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The Journal of Immunology, 2007, 178: 2853-2861.
Copyright © 2007 by The American Association of Immunologists, Inc.

Tumor Cells Loaded with {alpha}-Galactosylceramide Induce Innate NKT and NK Cell-Dependent Resistance to Tumor Implantation in Mice1

Kanako Shimizu*, Akira Goto*, Mikiko Fukui*, Masaru Taniguchi{dagger} and Shin-ichiro Fujii2,*

* Research Unit for Cellular Immunotherapy, and {dagger} Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology, Kanagawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DCs) loaded with {alpha}-galactosylceramide ({alpha}-GalCer) are known to be active APCs for the stimulation of innate NKT and NK cell responses in vivo. In this study, we evaluated the capacity of non-DCs to present {alpha}-GalCer in vitro and in vivo, particularly tumor cells loaded with {alpha}-GalCer (tumor/Gal). Even though the tumor cells lacked expression of CD40, CD80, and CD86 costimulatory molecules, the i.v. injection of tumor/Gal resulted in IFN-{gamma} secretion by NKT and NK cells. These innate responses to tumor/Gal, including the induction of IL-12p70, were comparable to or better than {alpha}-GalCer-loaded DCs. B16 melanoma cells that were stably transduced to express higher levels of CD1d showed an increased capacity relative to wild-type B16 cells to present {alpha}-GalCer in vivo. Three different tumor cell lines, when loaded with {alpha}-GalCer, failed to establish tumors upon i.v. injection, and the mice survived for at least 6 mo. The resistance against tumor cells was independent of CD4 and CD8 T cells but dependent upon NKT and NK cells. Mice were protected from the development of metastases if the administration of live B16 tumor cells was followed 3 h or 3 days later by the injection of CD1dhigh-{alpha}-GalCer-loaded B16 tumor cells with or without irradiation. Taken together, these results indicate that tumor/Gal are effective APCs for innate NKT and NK cell responses, and that these innate immune responses are able to resist the establishment of metastases in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A marine sponge-derived glycolipid, {alpha}-galactosylceramide ({alpha}-GalCer),3 has proven to be valuable for activating NKT cells in a CD1d-restricted manner. The activation of these NKT cells, which use an invariant TCR, subsequently leads to the transient bystander activation of NK, B, and T cells (1, 2). Activation of NKT cells also can lead to Ag-specific T cell responses by inducing the maturation of Ag-capturing dendritic cells (DCs), thus illustrating how {alpha}-GalCer and NKT cells play roles in linking innate and adaptive immunity (3). Invariant NKT cells have been implicated in both stimulation and inhibition of the immune response, including protective roles in microbial infection, tumor immunity, and prevention against various autoimmune diseases (4).

The immune responses of invariant NKT cells depend not only on the expression of CD1d molecules but also on the type of glycolipid that is presented. NKT cells recognize a limited number of synthetic and naturally occurring {alpha}-anomeric glycosphingolipids and, to a lesser extent, beta-anomeric glycosphingolipids in association with the CD1d on APCs, such as DCs (5, 6). Different types of glycolipids can induce distinct functional NKT cells, i.e., {alpha}-GalCer or isoglobotrihexosylceramide (7) has the potential to induce suppressive or stimulatory forms of immune responses, {alpha}-C-GalCer can strongly enhance Th1 type NKT cells (8), and OCH can induce Th2 type NKT cells (9).

The CD1 family of MHC-unlinked class Ib molecules is conserved across mammalian species (10, 11). CD1d expression is heterogeneous with respect to cell type and level of expression, but CD1d is expressed by many nonhemopoietic as well as hemopoietic lineages (12, 13). It is well known that NKT cells do not develop in CD1d-deficient mice and the expression pattern of CD1d is important for NKT cell development and selection of NKT cells in the thymus (4, 11). In the periphery, CD1d expression is also responsible for NKT cell recruitment and regulation. Natural up-regulation of CD1d in inflammation has been reported in hepatic cells in hepatitis C virus-infected patients (14), cardiac endothelial cells in coxsackievirus-induced myocarditis (15), B cells in gut-associated lymphoid tissues in the intestinal inflammation (16), and tumor cells in myeloma patients (17). However, the relation of CD1d expression on APCs to the initiation of NKT cell responses has not been well studied. It is possible that any CD1d+ cell is capable of eliciting some aspects of the NKT cell response.

We previously demonstrated that {alpha}-GalCer-loaded DC induced a prolonged IFN-{gamma}-producing NKT cell response in mice, whereas free {alpha}-GalCer induced long-term anergy of NKT cells (18). The anergy of NKT cells responding to {alpha}-GalCer has been confirmed (18, 19, 20, 21). In this study, we have compared different cell types, particularly tumor cells that lack costimulatory molecules like CD86 and CD40, for their capacity to stimulate innate NKT and NK cell responses following loading with {alpha}-GalCer ex vivo. When we studied tumor cells that expressed low levels of endogenous CD1d, or were transduced to express higher levels of CD1d in a stable fashion, we surprisingly found that tumor cells loaded with {alpha}- GalCer (tumor/Gal) induced strong NKT and NK responses in vivo, and that these innate lymphocytes could provide T cell-independent protection against the establishment of growing tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and cell lines

Pathogen-free C57BL/6 and BALB/c female mice at 6–8 wk from CLEA Japan, and CD4–/–, CD8–/–, CD40–/–, and CD80/86–/– mice from The Jackson Laboratory were purchased. The mice listed and J{alpha}18–/– mice were maintained under specific pathogen-free conditions and studied in compliance with institutional guidelines. B16, EL4, and J558 cell lines were obtained from the American Type Culture Collection, and WEHI-3B cells were from the Institute for Fermentation. The retroviral vector pMX-ORES-GFP and a Plat-E packaging cell line were provided by Dr. T Kitamura (University of Tokyo, Tokyo, Japan). After the introduction of full-length cDNA of murine CD1d to pMX-IRES-GFP, it was retrovirally transduced into tumor cells by lipofection, and the cells were subsequently sorted based on the expression of GFP by FACSVantage.

Reagents

{alpha}-GalCer was synthesized in RIKEN. {alpha}-GalCer and vehicle (0.4% DMSO) were diluted in PBS. This concentration of DMSO had no effect for loading on tumor cells as a control in our studies. The following mAbs were purchased from BD Pharmingen: anti-mouse CD1d (1B1), anti-CD3 (145-2C11), anti-CD19 (1D3), anti-CD40 (3 of 23), anti-CD80 (16-10A1), anti-CD86 (B7-2), anti-CD154 (MR1), anti-NK1.1 (PK136), anti-TCR-beta (H57-597), anti-V{alpha}2 TCR (B20.1), anti-H-2Kb (AF16-88.5), anti-I-Ab (KH74), anti-IFN-{gamma} (XMG1.2), anti-IL-4 (11B11), and mouse IgG1 (A85-1). Biotinylated mAbs were detected with streptavidin-allophycocyanin. For flow cytometry of invariant NKT cells, we used recombinant soluble dimeric mouse CD1d:Ig (BD Pharmingen). For analysis, FACSCalibur instrument and CellQuest (BD Biosciences) or FlowJo (Tree Star) software were used.

Cell preparation

Primary cells were isolated from spleen in C57BL/6 mice using magnetically beads (Miltenyi Biotec). DCs were isolated using CD11c-magnetic beads and subsequently other cells were isolated from CD11c– fraction. Macrophages (M{phi}) and NK cells were isolated using anti-biotin F4/80 Ab and anti-biotin magnetic beads or DX5-magnetic beads. T cells were isolated by negative selection using Abs and anti-biotin magnetic beads, and B cells were isolated using anti-CD19 magnetic beads. Bone marrow-derived DCs were generated in the presence of GM-CSF as previously described (22). On day 6, {alpha}-GalCer (100 ng/ml) was added to DCs for 40 h, and 100 ng/ml LPS was added for the last 16 h. For loading of {alpha}-GalCer, tumor cells were cultured for 48 h in presence of 500 ng/ml {alpha}-GalCer. These {alpha}-GalCer-loaded cells were washed three times before injection. To isolate mononuclear cells, the livers were teased into a single suspension and resuspended in a 40/60% Percoll solution (Amersham Biosciences) for centrifugation for 20 min at 900 x g.

Real-time quantitative RT-PCR

Total RNA was extracted using an RNeasy mini kit (Qiagen). Random hexamer (Applied Biosystems) and Superscript II reverse transcriptase (Invitrogen Life Technologies) were used for cDNA synthesis. Predesigned TaqMan probes for murine CD1d (Mm00783541-s1) and 18 S rRNA were purchased from Applied Biosystems. CD1d and 18 S transcripts were quantified by real-time quantitative PCR using TaqMan PCR Master Mix reagents and an ABI-Prism 700 Sequence Detector (Applied Biosystems) according to the manufacturer’s instructions. For each sample, the mRNA abundance was normalized to the amount of 18 S rRNA.

Cytokine assays

The serum concentrations of IFN-{gamma}, IL-4, and IL-12p70 were measured by sandwich ELISA (Opti EIA; BD Biosciences) 2, 6, 16, 24, and 48 h after administration of tumor, tumor/Gal, vehicle, or {alpha}-GalCer. ELISPOT assays for IFN-{gamma}-secreting cells were performed by culturing with or without {alpha}-GalCer for 16 h as previously described (18). The number of ligand-dependent IFN-{gamma} spots was analyzed with the series 3B ImmunoSpot Image Analyzer (Cellular Technology). For intracellular cytokine staining of NK or NKT cells by FACS, the cells were preincubated with 2.4G2 culture medium to block Fc{gamma}R, washed, incubated with anti-CD1d dimer-Gal followed by anti-mouse IgG1-biotin and streptavidin-allophycocyanin and CD19-FITC for NKT cells or NK1.1-allophycocyanin and CD3-FITC mAb for NK cells. In some experiments, mononuclear cells from spleen were cultured with brefeldin A (BD Biosciences) with or without in vitro stimulation with {alpha}-GalCer for 6 h, and stained for cell surface markers as described. After the cell surface was labeled with mAbs, cells were permeabilized in Cytofix-Cytoperm Plus (BD Biosciences) and stained with anti-IFN-{gamma} PE.

In vivo tumor studies

Mice were immunized i.v. with tumor/Gal (5 x 105) or 500 ng of free {alpha}-GalCer. Mice were killed 14 days after tumor inoculation, the lungs were removed and individual surface lung metastases were counted with the aid of a microscope. In some experiments, CD4–/–, CD8–/–, CD40–/–, or J{alpha}18–/– mice were used as recipient mice, or otherwise treated i.p. with 50 µl of polyclonal Ab to asialo-GM-1 (Wako Pure Chemical) 3 day before injection of B16 cells loaded with {alpha}-GalCer (B16/Gal) or CD1dhigh-B16/Gal and every other day until day 14. As previously described, to test the adjuvant effects for NK cells, we injected 2 x 105 B16 cells 3 h before administration of tumor/Gal to mice. In the tumor-bearing mouse models, mice were administered i.v. {alpha}-GalCer-loaded B16 melanoma, EL4 thymoma, or WEHI-3B leukemia cells and were evaluated for survival.

Statistical analysis

Differences in the survival of treatment groups were analyzed using the log-rank test. Differences in in vitro data were analyzed using Mann-Whitney U test. A value for p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Most types of leukocytes present {alpha}-GalCer to NKT cells in vitro

When we previously studied the capture of {alpha}-GalCer in vivo, we found that only CD11c+ DCs could successfully capture the glycolipid for stimulation of NKT cells to produce IFN-{gamma} over several days (18). We repeated this experiment and confirmed that CD11c+ cells, which were isolated from mice that had been given i.v. {alpha}-GalCer 16 h earlier, could be reinfused into new mice and elicit IFN-{gamma}-producing NKT cells in vivo, whereas CD11c– cells were inactive (Fig. 1A, right). However, when we did the same experiment by loading the CD11c+ and CD11c– ex vivo before infusion, then both cell fractions were able to elicit the NKT response (Fig. 1A, left). To pursue the capacity of different CD11c– leukocytes to present {alpha}-GalCer, we first evaluated expression of CD1d. T cells, B cells, NK cells, and M{phi} all expressed CD1d, and at substantial levels relative to DCs, both in the steady state as well as 16 h after i.v. injection of {alpha}-GalCer into mice (Fig. 1B). We then used these different cell types to stimulate liver mononuclear cells in vitro because liver is enriched in NKT cells relative to spleen. All populations (B, T, NK, M{phi}, DC) could induce IFN-{gamma} and IL-4 in a glycolipid-dependent manner, although B and T cells were less efficient (Fig. 1C). In contrast, only the DCs were active when we loaded the different cell types with {alpha}-GalCer in vivo rather than ex vivo, and then cocultured the in vivo loaded cells with liver mononuclear cells (Fig. 1D). These data suggested that as long as different types of CD1d-expressing leukocytes were able to capture glycolipid in vitro, they could stimulate NKT cells, and by extension, that the costimulatory properties of DCs were not essential. In fact, when we tested bone marrow-derived DCs from CD40 or CD80/CD86-deficient mice, we observed that these costimulatory molecules for T cell immunity were not required to stimulate cytokine production from NKT cells in vivo (Fig. 1E). Taken together, these observations indicate that many cell types are capable of presenting {alpha}-GalCer to NKT cells and that standard costimulators like CD40 and CD80/CD86 are not essential.


Figure 1
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  		FIGURE 1. Many types of leukocytes are able to capture {alpha}-GalCer ex vivo but not in vivo. A, Activation of splenic NKT cells by CD11c+ or CD11c– cells loaded in vivo (right) or ex vivo (left) with {alpha}-GalCer. CD11c+ and CD11c– cells were isolated from mice given i.v. {alpha}-GalCer 16 h beforehand (right), or alternatively, splenic CD11c+ or CD11c– cells were isolated from C57BL/6 mice and then were loaded with {alpha}-GalCer (100 ng/ml) in vitro for 16 h (left). The CD11c+ or CD11c– cells (1 x 106) were infused into naive mice, and 2 days later, spleen cells were harvested and analyzed for IFN-{gamma} production in the presence or absence of {alpha}-GalCer restimulation in an ELISPOT assay. B, Expression of CD1d on different cell types (T cells, B cells, NK cells, M{phi}, and DCs) isolated from spleen by magnetic beads (see Materials and Methods) in the steady state or 16 h after injection of {alpha}-GalCer. C, As in B but the different cell types were ex vivo loaded with {alpha}-GalCer for 16 h, and 5 x 104 cells were cocultured with liver mononuclear cells (5 x 104) from C57BL/6 mice for 48 h. The supernatants were collected and measured for IFN-{gamma} production by ELISA. D, As in C, but the cell types were magnetically isolated from spleen 16 h after {alpha}-GalCer administration in vivo. E, Bone marrow DCs were generated from wild-type, CD40–/–, or CD80/CD86–/– C57BL/6 mice, loaded with {alpha}-GalCer for 48 h and then administered to C57BL/6 mice (i.v.). Two days later, spleen cells were analyzed for IFN-{gamma} production by NKT cells by restimulation with {alpha}-GalCer in an ELISPOT assay. Data represent the mean obtained from three independent experiments.

 
Transduction of the CD1d gene into costimulation-poor tumor cells

The findings in Fig. 1 led us to hypothesize that tumor cells, which characteristically lack costimulatory molecules, might also be able to present {alpha}-GalCer to NKT cells. We verified that the EL4 thymoma and B16 melanoma lacked expression of CD40, CD70, CD86, and MHC class II, although EL4 cells expressed substantial levels of MHC class I (Fig. 2A). For CD1d, we examined the parental cell lines as well as stable variants that were transduced with a retrovirus to express high levels of murine CD1d. Because the retroviral vector contained both murine CD1d and GFP genes, the stable CD1dhigh-tumor cell lines were selected by sorting on a FACSVantage instrument (to a purity of >98%) (Fig. 2B). Before transduction, B16 melanoma cells and EL4 thymoma cells expressed lower levels of CD1d than J558 myeloma cells and WEHI-3B myelomonocytic leukemia cells, whereas bone marrow-derived DCs expressed the highest levels (Fig. 2C). When we quantified the expression of CD1d for all the transfectants by real-time RT-PCR and flow cytometric analysis, we found higher expression of CD1d by CD1dhigh-tumor cells than by any of the other cell lines or DCs (Fig. 2, C and D). Tumor cells were then cultured for 48 h in the presence of fluorescent {alpha}-GalCer (labeled with Cy3) or vehicle. We observed by microscopy the uptake of the glycolipid into the tumor cells (Fig. 2E). These observations set the stage to test different costimulator-poor, CD1d low and high expressing tumor cells as APCs for innate immune responses to {alpha}-GalCer.


Figure 2
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  		FIGURE 2. Flow cytometric analysis of CD1d expression on tumor cell lines. A, Surface markers on cell lines were analyzed by flow cytometry using anti-CD40, CD86, MHC class I (Kb), and MHC class II (I-Ab) mAbs. B, EL4 and B16 cell lines were transduced with CD1d and GFP doubly expressing retroviral vectors and analyzed by two-color flow cytometry. C and D, A murine CD1d cDNA was retrovirally transduced into the indicated cell lines. Endogenous or transduced CD1d was evaluated by real-time RT-PCR (C) and flow cytometry (D). E, EL4 and B16 cell lines were cultured in the presence of {alpha}-GalCer-Cy3 for 48 h and were then analyzed together with DAPI (4',6'-diamidino-2-phenylindole) staining by confocal microscopy. Data are representative of two independent experiments with similar results.

 
{alpha}-GalCer-loaded tumor cells stimulate innate lymphocytes in vitro

To test the ability of tumor/Gal to activate primary NKT cells and NK cells, we cocultured liver mononuclear cells with tumor/Gal or tumor alone for 48 h and measured the supernatants for IFN-{gamma} and IL-4 production in comparison to cytokine levels induced by {alpha}-GalCer-loaded DCs. The liver lymphocytes were activated by tumor/Gal to produce both IFN-{gamma} and IL-4 (Fig. 3A). The use of CD1d-transfected tumor/Gal cells resulted in a modest increase in IFN-{gamma} but not the IL-4 response, and the responses to tumor/Gal were similar to DCs loaded with {alpha}-GalCer (DC/Gal) (Fig. 3A). The production of cytokines was entirely dependent upon the presentation of {alpha}-GalCer and the presence of NKT cells. Cytokines were not produced when the tumor cells were not exposed to {alpha}-GalCer, or when the responding liver mononuclear cells were from NKT-deficient {alpha}18–/– mice (Fig. 3A). Also, as shown in Fig. 3A, when NK cell depletion was induced in mice by injections of anti-asialo GM-1 Ab, IFN-{gamma} production (but not IL-4 production) by liver mononuclear cells was reduced, indicating the involvement of NK cells, which are known to be recruited when {alpha}-GalCer stimulates NKT cells (23). These results indicate that CD1dhigh-B16/Gal activated not only NKT cells but NK cells as well. Blocking experiments with anti-CD1d Ab were also conducted (Fig. 3B) and confirmed that {alpha}-GalCer presentation on CD1d was required for tumor cells to trigger innate NKT responses. To further demonstrate glycolipid-dependent NKT cell responses without the need for costimulatory molecules that are species restricted, the mouse tumor cells or tumor/Gal were cocultured with an established human NKT cell line. As shown in Fig. 3C, human NKT cells specifically responded to tumor/Gal, with more IFN-{gamma} being induced by CD1d-transfected tumor cells. To evaluate whether tumor/Gal also served as targets for innate killer cells, we cultured bulk liver mononuclear cells with 51Cr-labeled B16/Gal, B16, CD1dhigh-B16/Gal, or CD1dhigh-B16 for 20 h (Fig. 3D) (24, 25). The glycolipid loaded tumor cells were targets, with CD1dhigh-B16/Gal being slightly better targets of activated innate lymphocytes than B16/Gal. There was no significant specific cell lysis seen in the control groups of B16 and GFP-transfected B16 cells (data not shown). Together, these findings indicate that tumor cells are capable of presenting {alpha}-GalCer on CD1d molecules and elicit combined NKT and NK responses.


Figure 3
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  		FIGURE 3. In vitro responses to {alpha}-GalCer-loaded tumor cells. A, A total of 3 x 104 liver mononuclear cells from C57BL/6, J{alpha}18–/–, or NK depleted mice were cocultured with 1 x 104 tumor/Gal or bone marrow DC/Gal cells in 96-well round plates, and the supernatants were collected after 48 h. Murine IFN-{gamma} (upper) or IL-4 (lower) was measured by ELISA. B, To block the function of CD1d, anti-mouse CD1d Ab or isotype control IgG (BD Pharmingen) were used. Tumor/Gal cells were incubated in the presence of 20 µg/ml anti-CD1d mAb or isotype control IgG for 2 h before coculturing with liver mononuclear cells. At 48 h later, the supernatants were collected. C, A human NKT-B1 NKT cell line was established from a healthy donor. A total of 1 x 104 NKT-B1 cells were cocultured with 1 x 104 tumor/Gal or DC/Gal cells in 96-well round-bottom plates, and the supernatants were collected after 48 h. Human IFN-{gamma} was measured by ELISA. D, 51Cr-labeled tumor cells as targets were mixed with liver mononuclear cells at various E:T ratios for 20 h (24 25 ). Data represent mean ± SD of triplicate wells from three independent experiments.

 
Tumor/Gal activate both NK and NKT cells in vivo, including prolonged expansion of IFN-{gamma}-producing NKT lymphocytes

Mice were then i.v. injected with either free {alpha}-GalCer or live tumor/Gal cells. Serum was collected at different intervals and evaluated for IFN-{gamma}, IL-12p70, and IL-4. We found higher serum levels of IL-12p70 in mice after injection with tumor/Gal, but lower levels of IL-4 and IFN-{gamma} as compared with mice injected with {alpha}-GalCer alone (Fig. 4A). The parental cell lines and CD1d transfected tumor cells were comparable (Fig. 4A). No cytokines were elicited in control groups administered B16 or CD1dhigh-B16 cells without {alpha}-GalCer (data not shown). Because we had previously shown that the expression of CD40L is an early activation marker of NKT cells (26), we evaluated up-regulation of this costimulatory molecule at 2 and 6 h after injection of tumor/Gal. Already at 2 h, we detected CD40L expression on NKT cells activated by tumor/Gal (Fig. 4B). At 6 h, we also used FACS assays to measure IFN-{gamma} production by both NKT cells and NK cells at the single cell level, and we noted comparable induction of IFN-{gamma} to that seen after injection of {alpha}-GalCer alone (Fig. 4B). These data indicate that tumor/Gal stimulate innate NKT and NK immunity in vivo.


Figure 4
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  		FIGURE 4. In vivo NKT cell responses to {alpha}-GalCer administered as a free drug, or loaded onto tumor cells. A, Mice were injected with 5 x 105 B16/Gal, CD1dhigh-B16/Gal, B16, or CD1dhigh-B16 cells or with 2 µg of {alpha}-GalCer (or vehicle as a control). At the indicated time points, sera were collected and measured for IFN-{gamma}, IL-4, and IL-12p70 by ELISA. The data represent the mean from three mice in three separate experiments. B, Groups of mice were immunized with tumor/Gal, and the livers were removed 2 and 6 h after injection. IFN-{gamma} production and CD40L expression on NKT cells was assessed by gating on CD19– CD1d dimer-GalCer+ binding cells using anti-CD19 FITC, anti-IFN-{gamma} PE or anti-CD154 PE, and anti-CD1d dimer-GalCer, and anti-mouse IgG1-biotin and streptavidin-allophycocyanin. NK cells were assessed by gating NK1.1+CD3– cells. Data represent three independent experiments with similar results. C, Graded doses of {alpha}-GalCer were used to load tumor cells for 48 h, the cells were washed, and then the tumor/Gal, free {alpha}-GalCer, or bone marrow-derived DC/Gal were i.v. injected IFN-{gamma}-producing NKT cells were measured in spleen 2 days later. D, Evaluation as shown in C of the effect of graded cell doses of tumor/Gal or DC/Gal. Two days after immunization, spleen cells were assessed by an IFN-{gamma} ELISPOT assay. Data shown represent the mean obtained from three mice per dose and are mean ± SD of triplicate wells from one of two independent experiments. E, To verify the production of IFN-{gamma} by NKT cells by intracellular cytokine staining, we injected mice with different forms of tumor cells on the top, and 5 days later, spleen cells were cultured with or without {alpha}-GalCer in the presence of brefeldin A for 6 h. The cells were stained with anti-CD19 FITC and anti-CD1d dimer-Gal PE, and then analyzed by intracellular cytokine staining for IFN-{gamma} production. Data are representative of two independent experiments with similar results.

 
To further evaluate the response of NKT cells in vivo, we administered DC/Gal or tumor/Gal, and 2 days later, we restimulated spleen cells in an IFN-{gamma} ELISPOT assay without or with {alpha}-GalCer in the assay (Fig. 4C). To determine the optimal dose of {alpha}-GalCer for loading tumor cells, we loaded various doses of {alpha}-GalCer on B16 and EL4 tumor cells and administered them to mice. The optimal dose of {alpha}-GalCer required to load onto the tumor cells ex vivo was 500 ng/ml. To more stringently compare the function of various APCs, graded cell doses of APCs loaded with {alpha}-GalCer were i.v. administered and then we assayed IFN-{gamma} production 2 days later with an ELISPOT assay of spleen cells. The optimal cell number to induce this response was 5 x 105 in tumor/Gal injected mice and DC/Gal injected mice, but again, both ex vivo loaded DC/Gal and tumor/Gal were able to stimulate an innate NKT cell response (Fig. 4D). To verify that the IFN-{gamma}-producing cells were NKT cells, we cultured the spleen cells from the immunized mice in the absence or presence of {alpha}-GalCer and performed intracellular staining rather than ELISPOT assays, after gating for cells that were CD19– and were able to bind {alpha}-GalCer-loaded CD1d-dimers. IFN-{gamma} producing invariant NKT cells were readily detected in mice given CD1dhigh-B16/Gal, and to a lesser extent in mice given B16/Gal at day 5 after immunization (Fig. 4E). These data further indicate that tumor/Gal act as APCs for innate NKT cell responses in vivo.

Antitumor effects of innate lymphocytes responding to {alpha}-GalCer-loaded tumor cells

We then assessed the antitumor effects of the innate immune response to injected tumor/Gal. We first used a lung metastasis model in which mice were i.v. injected with live tumor/Gal. Without exposure to {alpha}-GalCer, the tumor cells readily established metastases, but this did not occur if we used either live B16/Gal or CD1dhigh-B16/Gal (Fig. 5, first two rows). This resistance to the establishment of metastases did not require T cells because metastasis formation was largely resisted in CD4 and CD8 knockout mice (Fig. 5, third row). However resistance was reduced partially when we removed NK cells with anti-asialo GM-1 Ab, and largely abolished when NKT cells were absent in J{alpha}18–/– recipient mice (Fig. 5, fourth row). There was no contribution of CD40 in the recipient mice, and there was no resistance when we administered B16 tumor cells, followed by free {alpha}-GalCer (Fig. 5, fifth row). These results indicate that tumor/Gal activate innate lymphocytes in vivo sufficiently to block the establishment of lung metastases.


Figure 5
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  		FIGURE 5. Metastases on the surface of the lungs after injection of tumor/Gal. A total of 5 x 105 B16 melanoma tumor cells or B16/Gal or CD1dhigh-B16/Gal cells were i.v. administered. The number of B16 melanoma metastases in the lung was evaluated 14 days later. CD4–/–, CD8–/–, J{alpha}18–/–, CD40–/–, and wild-type (WT) mice were used as recipients. One group of mice was injected every other day with anti-asialo GM-1 mAb to deplete NK cells. The data are representative of five mice in each group.

 
Improved survival of mice to several tumors following vaccination with tumor cells loaded with {alpha}-GalCer

To extend the analysis of tumor resistance, and to consider other tumors than B16 melanoma, we conducted survival studies of mice injected with B16 melanoma, WEHI-3B myelomonocytic leukemia, and EL4 thymoma tumor cells. For each tumor, CD1d transfectants that were loaded with {alpha}-GalCer were resisted for over 6 mo (Fig. 6, A–C). In the case of EL4, it was necessary to use CD1d transfectants rather than native EL4 cells loaded with {alpha}-GalCer to observe such resistance, but CD1d transfectants were not required for B16/Gal and WEHI-3B/Gal (Fig. 6C vs A and B). The types of tumor cells in addition to the levels of CD1d expression, i.e., susceptibility to NK cells, may contribute to efficacy at the level of innate immune cell activation in vitro and in vivo. No significant survival was seen in the control groups, i.e., GFP-transfected tumor cells (data not shown). For CD1d transfected EL4, we verified that neither CD4+ nor CD8+ T cells were required for resistance (Fig. 6D). These results further demonstrate protection against {alpha}-GalCer-loaded tumor cells by innate immunity, but there is the value in CD1d transfection for some tumors like EL4.


Figure 6
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  		FIGURE 6. Survival rates in animals injected with tumor/Gal. A–D, {alpha}-GalCer-loaded melanoma (B16), lymphoma (EL4), or leukemia (WEHI-3B) cells (5 x 105 cells/mouse) were i.v. administered. Survival rates in each group of mice were evaluated. D, CD4–/– and CD8–/– mice were used as recipients. Data represent a minimum of two experiments, each with five mice per group. *, p < 0.001 (B16/Gal vs B16, CD1dhigh-B16/Gal vs CD1dhigh-B16) in A. **, p < 0.005 (WEHI-3B/Gal vs WEHI-3B, CD1dhigh-WEHI-3B/Gal vs CD1dhigh-WEHI-3B) in B. *, p < 0.001 (CD1dhigh-EL4/Gal vs CD1dhigh-EL4) and **, p < 0.01 (EL4/Gal vs EL4) in C.

 
{alpha}-GalCer-loaded tumor cells induce innate resistance to native tumor cells given 3 h to 3 days earlier

In contrast to the experiments described in this study in which we followed the development of metastases by tumor/Gal themselves, we turned to the capacity of tumor/Gal to provide a therapeutic effect on native tumor cells that were not loaded with glycolipid. We also compared tumor/Gal to DC/Gal, using bone marrow-derived DCs. We first i.v. injected live B16 melanoma cells, and 3 h later, we injected tumor/Gal (B16/Gal or CD1dhigh-B16/Gal) at low (5 x 104) and high (5 x 105) doses. We found that CD1dhigh-B16/Gal induced stronger antitumor effects than DC/Gal or B16/Gal against B16 melanoma cells in a cell dose-dependent manner (Fig. 7A). To assess the potential contribution of NK cells, we depleted mice of NK cells with anti-asialo GM-1 Ab and found that resistance was markedly reduced (Fig. 7B). In addition, we verified that irradiated tumor/Gal, which would be feasible to administer in clinical trials, also induced resistance to the establishment of metastases by live B16 melanoma (Fig. 7C). When the NK cell responses were assessed directly by FACS at 6 days after immunization with B16/Gal, CD1dhigh-B16/Gal cells, or DC/Gal, IFN-{gamma} production by CD3–NK1.1+ NK cells was greatest in CD1dhigh-B16/Gal immunized mice, relative to B16/Gal and DC/Gal (Fig. 7D). To extend the therapeutic model further, mice were injected with 1 x 105 live B16 cells, and 3 days later they were treated with a single dose of tumor/Gal or DC/Gal (Fig. 7E) or irradiated tumor/Gal (Fig. 7F). All forms of {alpha}-GalCer-loaded APC provided resistance to metastasis when live APCs were used (Fig. 7E), and likewise, irradiated {alpha}-GalCer-loaded tumor cells, especially CD1d transfectants, exerted a therapeutic effect (Fig. 7F). These results indicate the potential of tumor/Gal to elicit innate resistance to native tumor cells.


Figure 7
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  		FIGURE 7. Innate resistance to native tumor cells when NKT cells are activated by {alpha}-GalCer-loaded tumor cells. A and B, Low (5 x 104) or high (5 x 105) doses of tumor/Gal (B16/Gal or CD1dhigh-B16/Gal) were injected as a stimulus for innate immunity at 3 h after administration of 2 x 105 (A) or 1 x 105 (B) live B16 cells to C57BL/6 mice. A, A total of 5 x 105 DC/Gal were injected for comparison to tumor/Gal. B, Contribution of NK cells was assessed by prior i.v. treatment of the mice with anti-asialo GM-1 Ab. The number of metastases on the lung was counted 14 days later. *, p < 0.001 (B16 vs others); **, p < 0.01 (DC/Gal vs high number of B16/Gal, CD1dhigh-B16/Gal); and ***, p < 0.05 (DC/Gal vs low number of B16/Gal) and (high number of CD1dhigh-B16/Gal vs low and high number of B16/Gal, low number of CD1dhigh-B16/Gal). C, Same as in A, but irradiated tumor/Gal cells (5 x 105/mouse) were injected at 3 h after administration of 2 x 105 live B16 cells. *, p < 0.001. D, Mice were immunized with 5 x 105 B16/Gal, CD1dhigh-B16/Gal, or DC/Gal, and IFN-{gamma} production by NK cells was analyzed 6 days later by gating on CD3–NK1.1+ cells. E and F, Mice were i.v. injected with 1 x 105 live B16 melanoma cells as in A, but now we stimulated innate immunity in the mice 3 days later by treating with 5 x 105 B16/Gal, CD1dhigh-B16/Gal, or DC/Gal (E) or with low (5 x 104) or high number (5 x 105) of irradiated tumor/Gal (F). The number of metastases on the lungs was counted 14 days later. **, p < 0.01 and ***, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the current study, we demonstrate the capacity of tumor cells loaded with {alpha}-GalCer ex vivo (tumor/Gal) to induce strong innate immunity in a CD1d and NKT cell-dependent manner in mice. DCs are strong APCs for activating NKT cells. However, constitutive CD1d expression is found on many types of parenchymal cells, especially intestinal epithelial cells, hepatocytes and hemopoietic cells (11), and tumor cells (Figs. 1B and 2, C and D). Although these cells are not as potent as DCs, they are nonetheless active. We proved in this study that tumor/Gal-induced IFN-{gamma} production by both NKT and NK cells and that this was associated with significant innate resistance to the implantation of several tumors.

Once loaded onto CD1d, the binding of CD1d-glycolipid complexes to the {alpha}beta TCR has substantial affinity and is stable (27). However, it can take 12 h to load {alpha}-GalCer onto DCs (28). Also, as shown in Fig. 5, if we coinjected tumor cells with unbound {alpha}-GalCer, we could not protect against B16 metastases in the lung, whereas an injection of B16/Gal or CD1dhigh-B16/Gal was protective. Therefore, there is little possibility that {alpha}-GalCer-loaded tumor cells significantly transfer {alpha}-GalCer to unloaded tumor cells. In the case of the EL4 tumor, which required CD1d transduction to bind sufficient {alpha}-GalCer to elicit protective innate immunity, we were unable to induce resistance if we injected CD1dhigh-EL4 cells 3 h before injection with EL4 cells loaded with {alpha}-GalCer. In other words, the {alpha}-GalCer could not transfer from the EL4 cells to the CD1dhigh-EL4. In contrast, {alpha}-GalCer loaded CD1dhigh-EL4 induced strong resistance. These findings make it unlikely that elution of {alpha}-GalCer from the tumor/Gal could charge other tumor cells effectively in vivo. Instead the {alpha}-GalCer-loaded tumor needs to directly induce protective innate immunity.

Our data indicate that costimulatory molecules are not required on APCs to activate NKT cells in vivo. Previously Matsuda et al. (29) showed that IFN-{gamma} secretion by NKT cells is induced in CD40–/– mice after injection of {alpha}-GalCer. We also have previously shown that DC surface remodeling occurs in both CD80/CD86–/– and CD40–/– mice after administration of {alpha}-GalCer to the same extent as in wild-type mice (3, 26). Thus, CD40 and CD80/86 molecules are not essential in activating NKT cells in the primary response, but may act as supporting cofactors (20). In the current study, we demonstrated prominent innate immunity to APCs that lacked costimulatory molecules, particularly tumor cells (Fig. 2A).

As we discussed in Figs. 3D and 5, the tumor/Gal would be a target for killing by activated NKT cells soon after the NKT cells had encountered the injected tumor/Gal. Thus, both activated Th1 type NKT cells, as well as the inflammatory events that may take place following the killing of tumor cells, may enhance IL-12 production by DCs or M{phi}. The different kinetics and amounts of cytokines in the serum of mice given tumor/Gal compared with mice given free {alpha}-GalCer (Fig. 4A) could in part depend on the timing of NKT cell activation, that is, free {alpha}-GalCer can rapidly and systemically activate NKT cells, whereas in contrast, tumor/Gal requires more time to migrate and activate NKT cells in various organs. One critical variable that we are now assessing is that NKT activation by tumor/Gal may in turn lead to activation of DCs, including IL-12 production, followed by IFN-{gamma} production by NK cells. The kinetics of the two cytokines, IL-12 and IFN-{gamma}, may not be coordinated given the multiple pathways that can take place in vivo.

By several assays of the innate response, we assessed the value of CD1d transfection of tumor cells in presenting {alpha}-GalCer to NKT cells. As shown in Fig. 3B, anti-CD1d mAb blocks responses to {alpha}-GalCer-loaded tumor cells, indicating that NKT cells recognize the glycolipid on the CD1d of tumor cells. Increased numbers of NKT (Fig. 4E) and NK cells (Fig. 7D) capable of IFN-{gamma} production were detected by intracellular staining assays in response to CD1dhigh-B16/Gal when compared with B16/Gal cells. When we compared tumor cells to DCs, we found that tumor/Gal were more effective in inducing innate resistance, as long as we loaded the tumor cells ex vivo (Fig. 7). For example, even at 6 days after injection, NK production of IFN-{gamma} was much greater in mice treated with tumor/Gal than DC/Gal (Fig. 7D). The innate immunity including the adjunct effects were apparently correlated with antitumor effects. The strategy using tumor/Gal as APCs would appear to be an approach to mobilize both NKT and NK cells in resistance to tumors.

We therefore undertook experiments to use tumor/Gal to provide therapeutic innate resistance to tumors. Here CD1dhigh-B16/Gal cells were more effective than B16/Gal, but both induced significant antitumor effects when given 3 h or 3 days after an injection of tumor cells that were not loaded with {alpha}-GalCer (Fig. 7, A, E, and F). Because DC/Gal therapy was shown to be safe in recent clinical trials (30, 31, 32), the successful innate effects induced by irradiated tumor/Gal suggests the feasibility of our strategy for an immunotherapy. Our strategy might be pursued for example with hematological malignancies, such as acute myelogenous leukemia and acute lymphocyte leukemia. Because one could harvest many tumor cells at the onset of the diseases, the tumor cells could be loaded with {alpha}-GalCer and used as APCs in patients to activate NKT and NK cell-based resistance.


   Acknowledgments
 
We thank Hanae Fujimoto, Akiko Furuno, and Naoko Konishi for providing technical assistance, Drs. M. Harada and B. Meek for technical advice, and J. Adams for help with the figures. We thank Drs. T. Watanabe and T. Kurosaki for reviewing the manuscript and Dr. R. M. Steinman for thoughtful discussion and critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan (to K.S. and S.-i.F.). Back

2 Address correspondence and reprint requests to Dr. Shin-ichiro Fujii, Research Unit for Cellular Immunotherapy, RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. E-mail address: fujiis{at}rcai.riken.jp Back

3 Abbreviations used in this paper: {alpha}-GalCer, {alpha}-galactosylceramide; DC, dendritic cell; tumor/Gal, tumor cells loaded with {alpha}-GalCer; DC/Gal, DC loaded with {alpha}-GalCer; B16/Gal, B16 cells loaded with {alpha}-GalCer. Back

Received for publication April 25, 2006. Accepted for publication December 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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