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Cancer Research 66, 10497-10504, November 1, 2006. doi: 10.1158/0008-5472.CAN-06-1908
© 2006 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Combined Stimulation with Interleukin-18 and CpG Induces Murine Natural Killer Dendritic Cells to Produce IFN-{gamma} and Inhibit Tumor Growth

Umer I. Chaudhry, T. Peter Kingham, George Plitas, Steven C. Katz, Jesse R. Raab and Ronald P. DeMatteo

Hepatobiliary Service, Memorial Sloan-Kettering Cancer Center, New York, New York

Requests for reprints: Ronald P. DeMatteo, Memorial Sloan-Kettering Cancer Center, Box 203, 1275 York Avenue, New York, NY 10021. Phone: 212-639-5876; Fax: 212-639-4031; E-mail: dematter{at}mskcc.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Natural killer dendritic cells (NKDC) are a novel subtype of dendritic cells with natural killer (NK) cell properties. IFN-{gamma} is a pleiotropic cytokine that plays an important role in the innate immune response to tumors. Based on our previous finding that the combination of Toll-like receptor 9 ligand CpG and interleukin (IL)-4 stimulates NKDC to produce IFN-{gamma}, we hypothesized that NKDC are the major IFN-{gamma}-producing dendritic cell subtype and may play a significant role in the host antitumor response. We found that under several conditions in vitro and in vivo NKDC accounted for the majority of IFN-{gamma} production by murine spleen CD11c+ cells. IL-18 alone induced NKDC to secrete IFN-{gamma}, and the combination of IL-18 and CpG resulted in a synergistic increase in IFN-{gamma} production, both in vitro and in vivo. NK cells made 26-fold less IFN-{gamma} under the same conditions in vitro, whereas dendritic cells produced a negligible amount. The mechanism of IFN-{gamma} secretion by NKDC depended on IL-12. NKDC selectively proliferated in vitro and in vivo in response to the combination of IL-18 and CpG. Systemic treatment with IL-18 and CpG reduced the number of B16F10 melanoma lung metastases. The mechanism depended on NK1.1+ cells, as their depletion abrogated the effect. IL-18 and CpG activated NKDC provided greater tumor protection than NK cells in IFN-{gamma}–/– mice. Thus, NKDC are the major dendritic cell subtype to produce IFN-{gamma}. The combined use of IL-18 and CpG is a viable strategy to potentiate the antitumor function of NKDC. (Cancer Res 2006; 66(21): 10497-504)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IFN-{gamma} is a proinflammatory cytokine with pleiotropic functions (1). Its production is pivotal in initiating a Th1 immune response to pathogens and tumors. The importance of IFN-{gamma} is highlighted in mice and humans in that mutations in IFN-{gamma} or its receptor increase the susceptibility to infections by mycobacterium and other intracellular organisms (2). Additionally, a role for IFN-{gamma} in the host defense against tumors has been identified (3). IFN-{gamma} was initially considered to be produced solely by cells of lymphoid origin, particularly natural killer (NK) cells and T cells. However, there is a growing body of evidence suggesting that cells of the myeloid lineage (i.e., dendritic cells and macrophages) are also capable of secreting IFN-{gamma}. In 1985, Robinson et al. (4) showed that alveolar macrophages from patients with sarcoidosis can secrete IFN-{gamma}. Since then, multiple groups have reported IFN-{gamma} production by human macrophages in various disease states. Puddu et al. (5) reported IFN-{gamma} secretion by mouse peritoneal macrophages in response to interleukin (IL)-12. Munder et al. (6) later described that bone marrow–derived macrophages can be stimulated with IL-12 and IL-18 to produce IFN-{gamma}. Ohteki et al. (7) provided the first evidence that murine dendritic cells are capable of producing IFN-{gamma}. The mechanism was shown to be dependent on IL-12 and other groups have shown a synergistic effect with IL-2, IL-4, and IL-18 (8–11). To date, only one study has described IFN-{gamma} production by human dendritic cells in response to IL-12 and IL-18 stimulation (12).

The concept that cells of myeloid lineage can secrete IFN-{gamma} has been challenged recently. A study by Schleicher et al. (13) showed that IFN-{gamma} production ascribed previously to macrophages actually originates from contamination by either CD3+CD8+TCRß+ T cells or CD11b+CD11c+CD31+DX5+NK1.1+ cells. Although several independent investigators have published that dendritic cells can secrete IFN-{gamma}, we have reported that NK dendritic cells (NKDC; CD11c+NK1.1+CD3–) activated with CpG and IL-4 secrete high levels of IFN-{gamma} in vitro, whereas conventional dendritic cells (CD11c+B220–NK1.1–) make negligible amounts under the same conditions (14). Recently, several publications have reported on IFN-{gamma} production by cells with similar phenotype and function as NKDC. A study by Kamath et al. (15) showed that DX5+NK1.1+CD11c+ splenocytes are responsible for all lipopolysaccharide (LPS)–induced IFN-{gamma} production. Chan et al. (16) showed that IFN-producing killer dendritic cells (IKDC), defined as CD11cintB220+CD49b+, secrete IFN-{gamma} in response to combined stimulation with IL-12 and IL-15. However, to date, there is no detailed study of IFN-{gamma} secretion by NKDC and conclusive evidence that conventional dendritic cells make negligible amounts of IFN-{gamma} is lacking.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. Adult 6- to 8-week-old male C57BL/6 (B6, H-2Kb) mice were purchased from Taconic Farms (Germantown, NY) and B6.129S7-Ifngtm1Ts/J (IFN-{gamma}–/–) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained in the pathogen-free animal housing facility at Memorial Sloan-Kettering Cancer Center (New York, NY), and all procedures were approved by the Institutional Animal Care and Use Committee.

Cell isolation. Splenocytes were isolated as described previously (17). Briefly, animals were euthanized by CO2 inhalation. Spleens were mechanically disrupted before being passed through a sterile 70-µmol/L nylon mesh filter (BD Falcon). The resulting cell suspension was pelleted (300 x g for 7 minutes), RBC were lysed using a hypotonic solution, and the remaining cells were washed twice in complete medium (RPMI 1640, 10% FCS, 2 mmol/L L-glutamine, 0.1% 2-mercaptoethanol, 100 units/mL penicillin, and 100 units/mL streptomycin). Splenocytes were separated into CD11c+ and CD11c– fractions with immunomagnetic beads as per the manufacturer's protocol (Miltenyi Biotec, Auburn, CA). Before all immunomagnetic bead incubations, Fc{gamma} receptors were blocked with the monoclonal antibody 2.4G2 (Fc{gamma}III/IIR block; 1 µg/million cells; Monoclonal Antibody Core Facility, Memorial Sloan-Kettering Institute). Enriched CD11c+ cells were stained with fluorescently conjugated antibodies to B220, NK1.1, CD3, and CD11c (all were from BD PharMingen, San Diego, CA) for further separation of dendritic cell subtypes using a MoFlo cell sorter (DakoCytomation, Fort Collins, CO). Dead cells were excluded with 4',6-diamidino-2-phenylindole dilactate (Molecular Probes, Eugene, Oregon). Care was taken to exclude highly autofluorescent cells during fluorescence-activated cell sorting (FACS), and sorted cell populations were consistently >98% pure for the desired set of surface markers.

Flow cytometry. Five-color flow cytometry was done on a FACScan flow cytometer (BD Biosciences, San Jose, CA) with modifications by Cytek (Fremont, CA). Voltages were determined using unstained cells. Single-stained positive controls for each fluorochrome were used to set compensation. Samples were incubated with Fc{gamma}III/IIR block before staining. Approximately 5 x 105 cells were labeled with 0.1 µg FITC, phycoerythrin, peridinin chlorophyll-a protein (PerCP), allophycocyanin (APC), APC-Cy7, or biotin-conjugated antibody (all were from BD PharMingen). Biotinylated antibodies were secondarily stained with streptavidin-PerCP. Cells were stained for MHC I (H-2Kb) [AF6-88.5], MHC II (I-Ab) [AF6-120.1], CD3{varepsilon} [145-2C11], CD8{alpha} [53-6.7], CD11c [HL-3], CD11b [M1/70], CD40 [3/23], CD45R/B220 [RA3-6B2], CD49b [DX5], CD69 [H1.2F3], CD80 [16], CD86 [GL-1], CD122 [TM-ß1], NK1.1 [PK136], and IL-12Rß1 [114]. IL-15R{alpha} and IL-18R{alpha} (R&D Systems, Minneapolis, MN) were secondarily stained with anti-goat IgG FITC. Appropriate immunoglobulin isotype controls were used for phenotype analysis. Flow cytometry data were analyzed with FlowJo software (TreeStar, Ashland, OR).

Cytokine analysis. In vitro cytokine production was assessed by culturing 3 x 104 purified NKDC in a 96 well U-bottomed tissue culture plate (BD Falcon) in 100 µL medium for 72 hours. Supernatant IFN-{gamma} content was assayed using a cytometric bead array as per the manufacturer's protocol (BD Biosciences). IL-12 (1 ng/mL; BD PharMingen), IL-18 (20 ng/mL; Biosource, Camarillo, CA), Toll-like receptor (TLR) 3 ligand polyinosinic acid:poly-CMP (polyI:C; 10 µg/mL; Roche Applied Sciences, Chicago, IL), TLR4 ligand LPS (10 µg/mL; Chemicon International, Temecula, CA), TLR5 ligand flagellin (10 µg/mL; Calbiochem, San Diego, CA), TLR7 ligand loxoribine (10 µg/mL; InvivoGen, San Diego, CA), TLR9 ligand CpG ODN 1826 (CpG; 10 µg/mL; Oligos Etc., Wilsonville, OR), or {alpha}IL-12 (10 µg/mL; BD PharMingen) was added to some wells.

For in vivo cytokine analysis, mice were given various cytokines i.p. and sacrificed 8 hours later. Splenocytes were cultured for 5 hours with Brefeldin A (BFA; BD PharMingen) without restimulation. Cells were labeled with surface antibodies, fixed, and permeabilized. Next, cells were stained for intracellular IFN-{gamma} using an intracellular cytokine kit as per the manufacturer's protocol (BD PharMingen) and analyzed via flow cytometry.

Proliferation assays. In vitro proliferation of NK cells, NKDC, and dendritic cells was determined by culturing 3 x 104 FACS-purified cells with various stimuli for 3 days, at which time [3H]thymidine was added to the culture (1 µCi/well; Perkin-Elmer) and radioactive uptake was determined 20 hours later with a TopCount NXT microplate scintillation and luminescence counter (Perkin-Elmer). In vivo proliferation was assessed by administrating a single i.p. dose of CpG (100 µg), IL-18 (1 µg), or combination of CpG and IL-18 to mice and by analyzing splenocytes on days 3, 6, 9, and 12 by flow cytometry.

Quantitative reverse transcription-PCR. The expression of IFN-{gamma} transcripts was examined by Taqman real-time PCR. Briefly, total RNA was extracted from FACS-purified cells of both saline-treated [normal saline (NS)] and IL-18/CpG-treated mice and reverse transcribed using the Thermoscript reverse transcription-PCR system (Invitrogen, Carlsbad, CA) at 52°C for 1 hour. Resultant cDNA (20 ng) was used in a quantitative PCR using an iCycler (Bio-Rad, Hercules, CA) and predesigned Taqman Gene Expression Assays (Roche, Alameda, CA). Primers were chosen based on their ability to span the most 3' exon-exon junctions (Mm00801778_m1). Amplification was carried out for 40 cycles (95°C for 15 seconds and 60°C for 1 minute). IFN-{gamma} threshold cycle detection number was divided by the threshold cycle detection number for a constitutively expressed hypoxanthine guanine phosphoribosyl transferase (18). The ratios for NK cells and NKDC were divided by the ratio for dendritic cells, which expressed negligible levels of IFN-{gamma}.

Tumor model. The B16F10 melanoma cell line (American Type Culture Collection, Rockville, MD) was cultured in complete medium and tested negative for known mouse pathogens. Cultured tumor cells were washed twice in PBS, and 1 x 105 tumor cells, in 300 µL PBS, were injected via the lateral tail vein into B6 or IFN-{gamma}–/– mice. Spleen NK cells, NKDC, and dendritic cells from B6 donors were FACS purified and cultured with either IL-18 (20 ng/mL) and CpG (10 µg/mL) or medium for 2 hours. Eight hours after administration of tumor cells, 1 x 106 NK cells, NKDC, or dendritic cells in 300 µL PBS were adoptively transferred into the tumor-injected mice. Animals were sacrificed on day 15 and lung metastases were quantified with the aid of a dissecting microscope. NK cell depletion was done with three doses of PK136 (250 µg/dose) during the 3 days before administration of tumor cells. Some mice received i.p. injections of IL-18 (1 µg), CpG (100 µg), or a combination of IL-18 and CpG 8 hours after tumor cell administration. Control mice received PBS. Statistical analysis was done using one-way ANOVA test. All Ps < 0.05 were deemed statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NK1.1+CD11c+ cells are responsible for the majority of IFN-{gamma} secretion attributed previously to murine bulk dendritic cells. NKDC express both NK1.1 and CD11c cell surface molecules (Fig. 1A and B ). In the spleen, NKDC constitute approximately 10% to 15% of all CD11c+ cells (Fig. 1B). Although NKDC express low levels of CD11c (Fig. 1A), they copurify with conventional dendritic cells (NK1.1–CD11chiB220–) and plasmacytoid dendritic cells (pDC; NK1.1–CD11cintB220+) during standard dendritic cell enrichment techniques using anti-CD11c immunomagnetic beads (Fig. 1B). Up to 4.5% of CD11c-enriched cells are NKDC. To distinguish NKDC from conventional dendritic cells (hereafter referred to as dendritic cells), we showed that NKDC expressed high levels of all NK markers, including CD122 and IL-18R{alpha} (Fig. 1C).


Figure 1
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  		Figure 1. NKDC copurify with CD11c-enriched cells. A, splenocytes were analyzed for CD11c, NK1.1, B220, and CD3 expression. NKDC (NK1.1+CD11c+CD3–) expressed low to intermediate levels of CD11c. NK cells (NK1.1+CD11c–CD3–), pDC (NK1.1–CD11c+B220+), and conventional dendritic cells (DC; NK1.1–CD11c+B220–) are shown for comparison. B, CD11c+ cells from splenocytes and CD11c-enriched splenocytes were gated by flow cytometry. NKDC copurified with CD11chi dendritic cells during enrichment with anti-CD11c immunomagnetic beads. C, NKDC lacked CD8{alpha} and expressed high levels of all NK cell markers as well as IL-18R{alpha}. D, bulk dendritic cells (CD11c+) cultured with IL-12 and IL-18 produced high levels of IFN-{gamma}. Depletion of NKDC from bulk dendritic cells lead to a 340-fold decrease in IFN-{gamma} levels. Data are representative of a minimum of three separate experiments with similar results.

 
Previously, we found that, in response to CpG and IL-4, NKDC produced ~800 pg/mL IFN-{gamma}, whereas dendritic cells made negligible amounts (14). Therefore, we hypothesized that NKDC are responsible for the majority of IFN-{gamma} production ascribed previously to bulk murine dendritic cells (i.e., CD11c+ cells) under various other conditions. To test this, we used the cytokines IL-12 and IL-18, which in combination have been purported to induce maximal IFN-{gamma} secretion by murine dendritic cells (8, 9). To facilitate cell sorting, we first enriched freshly isolated splenocytes with anti-CD11c immunomagnetic beads and subsequently sorted bulk dendritic cells and bulk dendritic cells depleted of NKDC. When cultured with both IL-12 and IL-18, bulk dendritic cells produced large quantities of IFN-{gamma} (533 ± 35 ng/mL; Fig. 1D). However, when bulk dendritic cells were depleted of NKDC, IFN-{gamma} production decreased ~340-fold. Additionally, bulk splenocytes depleted of all NK1.1+ cells produced minute levels of IFN-{gamma} in response to IL-12 and IL-18 stimulation (data not shown), showing that IFN-{gamma} is a product of NK1.1+ cells. Thus, NKDC are responsible for the majority of IFN-{gamma} secretion by bulk dendritic cells in response to IL-12 and IL-18 stimulation. Furthermore, bulk dendritic cells stimulated with IL-18 alone produced a moderate amount of IFN-{gamma} (601 ± 140 pg/mL) but not when NKDC were depleted. Based on these data and the high expression of IL-18R{alpha} by NKDC (Fig. 1C), we investigated further the effect of IL-18 on NKDC production of IFN-{gamma}.

IL-18 stimulates NKDC to secrete IFN-{gamma}. To define the regulation of IFN-{gamma} secretion by NKDC, we FACS purified NK cells (NK1.1+CD3–CD11c–), NKDC, and dendritic cells from the spleens of normal B6 mice and cultured them for 72 hours with various cytokines believed to stimulate murine dendritic cells and NK cells to secrete IFN-{gamma} (Fig. 2A ). Notably, when stimulated with either IL-2 or IL-4, only NKDC secreted detectable levels of IFN-{gamma}. NK cells did make IFN-{gamma} when cultured with IL-18 alone, although not as much as NKDC. Dendritic cells made negligible amounts of IFN-{gamma} under all conditions tested.


Figure 2
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  		Figure 2. NKDC secrete IFN-{gamma} in vitro. Spleen NK cells, NKDC, and dendritic cells were FACS purified and cultured for 72 hours with proinflammatory cytokines (A), TLR ligands (B), a combination of IL-18 and TLR ligands (C), or a combination of IL-18 and other cytokines (D). At the end of culture, supernatant was analyzed for IFN-{gamma} content with a cytometric bead array. A blocking antibody to IL-12 ({alpha}IL-12) was added to some wells. An IL-18 dose-response assay was done with medium alone (E) or CpG 10 µg/mL (F). Representative of three separate experiments with similar results. Columns and points, mean of three replicates; bars, SE.

 
As it is known that dendritic cells and NK cells respond to a variety of TLR agonists (19, 20), we tested the response of NK cells, NKDC, and dendritic cells to polyI:C (TLR3), LPS (TLR4), flagellin (TLR5), loxoribine (TLR7), and CpG (TLR9). We found that only CpG induced substantial IFN-{gamma} by NKDC (Fig. 2B), although not to the level seen with cytokine stimulation (Fig. 2A). Because IL-18 was the most potent cytokine stimulator of IFN-{gamma} secretion by NKDC, we tested whether combined stimulation of IL-18 with various other agents could augment IFN-{gamma} production by NKDC. We discovered that IL-18 and CpG synergistically and selectively increased IFN-{gamma} production by NKDC (Fig. 2C). In contrast, NK cells made 26-fold less IFN-{gamma} than NKDC, and dendritic cells made negligible amounts under the same conditions. The combination of IL-12 and IL-18 was potent but not selective, resulting in the greatest amount of IFN-{gamma} by NKDC as well as NK cells (Fig. 2D). Adding an IL-12 blocking antibody abrogated the synergistic effect of IL-18 and CpG on NKDC production of IFN-{gamma} (Fig. 2C), raising the possibility that NKDC are secreting low levels (<10 pg/mL) of IL-12 because we were unable to detect supernatant IL-12 levels (data not shown). Overall, the combination of IL-18 and CpG was the most selective stimulant of IFN-{gamma} production by NKDC.

To optimize IL-18-induced IFN-{gamma} secretion by NKDC and to rule out the possibility that NK cells and perhaps dendritic cells may respond to a different concentration of IL-18, we did an IL-18 dose-response assay. FACS-purified NK cells, NKDC, and dendritic cells were cultured with a range of IL-18 concentrations. The threshold for IFN-{gamma} secretion by NKDC was 10 ng/mL IL-18, and secretion did not change above this dose (Fig. 2E). NK cell production of IFN-{gamma} was substantially lower and did not increase above 20 ng/mL IL-18. Dendritic cells made negligible levels of IFN-{gamma} in response to all concentrations of IL-18. In the presence of CpG (10 µg/mL), NKDC became more sensitive to IL-18 and a dose as low as 0.1 ng/mL was able to induce >2 ng/mL IFN-{gamma} (Fig. 2F). We chose 20 ng/mL IL-18 and 10 µg/mL CpG as the optimal culture conditions for the remainder of the in vitro experiments.

IL-18 and CpG synergistically stimulate NKDC to secrete IFN-{gamma} in vivo. Because NKDC were responsible for the majority of IFN-{gamma} secretion in vitro, we tested whether IL-18 and CpG induced IFN-{gamma} secretion in vivo. First, we determined the time course of IFN-{gamma} secretion in B6 mice after i.p. injection of either the combination of IL-12 and IL-18 or IL-18 and CpG. Serum IFN-{gamma} levels correlated with the serum levels of IL-12 (Fig. 3A ), with peak serum IFN-{gamma} levels occurring at 9 hours in both groups. At 12 hours, there was no detectable IL-12 and IFN-{gamma} in the serum of mice that had received IL-12 and IL-18. In contrast, IL-18 plus CpG lead to a continuous low level expression of serum IL-12 and IFN-{gamma} for up to 24 hours.


Figure 3
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  		Figure 3. NKDC secrete IFN-{gamma} in vivo. Mice were given a single i.p. injection of IL-12 (1 µg) and IL-18 (1 µg) or IL-18 and CpG (100 µg) and serum (A) IL-12 and IFN-{gamma} levels were quantified at serial time points. B and C, mice received a single i.p. dose of cytokines and/or CpG and were sacrificed 6 hours later. Splenocytes were cultured without further stimulation for 5 hours in BFA. After culture, cells were labeled with surface antibodies, fixed, and permeabilized for intracellular cytokine staining. Cells were analyzed by flow cytometry, and NK cells, NKDC, NKT cells (CD3+NK1.1+), other T cells (CD3+NK1.1–), other cells (CD3–NK1.1–CD11c–), and dendritic cells were gated on and screened for intracellular IFN-{gamma} production. Open histograms, gates were set based on isotype controls. D, eight hours after single i.p. injection of NS or IL-18/CpG, total RNA was extracted from FACS-purified cells and analyzed for IFN-{gamma} mRNA by Taqman real-time PCR. Data are normalized to NS dendritic cells. Representative of two mice per group and minimum of two separate experiments with similar results.

 
Because serum IFN-{gamma} levels peaked 9 hours after injection of IL-18 and CpG, we isolated splenocytes from treated animals 6 hours after i.p. injection and cultured them for 5 hours in the presence of BFA without any restimulation. By flow cytometry, we observed that 90% of NKDC were positive for intracellular IFN-{gamma} in mice that received the combination of IL-18 and CpG (Fig. 3B). In contrast, only 45% of NK cells stained positive. When compared with other cell types, NKDC constituted the greatest proportion of all IFN-{gamma}+ cells (Fig. 3C). IL-12 or IL-18 alone produced a negligible signal in each of the cell types, and we failed to detect intracellular IFN-{gamma} in dendritic cells under any of the conditions tested. Intracellular IFN-{gamma} analysis at 24 hours also failed to show a positive signal in dendritic cells (data not shown). Surprisingly, the combination of IL-12 and IL-18, which was the most potent stimulant for IFN-{gamma} production in vitro, resulted in only 45% of NKDC and 17% of NK cells staining positive for IFN-{gamma} in vivo. To rule out the possibility that dendritic cell production of IFN-{gamma} might depend on a factor secreted by another cell type, which would have been inhibited during culture in BFA, we also measured IFN-{gamma} mRNA in sorted cells 8 hours after treatment of mice with IL-18 and CpG. We found that NK cells and NKDC dramatically up-regulated IFN-{gamma} mRNA in response to IL-18 and CpG, whereas dendritic cells did not (Fig. 3D).

The combination of IL-18 and CpG induces NKDC proliferation. IL-18 has been implicated in the proliferation of NK1.1+ cells (21). Previously, we have reported that CpG expands NKDC in vivo (14). Therefore, we tested whether the combination of IL-18 and CpG also resulted in NKDC proliferation. We found that IL-18 or CpG alone had only slight proliferative effects in vitro (Fig. 4A ). However, IL-18 plus CpG resulted in a 36-fold increase in [3H]thymidine uptake by NKDC when compared with either agent alone (Fig. 4B). In contrast, NK cell proliferation increased 12- and 7-fold over IL-18 and CpG, respectively. Dendritic cell proliferation increased 9-fold when compared with IL-18 alone and 1.6-fold when compared with the CpG alone. The synergy of IL-18 and CpG in inducing NKDC proliferation depended on IL-12 secretion by NKDC, as shown with an IL-12 blocking antibody (Fig. 4B). Addition of an isotype control or a blocking antibody to IFN-{gamma} did not have any effects on proliferation (data not shown). The combination of IL-18 with other TLR ligands did not induce substantial proliferation.


Figure 4
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  		Figure 4. NKDC proliferate in response to IL-18 and CpG stimulation. FACS-purified spleen NK cells, NKDC, and dendritic cells were cultured with TLR ligands alone (A) or in combination with IL-18 (B) for 72 hours and proliferation was measured by [3H]thymidine uptake. C, in vivo expansion was assessed by administering a single i.p. dose of CpG (100 µg), IL-18 (1 µg), or a combination of IL-18 and CpG and by analyzing spleen NK cells, NKDC, and dendritic cells at various time points via flow cytometry. D, forward and side scatter plots are shown for day 6 IL-18/CpG-expanded NKDC. E, cell surface expression of classic dendritic cell maturation markers was analyzed by flow cytometry. Control mice received NS. Shaded histograms, surface expression of the indicated markers; open histograms, isotype controls. Columns and points, mean of two mice per time point.

 
Because the combination of IL-18 and CpG led to a dramatic expansion of NKDC in vitro, we tested our results in vivo. We treated mice with a single i.p. injection of IL-18 (1 µg) and CpG (100 µg) and analyzed splenocytes on days 3, 6, 9, and 12 following injection. Three days following IL-18 and CpG administration, the number of NKDC increased 6-fold above baseline from approximately 8.0 x 105 to 5.0 x 106 cells (Fig. 4C). In contrast, the number of NK cells decreased 2-fold at day 3 but returned to baseline by day 12, whereas dendritic cell counts did not change. NKDC remained elevated at day 6; however, the effects started to wane by day 9 and the numbers returned to baseline by day 12. NKDC stimulated with IL-18 plus CpG were larger in size and more granular as indicated by forward and side scatter analysis (Fig. 4D). Initially, NKDC from IL-18 plus CpG–treated mice were immature when compared with untreated mice (Fig. 4E), but by day 6, NKDC gained high expression of MHC II.

The combination of IL-18 and CpG prevents murine melanoma lung metastases. IFN-{gamma} is an important cytokine in tumor immunology. Blocking of endogenous IFN-{gamma} with neutralizing antibody inhibits tumor rejection (22), and mice deficient in IFN-{gamma} experience enhanced tumor growth (23). Because the combination of IL-18 and CpG activated NKDC to secrete IFN-{gamma} in vitro and in vivo, we investigated its antitumor potential in a melanoma lung metastasis prevention model. Systemic treatment of B6 mice with a single dose of IL-18 and CpG led to a synergistic decrease in the number of B16F10 lung metastases (Fig. 5A ). Depletion of NK1.1+ cells, including NKDC and NK cells (Fig. 5B), abrogated the protection afforded by the combination of IL-18 and CpG. To determine the relative antitumor effects of NKDC and NK cells, we performed adoptive transfer of B6 NK cells and NKDC into IFN-{gamma}–/– mice. Dendritic cells were injected as a control. The recipients had received an i.v. injection of B16F10 melanoma cells 8 hours before adoptive transfer. Animals were sacrificed on day 15 and the number of lung metastases was counted. Mice treated with IL-18/CpG-activated NKDC had a 4- to 5-fold decrease in the number of lung metastases and had smaller lesions when compared with PBS-treated and dendritic cell–treated control animals (Fig. 5C and D). Activated NK cells provided 2- to 3-fold less protection against tumor metastases than NKDC (Fig. 5C). Mice treated with nonactivated NK cells and NKDC had similar number of lung metastases as PBS control mice (data not shown).


Figure 5
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  		Figure 5. IL-18 and CpG activated NKDC inhibit tumor metastases. A, B6 mice were treated with a single i.p. dose of IL-18 (1 µg), CpG (100 µg), or a combination of IL-18 and CpG 8 hours after i.v. administration of 1 x 105 B16F10 tumor cells. Lung metastases were counted on day 15 with the aid of a dissecting microscope. B, NKDC depletion was done with PK136. FACS dot plots are gated on CD11c+ splenocytes and NK1.1 versus B220 expression is shown to distinguish NKDC from pDC and dendritic cells. C, on day 0, 1 x 105 B16F10 tumor cells were injected i.v. into B6 or IFN-{gamma}–/– mice. Eight hours later, FACS-purified spleen NK cells, NKDC, or dendritic cells, which had been activated in vitro with IL-18 (20 ng/mL) and CpG (10 µg/mL) for 2 hours, were adoptively transferred into the tumor recipients via the lateral tail vein. Some mice received PBS instead of effector cells. Animals were sacrificed on day 15 and lung metastases were quantified. D, lungs of representative IFN-{gamma}–/– mice from (C). Experiments consisted of five animals per group and were done a minimum of twice. *, P < 0.05.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NKDC are a novel dendritic cell subtype with NK cell properties (14, 17). These unique cells can lyse tumor cells, present tumor-derived antigen to naive T cells, and secrete IFN-{gamma} on activation. IFN-{gamma} is a pleiotropic cytokine that plays an important role in the innate and adaptive immune responses (1). The absence of IFN-{gamma} or cellular responsiveness to IFN-{gamma} in humans and experimental models significantly predisposes the host to microbial infection, validating the importance of this cytokine in preventing infectious disease. Endogenously produced IFN-{gamma} has also been shown to play a critical role in tumor immunosurveillance, preventing the development of primary and transplanted tumors (24, 25).

IL-12 and IL-18 in combination are known to induce maximal IFN-{gamma} production in macrophages and dendritic cells (6, 9). It has been shown that IL-12 can up-regulate IL-18 receptor expression on T and B cells (26). Additionally, IL-12-induced signal transducer and activator of transcription 4 expression contributes to IFN-{gamma} promoter activation by enhancing the binding activity of IL-18-induced activator protein-1 (27). We have found that NKDC account for 4% to 5% of CD11c+ splenocytes obtained by conventional enrichment methods (Fig. 1B). Previously published studies reporting a purity of 90% to 94% for CD11c+I-A+ dendritic cell preparations likely may have contained a significant proportion of NKDC (7, 8). Recently, murine pDC were shown to produce IFN-{gamma} in response to IL-4 stimulation (28). The pDC preparations may have included NKDC contamination, as isolation was done by immunomagnetic microbeads. In our study, conventional dendritic cells (CD11c+ splenocytes depleted of all NK1.1+ cells) produced 340-fold less IFN-{gamma} than nondepleted bulk dendritic cells when cultured in IL-12 and IL-18 (Fig. 1D), providing in vitro evidence that NKDC account for the majority of IFN-{gamma} production attributed previously to dendritic cells. Dendritic cells also failed to produce IFN-{gamma} in vivo when stimulated with IL-12 and IL-18 (Fig. 3B). Additionally, dendritic cells did not express IL-18R{alpha} (Fig. 1C) and failed to up-regulate IFN-{gamma} mRNA when stimulated in vivo (Fig. 3D). The in vivo assays were, however, limited to innate IFN-{gamma} production and it is plausible that dendritic cells might produce IFN-{gamma} during the adaptive response of the immune system.

NKDC made higher levels of IFN-{gamma} than NK cells, both in vitro and in vivo. In combination, IL-12 and IL-18 were the most potent stimulators of IFN-{gamma} production by NK cells and NKDC in vitro (Fig. 2D). In vivo, however, combined administration of IL-18 and CpG led to a higher percentage of IFN-{gamma}+ NK cells and NKDC than did IL-12 with IL-18. Rapid clearing of IL-12 from serum may account for these differences, as IL-12 has a reported and observed (Fig. 3A) half-life (t1/2) of 2 to 5 hours in rodents (29). The in vitro t1/2 of IL-12, however, may be higher, as we were able to detect high levels of supernatant IL-12 levels at 72 hours of in vitro culture with IL-12 and IL-18 (data not shown). In contrast, IL-18- and CpG-treated mice continued to secrete IL-12 in the serum for up to 24 hours, which correlated with sustained serum IFN-{gamma} levels. Although we did not identify the in vivo source of IL-12 in this study, B cells have been shown to secrete up to 90% of the IL-12 in vivo in response to CpG stimulation (30). The majority of the IFN-{gamma}, however, originated from NKDC as they comprised >50% of all IFN-{gamma}+ cells (Fig. 3C). In vitro, IL-18 and CpG selectively and synergistically activated NKDC to secrete IFN-{gamma} and this depended on low level IL-12 production by NKDC.

Ohteki et al. (7) reported that, in response to IL-12, IFN-{gamma} was produced predominantly by CD8{alpha}+ lymphoid dendritic cells. Subsequently, the same group published that CD8{alpha}– dendritic cells were also capable of secreting IFN-{gamma} when cultured with IL-12 in combination with IL-4 or IL-18 (8). Hochrein et al. (11) later showed that CD8{alpha}- dendritic cells were the major producers of IFN-{gamma} under a variety of conditions, whereas CD8{alpha}+ dendritic cells were responsible for secretion of IFN-{alpha}. Our data are consistent with that of Hochrein et al., as we show that NKDC lack CD8{alpha} expression (Fig. 1C) and dendritic cells, which include CD8{alpha}+ dendritic cells (Fig. 1D), produce negligible amounts of IFN-{gamma}.

The origin of NKDC has not yet been identified. There is evidence, however, that NK cells and dendritic cells share a common lineage. Murine fetal thymic progenitor cells have the capacity to differentiate into T cells, NK cells, or dendritic cells (31). A bipotential NK cell and dendritic cell precursor has also been identified in human thymus and human CD34+Lin–CD38+ progenitors can differentiate into NK cells, B-lineage cells, myeloid cells, and dendritic cells (32, 33). Additionally, the hematopoietic cytokine fms-like tyrosine kinase 3 ligand (Flt3L) has been shown to expand NK cells, NKDC, and dendritic cells (17, 34–36). The results of this study expand our previous observations that CpG expands NKDC in vivo (14), suggesting that NKDC may play an important role in the immune response to bacterial infections. We found that combining CpG with IL-18 led to a 6-fold expansion of NKDC in vivo, whereas NK cell numbers decreased by 2-fold (Fig. 4C). This raises the possibility that NKDC may originate from NK cells, as Hanna et al. (37) published that human NK cells can gain APC-like function after activation. Additionally, Werfel et al. (38) found that human NK cells can gain CD11b and CD11c expression on activation. A 2-fold decrease in NK cells, however, does not directly account for the magnitude of NKDC expansion. IL-18 and CpG also led to synergistic expansion of sorted NKDC in vitro, indicating that NKDC are capable of division and are not terminal cells. Recently, mature dendritic cells, which were initially considered a terminal cell type, were shown to proliferate for up to 12 days when cultured in vitro with spleen stromal cells (39).

Evidence for the existence of NKDC-like cells dates back to the mid-1990s, yet they have received little attention until recently. Our laboratory published that NKDC exist in lymphoid and nonlymphoid organs of mice and can stimulate naive T cells, lyse tumor cells, and secrete IFN-{gamma} on activation (14). We have also found that Flt3L expands and matures NKDC in vivo and Flt3L-expanded NKDC have increased T-cell stimulatory capacity (17). Chan et al. (16) reported that IKDC (CD11c+NK1.1+B220+) secrete type-I and type-II IFNs, lyse target cells, and stimulate naive T cells. To date, an NKDC counterpart in humans has not been identified, although Hanna et al. (37) reported that activated human NK cells can acquire APC-like function. Additionally, human pDC have been shown to acquire tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and lytic activity against TRAIL-sensitive target cells when activated with influenza virus or CpG (40). Furthermore, pDC found in the tumor-draining lymph nodes of breast cancer patients have been shown to produce innate cytokines, including IFN-{gamma} (41).

Systemic administration of IL-18 has been shown to potentiate antitumor responses in multiple murine models and recombinant human IL-18 is currently in clinical trials for the treatment of cancer (42). CpG is an effective adjuvant for immunotherapy of tumors in murine models and humans (43). We showed that the systemic administration of a single dose of IL-18 and CpG mediated antitumor effects via NK1.1+ cells. Using adoptive transfer into IFN-{gamma}–/– mice, we showed that activated NKDC were more potent than NK cells in preventing tumor metastases, which was consistent with the relative amounts of IFN-{gamma} they produce. The greater effects of NKDC compared with NK cells did not seem to be related to differences in their lytic activity, as combined activation with IL-18 and CpG resulted in increased but equal lytic capacity in vitro (data not shown). Hashimoto et al. (44) showed that NK cells, but not NKT cells, play an important role in the innate antitumor response induced by IL-18. Recently, cells with similar phenotype and function as NKDC were shown to be involved in tumor immunosurveillance as they prevented outgrowth of established tumors via a TRAIL-mediated mechanism (45).

In summary, NKDC account for the majority of IFN-{gamma} ascribed previously to dendritic cells. IL-18 and the TLR9 ligand CpG preferentially and synergistically stimulate NKDC to proliferate and produce large quantities of IFN-{gamma} in vitro and in vivo. NKDC activated with IL-18 and CpG have potent antitumor activity in a tumor prevention model. These findings imply that NKDC may play an important role in the innate immunity. The combination of IL-18 and CpG should be considered as a strategy to activate NKDC selectively for immunotherapy.


   Acknowledgments
 
Grant support: NIH grants R01 DK068346 and T32 CA09501 and the Charlotte Geyer Foundation.

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.

Received 5/24/06. Revised 8/14/06. Accepted 8/22/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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