Characterization of Structure and Direct Antigen Presentation by ...

ANTICANCER RESEARCH 33: 347-354 (2013)

Characterization of Structure and Direct Antigen Presentation by Dendritic/Tumor-fused Cells as Cancer Vaccines SHIGEO KOIDO2,3 and JIANLIN GONG1,3 1Department

of Medicine, Boston University School of Medicine, Boston, MA, U.S.A.; of Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University School of Medicine, Kashiwa, Chiba, Japan; 3Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, U.S.A.

2Division

Abstract. Background: Previous work has shown that fusion of dendritic cells (DCs) and tumor cells induces potent antitumor immune responses. However, little is known on whether fused cells directly present tumor-associated antigens (TAAs) through major histocompatibility complex (MHC) class I and II pathways in the context of costimulatory molecules. Materials and Methods: Fusion cells were generated between DCs and MC38 carcinoma cells stably expressing mucin-1 (MUC1) by polyethylene glycol. The characterization of structure and antigen presentation by fused cells was examined by immunoelectron microscopic and flow cytometric analyses. Results: The cytoplasm from both cellular entities was integrated, while their nuclei were independently preserved. Short-term culture gave fused cells sufficient time to integrate and directly display MUC1 through MHC class I and II pathways in the context of costimulatory molecules. Conclusion: DC-derived molecules and TAAs are presumably synthesized at separate sites of fused cells, to converge and complex with each other. Dendritic cells (DCs) are most potent antigen-presenting cells (APCs) that have been used in cancer vaccines because of their ability to initiate Cluster of Differentiation (CD)8+ cytotoxic T-lymphocyte (CTL)-mediated immune responses (1). Thus, DC-based cancer vaccines are considered to be a promising approach for boosting antitumor responses (1). Various strategies have been developed to deliver tumorassociated antigens (TAAs) into DCs with tumor RNA, tumor lysates, or apoptotic tumor cells to elicit and boost antitumor

Correspondence to: Shigeo Koido, Department of Gastroenterology and Hepatology, The Jikei University School of Medicine, 163-1 Kashiwa-shita, Kashiwa, Chiba 277-8564, Japan. Tel: +81 471641111, Fax: +81 471633488, e-mail: [email protected] Key Words: Dendritic cell, cancer vaccine, antigen presentation, fusion.

0250-7005/2013 $2.00+.40

immune responses (2-5). However, a major drawback of these strategies comes from the limited number of known tumor peptides available in many human leukocyte antigen (HLA) contexts. An alternative strategy for inducing antitumor immunity is the use of cells derived from fusion of DCs and tumor cells (6). The fusion of DCs and tumor cells through chemical (7-10), electrical (11-13), physical (14-16), or viral (17-19) means creates heterokaryons, combining the machinery needed for immune stimulation with presentation of a large repertoire of TAAs. The fusion approach offers several advantages for tumor-peptide presentation and subsequent induction of antitumor immunity including (2022): (i) processing and presentation of unidentified TAAs, thus circumventing the daunting task of identifying individual TAAs; (ii) presentation of multiple TAAs, thus increasing the frequency of responding T-cells and maximizing antitumor immunity; (iii) presentation of TAAs in the context of abundant co-stimulatory molecules, thus avoiding the potential induction of tolerance; and (iv) activation of polyclonal CD4+ and CD8+ T-cells, thus providing T-cell help for the induction of CTL responses. In DC/tumor-fused cells, efficient CTL induction is closely correlated with the level of the fusion efficiency (23). Therefore, we attempted to apply simple techniques to enrich DC and tumor cell fusion and examined the events surrounding the direct presentation of TAAs by immunoelectron microscopic analysis.

Materials and Methods Cell culture. Murine MC38 colon adenocarcinoma (C57BL/6) cell line stably transfected with a MUC1 cDNA (MC38/MUC1) (6, 24) was maintained in Dulbecco's modified Eagle’s minimal essential medium (DMEM), supplemented with 10% heat-inactivated (Fetal Calf Serum) FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 400 μg/ml geneticin (G418; Life Technologies, Tokyo, Japan). DCs were generated from the bone marrow of wild-type C57BL/6 mice by culture in 20 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF) (Sigma, St. Louis, MO, USA) for five days, as described elsewhere (25).

347

ANTICANCER RESEARCH 33: 347-354 (2013) Fusion of DCs and tumor cells. Fused cells were generated with purified DCs and MC38/MUC1 in the presence of polyethylene glycol (PEG) (molecular weight=1,450) in dimethyl sulfoxide (DMSO) solution (Sigma-Aldrich, St. Louis, MO, USA), as described elsewhere (6). Briefly, DCs and MC38/MUC1 cells were mixed at a 10:1 ratio in serum-free pre-warmed RPMI-1640. After centrifugation, the mixed cell pellets were gently resuspended in pre-warmed 50% PEG solution (1 ml per 5×107 cells) for 5 min at room temperature. Subsequently, the PEG solution was diluted by slow addition and mixing with 1, 2, 4, 8, and 16 ml of serum-free pre-warmed medium to a volume of 50 ml. Cell pellets obtained after centrifuging at 170 ×g (1,000 rpm) were resuspended in RPMI1640 medium supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 10 mM nonessential amino acids, 1 mM sodium pyruvate, 10% NCTC-109 medium, 10 U/ml penicillin, 100 μg/ml streptomycin, and 10 ng/ml recombinant murine GM-CSF, and cultured for eight days. Unfused tumor cells grow firmly attached to the plates, whereas fused DC/tumor cells (DC/MUC1) grow loosely in the wells and are suspended in the medium. Fused cells were selected and purified by gentle pipetting, and firmly attached tumor cells were discarded. Flow cytometry. DCs, MC38/MUC1, and DC/MUC1 were incubated with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAb) against MUC1 (HMPV; BD Pharmingen, San Diego, CA, USA) (26-28) and phycoerythrin (PE)-conjugated mAb against MHC class II (M5/114; BD Pharmingen) for 45 min on ice. Cells were washed, fixed, and analyzed by flow cytometry (FACScan; BD Immunocytometry System, NJ, USA) with the CellQuest software (BD Biosciences, NJ, USA). The cell aggregates were eliminated by gating-out before flow cytometric analysis. The fusion efficiency was determined by dual expression of tumor marker, MUC1, and DC marker, MHC class II molecules. Transmission electron microscopy (TEM). For observation of cell morphology and intracellular structure, DC/MUC1 cells were stained with FITC-conjugated mAb against MUC1 (HMPV; BD Pharmingen) and PE-conjugated mAb against MHC class II (M5/114; BD Pharmingen), then sorted by MoFlo (Cytomation, Fort Collins, CO, USA) with Summit v3.0 analysis software. DCs, MC38/MUC1, and sorted DC/MUC1 fused cells were fixed with 1.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 1 h at 48˚C. The specimens were washed, treated with 1% osmium tetroxide in 0.1 M cacodylate buffer, and passed through an alcohol gradient. They were further treated with propylene oxide and then embedded in plastic resin. Ultrathin sections were cut with an MT2 Sorvall ultra microtome (Thermo Fisher Scientific, MA, USA) and examined with a JEOL 100 CX TEM (JEOL USA, Inc., MA, USA) (29). Immunoelectron microscopic analysis. Pre-embedded immunogold labeling techniques were used to determine the antigen-presentation on the sorted DC/MUC1 cell surface. Cells were pelleted in phosphate-buffered saline (PBS) containing 0.5 ml 1% egg albumin for 30 min and then incubated with a 1:100 dilution of mAb against MUC1 (HMPV; BD Pharmingen) for 45 min at 4˚C. Gold-conjugated mAb against mouse IgG (AutoProbe EM-G5, 1:10 dilution; Amersham Life Sciences, Arlington Heights, IL, USA) was added for 30 min at 4˚C. After washing twice with PBS, the cells were incubated with a 1:100 dilution of biotinylated mAb against MHC class II mAb (M5/114; BD Pharmingen), MHC class I (M1/42.3.9.8;

348

BD Pharmingen), or CD86 (GL1; BD Pharmingen) and then with streptavidin-gold-anti-rat IgG (AuroProbe EM-G10, 1:10 dilution; Amersham Life Sciences). The specimens were processed, sectioned, and examined with a JEOL100CX TEM to determine gold particlelabeled molecules on the cell surface. For subcellular localization of MHC class II and MUC1 antigenic peptides, the sorted DC/MUC1 cells were prepared for ultrathin cryosectioning and immunogold labeling (30). Briefly, cells were fixed in 2% paraformaldehyde and 1% acrolein for 3-4 days, washed twice in PBS with 0.15 M glycine, and finally embedded in 10% gelatin, which was solidified on ice. Small gelatin blocks were infiltrated with 2.3 M sucrose for 3 h at 4˚C and then frozen in liquid nitrogen. Ultrathin cryosections were cut and picked-up on carbon-coated gold grids. Immunogold labeling was then performed as described elswhere (18). Briefly, the ultrathin cryosections were carefully washed with PBS containing 0.5% bovine serum albumin (BSA) and 0.15% glycine, pH 7.4, blocked with 1% egg albumin in PBS, incubated with a 1:100 dilution of anti-MUC1 mAb for 30 min, and then washed and incubated with a 1:10 dilution of gold-conjugated anti-mouse IgG (5-nm particles). The sections were washed six times with PBS and stained with a 1:100 dilution of biotinylated-mAb against MHC class II mAb for 30 min, and incubated with a 1:10 dilution of gold-conjugated-streptavidin mAb against rat IgG (10-nm particles). The cryosections were washed and mounted on a thin film of 1.25% methylcellulose, and examined with a JEOL 100 CX TEM.

Results Characterization of DC/MUC1 cell preparations. Bone marrow-derived murine DCs were generated in the presence of GM-CSF for five days. DCs displayed a characteristic phenotype with expression of MHC class II molecules (Figure 1A). Moreover, MC38/MUC1 cells expressed high levels of MUC1 antigen (Figure 1B). In this study, fusions of DCs and MC38/MUC1 cells were generated through PEG treatment, which is a straightforward procedure (31). Generation of DCs with GM-CSF for more than seven days’ culture resulted in cell death and consequently reduced fusion efficiency through PEG treatment (data not shown). Therefore, DCs were generated in five days’ culture and immediately used for fusion in this study. After initiation of the fusion process, some DCs were fused to MC38/MUC1 cells, while most of the PEG-treated cells remained as cell aggregates (Figure 1C). When the cell aggregates were gated out, a small number of cells were double-positive for MUC1 and MHC class II. However, after eight days’ culture, the proportion of cell aggregates had decreased, whereas the double-positive populations were drastically increased (Figure 1D), suggesting that most of the cell aggregates are precursors of fused cells. After eight days’ culture, DC/MUC1 cells were integrated to a single entity and were loosely adherent to the culture dish, whereas tumor/tumor fusions and unfused tumor cells were attached firmly to the dish. Thus, it was possible to select the DC/MUC1 cells by gentle pipetting and then collecting the loosely adherent cells. In contrast, incubating the mixture of DCs and

Koido et al: Direct Antigen Presentation by DC/Tumor Fusions

Figure 1. Characterization of fused DC/tumor cell preparations. Cell morphology of DCs (A), MC38/MUC1 cells (B), fused DC/MUC1 cell preparations cultured for two days, after initiating the fusion process (C), DC/MUC1 fusion cell preparations cultured for eight days after initiating fusion process (D), and DCs mixed with MC38/MUC1 cells and cultured for eight days (E) were examined under an inverted microscope (left hand panel). Cells were stained with fluorescein isothiocyanate-anti-MUC1 and phycoerythrin-anti-MHC class II mAb. Cell aggregates were gated out (middle panel) at the time of fluorescence-activated cell sorting analysis (right hand panel). Similar results were obtained in repeated experiments. Magnification A-D, ×40; E, ×20.

MC38/MUC1 cells for eight days resulted in a small number of cells being positive for MUC1 and MHC class II (Figure 1E). These cells may have been DCs that had phagocytosed MC38/MUC1 fragments. The data indicate that physical contact is not sufficient to generate fused DC/tumor cells. Interestingly, viewing under the inverted microscope revealed morphological changes in DC/MUC1 cells (Figure 1D).

Morphology of DC/MUC1 cells. To examine the global and intracellular morphology, DC, MC38/MUC1, and sorted DC/MUC1 cells were preparad and viewed under TEM. DCs exhibited characteristic morphology, with a lobulated nucleus and a distinctive pattern of euchromatin and heterochromatin, and displayed short cytoplasmic processes (Figure 2A, left panel). In contrast, MC38/MUC1 cells had a large nucleus

349


Figure 2. Cell surface and intracellular structure of fused DC/MUC1 cells. Preparations of fused DC/MUC1 cells were stained with fluorescein isothiocyanate-anti-MUC1 and phycoerythrin-anti-MHC class II mAbs and sorted by MoFlo. Intracellular structure of DCs (left hand panel), MC38/MUC1 cells (middle panel), and sorted fused DC/MUC1 cells (right hand panel) were examined by immunoelectron microscopy (A, ×4,800; B, ×72,500). The lower right hand panel shows the integration of DCs and tumor cells (arrow). T-N, tumor nucleus; DC-N, DC nucleus.

with conspicuous chromatin and a relatively smooth cell surface (Figure 2A, middle panel). Compared with DCs and MC38/MUC1 cells, sorted DC/MUC1 cells had multiple nuclei derived from DCs and MC38/MUC1. Moreover, DC/MUC1 cells had short cytoplasmic processes (Figure 2A, right panel). Interestingly, the cytoplasm of DCs and MC38/MUC1 cells was integrated in sorted DC/MUC1 cells (Figure 2B, right panel, arrows), whereas their nuclei remained separate entities. Antigen presentation through MHC class I and class II pathways by DC/MUC1 cells. As previously reported (12, 22), both CD4+ and CD8+ T-cells are involved in induction of antitumor immunity by fused DC/tumor cells. Therefore, we hypothesize that the fused DC/tumor cells have the

350

ability to present TAAs directly through MHC class I and class II pathways in the presence of co-stimulatory molecules. To test this hypothesis, we used an immunoelectron microscopic technique that is able to bridge the information gap between molecular biology and ultrastructural studies, placing macromolecular functions within a cellular context. DCs, MC38/MUC1, and sorted DC/MUC1 cells were dually-stained with pre-embedded immunogold-labeled anti-MUC1 mAb (5-nm particles) and anti-MHC class I, class II, or CD86 mAb (10-nm particles), and examined by immunoelectron microscopy. DC/MUC1 cells expressed both MUC1 and MHC class II molecules on the surface (Figure 3A, right panel). Importantly, MUC1 and MHC class II molecules were co-localized on the DC/MUC1 cell surface, suggesting the physical association of these two


Figure 3. Co-localization of MUC1 and MHC class I and class II molecules in fused DC/MUC1 cells. Sorted fused DC/MUC1 cells were prepared, stained with pre-embedded immunogold-labeled anti-MUC1 mAb (gold 5-nm size) and anti-MHC class II mAb (gold 10-nm size) (A), anti-MHC class I mAb (gold 10-nm size) (B), or anti-CD86 mAb (gold 10-nm size) (C) and examined by immunoelectron microscopy (×47,500). The middle and right hand panels are enlarged views of the area shown in the inset of images in the left hand and middle panels, respectively, showing colocalization of MUC1 (narrow arrow) and MHC class II, class I, or CD86 (wide arrow) on the surface of fused DC/MUC1 cells.

molecules (Figure 3A, right panel). MUC1 and MHC class I molecules were also co-localized on the DC/MUC1 cell surface (Figure 3B, right panel). Although CD86 molecules were expressed on the fusion cells with MUC1 antigen, they were not as closely associated as those of MHC class I and class II with MUC1 (Figure 3C, right panel). These results suggest that the fused DC/MUC1 cells possess the ability to present MUC1 through both MHC class I and class II pathways in the presence of CD86. In contrast, DCs expressed MHC class II, CD86, and not MUC1 on the surface, while MC38/MUC1 were positive for MUC1 but not MHC class II and CD86 (data not shown).

Subcellular co-localization of MUC1 and MHC class II molecules in fused DC/MUC1 cells. Given the observation that co-localization of MUC1 and MHC class II molecules expression is found on the DC/MUC1 cell sufrace, it is of interest to determine how this antigen processing and presentation pathway is preserved in the fused cells. To address this issue, sorted DC/MUC1 cells were prepared for immunoelectron microscopic analysis. The labeling for MUC1 (5-nm particles) and MHC class II (10-nm particles) were co-localized in the subcellular structures of DC/MUC1 cells (Figure 4). The expression of MUC1 and MHC class II was well-preserved in the fused cells. In contrast, only MHC

351


Figure 4. Intracellular distribution of MUC1 and MHC class II molecules in fused DC/MUC1 cells. Ultrathin cryosections were stained with an immunogold-labeled anti-MUC1 (gold 5-nm size) mAb and an anti-MHC class II (gold 10-nm size) mAb and examined by immunoelectron microscopy (×72,500). The right hand panels are an enlarged view of vesicles in fused DC/MUC1 cells showing co-localization of MUC1 (narrow arrow) and MHC class II (wide arrow).

class II expression and MUC1 was detected in the subcellular structures of DCs and MC38/MUC1 cells, respectively (data not shown). This finding suggests that direct antigen presentation through MHC class II molecules can be sustained in fused DC/tumor cells.

Discussion Although the level of fusion efficiency is closely correlated with antitumor immunity (23), a variation of fusion efficiency has been reported (17, 32). Fusion efficiency, at least in part, depends on the cell types used for fusion (13) as well as the fusion methods used (33). There are some differences in the sensitivity of cells to PEG treatment (data not shown), thus, it is desirable to perform a dose–response test to evaluate the conditions of PEG treatment for cells and to determine the optimal exposure time. Moreover, any improper handling of cells, such as excessive force or deprivation of culture medium prior to fusion, may also have a detrimental effect on the health of cells and subsequently on fusion efficiency.

352

Unlike electrofusion, fusion by PEG treatment is an active and evolving process, thus, it is conceivable that a larger initial contact surface between the DC and tumor cell leads the integration of these cells. In this study, short-term culture of DC and tumor cells promoted fusion efficiency. The duration of culture of PEG-mediated cells may allow for the cells undergoing fusion sufficient time to integrate and display TAAs through MHC class I and class II pathways. Indeed, the fusion efficiency was lower immediately after the fusion process was initiated, however, a short-duration culture resulted in more than a 10-fold increase in fusion efficiency. Prolonged culture should be avoided since unfused tumor cells overgrow. Previously, most investigators vaccinated the fused cell preparations into patients shortly after the fusion process (34, 35). It is likely that such cell preparations may not at this time be in an optimal form to present TAAs to the host immune cells. Moreover, it is also useful to activate DCs by Toll-like receptor agonists before fusion (23). There are two different pathways for antigen presentation. Endogenously-synthesized proteins are processed and


presented through the MHC class I-restricted pathway. In contrast, exogenous proteins are processed and displayed in association with MHC class II molecules through the endocytic pathway (36). However, little is known whether fused DC/tumor cells can process and present TAAs directly through MHC class I and class II pathways in the presence of co-stimulatory molecules. By immunoelectron microscopic technique, we demonstrated structural evidence that the cytoplasm from both DCs and tumor cells were integrated, while their nuclei remained separate entities. Such a characteristic structure may make it possible to maintain the functions of both original cell types, including synthesis of TAAs and co-stimulatory molecules. Interestingly, MUC1 was co-localized with MHC class I, class II, and CD86 molecules on the surface of fused cells. Moreover, co-localization of MUC1 and MHC class II was also detected in the subcellular structures of fused DC/MUC1 cells. These findings suggest that the endocytic pathway of antigen processing and presentation in fused DC/tumor cells is not interrupted by the fusion process. DC-derived MHC class II molecules and tumor-derived antigenic peptides may travel by separate routes and converge to form MHC class II/peptide complexes inside the fused DC/tumor cells. Moreover, the co-localization of MHC class I molecules and MUC1 on the surface of fused cells also suggests that the endogenous pathway of direct antigen processing and presentation in fused cells is preserved. It is also possible that the induction of CTL in a vaccinated host is the result of cross-presentation of TAAs expressed by fused cells and mediated by host DCs. Both direct antigen presentation by fused cells and cross-presentation by the host DCs may participate in T-cell activation. In summary, we demonstrated that a short-duration culture of PEG-treated cells was helpful in the fusion of DCs and tumor cells. DC and tumor components in fused DC/tumor cells presumably synthesized their separate molecules and TAAs at separate sites in fused cells that may be converged at the end of assembly line. Such properties of fused DC/tumor cells may explain the direct presentation of TAAs through MUC class I and class II pathways in the presence of co-stimulatory molecules and may result in simultaneous activation of CD4+ and CD8+ T-cells.

Competing Interests The Authors declare that they have no competing interests.

Acknowledgements This work was supported by National Cancer Institute Grant R01 CA87057; by the US Department of Defense Breast Cancer Research programs, grant 990344; by the Susan G. Komen Breast Cancer Foundation, grant 9825; and by Grants-in-Aid for Scientific Research (C) from the Ministry of Education, Cultures, Sports, Science and Technology of Japan.

References 1 Steinman RM: The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 9: 271-296, 1991. 2 Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G and Schadendorf D: Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 4: 328-332, 1998. 3 Koido S, Kashiwaba M, Chen D, Gendler S, Kufe D and Gong J: Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA. J Immunol 165: 5713-5719, 2000. 4 Paglia P, Chiodoni C, Rodolfo M and Colombo MP: Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vivo. J Exp Med 183: 317-322, 1996. 5 Russo V, Tanzarella S, Dalerba P, Rigatti D, Rovere P, Villa A, Bordignon C and Traversari C: Dendritic cells acquire the MAGE-3 human tumor antigen from apoptotic cells and induce a class I-restrected T-cell response. Proc Natl Acad Sci USA 97: 2185-2190, 2000. 6 Gong J, Chen D, Kashiwaba M and Kufe D: Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells. Nat Med 3: 558-561, 1997. 7 Gong J, Chen L, Chen D, Kashiwaba M, Manome Y, Tanaka T and Kufe D: Induction of antigen-specific antitumor immunity with adenovirus-transduced dendritic cells. Gene Ther 4: 10231028, 1997. 8 Gong J, Koido S, Chen D, Tanaka Y, Huang L, Avigan D, Anderson K, Ohno T and Kufe D: Immunization against murine multiple myeloma with fusions of dendritic and plasmacytoma cells is potentiated by interleukin 12. Blood 99: 2512-2517, 2000. 9 Liu Y, Zhang W, Chan T, Saxena A and Xiang J: Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity. Leuk Res 26: 757-763, 2002. 10 Wang JL, Saffold S, Cao XT, Krauss J and Chen W: Eliciting Tcell immunity against poorly immunogenic tumors by immunization with dendritic cell tumor fusion vaccines. J Immunol 161: 5516-5524, 1998. 11 Kjaergaard J, Shimizu K and Shu S: Electrofusion of syngeneic dendritic cells and tumor generates potent therapeutic vaccine. Cell Immunol 225: 65-74, 2003. 12 Parkhurst MR, DePan C, P. RJ, Rosenberg SA and Shu S: Hybrids of dendritic cells and tumor cells generated by electrofusion simultaneously present immunodominant epitopes from multiple human tumor-associated antigens in the context of MHC class I and class II molecules. J Immunol 170: 5317-5325, 2003. 13 Salomskaite-Davalgiene S, Cepurniene K, Satkauskas S, Venslauskas MS and Mir LM: Extent of cell electrofusion in vitro and in vivo is cell line dependent. Anticancer Res 29: 31253130, 2009. 14 Lindner M and Schirrmacher V: Tumour cell dendritic cell fusion for cancer immunotherapy: Comparison of therapeutic efficiency of polyethylen-glycol versus electro-fusion protocols. Eur J Clin Invest 32: 207-217, 2002. 15 Siders WM, Vergilis KL, Johnson C, Shields J and Kaplan JM: Induction of specific antitumor immunity in the mouse with the electrofusion product of tumor cells and dendritic cells. Mol Ther 7: 498-505, 2003.

353

ANTICANCER RESEARCH 33: 347-354 (2013) 16 Jantscheff P, Spagnoli G, Zajac P and Rochlitz CF: Cell fusion: An approach to generating constitutively proliferating human tumor antigen-presenting cells. Cancer Immunol Immunother 51: 367-375, 2002. 17 Busch R, Doebele RC, Patil NS, Pashine A and Mellins ED: Accessory molecules for MHC class II peptide loading. Curr Opin Immunol 12: 99-106, 2000. 18 Phan V, Errington F, Cheong SC, Kottke T, Gough M, Altmann S, Brandenburger A, Emery S, Strome S, Bateman A, Bonnotte B, Melcher A and Vile R: A new genetic method to generate and isolate small, short-lived but highly potent dendritic cell tumor cell hybrid vaccines. Nat Med 9: 1215-1219, 2003. 19 Hiraoka K, Yamamoto S, Otsuru S, Nakai S, Tamai K, Morishita R, Ogihara T and Kaneda Y: Enhanced tumor-specific long-term immunity of hemagglutinating (correction of hemaggluttinating) virus of Japan-mediated dendritic cell tumor fused cell vaccination by co-administration with CpG oligodeoxynucleotides. J Immunol 173: 4297-4307, 2004. 20 Gong J, Chen D, Kashiwaba M, Li Y, Chen L, Takeuchi H, Qu H, Rowse GJ, Gendler SJ and Kufe D: Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells. Proc Natl Acad Sci USA 95: 6279-6283, 1998. 21 Gong J, Avigan D, Chen D, Wu Z, Koido S, Kashiwaba M and Kufe D: Activation of antitumor cytotoxic T-lymphocytes by fusions of human dendritic cells and breast carcinoma cells. Proc Natl Acad Sci USA 97: 2715-2718, 2000. 22 Koido S, Tanaka Y, Chen D, Kufe D and Gong J: The kinetics of in vivo priming of CD4 and CD8 T-cells by dendritic/tumor fusion cells in MUC1-transgenic mice. J Immunol 168: 21112117, 2002. 23 Koido S, Hara E, Homma S, Torii A, Mitsunaga M, Yanagisawa M, Toyama Y, Kawahara H, Watanabe M, Yoshida S, Kobayashi S, Yanaga K, Fujise K and Tajiri H: Streptococcal preparation OK-432 promotes fusion efficiency and enhances induction of antigen-specific CTL by fusions of dendritic cells and colorectal cancer cells. J Immunol 178: 613-622, 2007. 24 Siddiqui J, Abe M, Hayes D, Shani E, Yunis E, and Kufe D: Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen. Proc Natl Acad Sci USA 85: 2320-2323, 1988. 25 Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, Muramatsu S and Steinman RM: Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 176: 1693-1702, 1992. 26 Petrakou E, Murray A and Price M: Epitope mapping of antiMUC1 mucin protein core monoclonal antibodies. Tumour Biol 19: 21-29, 1998.

354

27 Gong J, Apostolopoulos V, Chen D, Chen H, Koido S, Gendler SJ, McKenzie IF and Kufe D: Selection and characterization of MUC1-specific CD8+ T-cells from MUC1 transgenic mice immunized with dendritic carcinoma fusion cells. Immunology 101: 316-324, 2000. 28 Vlad AM, Muller S, Cudic M, Paulsen H, Otvos L Jr., Hanisch FG and Finn OJ: Complex carbohydrates are not removed during processing of glycoproteins by dendritic cells: Processing of tumor antigen MUC1 glycopeptides for presentation to major histocompatibility complex class II-restricted T-cells. J Exp Med 196: 1435-1446, 2002. 29 Koido S, Ohana M, Liu C, Nikrui N, Durfee J, Lerner A and Gong J: Dendritic cells fused with human cancer cells: Morphology, antigen expression and T-cell stimulation. Clin Immunol 113: 261-269, 2004. 30 Geuze HJ, Slot JW, van der Ley PA and Scheffer RC: Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections. J Cell Biol 89: 653-665, 1981. 31 Koido S, Hara E, Homma S, Namiki Y, Ohkusa T, Gong J and Tajiri H: Cancer vaccine by fusions of dendritic and cancer cells. Clin Dev Immunol 2009: 657369, 2009. 32 Gottfried E, Krieg R, Eichelberg C, Andreesen R, Mackensen A and Krause SW: Characterization of cells prepared by dendritic cell-tumor cell fusion. Cancer Immun 2: 15, 2002. 33 Shu S, Zheng R, Lee WT and Cohen PA: Immunogenicity of dendritic tumor fusion hybrids and their utility in cancer immunotherapy. Crit Rev Immunol 27: 463-483, 2007. 34 Haenssle HA, Krause SW, Emmert S, Zutt M, Kretschmer L, Schmidberger H, Andreesen R and Soruri A: Hybrid cell vaccination in metastatic melanoma: Clinical and immunologic results of a phase I/II study. J Immunother 27: 147-155, 2004. 35 Krause SW, Neumann C, Soruri A, Mayer S, Peters JH and Andreesen R: The treatment of patients with disseminated malignant melanoma by vaccination with autologous cell hybrids of tumor cells and dendritic cells. J Immunother 25: 421-428, 2002. 36 Cresswell P: Assembly, transport, and function of MHC class II molecules. Annu Rev Immunol 12: 259-293, 1994.

Received November 24, 2012 Revised December 21, 2012 Accepted December 21, 2012