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. 2011 Apr 26;60(7):1029–1038. doi: 10.1007/s00262-011-1015-5

Enhanced anti-tumor immunity by superantigen-pulsed dendritic cells

Masato Kato 1, Yutaro Nakamura 1,✉, Takafumi Suda 1, Yuichi Ozawa 1, Naoki Inui 1,2, Naohiro Seo 3, Toshi Nagata 4, Yukio Koide 5, Pawel Kalinski 6, Hirotoshi Nakamura 1, Kingo Chida 1
PMCID: PMC11029592  PMID: 21519830

Abstract

Staphylococcal enterotoxins A (SEA) and B (SEB) are classical models of superantigens (SAg), which induce potent T-cell-stimulating activity by forming complexes with MHC class II molecules on antigen-presenting cells. This large-scale activation of T-cells is accompanied by increased production of cytokines such as interferon-γ (IFN-γ). Additionally, as we previously reported, IFN-γ-producing CD8+ T cells act as “helper cells,” supporting the ability of dendritic cells to produce interleukin-12 (IL-12)p70. Here, we show that DC pulsed with SAg promote the enhancement of anti-tumor immunity. Murine bone marrow-derived dendritic cells (DC) were pulsed with OVA257–264 (SIINFEKL), which is an H-2Kb target epitope of EG7 [ovalbumin (OVA)-expressing EL4] cell lines, in the presence of SEA and SEB and were subcutaneously injected into naïve C57BL/6 mice. SAg plus OVA257–264-pulsed DC vaccine strongly enhanced peptide-specific CD8+ T cells exhibiting OVA257–264-specific cytotoxic activity and IFN-γ production, leading to the induction of protective immunity against EG7 tumors. Furthermore, cyclophosphamide (CY) added to SAg plus tumor-antigens (OVA257–264, tumor lysate, or TRP-2) pulsed DC immunization markedly enhanced tumor-specific T-cell expansion and had a significant therapeutic effect against various tumors (EG7, 2LL, and B16). Superantigens are potential candidates for enhancing tumor immunity in DC vaccines.

Keywords: Superantigen, Dendritic cell, Low-dose cyclophosphamide, Cancer vaccine, Cellular immunotherapy

Introduction

Staphylococcal enterotoxins A (SEA) and B (SEB) are classical models of bacterial superantigens (SAg). Unlike conventional antigens, SAg bind directly to the lateral surfaces of MHC class II molecules and to the Vβ domain of the TCR; therefore, they do not need to be processed and presented like conventional antigens [1–4]. This ability enables SAg to induce large-scale activation of the immune system by stimulating the large number of T-cells that express β-chains of the T-cell antigen receptor, which contain variable regions coded for by specific families of Vβ genes. Furthermore, this large-scale activation of T-cells is accompanied by the increased production of tumoricidal cytokines such as interferon-γ (IFN-γ) [5–7], although a majority of the expanding cells will subsequently be deleted by the induction of apoptosis, and the remaining CD4+ T cells will become anergic upon subsequent exposure to the SAg [8–10].

DC are specialized APC, which exist in virtually every tissue, capture antigens in situ, and migrate to lymphoid organs to activate naive T cells. DC are able to bind to much higher levels of SAg than other APC, and very small numbers of superantigen-MHC class II complexes are sufficient to trigger resting primary T cells [11, 12]. Recently, it became apparent that CD8+ T cells can also activate DC and support type-1 immunity as well as CD4+ T cells [13–15]. This “helper” function of CD8+ T cells depends on their ability to produce IFN-γ and to promote the dendritic cell production of interleukin-12 (IL-12) p70, the key factor supporting T helper 1 responses and CTL responses [16].

DC-based tumor vaccines have demonstrated effective antitumor immune responses in vitro and after adoptive transfer in mice and humans. However, difficulties still remain to be overcome when they are used clinically [17, 18]. We evaluated the capacity of SAg to enhance DC vaccines in the setting of some tumors including poorly immunogenic B16 melanoma. We demonstrate here that simultaneous pulsing of DC with SAg and tumor antigen results in pronounced enhancement of vaccine-mediated immune priming. Furthermore, tumor antigen and SAg-pulsed DC are capable of significantly suppressing the growth of established tumors and inducing a strong antitumor T-cell response after pretreatment with cyclophosphamide (CY) in a therapeutic setting [19].

Materials and methods

Mice

Six to 8-week-old male C57BL/6 mice purchased from Japan SLC, Inc. and C57BL/6-IL12tmljm (p40-knockout) mice purchased from the Jackson Laboratory were maintained in microisolator cages and used for all experiments at 8–10 weeks of age. All experimental procedures were approved by our institutional Animal Care and Use Committee.

Cell line, cell isolation, and culture

Lewis Lung Carcinoma cells (2LL), EG7 [ovalbumin(OVA)-expressing EL4] murine lymphoma, B16-F10 murine melanoma, and J774 cells were purchased from the American Type Culture Collection. Spleen CD8+ T cells were negatively selected using MACS columns (Miltenyi Biotech) with 90% to 95% purity. LCD40L cells expressing CD40L on their cell surface and derived from murine Langerhans cells were kindly provided by Dr. Seo (Hamamatsu University, Japan) [20]. All cells were maintained in RPMI 1640 with 10% heat-inactivated fetal bovine serum (Invitrogen), glutamine, streptomycin, and penicillin (Invitrogen).

Superantigens

SEA and SEB were purchased from Toxin Technology. Naïve splenocytes from C57BL/6 mice were co-cultured with SEA or SEB or both for 3 days. The IFN-γ concentrations of the supernatants were determined by ELISA.

Dendritic cells

Bone marrow-derived dendritic cells (DC) were generated in cultures supplemented with granulocyte macrophage colony-stimulating factor and IL-4 (both 1,000 units/ml; R&D Systems), as described in our previous reports [21–23]. On day 7, CD11c+ DC were isolated using anti-mouse CD11c-coated magnetic beads (Miltenyi Biotech) and were treated with lipopolysaccharide (250 ng/ml; Sigma–Aldrich) for 16 h. Then, the DC were collected and prepared for immunization.

Induction of IL-12p70

Antigen-free or SEA or SEB (SAg)-pulsed DC (2 × 104/0.2 ml per well) were first co-cultured for 48 h with CD8+ T cells (3 × 105 from the spleens of wild-type mice), harvested, washed, counted, and stimulated (at 2 × 104/0.2 ml) with 5 × 104 CD40L-transfected L cells [22] for 24 h. The IL-12p70 concentrations of the supernatant fluid were determined by ELISA (R&D systems).

Tumor vaccines

To prepare the vaccine against EG7, after treatment with lipopolysaccharide (LPS), the DC were resuspended in RPMI 1640 and pulsed with the dominant H-2Kb-restricted ovalbumin (OVA) epitope, OVA257–264 (SIINFEKL; synthesized by Invitrogen), alone or in combination with SAg or remained untreated (none). To prepare the B16-F10 vaccine, tyrosinase-related protein 2 (TRP-2) 180–188 (SVYDFFVWL; synthesized by Invitrogen) was used as a CTL-epitope instead of OVA257–264. Pulsing with OVA257–264 and TRP-2180–188 was performed at a concentration of 10 μg/ml for 60 min. To prepare the 2LL vaccine, DC were loaded overnight with 2LL tumor cells lysates (freeze-thawed thrice, centrifuged, and supernatant collected), at a concentration of three tumor cell equivalents to one DC, in the presence of LPS. The SAg pulsing was performed at a concentration of 1 μg/ml for 60 min. The expression of CD11c, MHC II, CD86, CD80, and CD40 was analyzed by flow cytometry before and after the SAg pulsing. All vaccines were washed twice and suspended in PBS (1 × 106 per 0.2 ml).

Induction of OVA-specific immune response and protective immunity against EG7 tumor

LPS-treated DC were pulsed with OVA257–264 (5 μg per 1 × 106 DC) in the presence or absence of SAg (1 μg per 1 × 106 DC, respectively) and were washed twice and resuspended in PBS (1 × 106 in 0.2 ml). Wild-type C57BL/6 mice (12 per group) were subcutaneously vaccinated (day-28) once. Cells were then harvested from the spleens of 4 week-immunized mice (half of each group) and cultured in the presence of OVA257–264 (10 μg per well) for 3 days. The IFN-γ concentrations of the supernatants were determined by ELISA. The remaining 4 week-immunized mice had high numbers of EG7 cells (5 × 106/mouse) inoculated into their right flank (day 0) in order to evaluate the protective immunity induced by the vaccine. Tumors were measured using vernier calipers every 3–4 days. Data are reported as the mean ± SE of tumor area (product of the largest perpendicular diameters). Furthermore, survival was recorded as the percentage of surviving animals after tumor injection and analyzed in EG7 tumor model.

CTL activity

Fourteen days after the tumor challenge, splenocytes were harvested from two animals per group. They were restimulated in vitro (8 × 106 per well) for 5 days with OVA257–264-pulsed syngeneic splenocytes that had been treated with 50 μg/ml mitomycin C (Roche Diagnostics GmbH) beforehand (two responder cells to one stimulator cell) in the presence of 10 IU/ml recombinant human IL-2 (R&D Systems) in 24-well culture plates. After the collection of viable effector cells, various numbers of effector cells were co-cultured with J774 cells that had been pulsed with OVA257–264, which were used as the target cells in this study, (at 1 × 104 cells/well) for 5 h. The cell-mediated cytotoxicity of OVA257–264 peptide was measured using a lactate dehydrogenase (LDH) cytotoxicity detection kit (Roche Diagnostics). The LDH released into the medium was measured, and the specific lysis of the target cells was calculated as 100 × ([experimental release]−[spontaneous release])/([maximal release]−[spontaneous release]) according to the manufacturer’s instructions. Spontaneous and maximal release were determined in the presence of medium alone and 1% Triton X-100, respectively. For 2LL tumors, 2LL cells treated with mitomycin C were used for in vitro stimulation, and untreated 2LL cells were used as the target cells. For B16-F10 tumors, TRP2180–188 peptides were used for in vitro stimulation, and B16-F10 cells were used as the target cells.

Therapeutic model of DC vaccine involving pretreatment with low-dose cyclophosphamide

Wild-type C57BL/6 mice (6 per group) had tumor cells s.c. inoculated into their right flank (EG7: 1.5 × 106, 2LL: 3 × 105, or B16-F10: 1.5 × 105 per mouse) (day 0). The mice were i.p inoculated with CY (1 mg/0.1 ml PBS, Sigma–Aldrich) or PBS on day 2 and s.c. vaccinated (1 × 106 DC) into the same flank on day 5. Tumor area was measured every 3–4 days. Tumor size was monitored for 14 days, and then the splenocytes from two animals per group were harvested for the CTL assay. Furthermore, survival was recorded as the percentage of surviving animals and analyzed. Survival was followed as described above in EG7 tumor model.

Flow cytometory

To determine the phenotype of cultured DCs, we stained them with phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against cell surface molecules (CD11c and MHC classII; obtained from Miltenyi Biotec, CD86, CD80, and CD40; obtained from Pharmingen) and analyzed on a flow cytometry (Beckman Coulter EPICS XL). To enumerate OVA257–264-specific CD8+ T cells, spleen cells from immunized mice were stained by PE-conjugated tetrameric H-2Kb/OVA257–264 peptide complex (synthesized by MBL) and FITC-conjugated anti-CD8α monoclonal antibody (MBL) and analyzed on a flow cytometry (Beckman Coulter EPICS XL). Intracellular forkhead/winged helix transcription factor (Foxp3) staining was performed according to the manufacturer’s instructions. Briefly, spleen cells from vaccinated tumor-bearing mice were firstly surface-labeled with FITC-conjugated anti-CD4 monoclonal antibody and allophycocyanin (APC)-conjugated anti-CD25 monoclonal antibody. Then, after fixation and permeabilization, the cells were stained with anti-Foxp3 PE (mouse regulatory T-cell staining kit; eBioscience) and analyzed on a flow cytometry (BD FACSAria™).

Statistical analysis

Data from multiple experiments were expressed as the mean ± SEM. Statistical evaluation of the differences between the experimental group means was performed by the Mann–Whitney U test, and time series analyses of tumor size between the experimental groups were performed by repeated measurements of ANOVA. Survival was analyzed by log-rank test on Kaplan–Meier survival curves using JMP (SAS Institute). All tests were two-sided and performed at the 0.05 significance level.

Results

Naive splenocytes strongly express IFN-γ in the presence of both SEA and SEB

To test which SAg is able to efficiently generate IFNγ-producing cells in vivo, we evaluated IFN-γ production by spleen cells from naive mice. Upon stimulation with SEA or SEB, or both, the spleen cells stimulated with a combination of SEA and SEB produced significantly higher levels of IFN-γ than spleen cells treated with either SEA or SEB (Fig. 1a).

Fig. 1.

Fig. 1

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DC and T-cell activation of SAg. a Naïve splenocytes stimulated with SEA/SEB produced significantly more IFN-γ than those stimulated with either SEA or SEB alone. Splenocytes from naïve mice were incubated in the absence or presence of SEA or SEB for 48 h. Columns, mean from one experiment of three that yielded similar results; bars, SD. b Interaction with CD8+ T cells primes dendritic cells for high IL-12p70 production. Dendritic cells were co-cultured for 48 h with CD8+ T cells from wild-type B6 mice, in the absence or presence of SEA/SEB, before being washed and stimulated with CD40L. Similar results were obtained in one additional experiment. c Phenotypic characteristics of SEA/SEB-pulsed DC and non-pulsed DC as determined by flow cytometry. The addition of SAg had no or a marginal effect. The expression of cell surface molecules on DC (CD11c, MHC class II, CD86, CD80, and CD40) was evaluated by flow cytometry. The data are representative of three independent experiments in which similar results were obtained

Dendritic cell activation of CD8+ T cells with SAg and their phenotype

To analyze the ability of mouse CD8+ T cells to affect dendritic cell functions in vitro, we used the previously established model of SEA/SEB-driven stimulation of T cells from wild-type C57BL/6 mice. This model allowed us to promote the interaction of dendritic cells with high numbers of CD8+ T cells without the need for prior T-cell activation and clonal expansion. In accordance with our previous data, freshly isolated mouse CD8+ T cells primed the SEA/SEB-loaded dendritic cells for high IL-12p70 production during their subsequent interaction with CD40L-expressing L cells, which were used as CD4+ T-cell surrogates (Fig. 1b).

Then, we examined the expression patterns of various cell surface molecules by flow cytometry. DC pulsed with SAg (SEA and SEB) and non-pulsed DC expressed similar amounts of CD11c, MHC class II molecules, CD86, CD80, and CD40, (Fig. 1c), indicating that pulsing SAg into DC does not affect their phenotype.

SAg/OVA-pulsed DC vaccination generates OVA257–264-specific CTL

We determined the ability of SAg-pulsed DC to induce epitope-specific CD8+ T cells in the spleen. Using an OVA257–264 peptide-specific H-2kb tetramer, we evaluated the frequencies of tetramer-bound CD8+ T cells by flow cytometry in the spleen cells from mice that had been immunized with SAg/OVA-pulsed DC (SAg/OVA-DC) 4 week before, and compared it with that by spleen cells from mice that had been immunized with OVA257–264-pulsed DC (OVA-DC), SAg-pulsed DC (SAg-DC), none-pulsed DC (con-DC) or PBS. Immunization with SAg/OVA-DC resulted in significantly higher numbers of OVA257–264-specific CD8+ cells than immunization with OVA-DC, SAg-DC, con-DC or PBS (P = 0.0157, SAg/OVA-DC vs. OVA-DC, Fig. 2a). Furthermore, upon stimulation with OVA257–264 peptide, the spleen cells from SAg/OVA DC-immunized mice produced significantly higher levels of IFN-γ than spleen cells from mice immunized with OVA-DC, SAg-DC or con-DC (P=0.0200, SAg/OVA-DC vs. OVA-DC, Fig. 2a inset). Collectively, these data suggest that immunization with SAg/OVA-pulsed DC efficiently generates OVA257–264-specific CTL and IFN-γ-producing cells in vivo.

Fig. 2.

Fig. 2

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SAg(SEA/SEB)-pulsed DC to induce potent anti-tumor protective immunity. a OVA257–264-specific CD8+ T cells in spleens of 4 week-immunized mice. Spleen cells from mice immunized with OVA257–264 or SAg plus OVA257–264-pulsed DC (OVA-DC and SAg/OVA-DC, respectively) or SAg-pulsed DC (SAg-DC) or none-pulsed DC (Con-DC), or from PBS immunized mice (PBS) were harvested 4 weeks after immunization and OVA257–264-specific CD8+ T cells were evaluated by flow cytometry with a tetrameric OVA257–264 H-2kb/OVA257–264 peptide complex. Percentage of OVA257–264 tetramer positive cells among the total CD8+ T cells was shown. The data are means and standard deviations for four mice in each experimental group. Inset, IFN-γ secretion by OVA257–264-stimulated splenocytes from mice immunized with SEA/SEB plus OVA257–264-pulsed DC or OVA257–264-pulsed DC. The concentrations of IFN-γ in the culture supernatants were determined by a sandwich enzyme-linked immunosorbent assay. The data are means and standard deviations for six mice in each experimental group. b Induction of CTL specific to EG7 lymphoma. Spleen cells from mice immunized with both SAg and OVA257–264, or either of the other pulsed DC (SAg/OVA-DC, SAg-DC, or OVA-DC) or non-pulsed DC (Con-DC), or PBS were harvested 4 weeks after immunization and stimulated in vitro with OVA257–264 peptide-pulsed spleen cells for 5 days. The percentage of specific lysis was determined using J774 cells (H2d) that had been pulsed with OVA257–264 peptide as target cells. Immune spleen cells (effectors) were incubated with target cells at the effector-to-target-cell ratios (E/T Ratio) indicated on the x-axis. The data are representative of data obtained in four independent experiments. c Induction of protective immunity against tumor growth by immunization with SAg plus OVA257–264 DC. The mice were immunized with the indicated vaccines. Four weeks after immunization, the mice were challenged with 1.5 × 106 of EG7 lymphoma. The data are the means and standard errors for six mice in each experimental group. d Survival was recorded as the percentage of surviving animals. Data represent one of two independent experiments. e, f IL-12p40–deficient dendritic cells (unable to produce IL-12p70 and IL-23) do not mediate heterologous CD8 help via SAg. Mice received single s.c. injections of dendritic cells (generated from wild-type or IL-12p40–knockout mice) loaded with OVA257–264 alone or in combination with SAg. The differences between the treatment groups were evaluated by repeated measurements of ANOVA. An exact log-rank test was used to analyze the survival differences between the SAg/OVA-DC and OVA-DC treatment groups (P = 0.0010)

Next, we determined the cytolytic activities of OVA257–264-specific CTL induced by immunization with SAg/OVA-pulsed DC. After in vitro restimulation of immune spleen cells with the OVA257–264 peptide, the cells from SAg/OVA-pulsed DC-immunized mice lysed the peptide-pulsed J774 cells more effectively than the cells from OVA-pulsed DC-immunized mice (Fig. 2b). The CTL activity of the spleen cells from the mice immunized with SAg-pulsed DC was similar to that of the spleen cells from the mice immunized with OVA-pulsed DC. These CTL activities correlated well with the frequencies of OVA257–264-specific tetramer-bound CD8+ T cells and the levels of OVA257–264-specific IFN-γ (Fig. 2a, b).

SAg/OVA-pulsed DC vaccination provides protective immunity against a subsequent challenge with an EG7 OVA257–264 expressing tumor

To ascertain whether the immune responses induced in the mice immunized with the DC vaccines efficiently protected against tumors, the vaccinated animals were subcutaneously challenged with EG7 tumors. The suppression of tumor growth was significantly higher in the mice that received SAg/OVA-pulsed DC than in the mice that had received OVA-pulsed DC, SAg-pulsed DC, or untreated control DC (P = 0.0048, SAg/OVA-DC vs. OVA-DC, Fig 2c). Subsequently, survival analysis revealed that, although all of the mice immunized with SAg-DC, OVA-DC, or control DC died within 30 days after tumor inoculation, 80% of mice immunized with SAg/OVA-pulsed DC survived (Fig. 2d). The differences in the survival curves were statistically significant between SAg/OVA-pulsed DC-immunized mice and OVA-pulsed DC-immunized mice (P = 0.0101).

Critical role of dendritic cells in mediating CD8 help

Whereas the inclusion of SAg strongly enhanced the immunologic and antitumor effects of vaccination in the animals, the heterologous help from CD8+ T cells induced by SAg was not mediated by IL-12/IL-23-deficient dendritic cells generated from the bone marrow of p40-knockout animals (Fig. 2e, f).

SAg enhance the therapeutic efficacy of DC-based immunization against established tumors in combination with low-dose CY

To determine the therapeutic potential of immunization with SAg/OVA-pulsed DC, we attempted to induce tumor rejection in mice with EG7 tumors. In a 5-day treatment model, B6 mice harboring EG7 were treated with SAg/OVA-pulsed DC with or without low-dose CY. As shown in Fig. 3a and b, although we could not see any potential effect of this vaccine in terms of the induction of OVA257–264-specific CTL or tumor size reduction, treatment with a combination of SAg/OVA-pulsed DC and low-dose CY markedly generated OVA257–264-specific CTL. Furthermore, combined treatment with SAg/OVA-pulsed DC and low-dose CY resulted in a significant reduction of tumor growth (P = 0.0015; compared with mice receiving OVA-pulsed DC). Similar effects were observed in other two cell line two poorly immunogenic tumors (2LL and B16, respectively) (Fig. 3c–f). Collectively, these findings demonstrated that SAg enhanced the therapeutic efficacy of DC immunization and induced protective immunity against poorly immunogenic tumors, which was achieved in combination with the systemic administration of low-dose CY. Furthermore, survival analysis revealed that survival of mice vaccinated with SAg/OVA-pulsed DC were improved in EG7 model (Fig. 3g). The differences in the survival curves were statistically significant between SAg/OVA-pulsed DC-immunized mice with CY and OVA-pulsed DC-immunized mice with CY (P = 0.0317).

Fig. 3.

Fig. 3

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SAg plus OVA257–264-pulsed DC mediate significant therapeutic antitumor effects in combination with low-dose cyclophosphamide. a-f Three days after tumor inoculation, B6 mice bearing established EG7 (a, b), B16 (c, d), or 2LL (e, f) tumors were treated with or without cyclophosphamide followed by the indicated vaccines. a, c, e SAg plus OVA257–264-pulsed DC efficiently enhance the induction of CTL specific to the individual tumors. b, d, f SAg with low-dose cyclophosphamide supports the therapeutic activity of cancer vaccines. The open arrowhead shows the day of CY treatment. The closed arrow shows the day of DC vaccination. Similar results were obtained in two separate experiments. Data are presented as the mean ± SE. g Survival of B6 mice bearing established EG7 treated with cyclophosphamide followed by the indicated vaccine was recorded as the percentage of surviving animals. An exact log-rank test was used to analyze the survival differences between the SAg/OVA-DC with CY i.p. and OVA-DC with CY i.p. treatment groups (P = 0.0317). Data represent one of two independent experiments

Low-dose CY support the therapeutic effect of SAg/OVA-pulsed DC vaccine by inhibiting the increase of CD4+CD25+Foxp3+ cells

To ascertain the role of low-dose CY in the therapeutic model, the proportion of regulatory T cells of the splenocytes from vaccinated mice was investigated using flow cytometry (Fig. 4a). On day 5, after tumor inoculation, B6 mice harboring EG7 were treated with SAg/OVA-pulsed DC with or without low-dose CY. Three days after vaccination, the percentage of CD4+CD25+Foxp3+ cells among the total CD4+ cells in the spleen was increased in the tumor-bearing mice (P = 0.0001, Naïve vs. Tumor bearing, Fig. 4b), but was reduced by low-dose CY. Furthermore, SAg/OVA-pulsed DC vaccine induced the increase in CD4+CD25+Foxp3+ cells and was reduced similarly by low-dose CY (P=0.0118, Tumor bearing with CY and DC vs. Tumor bearing with DC, Fig. 4b). These findings suggest that inhibition of regulatory T cells by systemic administration of low-dose CY support the therapeutic effect of SAg/OVA-pulsed DC vaccine.

Fig. 4.

Fig. 4

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CD4+CD25+Foxp3+cells (Treg) is increased in spleen of tumor-bearing mice, which is enhanced by SAg/OVA-DC vaccine but reduced by pretreatment with low-dose cyclophosphamide. As Fig. 3a, three days after tumor inoculation, B6 mice bearing established EG7 were treated with or without CY and followed by SAg/OVA-DC vaccine or PBS. On day 8 (3 days after vaccination), the spleens were harvested and analyzed by flow cytometry. a Representative profiles of the flow cytometry analysis. b Percentage of CD4+CD25+Foxp3+ regulatory T cells among the total CD4+ T cells. The data are means and standard deviations for four mice in each experimental group

Discussion

Superantigens cause intense T-cell proliferation and the production of cytokines. The predominant cytokines produced and released during superantigen activation are IL-2 and IFN-γ [5–7], both of which are intimately involved in the cytokine cascade during immune responses. One of these cytokines, IFN-γ, possesses immunostimulatory activity in that it activates CD8+ T cells, macrophages, and NK cells so that they become cytolytic. It was also reported that the high cytolytic activity detected in vaccinated mice given SEA/SEB was predominantly due to CD8+ T cells [24]. T cells activated by superantigen promotes the maturation of DC mainly via MHC classII molecule, which contributes to induce tumor-specific CTLs [25]. Other cell types have been found to play roles in antitumor immunity, such as NK cells or activated eosinophils, which may be marginally involved. Thus, as various cancer patients have been shown to possess low levels of tumor-specific T cells, superantigens may be able to expand these small populations to produce populations that are effective at fighting the tumor. IFN-γ has been shown to elicit direct anticellular effects on a variety of tumor cell lines. Furthermore, IFN-γ-induced cell growth inhibition was accompanied by the increased production of the tumor suppressor gene products p21 and p27 [26, 27]. This suggests that the direct anticellular effects of IFN-γ also contribute to the antitumor protection seen in response to vaccination followed by superantigen administration.

The ability of DC to generate potent type-1 immune responses has made them highly attractive for use as cancer vaccines where cytolytic immunity is desired for the clearance of tumor cells. IL-12 enhances natural killer cell and CTL activities and plays a key role in the induction of type-1 immune responses. Numerous studies have demonstrated that the co-administration of IL-12 or genetic engineering of DC to produce high levels of IL-12 strongly enhances the antitumor efficacy of cancer vaccines [28–30]. Recently, we found that the ability of class I restricted CD8+ T cells to coactivate and polarize DC supports the induction of type-1 responses using ovalbumin or superantigen mice model [16]. Consistent with previous studies using the OVA model, heterologous help from CD8+ T cells could not be mediated by IL-12/IL-23-deficient dendritic cells generated from the bone marrow of p40-knockout animals in this study, suggesting that dendritic cells play a critical role in mediating CD8 help for anticancer immunity.

Superantigenic stimulation of T cells prior to their activation by vaccination may ultimately lead to the induction of anergy [9, 10]. Furthermore, tumor-specific anergy has been shown to be an early event in the tumor-bearing host. Although the CD4+ CD25+ T-cell number is tightly regulated in normal conditions, they accumulate in tumor-bearing hosts [31, 32]. Valzasina et al. [32] reported CD4+CD25− T-cell conversion into regulatory T cells (Treg) as the mechanism of Treg replenishment and expansion in tumor-bearing mice. Furthermore, therapeutic vaccination of the tumor-bearing host expanded both tumor-induced Treg and effector cells, but suppression was dominat, blunting the expansion of naïve tumor-specific T cells and blocking the execution of effector function [33]. Cyclophosphamide (CY) is an alkylating agent that is widely used in the treatment of malignancies as well as autoimmune disorders. As an immunomodulatory agent, CY increases the efficacy of various kinds of antitumor immunotherapeutic regimens, including active or adoptive immunotherapies. The administration of low-dose CY prior to DC immunization resulted in a significant increase in antitumor Th1 immune responses and a decrease in the number of CD4+ CD25+ Treg cells in the tumor-bearing host [34]. Our data demonstrate that although the efficacy of tumor antigen and SAg-pulsed DC was abolished in a therapeutic tumor-bearing model, significant antitumor effects were observed in animals bearing various established tumors (EG7, B16, and 2LL) after low-dose CY treatment. Complete tumor eradication was observed in 30–60% of mice treated with a single injection of SAg-pulsed DC, even when a weakly immunogenic B16 or 2LL tumor was used. Consistent with previous reports [31, 32, 34], our data also demonstrated that these efficacy might be due to an ability of CY to control the number of Treg in tumor-bearing animals.

Few studies have utilized SAg as an immune adjuvant for anti-cancer therapy. Wirth et al. [35] investigated whether the induction of a CTL response to HLA-A2-Melan-A was improved by using rVV expressing the CTL defined epitope alone or in combination with SAg. They showed that the anti-Melan-A response was efficiently induced but that tumor suppression was not significantly enhanced by coexpression of the SAg in a tumor protective model. However, the host dendritic cells might not have worked efficiently in their model. Kominsky et al. [24] reported that the vaccination of mice with irradiated B16F10 cells followed by treatment with a combination of SEA and SEB led to significant and specific protection against subsequent challenge with viable B16F10 cells. Here, we showed that tumor antigen and SAg-pulsed ex vivo generated dendritic cells efficiently induced antitumor immunity in therapeutic models (tumor-bearing state) as well as the prophylactic setting. Although SAg is known to cause toxicity through excessive immune responses, SAg was administered in several human clinical studies as well as animal studies, and no serious adverse events were reported [36–38]. Indeed, we observed five non-tumor-bearing animals that were injected with the same dose of SAg-pulsed DC, and they remained alive for at least a year.

In summary, systemic immune responses, as demonstrated by CTL activity and IFN-γ production, were significantly higher and tumor specific when SAg-pulsed DC were used. High cytolytic activity in association with a type-1 response may have contributed to the profound in vivo anti-tumor effects that we observed. These results strongly suggest that treatment with SAg-pulsed DC in combination with low-dose CY should be considered in humans, in order to facilitate the development of effective strategies for therapeutic vaccination of patients with cancer and chronic infections.

Acknowledgments

We thank Drs. T. Uchiyama and H. Kato (Tokyo Women's Medical University) for providing SEA used in preliminary experiments, K. Shibata for operating flow cytometry. This work was supported by grants-in-aid for scientific research from the Japanese Society for the Promotion of Science (grant 19590889 to Y.N.).

Conflict of interest

None of the authors has any financial relationships with commercial entities that have an interest in the subject of this manuscript.

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