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. 2005 May 13;55(2):131–139. doi: 10.1007/s00262-005-0684-3

Allogeneic gastric cancer cell-dendritic cell hybrids induce tumor antigen (carcinoembryonic antigen) specific CD8+ T cells

Sachiko Matsumoto 1, Hiroaki Saito 1,✉, Shunichi Tsujitani 1, Masahide Ikeguchi 1
PMCID: PMC11030765  PMID: 15891883

Abstract

The development of protocols for the ex vivo generation of dendritic cells (DCs) has led to intensive research of their potential use in immunotherapy. Accumulating results show the efficacy of this treatment on melanomas which are highly immunogenic. However, its efficacy remains unclear in other tumors. In this study, allogeneic gastric cancer cell–DC hybrids were used to determine the efficacy of this type of immunotherapy in gastric cancer. Fusion cells of DC and allogeneic gastric cancer cells were generated by polyethylene glycol (PEG) and electrofusion. These hybrids were used to induce tumor associated antigen (TAA) specific cytotoxic T lymphocytes (CTLs). The DCs were successfully fused with the allogeneic gastric cancer cells resulting in hybrid cells. These hybrid cells were functional as antigen-presenting cell because they induced allogeneic CD4+ T cells proliferation. CD8+ T cells stimulated by the MKN-45-DC hybrid cells were able to kill MKN-45 when used for immunization. The CTLs killed another gastric cancer cell line, MKN-1, as well as a melanoma cell line, 888mel, suggesting the recognition of a shared tumor antigen. MKN-45 specific CTLs can recognize carcinoembryonic antigen (CEA), indicating that the killing is due to tumor antigens as well as alloantigens. This approach suggests the possible use of allogeneic gastric cancer cell–DC hybrids in DC based immunotherapy for gastric cancer treatment.

Keywords: Fusion, Tumor immunity, Tumor vaccine, Immunotherapy

Introduction

Gastric cancer is one of the most common cancers in Asia. In Japan, it was estimated that more than 17,000 individuals died of gastric cancer in 2002. Despite the recently reduced mortality rates due to both earlier detection and improved therapy, gastric cancer death still ranks second among all cancer deaths worldwide [26]. To improve the survival rates of gastric cancer patients, the development of new treatments are therefore crucial.

Although immunotherapy has been shown to be a promising therapy for melanomas [4, 25, 30], it has not been considered seriously in gastric cancer treatment because of the common conception that gastric cancer is weakly immunogenic like colorectal, breast, and ovarian cancer. However, there are accumulating evidences showing the presence of naturally occurring immunity against tumor antigens even in patients with weakly immunogenic cancer. Disis et al. [8–10] revealed the presence of T cells and antibody immunity to the HER-2/neu oncogenic protein in patients with HER-2/neu-overexpressing breast cancer. In addition, it was recently reported that the peripheral activation of cerebellar degeneration-related 2 (cdr2) specific CTLs is likely to contribute to the subsequent development of autoimmune neuronal degeneration, paraneoplastic cerebellar degeneration, in patients with breast or ovarian cancer [1]. Based on these facts, it was hypothesized that immunotherapy was also a promising therapy for weakly immunogenic cancers. In this study, therefore, we tried to develop effective dendritic cell (DC) based immunotherapy for gastric cancer.

Dentritic cells are considered the most powerful antigen-presenting cells, enabling the induction of and maintaining immune responses [5]. The development of protocols for ex vivo DC generation has led to intensive research on their potential use in immunotherapy. Some clinical studies have been commenced in which DCs are generated ex vivo, charged with tumor-associated antigens (TAA), exposed to maturation stimuli and reinforced for immunization. One of the most commonly used, clinically approved approach is to load empty major histocompatibility complex (MHC) class I molecules with exogenous tumor associated peptides. This approach is limited, however, to patients who express certain human leukocyte antigen (HLA) haplotypes and needs characterization of the targeted TAA. However, TAA have not been clearly identified in gastric cancer compared to melanomas. Furthermore, this approach ignores the important role of HLA class II-restricted helper T cells in initiating and maintaining effective immune responses. Therefore, alternative strategies were developed as follows: (1) DCs loaded with full-length recombinant proteins and dead tumor cells (apoptotic bodies, necrotic cells) [22]; (2) DCs fused with tumor cells [24]; (3) DCs transfected with TAA encoding mRNA [21] or total tumor RNA [14]; and (4) gene-based delivery of TAA into the DCs [20]. Recently, it has been shown that tumor cell–DC hybrids induce strong immune responses [11, 12]. Moreover, Avigan et al. [3] reported that fusion cell vaccination of patients with metastatic breast and renal cancer induces immunological and clinical responses. However, little is known about immunotherapy for gastric cancer.

In this study, allogeneic gastric cancer cell–DC hybrids were generated and shown to induce gastric cancer specific CD8+ T cells. In vitro-induced CTLs, specific for the gastric cancer cell line MKN-45, were also shown to selectively recognize carcinoembryonic antigen (CEA), indicating that this cytotoxicity is directed not only to the alloantigens but also to the tumor antigens.

Materials and methods

Media and reagents

A complete culture medium (CM) consisting of RPMI 1640 (Gibco-BRL, Grand Island, NY, USA), 1%-L-glutamine (Invitrogen, Carlsbad, CA, USA), 1% penicillin/streptomycin (Invitrogen), 2.5% HEPES solution buffer (Gibco BRL), 1% sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 1% essential amino acids (Sigma-Aldrich), and 10% heat-inactivated FCS (Thermo Trace Ltd. Melbourne, Australia) was used. For the T cell cultures, the FCS was replaced by 10% human serum AB (Gemini Bio-Products, Woodlands, CA, USA). Human GM-CSF (Kirin Brewery Co. Ltd., Tokyo, Japan), IL-2 (Immunace35, Shionogi Pharmaceutical Co., Tokyo, Japan), and IL-7 (R&D Systems, Cambridge, UK) were used at respective concentrations of 100 ng/ml, 10 IU/ml, and 10 IU/ml, and human IL-4 (Ono Pharmaceutical Co., Osaka, Japan) and TNF-α (Genzyme, Cambridge, MA, USA) were used at concentrations of 50 and 100 ng/ml, respectively.

Cell lines

The gastric cancer cell lines, MKN-1 (HLA-A24+, A26+, CEA−) and MKN-45 (HLA-A24+, CEA+), and human chronic myelogenous leukemia cell line, K562, were purchased from the RIKEN Cell Bank (Tsukuba, Japan), and the melanoma cell line, 888mel (HLA-A1+, A24+, CEA+), was provided by Dr Y. Kawakami (Keio University, Tokyo, Japan). The TAP-deficient BxT hybrid cell line T2 transfected with the HLA-A2402 gene (referred to as T2-A24) was donated by Dr K. Kuzushima (Aichi Cancer Center Research Institute, Japan) [19]. The T2-A24 cell line was cultured in CM with 0.8 mg/ml G418 (Gibco BRL), while the other cell lines were maintained in CM.

Generation of the DCs

Immature monocyte-derived DCs were generated from an adherent fraction of peripheral blood mononuclear cells (PBMCs) [6]. Briefly, PBMCs were isolated from a donor who was known to be HLA-A24+, suspended in serum-free medium and allowed to adhere to plastic dishes (100 mm dishes; IWAKI, Tokyo, Japan). After 2-h incubation at 37°C, the nonadherent cells were gently washed out with phosphate buffer saline (PBS) three times. The adherent cells were cultured in CM with 100 ng/ml of GM-CSF and 50 ng/ml of IL-4. On day 7, DC maturation was induced by the addition of 100 ng/ml of TNF-α and nonadherent cells were harvested on day 9.

Hybrid Generation

On day 9, cell fusion of the mature DCs and MKN-45 cells was performed. Briefly, the DCs and MKN-45 cells were labeled with PKH-26 and PKH-67 (Sigma-Aldrich), respectively, and the MKN-45 cells were irradiated with 50 Gy before cell fusion. To induce cell fusion, the DCs and irradiated tumor cells were mixed at a ratio of 1:1 and incubated in serum-free RPMI 1640 medium containing 50% polyethylene glycol (PEG) for 1 min. After slowly diluting with serum-free RPMI 1640 medium, the cells were washed three times, resuspended in CM, and cultured at 37°C for 2 h. The cells were then suspended in a 0.3 M sucrose solution (pH 7.2–7.4). After centrifugation, the cells were resuspended in the same solution. Routinely, 1 ml of cell suspension containing 1×106 DCs and 1×106 tumor cells was processed using a 4-mm Gapped fusion chamber. During electrofusion, a pulse generator (model ECM 2001; BTX Instruments, Genetronics, San Diego, CA, USA) was used for application of the field pulses. Cell alignment was first induced by electrophoresis with an alternating current (ac) pulse of 100 V/cm for 10 s. Subsequently, cell fusion was triggered by application of a single square wave direct current (dc) pulse of 1,200 V/cm for 30 μs. The fusion mixture was allowed to rest for 5 min before suspension in CM.

Synthetic peptides

HLA-A24 restricted CEA268 (10) (QYSWFVNGTF), CEA652 (9) (TYACFVSNL) and PMSA178 (9) (NYARTEDFF) peptides were >90% pure as indicated by high-pressure liquid chromatography (HPLC) analysis (TaKaRa Biomedicals, Otsu, Japan). Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO), diluted to 1 mg/ml in distilled water, and stored at −80°C.

T cell purification

For the T cell proliferation assay, purified CD4+ T cells were positively selected using anti-CD4 Micro Beads (MACS system, Miltenyi Biotec, Bergisch Gladbach, Germany), respectively, from Ficoll-separated PBMCs of a healthy donor who was known to be HLA-A24+. To generate the CTLs, autologous CD8+ T cells were prepared by depleting the other cells using a cocktail of biotin-conjugated antibodies against CD4, CD14, CD16, CD19, CD36, CD56, CD123, TCR γ/δ, glycophorin A, and anti-biotin Microbeads (MACS system).

Flow Cytometry Analysis

FACS analysis was performed on a FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA), and the following antibodies were used to classify the cells: anti-CD1a-PE or Cy-chrome, anti-CD3-FITC, anti-CD11c-PE or Cy-Chrome, anti-CD14-FITC or PerCP, anti-CD40-PE or Cy-Chrome, anti-CD56-PE, anti-CD80-FITC or Cy-Chrome, anti-CD83-FITC, anti-CD86-PE or Cy-Chrome, anti-HLA-ABC-PE or Cy-Chrome, anti-HLA-DR-FITC or PerCP, anti-CD4-FITC, and anti-CD8-PE (Becton Dickinson), anti-CD83-PE-Cy5 (Beckman Coulter, Fullerton, CA, USA), and anti-CD66e (CEA)-PerCP (PeliCluster, Cell Science, Norwood, MA, USA)

Confocal staining

Mature DCs were labeled with a red fluorescent cell linker (PKH26) and MKN45 cells were labeled with a green fluorescent cell linker (PKH-67) (Sigma-Aldrich). After fusion, the cells were assessed by fluorescent microscopy to confirm successful fusion of DC and MKN-45.

T cell proliferation assay

Hybrids of the mature DCs and MKN-45 cells were cultured at a graded dose in CM with 10% human AB serum with purified allogeneic CD4+ T cells (1×105/well/200 μl). A small amount of the CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI, USA) was added directly to the culture wells on day 5. After 4 h incubation, proliferation was determined by recording the absorbance at 490 nm with a 96-well plate reader.

Generation of specific CTLs

DC-MKN-45 hybrids were used as stimulatory cells, while autologous purified CD8+ T cells were used as responders. Cultures were prepared in 24-well plates (Falcon) by plating loaded DCs at 1×105 cells with 1×106 T cells in a final volume of 2 ml. CD8+ T cells were stimulated by MKN-45-DC hybrids once a week for 3 weeks. MKN-45-DC hybrids were made fresh for every stimulation. The CM was supplemented with 10% AB serum, IL-7 (10 IU/ml in weeks 1, 2, and 3), and IL-2 (10 IU/ml in weeks 2 and 3). Five to six days after the last stimulation, cells were harvested and their cytotoxic activity was tested.

51 Cr cytotoxicity assay

Cytotoxicity was measured in a standard 4-h 51 Cr-release assay. The different targets were then labeled with 51 Cr (Perkin Elmer, Wellesley, MA, USA) and washed three times with PBS. CTLs were cocultured at 37°C for 4 h with 103 51Cr-labeled target cells in 200 μl of CM supplemented with 10% AB serum in 96-well culture plates. After 4 h, 50 μl of supernatant was collected and the percentage of killed cells was calculated using the following formula: % release=100×(cpm experiment − cpm spontaneous release)/(cpm maximum release - cpm spontaneous release). For the monoclonal antibody (mAb) blocking assays, anti-HLA-ABC antibody (W6/32, DAKO, Carpenteria, CA; 5 μg/ml) was added 30 min before the addition of T cells, and the mAbs were left throughout the culture period. Irrelevant mAbs were used as isotype controls.

Enzyme-linked immunospot assay for IFN-γ release

To quantitate the antigen-specific IFN-γ-releasing effector T cells, an enzyme-linked immunospot (ELISPOT) assay was performed according to the manufacturer’s protocol (R&D systems). CD8+ T cells (5×104 /well) were added in triplicate to nitrocellulose-bottomed 96-well plates at 100 μl of CM per well. For the detection of specific reactive T cells, T2-A24 cells pulsed with HLA-A24 restricted peptides were added at 5×103 per well (final volume, 100 μl/well). After 4 h, the wells were washed three times, incubated with biotinylated second mAb to IFN-γ for 2 h, washed again, and stained with alkaline phosphatase-conjugated streptavidin. The number of spots was counted with microscopy.

ELISA for IFN-γ and IL-4

Commercial ELISAs (R&D Systems) were used to quantitate human IFN-γ and IL-4. ELISAs were performed in triplicate and laboratory standards were included on each plate.

Tetramer binding assay

After three stimulations, the T cells were labeled with anti-CD3-Cy-Chrome (Becton Dickinson) and anti-CD8-FITC for 30 min at 4°C, then washed and stained with a PE-conjugated CEA/HLA-A24 class I tetramer (ProImmune Limited, Oxford, UK) for 30 min at 4°C. Samples were analyzed using a FACSCalibur (Becton Dickinson).

Statistical analysis

Differences were determined using the unpaired t-test, with P<0.05 considered significant.

Results

Generation of allogeneic gastric cancer cell line MKN-45-DC hybrid cell

Successful fusion of the DCs with the allogeneic gastric cancer cells was assessed. Before cell fusion, the DCs and MKN-45 cells were stained with red and green fluorescent dyes, respectively, and the frequency of double positive cells was checked by flow cytometry and fluorescence microscopy after the fusion process. Approximately 20% of the cells were double positive and supposed to be fusion cells (Fig. 1a). Phenotype analyses showed that these double positive cells expressed not only DC markers such as CD1a, CD80, CD86, and MHC classes I and II, but also CEA that the original MKN-45 cells express (Fig. 1b). These results indicate that the fluorescent double positive cells are MKN-45-DC hybrid cells.

Fig. 1.

Fig. 1

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Characterization of the hybrid cell vaccine. a Generation of hybrid cells. DCs and tumor cells were labeled with red (PKH26) and green fluorescent dyes (PKH67), respectively. The tumor cells fused with DCs were analyzed by flow cytometry. b Phenotype of the DCs and tumor cells before and after the fusion process as determined by flow cytometry. After fusion process, only PKH26 and PKH67 double positive cells were gated and their phenotypes were analyzed. c The MKN45-DC hybrid cells induced allogeneic CD4+ T cell proliferation. DCs or MKN45-DC hybrid cells were added at the indicated ratios to 1×105 allogeneic CD4+ T cells. After 5 days of incubation, the reagent was added, and 4 h later proliferation was measured. Each value represents the mean from triplicate wells. P<0.05 at DC number of 5,000, 2,500, and 1,250 for mature DC versus hybrid cell. All results are representative of three experiments

As PEG, followed by electrofusion, was applied to generate more fusion cells in this study, we were afraid that this negatively impact on cell viability. Therefore, we determined if these hybrids were able to induce allogeneic CD4+ T cell proliferation to see if they were still alive and function as antigen-presenting cells even after fusion process, as DCs are potent stimulators of primary mixed lymphocyte reactions (MLRs). Irradiated MKN-45 did not induce any proliferation of allogeneic CD4+ T cells (data not shown). The MKN-45-DC hybrids induce even more allogeneic CD4+ T cell proliferation than DC (Fig. 1c), indicating that these hybrids are still alive and function as antigen-presenting cells.

MKN-45-DC hybrid cells induce CEA specific CD8+ cytotoxic T cells

Since the goal of tumor immunotherapy is to elicit specific immune responses against tumors, we assessed whether the MKN-45-DC hybrid could induce tumor specific CTLs. Autologous CD8+ T cells (>90% purity, HLA-A24+) were stimulated with either DCs or MKN-45-DC hybrid cells once a week for 3 weeks. First, the concentration of IFN-γ and IL-4 in the supernatant of the CD8+ T cell culture was determined after three round stimulations (Fig. 2a). The concentration of IFN-γ in the T cell supernatant stimulated by the MKN-45-DC hybrid cells was much higher than that stimulated by the DCs, indicating that the MKN-45-DC hybrid strongly drives CD8+ T cells into gamma interferon-producing CD8+ T cells (Tc1).

Fig. 2.

Fig. 2

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Allogeneic gastric cancer cell-DC hybrid cells induce CTLs specific for gastric cancer cell. Autologous CD8+ T cells were negatively selected using microbeads and MKN45-DC hybrids were used as stimulator cells. The cultures were set in 24-well plates by plating stimulator cells with autologous CD8+ T cells. CD8+ T cells were stimulated by MKN45-DC hybrid cells once a week for 3 weeks. a Concentrations of IFN-γ and IL-4. The supernatant was collected 5 days after the third stimulation. The concentration of IFN-γ in the T cell supernatant stimulated by the MKN-45-DC hybrid cells was much higher than that stimulated by the DCs (P<0.05). b Five to six days after the third stimulation, the cytotoxic activity of the expanded CD8+ T cells was assessed with a standard 51Cr release assay using sensitizing MKN-45, MKN-1, 888mel, and K562 as targets. CD8+ T cells primed with MKN45-DC hybrid cells display cytotoxic activity against MKN-45 when used for priming, but not against NK-sensitive K562 cells (P<0.05 at the all E:T ratio for MKN-45 versus K562). The CTLs also killed another gastric cancer cell line, MKN-1 (P<0.05 at the E:T ratio of 60:1, 30:1, and 15:1 for MKN-1 versus K562), and a melanoma cell line, 888mel (P<0.05 at the E:T ratio of 60:1 and 30:1 for 888mel versus K562). c The cytotoxic activity was blocked by adding an anti-MHC class I antibody (P<0.05 at the E:T ratio of 60:1 and 30:1 for none or anti-IgG2a versus anti class I). The percent cytotoxicity was measured as a function of spontaneous and total release and each value represents the mean from triplicate well. All results are representative of three experiments by using three different donors

Next, the cytotoxic activity against MKN-45 (HLA-A24+) by the CTLs stimulated with MKN-45-DC hybrid cells was determined. Figure 2b shows that CD8+ T cells stimulated by the MKN-45-DC hybrid could kill MKN-45 when used for immunization. This killing was dependent on the CD8+ T cells because it could be blocked with an anti-MHC class I antibody (Fig. 2c). The CTLs could also kill a melanoma cell line, 888mel (HLA-A24+), as well as another gastric cancer cell line, MKN-1 (HLA-A24+).

To ensure that the killing observed was due not only to the alloantigens but also to tumor related antigens, we determined if the CD8+ T cells generated using the MKN-45-DC hybrid cells could recognize any tumor antigens by ELISPOT assay and tetramer staining. We focused on CEA because it’s one of the most common gastric cancer antigens and expressed in MKN-45. The ELISPOT assay showed that the number of IFN-γ spots produced in response to TAP–deficient T2-A24 cells expressing HLA-A24 loaded with CEA peptides (CEA652 or CEA268) was more than those produced in response to T2-A24 cells loaded with control peptides (PSMA) (Fig. 3a). Figure 3b shows that CD8+ T cells stimulated by MKN-45-DC hybrid cells could kill T2-A24 cells loaded with CEA peptides, but not T2-A24 loaded with control peptides (PSMA). Moreover, the CEA/HLA-A24 tetramer staining showed that the MKN-45-DC hybrids induced CEA-specific CD8+ T cell expansion (Fig. 3c). These results indicate the presence of CEA specific CD8+ T cells induced by MKN-45-DC hybrid cells.

Fig. 3.

Fig. 3

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MKN45-DC hybrid cells induce CEA-specific CTLs. a ELISPOT assay using T2-A24 loaded with either CEA peptides or control peptides (PSMA). P<0.05 for CEA268 or CEA652 versus PSMA. b The CTLs killed T2-A24 loaded with CEA peptides, indicating the presence of CEA-specific CD8+ T cells. P<0.05 at the E:T ratio of 30:1 and 15:1 for CEA268 versus PSMA. The percent cytotoxicity was measured as a function of spontaneous and total release and each value represents the mean from triplicate well. c The presence of CEA-specific CD8+ T cells as determined by tetramer staining. MKN45-DC hybrid cells induce expansion of CEA-specific CD8+ T cell. All results are representative of three experiments by using three different donors

Discussion

The researches and clinical trials of DC based immunotherapy have mainly been focused on melanomas because of their high immunogenecity and the presence of many known melanoma related antigens such as MART-1, tyrosinase, MAGE-3, and gp-100. Accumulating results have shown the efficacy of this treatment in melanomas. On the other hand, this therapy has not been seriously considered in the treatment of gastric cancer due to the common conception that gastric cancer is less immunogenic than melanomas. One major problem of immunotherapy against gastric cancer is the limited number of well-defined TAA. In this study, an attempt was made to develop DC based immunotherapy using allogeneic gastric cancer cell–DC hybrids. It is believed that tumor cells themselves have many unknown TAA that this polyvalent vaccine is able to induce T cell responses against multiple naturally processed and presented immunodominant epitopes, therefore, it might reduce the occurrence of clonal tumor escape phenomena. Moreover, it has been recently shown that the fusion of tumor cells and DCs induce strong immune responses [11, 12].

Hybrids of DCs and tumor cells have been produced using either PEG or electrofusion [12, 13, 28]. In preclinical murine models, DC–tumor cell hybrids have stimulated potent, protective, and therapeutic immune responses to carcinomas, lymphomas, melanomas, and gliomas. In two recent human clinical trials, however, fusions of DCs and autologous tumor cells were primarily ineffective in the treatment of patients with melanomas and gliomas [17, 18]. This might be due to small number of fusion cells. In both of these protocols, PEG was used as the fusogenic reagent. In one previous investigation, electrofusion was preferred over PEG-mediated fusion specifically because fusion efficiency is very low with PEG, ranging from only 0.5% to 4.5% [15]. Our preliminary data also showed that the fusion frequency ranges from 6% to 12.8% with PEG and from 2.1% to 9.9% with electrofusion. In this study, therefore, both PEG and electrofusion were used to obtain more hybrid cells (ranging from 10.4% to 24.5%). This procedure might improve the clinical effects because more hybrid cells might induce more T cell immunity.

Since DCs are potent stimulators of primary mixed lymphocyte reactions (MLRs), allogeneic CD4+ T cell proliferation was determined using MKN-45-DC hybrids to characterize in part, the function of MKN-45-DC hybrids. The results indicated that MKN-45-DC hybrids function as antigen-presenting cells, because they induced allogeneic CD4+ T cell proliferation. Fusion cells induce more proliferation of allogeneic CD4+ T cells than DCs. This can be explained by the upregulation of costimulatory factors on fusion cells, such as CD80. We suppose that this may be induced by fusion procedures.

The present study showed for the first time that gastric cancer cell line MKN-45 specific CD8+ T cells could be generated using MKN-45-DC hybrid cells. We determined if MKN-45 specific CD8+ T cells could recognize any TAAs to ensure that the killing observed was due not only to alloantigens but also to TAAs. To this end, we focused on CEA because it was expressed by MKN-45. CEA is a 180 kDa glycoprotein that is extensively expressed in the vast majority of colorectal, gastric, and pancreatic carcinomas, approximately 50% of breast cancers and 70% of non-small-cell lung cancers [29]. It is also present, but usually at lower concentrations, in normal colon epithelium and in some fetal tissues. Therefore, CEA could be seen as a selfantigen by the immune system, and thus most individuals including cancer patients could be immunologically tolerant to this TAA. However, recent studies have shown that, despite being a selfantigen, CEA is capable of generating an immunological response in humans without evoking autoimmune toxicity [16]. Two HLA-A24 restricted CEA peptides have been identified so far [23]. We confirmed the presence of CTLs that recognize CEA peptide by CTL assay, ELISPOT assay, and tetramer staining in the current study.

The CTLs generated by MKN-45-DC hybrid cells are also able to kill 888mel as well as MKN-1. Because both MKN-45 and 888mel are both HLA-A24 and CEA positive, cross killing between MKN-45 and 888mel, we have seen in the current experiment, might be due to the recognition of HLA-A24 restricted CEA peptide by CTL specific for MKN-45. However, MKN-1, which is HLA-A24 positive cell line, does not express CEA. As the killing of MKN-1 by CTLs specific for MKN-45 is MHC class I restricted, this killing must be due to the recognition of other HLA-A24 restricted peptide or other HLA-B or C restricted peptide by CTLs specific for MKN-45. These data show the possibility that allogeneic gastric cancer cell–DC fusion cells are able to generate various kinds of CTLs, which are able to recognize known and unknown tumor antigens. This is the most effective point using cancer cell–DC fusion cells instead of peptide.

It has been shown that DCs can capture dead cells and present their antigens in the context of class I and II MHCs [2, 7]. Therefore, there is the possibility that DCs, which capture dead MKN-45, but not MKN-45-DC hybrid cells, induce MKN-45 specific CTLs and CEA specific CTLs. In this regard, mature rather than immature DCs were used in this study because mature DCs are reportedly not able to capture dead cells [27]. In fact, mature DCs cannot uptake FITC-dextran although immature DCs could uptake FITC-dextran in this study (data not shown). This indicates that the MKN-45 specific CTLs were induced by MKN-45-DC hybrid cells, not by DCs that capture dead MKN-45. In the current study, DC maturation was induced by TNF-α. However, CD83 expression was sometime not so high (33.6 to 80.3%, 63±17.3; n=7), indicating that DC maturation might not be enough. Therefore, improvement of maturation method, such as a mixture of IL-1ß, IL-6, TNF-α, and prostaglandin E2 might improve the generation of CTLs.

Important for the clinical application of DC-tumor hybrids is the fate of hybrids at later times postfusion, both with respect to the retention of hybrids and viability. Trevor et al. [31] reported that hybrids were viable and retained in the population for 48 h postfusion, irrespective of the tumor type used. A further consideration for clinical trial is the number of hybrids infused to patients. In this regard, Avigan et al. [3] demonstrated that small number of hybrid cells (3x105 cells) was enough to induce disease regression in patients with metastatic breast cancer. These evidences show that fusion cell vaccination of patients with gastric cancer might be practical.

In conclusion, this study shows for the first time that allogeneic gastric cancer cell–DC hybrid cells induce TAA specific CTLs. Only immunogenic tumors, such as melanomas, have previously been focused on in terms of immunotherapy. Our approach suggests the possible use of allogeneic gastric cancer cell–DC hybrid cells in DC based immunotherapy for gastric cancer treatment.

Acknowledgments

We thank Drs. Shinichi Hayashi, Michio Maeta and Nobuaki Kaibara for critically reviewing the manuscript, Dr Yutaka Kawakami for providing the 888mel cell line and Dr Kiyotaka Kuzushima for providing the T2-A24 cell line. We also thank Kirin Brewery Co. for providing the recombinant human GM-CSF, Ono Pharmaceutical Co. for providing the recombinant human IL-4, and Shionogi Pharmaceutical Co. for providing the recombinant human IL-2.

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