Clinical Cancer Research Bridging the Lab and the Clinic in Cancer Medicine Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Suzuki, T.
Right arrow Articles by Nukiwa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Suzuki, T.
Right arrow Articles by Nukiwa, T.
Clinical Cancer Research Vol. 11, 58-66, January 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

Vaccination of Dendritic Cells Loaded with Interleukin-12-Secreting Cancer Cells Augments In vivo Antitumor Immunity: Characteristics of Syngeneic and Allogeneic Antigen-Presenting Cell Cancer Hybrid Cells

Takuji Suzuki1, Tatsuro Fukuhara1, Masashi Tanaka1, Akira Nakamura1, Kenichi Akiyama1, Tomohiro Sakakibara1, Daizo Koinuma1, Toshiaki Kikuchi1, Ryushi Tazawa1, Makoto Maemondo1, Koichi Hagiwara3, Yasuo Saijo2 and Toshihiro Nukiwa1

1 Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging, and Cancer; 2 Department of Molecular Medicine and Gene Transfer Research, Graduate School of Medicine, Tohoku University, Sendai, Japan; and 3 Department of Respiratory Medicine, Saitama Medical School, Saitama, Japan

Requests for reprints: Toshihiro Nukiwa, Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging, and Cancer, Tohoku University, 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22-717-8539; Fax: 81-22-717-8549; E-mail: toshinkw{at}idac.tohoku.ac.jp.


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer immunotherapy by fusion of antigen-presenting cells and tumor cells has been shown to induce potent antitumor immunity. In this study, we characterized syngeneic and allogeneic, murine macrophage/dendritic cell (DC)-cancer fusion cells for the antitumor effects. The results showed the superiority of allogeneic cells as fusion partners in both types of antigen-presenting cells in an in vivo immunotherapy model. A potent induction of tumor-specific CTLs was observed in these immunized conditions. In addition, the immunization with DC-cancer fusion cells was better than that with macrophage-cancer fusion cells. Both syngeneic and allogeneic DC-cancer fusion cells induced higher levels of IFN-{gamma} production than macrophage-cancer fusion cells. Interestingly, allogeneic DC-cancer fusion cells were superior in that they efficiently induced Th1-type cytokines but not the Th2-type cytokines interleukin (IL)-10 and IL-4, whereas syngeneic DC-cancer fusion cells were powerful inducers of both Th1 and Th2 cytokines. These results suggest that allogeneic DCs are suitable as fusion cells in cancer immunotherapy. To further enhance the antitumor immunity in the clinical setting, we prepared DCs fused with IL-12 gene-transferred cancer cells and thus generated IL-12-secreting DC-cancer fusion cells. Immunization with these gene-modified DC-cancer fusion cells was able to elicit a markedly enhanced antitumor effect in the in vivo therapeutic model. This novel IL-12-producing fusion cell vaccine might be one promising intervention for future cancer immunotherapy.

Key Words: fusion cell • cancer immunotherapy • allogeneic • DC • IL-12


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer cells evade host immune surveillance at least in part due to their low immunogenicity (1). Reduced or absent expression of MHC class I (2), TAP-1 and TAP-2 (3, 4), or costimulatory molecules (5, 6) have been reported in malignant tumors. Strategies to augment the host immune response against cancer cells include vaccinations by gene-modified tumor cells or pulsing antigen-presenting cells (APC) with tumor-associated antigens (7). Recent studies have shown that vaccination of tumor cells fused with APCs such as dendritic cells (DC) or B cells could effectively elicit a host T-cell–mediated antitumor response (8–10). Such fusion cells present tumor-specific antigens, including known and unknown molecules, together with costimulatory molecules. Furthermore, fusion cells of allogeneic DCs and tumor cells express allogeneic MHC molecules, which would also stimulate alloreactive T cells (11–14) and support the induction of a potent reaction against tumor cells. These advantages of allogeneicity prompted us to investigate whether the allogeneic APC-cancer fusion cells induce a stronger reaction than syngeneic fusion cells.

Interleukin (IL)-12 is a heterodimeric proinflammatory cytokine produced by macrophages, DCs, and B cells, which stimulates T lymphocytes and natural killer cells to release IFN-{gamma} and promotes an antitumor response (15, 16). Recent studies fromus as well as others have shown that IL-12 has powerful adjuvant properties, and the immune-modulating characteristics of IL-12are considered to be responsible for such adjuvant effects (17–20).

In this study, we first compared the antitumor effect of syngeneic and allogeneic APC-cancer fusion cells. Secondly, we investigated the adjuvant effect of secreted form of IL-12 on fusion cell immunotherapy. A combination of allogeneic APCs and IL-12-secreting cancer cells may provide us with a promising strategy for cancer therapy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice. C57BL/6 (H-2b) and BALB/c (H-2d) female mice, 6 to 8 weeks old, were obtained from Japan Charles River (Atsugi, Japan).

Cell Culture. The colon-26 adenocarcinoma cell line (C26) (H-2d) and BALB/3T3 fibroblast cell line (H-2d) were obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). The cell lines were maintained in complete RPMI 1640 (10% fetal bovine serum, 100 µg/mL streptomycin, and 100 units/mL penicillin). DCs were generated from mouse bone marrow precursors in complete RPMI 1640 with 10 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN) and 2 ng/mL recombinant mouse IL-4 (R&D Systems) as described previously (21). Briefly, on days 2 and 4, nonadherent cells were gently removed and fresh medium was added. On day 6, loosely adherent proliferating DC aggregates were dislodged and replated. On day 8 of culture, released nonadherent cells with the typical morphologic features of DCs were used for the experiment. Macrophages were harvested by peritoneal lavage with ice-cold PBS 4 days after i.p. injection with 2 mL sterile 4% thioglycollate broth. For phenotypic analysis, DCs and macrophages were stained with a panel of antibodies, including MHC class I and II, CD80, CD86, CD11b, and CD11c (PharMingen, San Diego, CA) and quantified by flow cytometry (Becton Dickinson, San Jose, CA).

Fusion Cells. The fusion cells were generated by electrical pulse (electrofusion; refs. 22, 23). Cells were mixed in 0.3 mol/L sucrose and first aligned by direct current at 100 V for 10 seconds. Electrofusion was driven by a Bio-Rad gene pulser (Bio-Rad, Hercules, CA; 600 V, 25mFd). DC and C26 were stained with CellTracker green CMFDA and orange CMTMR fluorescent dyes (Molecular Probes, Eugene, OR), respectively. DC, C26, mixture of DC and C26 (Mix), and DC-C26 fused cells (Fusion) were analyzed by flow cytometry. To verify surface phenotype of the fusion cells, C26 tumor cells were prelabeled with CMTMR. After electrofusion, both syngeneic and allogeneic DC-cancer fusion cells were detected by staining with FITC-conjugated antibodies against MHC class II, CD80, CD86, CD11c, and ICAM-I.

Generation of IL-12-Secreting C26 Cells. The retroviral vector, TFG-m-IL-12 (kindly provided by Dr. H. Tahara, University of Tokyo, Tokyo, Japan), encodes both p35 and p40 murine IL-12 subunits with the IRES construct and selectable neomycin phosphotransferase gene under long terminal repeat control (24). C26 cells (5 x 105 per six-well dish) were infected with the viral supernatant of the producer cell lines in the presence of polybrene (8 µg/mL; ref. 17). Infected cells were subsequently selected by G418 (600 µg/mL). These cells were continuously cultured in bulk and used with DCs for the generation of fusion cells.

Tumor Model and Immunization. Tumor cells (C26, 5 x 105) were implanted s.c. in the midflank of BALB/c mice. On days 3 and 10, tumor-bearing mice were injected i.p. with 2 x 106 syngeneic DC-C26 mixed cells, syngeneic and allogeneic DC-C26 fusion cells, or syngeneic and allogeneic macrophage-C26 fusion cells. Tumor-bearing control mice were injected i.p. with PBS instead of cells on days 3 and 10. All cells used for immunization were irradiated (40 Gy) before injection. Two perpendicular diameters of tumors were measured using calipers. Tumor volumes were calculated as described previously (17). In preliminary experiments to address the different routes of vaccine administration to induce antitumor immunity, tumor-bearing mice were immunized s.c. or i.p. with allogeneic DC-cancer fusion cells. Because the tumor size showed no significant difference between these two groups, we did i.p. injection of fusion cells in this study.

CTL Assays. Procedures to measure CTL activity have been described previously (21). In brief, splenocytes were isolated 10 days after the last immunization (day 20). Effector cells were restimulated by coculturing splenocytes (3 x 106 cells) with mitomycin C–treated tumor cells (106 cells) in 24-well culture plates. The mitomycin C treatment (100 µg/mL) was done to block the cell division of the tumor cells before coculture with splenocytes. After restimulation, viable effector cells were collected and tested for their ability to lyse target cells by quantitatively measuring lactate dehydrogenase release using a CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). C26 tumor cells (H-2d) and BALB/3T3 fibroblast cells (H-2d) were used for tumor-specific and nonspecific target cells, respectively. The percentage of cytotoxicity was calculated as 100 x ([experimental release] – [effector spontaneous release] – [target spontaneous release]) / ([target maximum release] – [target spontaneous release]). Results are representative of three independent experiments.

Cytokine Assays. Splenocytes (3 x 106) obtained from immunized mice on day 20 were cultured with mitomycin C–treated tumor cells (1x106) in 24-well culture plates. Secretion of IFN-{gamma}, tumor necrosis factor-{alpha} (TNF-{alpha}), IL-4, IL-10, and transforming growth factor-ß (TGF-ß) from splenocytes was determined by ELISA kits (IFN-{gamma}, TNF-{alpha}, IL-4, and IL-10, Biosource International, Camarillo, CA; TGF-ß1Emax Immunoassay System, Promega). The levels of murine IL-12 p40 released from retrovirus-transfected cells and fusion cells were assessed by an ELISA kit(Biosource International; pg/106 cells/24 hours). CD4+ and CD8+ enriched T cells were positively selected from splenocytes by paramagnetic beads conjugated with anti-CD4 and anti-CD8a monoclonal antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively. Each purified CD4+ or CD8+ cells (3x 105) were cultured with mitomycin C–treated tumor cells (1 x 105). Secretion of IFN-{gamma} and IL-4 by purified cells was determined by ELISA as described above. All cytokines were measured for five samples each group.

Statistical Analysis. All data are reported as mean ± SE. Statistical comparison was done using the two-tailed Student t test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of DC-Tumor Fusion Cells and Antitumor Effect. DCs were stained with CellTracker green CMFDA and cancer cells (C26) with orange CMTMR. Both cells were then subjected to electrofusion, which generated CMFDA-CMTMR double-positive fusion cells at consistent yield of ~30% (Fig. 1A). The fusion cells expressed both tumor and DC surface molecules, such as MHC class I (H-2Kd on C26 cancer cells and H-2Kb on C57BL/6 mice-derived allogeneic DC) and class II (I-Ab), CD80, CD86 (Fig. 1B), CD11c, andICAM-I (data not shown). Both syngeneic and allogeneic DC-cancer fusion cells showed comparable phenotype (syngeneic DC-cancer fusion cells: MHC class II 23.7 ± 3.1%, CD80 33.9 ± 3.8%, CD86 28.8 ± 2.4%, CD11c 23.1 ± 1.5%, ICAM-I 23.9 ± 2.7%; allogeneic DC-cancer fusion cells: MHC class II 20.4 ± 4.8%, CD80 35.2 ± 4.5%, CD86 25.9 ± 2.9%, CD11c 21.9 ± 3.1%, ICAM-I 24.4 ± 4.5%). Mice treated with only irradiated cancer cells or with simple mixture of syngeneic DCs and cancer cells did not show significant antitumor effects compared with that with PBS control. Immunization with the syngeneic DC-cancer fusion cells significantly more suppressed the tumor volume than immunization with PBS or mixture of syngeneic DCs and cancer cells (Fig. 1C).



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 1 Generation and functional analysis of DC-cancer fusion cells. A, generation of fusion cells. DCs and C26 cells were stained with CellTracker green CMFDA and orange CMTMR fluorescent dyes, respectively. The fusion cells were generated by electrical pulse (electrofusion). DC, C26, mixture of DC and C26 (Mix), and DC-C26 fused cells (Fusion) were analyzed by flow cytometry. Note that a typical example in A shows a yield of 31.9%. B, phenotype of electrofusion cells. Mixture (top) and electrofusion (bottom) of allogeneic DCs and C26 cancer cells were analyzed by bidimensional flow cytometry for the expression of MHC class I (H-2Kd) and MHC class I (H-2Kb), MHC class II (I-Ab), CD80, or CD86. Note that fusion cells express both molecules of DCs and C26 cells. C, in vivo antitumor immunity by fusion cells. Tumor cells (C26, 5 x 105) were implanted s.c. in the midflank of BALB/c mice. On days 3 and 10, tumor-bearing mice were treated by i.p. injection of syngeneic DC-C26 mixture cells ({bullet}), syngeneic DC-C26 fusion cells ({blacksquare}), PBS control ({blacklozenge}), or only C26 cancer cells ({triangleup}). All immunized cells were used after irradiation (40 Gy). Tumor volumes were presented as the mean ± the standard error (n = 10 mice in each group). The data shown were representative of three independent experiments. *, P < 0.01 versus PBS control.

 
Antitumor Effect by Syngeneic and Allogeneic APC-Tumor Fusion. Examination of the syngeneic and allogeneic APCs as fusion partners revealed significant antitumor effect with the use of allogeneic APCs (Fig. 2). We compared the antitumor immunity elicited by syngeneic DC-cancer fusion cells and allogeneic DC-cancer fusion cells, where tumor inoculated mice were immunized on days 3 and 10 with syngeneic (BALB/c) DC-cancer fusion or allogeneic (C57BL/6) DC-cancer fusion cells. As shown in Fig. 2A, allogeneic DC-cancer fusion cells exerted a significantly more antitumor effect than the syngeneic DC-cancer fusion cells (tumor volume on day 19: PBS control 839 ± 69 mm3, syngeneic DC-cancer fusion 333 ± 81 mm3, and allogeneic DC-cancer fusion 215 ± 40 mm3). To confirm the induction of tumor-specific CTL, the splenocytes from mice immunized with both fusion cells were analyzed after restimulation with cancer cells. Splenocytes immunized by either syngeneic or allogeneic DC fusion cells showed tumor-specific cytotoxicity against C26 target cells (Fig. 2B). The cell lysis was not observed for BALB/3T3 fibroblast target cells (data not shown), indicating the lysis is C26 cancer cell specific.



View larger version (29K):
[in this window]
[in a new window]
  		Fig. 2 Antitumor immunity by syngeneic and allogeneic DC-Macrophage-cancer fusion cells. A, antitumor immunity induced by DC-cancer fusion cells. Tumor cells (C26, 5 x 105) were implanted s.c. in the midflank of BALB/c mice. On days 3 and 10, tumor-bearing mice were treated by i.p. injection of syngeneic DC-C26 fusion cells ({bullet}), allogeneic DC-C26 fusion cells ({blacksquare}), or PBS control ({blacklozenge}). All cells were immunized after irradiation (40 Gy). Tumor volumes were presented as the mean ± the standard error (n = 10 mice in each group). The data shown were representative of three independent experiments. *, P < 0.01 versus PBS control. B, tumor-specific CTL induction by DC-cancer fusion cells. BALB/c mice implanted with C26 tumor cells were immunized by i.p. injection of syngeneic DC-C26 fusion cells ({bullet}), allogeneic DC-C26 fusion cells ({blacksquare}), or PBS control ({blacklozenge}) on days 3 and 10. Splenocytes were isolated 10 days after the last vaccination (day 20). After restimulation in vitro for 5 days with mitomycin C–treated tumor cells, viable effector cells were collected and incubated at the indicated E:T with C26 cells for cytolytic assay. In this and other CTL assay experiments, splenocytes stimulated in the same manner failed to show any significant cytotoxic activity against BALB/3T3 fibroblast (nonspecific) target cells (%Lysis < 10). C, antitumor immunity induced by macrophage-cancer fusion cells. Tumor cells (C26, 5 x 105) were implanted s.c. in the midflank of BALB/c mice. On days 3 and 10, tumor-bearing mice were treated by i.p. injection of syngeneic macrophage (M{Phi})-C26 fusion cells ({bullet}), allogeneic macrophage-C26 fusion cells ({blacksquare}), or PBS control ({blacklozenge}). All cells used for immunization were irradiated (40 Gy). Points, mean tumor volumes (n = 10 in each group); bars, SE. Representative of three independent experiments. *, P < 0.01 versus PBS control. D, tumor-specific CTL induction by macrophage-cancer fusion cells. Splenocytes isolated on day 20 from mice immunized with syngeneic macrophage-C26 fusion ({bullet}), allogeneic macrophage-C26 fusion cells ({blacksquare}), or PBS control ({blacklozenge}) on days 3 and 10 were assessed for the cytolytic function as described above.

 
We then examined the effects of allogeneic or syngeneic fusion cells with other APCs on tumor immunity. Peritoneal macrophages generated fusion cells at a similar yield with DCs (~30%), and the fusion cells expressed MHC class I and II, CD11b, CD80, and CD86 (data not shown). Mice treated with the macrophage-cancer fusion cells showed significant antitumor effects in the therapeutic model (tumor volume on day 19: PBS control 839 ± 69 mm3, syngeneic macrophage-cancer fusion 477 ± 95 mm3, and allogeneic macrophage-cancer fusion 316 ± 70 mm3; Fig. 2C). The allogeneic macrophage-cancer fusion cells showed stronger antitumor immunity than did the syngeneic macrophage-cancer fusion cells. Tumor-specific CTLs against C26 target cells were induced by syngeneic and allogeneic macrophage-cancer fusion cells (Fig. 2D), although nonspecific cell lysis against BALB/3T3 cells was not observed (data not shown).

Functional Analysis of Splenocytes from Immunized Mice. Analysis of the cytokine production of splenocytes from mice immunized with APC-cancer fusion cells revealed significant differences according to the DC-macrophage and the syngeneic or allogeneic background (Fig. 3). IFN-{gamma} production from splenocytes was twice as high in mice immunized with DC-cancer fusion cells than in those immunized with macrophage-cancer fusion cells (Fig. 3A). The TNF-{alpha} production showed similar profiles in the mice with the DC-cancer combination and those with the macrophage-cancer combination (Fig. 3C). In marked contrast, both IL-4 and IL-10 production were high in splenocytes from mice immunized with syngeneic DC-cancer fusion cells but not in those with the other combinations (Fig. 3B and D). TGF-ß production was not affected in the mice with the syngeneic or allogeneic and DC-macrophage combination (Fig. 3E). To characterize the cytokine-producing cells, we specifically analyzed IFN-{gamma} and IL-4 secretion by purified CD4+ or CD8+ T cells. Whereas IFN-{gamma} was secreted from mainly CD4+ and partly CD8+ T cells, IL-4 was produced only by CD4+ T cells but not by CD8+ T cells (Fig. 3A and B). The differences in these cytokine profiles might have affected the antitumor activity.



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 3 Cytokine production from immunized splenocytes. Tumor-bearing mice were treated with syngeneic DC-C26 fusion, allogeneic DC-C26 fusion, syngeneic macrophage-C26 fusion, allogeneic macrophage-C26 fusion, or PBS control cells on days 3 and 10, and the splenocytes harvested on day 20 were cocultured with mitomycin C–treated C26 cells. Secretion of (A) IFN-{gamma}, (B) IL-4, (C) TNF-{alpha}, (D) IL-10, and (E) TGF-ß from splenocytes was determined by ELISA (n = 5 in each group). Analysis of IFN-{gamma} and IL-4 secretion by purified CD4+ or CD8+ T cells from immunized splenocytes were shown in A and B. Light gray bars, cytokine secretion by CD4+ T cells; dark gray bars, CD8+ T cells.

 
In vivo Functional Analysis of IL-12-Secreting DC-Tumor Fusion Cells. IL-12 is a pivotal cytokine that elicits strong antitumor effects by stimulating immune cells, including T cells and natural killer cells (15). To enhance the antitumor immunity by DC-cancer fusion cells, cancer cells that had been retrovirally transferred the secreted form of IL-12 gene were fused with syngeneic or allogeneic DCs (Fig. 4). The IL-12 transfected C26 cells expressed murine IL-12 (2,796 ± 122 pg/mL/106 cells/24 hours), and the derived fusion cells showed large amounts of IL-12 production (syngeneic DC-IL-12 C26 fusion: 1,548 ± 265 pg/mL/106 cells/24 hours; allogeneic DC-IL-12 C26 fusion: 1,593 ± 289/mL/106 cells/24 hours; Fig. 4A). The IL-12 production was not affected by 40 Gy irradiation before vaccination (data not shown). When tumor-bearing mice were immunized with IL-12-secreting fusion cells on days 3 and 10, augmented antitumor immunity was observed compared with the mice immunized with allogeneic DC-cancer fusion cells (tumor volume on day 19:PBS control 839 ± 69 mm3, syngeneic DC IL-12 fusion 169 ± 30mm3, and allogeneic IL-12 DC fusion 157 ± 15 mm3; Fig. 4B). Although IL-12-secreting allogeneic DC-cancer fusion cells showed no stronger effect than IL-12-secreting syngeneic ones in vivo, tumor-specific CTL induction was more powerful in IL-12-secreting allogeneic DC-cancer fusion cells (Fig. 4C). The cytokine production by splenocytes immunized with IL-12-secreting fusion cells showed a similar profile to that by immunized with DC-cancer fusion cells without IL-12 secretion (Fig. 4D). Whereas IFN-and TNF-secretion were not affected byIL-12 gene transfer, both IL-4 andIL-10 secretion were significantly suppressed by IL-12 expression (Figs. 3C and D and 4D). Specific analysis showed that CD4+ T cells were involved in the secretion of IFN-and IL-4 and CD8+ T cells also have some role of producing IFN-(Fig. 4D). IL-12-secretingDC-cancer fusion cells most suppressed the tumor growth (Fig. 5).



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 4 Generation and functional analysis of IL-12-secreting DC-cancer fusion cells. (A) IL-12 production by fusion cells. Murine (IL-12) gene was introduced into C26 cells via retroviral vector. These IL-12 gene-modified C26 cells (IL-12-C26) were fused with syngeneic (BALB/c) DCs and allogeneic (C57BL/6) DCs by electrofusion. Secretion of IL-12 from these cells (pg/106 cells/24 hours) was measured by ELISA. B, enhanced antitumor effect by IL-12-secreting DC-cancer fusion cells. Tumor cells (C26, 5 x 105) were implanted s.c. in the midflank of BALB/c mice. On days 3 and 10, tumor-bearing mice were treated by i.p. injection of syngeneic DC-IL-12 C26 fusion cells ({circ}), allogeneic DC-IL-12 C26 fusion cells ({square}), or PBS control ({blacklozenge}) after irradiation (40 Gy). Tumor volumes were presented as the mean ± the standard error (n = 10 mice in each group). The data shown were representative of three independent experiments. *, P < 0.01 versus PBS control. C, induction of tumor-specific CTL by IL-12-secreting DC-cancer fusion cells. Splenocytes isolated from mice immunized with syngeneic DC-IL-12 C26 fusion ({circ}), allogeneic DC-IL-12 C26 fusion cells ({square}), or PBS control ({blacklozenge}) were assessed for the cytolytic function. D, cytokine production from immunized splenocytes. Tumor-bearing mice were immunized with syngeneic DC-IL-12 C26 fusion, allogeneic DC-IL-12 C26 fusion cells, or PBS control on days 3 and 10, and the splenocytes harvested on day 20 were cocultured with mitomycin C–treated C26 cells. Secretion of IFN-{gamma}, IL-4, TNF-{alpha}, IL-10, and TGF-ß in the culture medium was measured by ELISA (n = 5 in each group). Light gray bars, analysis of IFN-{gamma} and IL-4 secretion by purified CD4+ T cells; dark gray bars, analysis of IFN-{gamma} and IL-4 secretion by purified CD8+ T cells.

 


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 5 Summary of antitumor effect by immunization with fusion cells. Average tumor volume on day 19 immunized with PBS control, syngeneic macrophage-cancer fusion, allogeneic macrophage fusion, syngeneic DC fusion, allogeneic DC fusion, syngeneic DC IL-12 fusion, and allogeneic DC IL-12 fusion cells were plotted (n = 10 in each group).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Without destroying the immune barrier mechanisms of established solid tumors, there can be no effective therapy to eradicate cancer cells (25). Vaccination of tumor cells artificially fused with APCs is an attractive approach to overcome the immune escape mechanism that results from the reduced MHC expression or suppressed T-cell activation in the tumor-bearing condition (8–10, 22, 23, 26–34) . The theoretical advantages of immunogenic fusion cells include the possible involvement of both known and unidentified tumor antigens and further immune activation. Among several kinds of cancer immunotherapy, the use of synthetic short peptide antigens has been widely applied (7). A recent report, however, described the occurrence of post-translational protein splicing in a human renal cancer (32) and a melanoma cell (33), suggesting yet unknown immune escape mechanism. A method using whole tumor cells with APCs, including the tumor-DC fusion strategy, is possibly effective against such cancer cells.

In this study, we showed for the first time that macrophages as well as DCs are a promising fusion partner for generating a hybrid vaccine to be used in the treatment of cancer. Originally, B cells were used as a fusion partner for an effective tumor vaccine by Guo et al. (8). Gong et al. first used DCs as a fusion partner and reported that immunization with DC-cancer fusion cells induced the rejection of established metastasis in mice experiments (10). Several reports showed intriguing results with human DC-cancer fusion cells, demonstrating a regression of metastatic renal cell carcinoma (22), metastatic melanoma (26), and glioma (27). However, macrophages have never been proposed as a fusion partner. Because macrophages play an important role in the host defense against pathogens, wound healing, angiogenesis, various types of acute and chronic inflammation, and cancer immunity, the utilization of macrophages for fusion with cancer cells seemed worth investigating. Although we directly induced electrofusion after harvesting thioglycollate-stimulated peritoneal macrophages without any further in vitro treatment, the vaccination with these products showed effective antitumor activity in our therapeutic mouse model (Fig. 2C and D). Differences in the effects between DCs and macrophages might have been due to the lower production of IFN-{gamma} shown in the macrophage-cancer cell fusion experiment (Fig. 3A). However, a possible advantage of the use of macrophages is the availability and abundance of such cells: human alveolar macrophages can be harvested by bronchoalveolar lavage and readily transformed into fusion cells, sterilized by irradiation, and vaccinated.

We next showed allogeneic APCs were better fusion partner with cancer cells than syngeneic APCs. Although several studies of cancer immunotherapy using fusion cells with allogeneic DCs and autologous tumor cells have been conducted in clinical settings (22), there has been little evidence supporting the validity of using allogeneic cells (28, 29). Recent reports on the existence of peripheral T cells responsive to alloantigen provide convincing support for the notion of using allogeneic DC-cancer fusion cells to induce a stronger reaction than that by syngeneic fusion cells. The frequency of peripheral T cells that recognize alloantigen has been reported to be between 1% and 10%, higher than the percentage of T cells that respond to foreign antigens (11–13), suggesting the advantage of allogeneicity as a vaccination compartment.

To clarify the underlying mechanism of the superior antitumor effect by allogeneic DC-cancer fusion cells, we examined the tumor-specific CTL activities and the cytokine profiles of splenocytes from immunized mice. Although there were no different CTL activities between mice treated with syngeneic and allogeneic DC-cancer fusion cells (Fig. 2B), several different cytokine profiles were detected between them (Fig. 3). Allogeneic DC-cancer fusion cells elicited a stronger antitumor effect because they induced sufficient secretion of Th1 cytokine IFN-{gamma} and lower production of Th2 cytokines IL-4 and IL-10 than syngeneic DC-cancer fusion cells did (Figs. 3 and 4). In contrast, immunization with syngeneic DC-cancer fusion cells efficiently induced the production of both Th1- and Th2-type cytokines (Fig. 3). Further analysis revealed that CD4+ T cells mainly secreted IFN-{gamma}, suggesting the important role of Th1 CD4+ T cells, and CD8+ T cells also contribute to produce IFN-{gamma} (Fig. 3A). IL-4 was secreted only by CD4+ Th2 cells in mice treated with syngeneic DC-cancer fusion cells (Fig. 3B). Tanaka et al. (30) reported that IL-10 production induced by syngeneic DC-cancer fusion cells caused insufficient tumor eradication and that systemic IL-12 administration was required for effective antitumor effects in mice experiment. In contrast, we showed a significant therapeutic antitumor effect especially by allogeneic DC-cancer fusion cells, which could effectively elicit a cytokine profile of high IFN-{gamma} and TNF-{alpha} and low IL-4 and IL-10 (tumor growth suppression compared with tumor volume of PBS control on day 19: syngeneic DC-cancer fusion 60% reduction and allogeneic DC-cancer fusion 74% reduction; Figs.2A and 5). These findings showing allogeneic DC as a superior fusion partnerare consistent with those of a recent report of on the use of allogeneic DCs by Siders et al. (29).

Although the method of fusing APCs and cancer cells can be used to suppress tumor growth, additional immunomodulation will be required to further potentiate the immune response. For this purpose, we investigated the use of IL-12 gene transfer to cancer cells and examined the effect of IL-12 secretion in the local milieu by gene-modified fusion cells. Immunization with both syngeneic and allogeneic IL-12 gene-modified fusion cells showed highly augmented antitumor effects in our therapeutic model (tumor growth suppression compared with tumor volume of PBS control on day 19: syngeneic DC-IL-12-cancer fusion 76% reduction and allogeneic DC-IL-12-cancer fusion 78% reduction; Figs. 4B and 5). In addition, DC-IL-12-cancer fusion cells induced stronger tumor-specific CTL activities compared with DC-cancer fusion cells (no gene-modified fusion cells; Figs. 2B and 4C). Furthermore, IL-12 gene-modified fusion cells elicited strong Th1 response and enhanced the suppression of the Th2 response (Figs. 3 and 4D), thus inducing augmented antitumor immunity (Fig. 4B). Although DC-IL-12-cancer fusion cells strategy led to the good results, no difference was observed between antitumor effects by using syngeneic and allogeneic DCs as fusion partners. This is partly because that the enough effect by IL-12 canceled out the advantage of allogeneicity.

Studies on fusion cells composed of DCs and gene-modified cancer cells have provided further insight in tumor immunology. Kao et al. (34) reported that mice vaccinated with TGF-ß-secreting fusion cells had lower tumor-specific CTL activity and had lower survival after tumor challenge as compared with control animals. Liu et al. (35) showed that IL-4 gene-modified fusion vaccine enhanced antitumor immunity in a tumor protection model, suggesting the possible importance of stimulating the DC maturation mechanism. In contrast, the present study showed that IL-12-secreting fusion cells directly enhanced tumor-specific CTL activity, induced stronger Th1 but suppressed Th2 cytokine production, and exerted potent antitumor effects in vivo. Among the variety of the choices of immune modulatory molecules used in preclinical animal models, the IL-12 gene-modified vaccine through affecting the accumulating host immune cells shows promise for future clinical use.


   ACKNOWLEDGMENTS
 
We thank Dr. H. Tahara for providing the retroviral vector TFG-m-IL-12, Dr. T. Takai for supporting flow cytometry analysis, and Brent Bell for reading the article.


   FOOTNOTES
 
Grant support: Ministry of Education, Science, Sports, Culture, and Technology of Japan Grants-in-Aid for Scientific Research 13877090 and 15790406.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/20/04; revised 9/28/04; accepted 10/ 7/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Marincola FM, Jaffee EM, Hicklin DJ, Ferrone S. Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance. Adv Immunol 2000;74:181–273.[Medline]
  2. Elliott BE, Carlow DA, Rodricks AM, Wade A. Perspectives on the role of MHC antigens in normal and malignant cell development. Adv Cancer Res 1989;53:181–245.[Medline]
  3. Maeurer MJ, Gollin SM, Martin D, et al. Tumor escape from immune recognition: lethal recurrent melanoma in a patient associated with downregulation of the peptide transporter protein TAP-1 and loss of expression of the immunodominant MART-1/Melan-A antigen. J Clin Invest 1996;98:1633–41.[Medline]
  4. Seliger B, Maeurer MJ, Ferrone S. TAP off-tumors on. Immunol Today 1997;18:292–9.[CrossRef][Medline]
  5. Chen L, Ashe S, Brady WA, et al. Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4. Cell 1992;71:1093–102.[CrossRef][Medline]
  6. Townsend SE, Allison JP. Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells. Science 1993; 259:368–70.[Abstract/Free Full Text]
  7. Ribas A, Butterfield LH, Glaspy JA, Economou JS. Current developments in cancer vaccines and cellular immunotherapy. J Clin Oncol 2003;21:2415–32.[Abstract/Free Full Text]
  8. Guo Y, Wu M, Chen H, et al. Effective tumor vaccine generated by fusion of hepatoma cells with activated B cells. Science 1994;263:518–20.[Abstract/Free Full Text]
  9. Stuhler G, Walden P. Recruitment of helper T cells for induction of tumour rejection by cytolytic T lymphocytes. Cancer Immunol Immunother 1994;39:342–5.[Medline]
  10. Gong J, Chen D, Kashiwaba M, Kufe D. Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells. Nat Med 1997;3:558–61.[CrossRef][Medline]
  11. Ford WL, Atkins RC. The proportion of lymphocytes capable of recognizing strong transplantation antigens in vivo. Adv Exp Med Biol 1973;29:255–62.[Medline]
  12. Ford WL, Simmonds SJ, Atkins RC. Early cellular events in a systemic graft-vs.-host reaction. II. Autoradiographic estimates of the frequency of donor lymphocytes which respond to each Ag-B-determined antigenic complex. J Exp Med 1975;141:681–96.[Abstract/Free Full Text]
  13. Sherman LA, Chattopadhyay S. The molecular basis of allorecognition. Annu Rev Immunol 1993;11:385–402.[CrossRef][Medline]
  14. Suchin EJ, Langmuir PB, Palmer E, Sayegh MH, Wells AD, TurkaLA. Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question. J Immunol 2001;166:973–81.[Abstract/Free Full Text]
  15. Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3:133–46.[CrossRef][Medline]
  16. Trinchieri G. Interleukin-12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 1995;13:251–76.[Medline]
  17. Tanaka M, Saijo Y, Sato G, et al. Induction of antitumor immunity by combined immunogene therapy using IL-2 and IL-12 in low antigenic Lewis lung carcinoma. Cancer Gene Ther 2000;7:1481–90.[CrossRef][Medline]
  18. Tahara H, Zeh HJ III, Storkus WJ, et al. Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo. Cancer Res 1994;54:182–9.[Abstract/Free Full Text]
  19. Salgaller ML, Lodge PA. Use of cellular and cytokine adjuvants in the immunotherapy of cancer. J Surg Oncol 1998;68:122–38.[CrossRef][Medline]
  20. Gong J, Koido S, Chen D, et al. Immunization against murine multiple myeloma with fusions of dendritic and plasmacytoma cells is potentiated by interleukin 12. Blood 2002;99:2512–7.[Abstract/Free Full Text]
  21. Kikuchi T, Maemondo M, Narumi K, Matsumoto K, Nakamura T, Nukiwa T. Tumor suppression induced by intratumor administration of adenovirus vector expressing NK4, a 4-kringle antagonist of hepatocyte growth factor, and naive dendritic cells. Blood 2002;100:3950–9.[Abstract/Free Full Text]
  22. Kugler A, Seseke F, Thelen P, et al. Autologous and allogenic hybrid cell vaccine in patients with metastatic renal cell carcinoma. Br J Urol 1998;82:487–93.[Medline]
  23. Scott-Taylor TH, Pettengell R, Clarke I, et al. Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines. Biochim Biophys Acta 2000;1500:265–79.[Medline]
  24. Tahara H, Zitvogel L, Storkus WJ, et al. T. Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector. J Immunol 1995;154:6466–74.[Abstract]
  25. Ochsenbein AF, Sierro S, Odermatt B, et al. Roles of tumour localization, second signals and cross priming in cytotoxic T-cell induction. Nature 2001;411:1058–64.[CrossRef][Medline]
  26. Trefzer U, Weingart G, Chen Y, et al. Hybrid cell vaccination for cancer immune therapy: first clinical trial with metastatic melanoma. Int J Cancer 2000;85:618–26.[CrossRef][Medline]
  27. Kikuchi T, Akasaki Y, Irie M, Homma S, Abe T, Ohno T. Results of a phase I clinical trial of vaccination of glioma patients with fusions of dendritic and glioma cells. Cancer Immunol Immunother 2001; 50:337–44.
  28. Gong J, Nikrui N, Chen D, et al. Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity. J Immunol 2000;165:1705–11.[Abstract/Free Full Text]
  29. Siders WM, Vergilis KL, Johnson C, Shields J, Kaplan JM. Induction of specific antitumor immunity in the mouse with the electrofusion product of tumor cells and dendritic cells. Mol Ther 2003;7:498–505.[CrossRef][Medline]
  30. Tanaka H, Shimizu K, Hayashi T, Shu S. Therapeutic immune response induced by electrofusion of dendritic and tumor cells. Cell Immunol 2002;220:1–12.[CrossRef][Medline]
  31. Phan V, Errington F, Cheong SC, et al. A new genetic method to generate and isolate small, short-lived but highly potent dendritic cell-tumor cell hybrid vaccines. Nat Med 2003;9:1215–9.[CrossRef][Medline]
  32. Hanada K, Yewdell JW, Yang JC. Immune recognition of a human renal cancer antigen through post-translational protein splicing. Nature 2004;427:252–6.[CrossRef][Medline]
  33. Vigneron N, Stroobant V, Chapiro J, et al. An antigenic peptide produced by peptide splicing in the proteasome. Science 2004; 304:587–90.
  34. Kao JY, Gong Y, Chen CM, Zheng QD, Chen JJ. Tumor-derived TGF-ß reduces the efficacy of dendritic cell/tumor fusion vaccine. J Immunol 2003;170:3806–11.[Abstract/Free Full Text]
  35. Liu Y, Zhang W, Chan T, Saxena A, Xiang J. Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity. Leuk Res 2002;26:757–63.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
B. Vasir, Z. Wu, K. Crawford, J. Rosenblatt, C. Zarwan, A. Bissonnette, D. Kufe, and D. Avigan
Fusions of Dendritic Cells with Breast Carcinoma Stimulate the Expansion of Regulatory T Cells while Concomitant Exposure to IL-12, CpG Oligodeoxynucleotides, and Anti-CD3/CD28 Promotes the Expansion of Activated Tumor Reactive Cells
J. Immunol., July 1, 2008; 181(1): 808 - 821.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Koido, E. Hara, S. Homma, M. Mitsunaga, A. Takahara, E. Nagasaki, H. Kawahara, M. Watanabe, Y. Toyama, S. Yanagisawa, et al.
Synergistic Induction of Antigen-Specific CTL by Fusions of TLR-Stimulated Dendritic Cells and Heat-Stressed Tumor Cells
J. Immunol., October 1, 2007; 179(7): 4874 - 4883.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
I. I. Slukvin, M. A. Vodyanik, J. A. Thomson, M. E. Gumenyuk, and K.-D. Choi
Directed Differentiation of Human Embryonic Stem Cells into Functional Dendritic Cells through the Myeloid Pathway.
J. Immunol., March 1, 2006; 176(5): 2924 - 2932.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Suzuki, T.
Right arrow Articles by Nukiwa, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Suzuki, T.
Right arrow Articles by Nukiwa, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation