, as described previously,28 mice were given injections of 30 µg lipopolysaccharide (Sigma-Aldrich), then 1 hour later with 3 mg ovalbumin (Sigma-Aldrich), then the cDC subsets were isolated 19 hours later. Cross-presentation was assessed by the ability to stimulate ovalbumin-specific TCR-transgenic naive CD8+ OT-I T cells, isolated as described previously28 from lymph nodes. Cultures were set up in V-bottom 96-well plates in 0.2 mL modified RPMI-1640/10% FCS medium at 37°C in 10% CO2-in-air. The cDC subsets were plated at 0.5 x 103 to 5 x 103 cells per well and the OT-I T cells were added at 2 x 104 cells per well. For the in vitro presentation only, ovalbumin was added to the cultures at 250 µg/mL. After 2 days in culture, the wells were pulsed with 1 µCi (37 (kBq)/well (3H) thymidine for 8 hours, the cells harvested onto glass fiber filters, and the incorporation into cellular DNA determined by liquid scintillation counting.
Spleen cDC preparations, cDC subsets, and IKDC contaminants
Our standard protocol for isolation of cDCs is given in Figure 1. The DCs were first enriched by selecting the 3% to 5% lightest density cells, which removed the bulk of B cells, T cells, and NK cells. Subsequent immunomagnetic bead depletion of the light density fraction then eliminated most pDCs, and residual B cells, T cells, and other lineages. Finally, sorting for cells bearing high levels of CD11c strongly selected for cDCs and against any residual pDCs, T cells, or NK cells. Additional sorting for CD4 or CD8 expression allowed segregation of the 3 main cDC subtypes, namely those that were CD4+8–, CD4–8+, and CD4–8– (also termed CD4+, CD8+, and DN, respectively; Figure 1). Recently we have shown,29 using the newer markers CD172a (Sirp- This standard protocol produced cDC subtypes that were around 99% pure on reanalysis, but we could not eliminate the possibility of contamination below the 1% level. It became clear that our enriched cDC preparations, prior to sorting, contained around 0.5% of cells that were light in density, expressed moderate levels of CD11c, and that also expressed the NK marker CD49b, revealed by the mAb DX5 (Figure 3A). These resemble IKDCs, the recently described "hybrid" NK-DCs.22,23 Most but not all of these IKDCs were eliminated by our gating for CD11chi cDCs (Figure 3A). As an approach to eliminating all IKDCs, as well as any residual conventional NK cells, we introduced an additional MACS depletion step using antibodies selective for NK cells (Figure 1). As shown in Figure 3A, this resulted in a very marked reduction of cells bearing the NK marker CD49b in both the cDC-enriched preparation and the sorted cDC fractions. Particularly important was the elimination of NK cells and/or IKDCs from the CD4–8– cDC fraction, since selection against CD4 and CD8 expression would concentrate any NK-like cells that had not been removed by the CD11c gating.
IFN- production by cDC subtypes and by IKDCs
We examined the possibility that the IFN-
Using the ELISA, we confirmed the production of IFN-
To determine in a direct way the possible contribution of IKDCs to the IFN- production by our isolated cDC subpopulations, we carefully analyzed the level of cells bearing CD49b in the various fractions, comparing the standard cDC subset preparations with those from cDCs predepleted of cells bearing NK markers (Figure 3A). Although our standard enriched cDCs contained around 0.5% putative IKDCs, most but not all of these were depleted by the CD11chi gate we routinely used to select cDCs. Predepletion of cells bearing NK markers reduced this initial level of putative IKDCs in the cDC preparation around 250-fold (Figure 3A). This predepletion of cells bearing NK markers did result in some reduction (around 2-fold) in the final IFN- production by the cDC subsets, suggesting that traces of contaminating IKDCs had made some contribution to the measured IFN- production by the original cDC subset preparations (Figure 3B). However, significant IFN- production was still obtained when IKDCs or NK cells were below detectable levels. Importantly, the CD4–8– cDC subset was still a significant producer of IFN- when not even a single CD49b-bearing cell could be detected on analysis of 105 cells (Figure 3A-B). Thus, even though NK cells and IKDCs were around 100-fold more efficient as IFN- producers, they could not explain this level of IFN- production. These results support our previous conclusion,9 and the conclusion from Table 1, that the major source of IFN- by spleen cDC preparations is the CD4–8– cDC fraction. Developing cDCs within the CD4–8– cDC preparation
We have recently demonstrated that the spleen contains a substantial pool of immediate precursors of cDCs.30 Most of these are of medium rather than low buoyant density, lack surface expression of MHC II, and express only moderate levels of CD11c. However, some of these cDC precursors appear to be in transit toward the fully developed steady-state spleen cDCs, having already acquired the light density and higher surface CD11c characteristics, and having already begun surface MHC II expression. These cDC precursors have not yet acquired surface expression of CD4 or CD8, although some have already demonstrated commitment toward particular cDC subtypes by the expression of the CD172a– (Sirp
As shown in Figure 4A, although fully developed CD4–8– cDCs should be CD172a+, the CD4–8– cDC preparation did include a minor group of cells (10%-11%) that were CD172a–. Further analysis showed these CD172a– cells were lower in surface expression of MHC II, and were positive for CD24 expression (Figure 4A). This suggested they were indeed earlier cells en route to the CD4–8+ cDC subtype. Indeed, when these were cultured overnight in a conditioned medium, 42% then expressed CD8
Production of IFN- by the cDC precursors within the CD4–8– cDC fraction
A preparation of spleen cDCs, once free of IKDCs or NK cell contaminants, showed only a low capacity to produce IFN- Lack of cytotoxic activity by the cDC precursors within the CD4–8– cDC fraction
Since the CD172a– precursors of CD4–8+ cDCs within the CD4–8– population produced IFN-
Spleen pDC preparations, pDC subsets, and IKDC contaminants Since IKDCs appear closer to pDCs than to cDCs in surface phenotype,22,23 we examined their contribution to pDC preparations. Our protocol for the isolation of pDCs is given in Figure 2A. The approach was similar to that used for cDCs, but the mAbs used for immunomagentic bead depletion differed. B cells were depleted with anti-CD19, rather than with anti-CD45R (B220), which also depletes pDCs. Granulocytes were depleted with a mAb specific for Ly6G that has no reactivity with Ly6C, to avoid any possible pDC depletion. Some mAbs reactive with cDCs were included to reduce the load of cDCs, although they did not give complete removal. However, since we wished to examine the relationship of IKDCs to the pDC subsets, no depletion of cells bearing NK markers was applied. The pDCs were finally sorted and separated from residual cDCs based on their high expression of CD45RA and intermediate rather than high levels of CD11c (Figure 2A). Very similar results were obtained if anti-CD45R (B220) was used as a pDC\E marker instead of CD45RA (data not shown). Since cells bearing intermediate levels of CD45RA can overlap the CD45RAhi fraction, and since IKDCs are found with intermediate as well as high levels of CD45R,22,23 CD45RAint cells were also sorted for analysis. The pDC fraction, CD45RAhi CD11cint, could be further subdivided based on Ly6C expression into a major group (91% ± 4%) of Ly6C+ cells, considered as the typical spleen pDCs, and a minor (9% ± 4%) subgroup of Ly6C– cells (Figure 2A). The Ly6C+ cells also stained weakly with the anti-Ly6G mAb Gr-1, which is believed to cross-react with Ly6C; however, in our hands the staining was too weak to allow the clear segregation seen with mAb directed to Ly6C itself. In contrast to these results with spleen pDCs, when the equivalent CD45RAhi CD11cint pDC fraction was isolated from bone marrow (BM), it consisted of 57% Ly6C+ cells and 43% Ly6C– cells (data not shown). In addition, cultures of BM stimulated by Flt-3 ligand29 produced CD45RAhi pDCs, which consisted of 32% Ly6C+ and 68% Ly6C– cells (data not shown). This segregation of pDCs based on Ly6C expression recalled recent studies in which segregation of pDCs using Ly49Q expression produced Ly49Q– and Ly49Q+ subtypes, the Ly49Q– subtype being predominant in BM and the Ly49Q+ subtype being predominant in spleen.32,33 The Ly49Q– pDCs have been reported to develop into Ly49Q+ pDCs in culture.33 Accordingly, we stained spleen pDC preparations for both Ly6C and Ly49Q and cross-correlated the expression of the 2 markers. Indeed, there was an extensive correlation, the Ly6C– pDCs being mainly Ly49Q–, and the Ly6C+ pDCs being mainly Ly49Q+ (Figure 2B). To determine if any IKDCs were present within our spleen pDC preparations, we stained for CD49b using the mAb DX5. As shown in Figure 2A, a small proportion of cells in the CD45RAhi CD11cint pDC fraction were strongly positive for CD49b. These were entirely within the Ly6C– subset, which could be clearly separated into Ly6C– CD49b+ cells and Ly6C– CD49b– cells. The CD49b+, putative IKDCs showed a wider range of CD45RA expression than pDCs and some were also present in the CD45RAint fraction of the enriched pDC preparation (Figure 2A). Such cells would not readily be depleted using anti-CD45R mAb, so probably contributed to the few IKDCs that contaminated our standard cDC preparations. NK-like killing by IKDCs To check that the CD49b+ cells isolated from our pDC preparations were equivalent to the IKDCs described by others,22,23 we tested their cytotoxic activity on the NK target cell YAC-1. Indeed, these CD49b+ Ly6C– CD11c+ CD45RA+ putatative IKDCs showed an immediate strong cytotoxic activity on YAC-1 cells in a 4-hour assay (Figure 6B). This was similar to the activity of NK cells isolated from spleen suspensions (Figure 6A). In contrast, the CD49b-depleted pDC and cDC preparations showed no killing of YAC-1 targets (Figure 6B). The extent of killing was not notably increased by including CpG1668 in the cytotoxic assays to activate the IKDCs (data not shown). When these putative IKDCs were first preactivated by overnight culture with CpG1668 together with GM-CSF, duplicating the approach of others,22 the viable cells that survived were also cytotoxic to YAC-1; however, under these conditions viable cell survival was only 10% to 30% (data not shown). By the criterion of NK-like killing, the CD49b+ cells in our pDC preparations could be classified as IKDCs. Production of interferons by pDCs and IKDCs
To determine which interferons were produced by the "true" pDCs and which were produced by the putative IKDC "contaminants," the sorted fractions of Figure 2A were assayed for the production in culture of IFN-
IFN-
In contrast to the results on IFN- production, IFN- was produced at high levels by the CD49b+ cells within the pDC preparations (Figure 7), indicating that these could indeed be called IKDCs. However, no IFN- was produced by the typical CD45RAhi CD11cint Ly6C+ spleen pDCs, and only moderate amounts were produced by the presumed earlier pDC form, the Ly6C– CD49b– subset. Thus, there was little overlap in the capacity to produce these 2 interferons in response to the stimuli used, the pDCs producing IFN-, the IKDCs producing IFN-.
The main objective of this study was to determine the extent of production of interferons by fully developed, steady-state DCs of the type found in mouse spleen. The existence of multiple subtypes of DCs and of other cells with properties that overlap those of DCs, made this a demanding exercise. The problems are emphasized by the recent description of a cell type termed IKDC22–24 which, like DCs, is light in density and expresses surface CD11c, CD45R (B220), and MHC II, but which also has the cytotoxic potential and surface markers of NK cells. The true lineage origins of these IKDCs are yet to be determined. Such IKDCs are likely to be a contaminant in most cDC preparations where expression of CD11c is the main selection criterion. Because many IKDCs express relatively high levels of CD45R and CD45RA, they will be a component of pDC preparations selected by these criteria. Stringent exclusion of cells bearing NK markers, such as CD49b (DX5), is required to eliminate these cells.
Since IKDCs are capable of producing large amounts of IFN-
We have found that spleen contains a reservoir of precursors of cDCs, but not of pDCs.30 Most of these pre-cDCs lack surface MHC II, express only intermediate levels of CD11c and moderate buoyant density, so are separable from fully developed steady-state cDCs. However, because of the rapid turnover of spleen cDCs,34 spleen must contain cells in transit between pre-cDCs and fully developed cDCs. We have now identified such a minor population of cDCs within the CD4–8– fraction of our cDC preparations. Although these cells lack surface CD8
The immunologic significance of the potential of splenic early CD8+ cDCs to produce moderate amounts of IFN-
Amongst DCs, the pDCs are considered to be the major producers of type 1 interferons in response to viral and other infections and in response to the TLR-9 stimulus CpG.1–5 We have noted that CD4–8+ cDC preparations are capable of some IFN-
A striking finding in our study is the dichotomy between IFN-
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Abstract
Introduction
