Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity

Leukemia Research 26 (2002) 757–763

Engineered fusion hybrid vaccine of IL-4 gene-modified myeloma and relative mature dendritic cells enhances antitumor immunity Yongqing Liu a , Weidong Zhang a , Tim Chan a , Anurag Saxena b , Jim Xiang a,∗ a

Research Unit, Saskatchewan Cancer Agency, Departments of Oncology and Pathology, College of Medicine, University of Saskatchewan, 20 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 4H4 b Department of Pathology, College of Medicine, University of Saskatchewan, 103 Hospital Drive, Saskatoon, Saskatchewan, Canada S7N 0W8 Received 17 September 2001; accepted 4 December 2001

Abstract Dendritic cell (DC)-tumor fusion hybrid vaccine which facilitates antigen presentation represents a new powerful strategy in cancer therapy. In the present study, we investigated the antitumor immunity derived from vaccination of fusion hybrids between wild-type J558 or engineered J558-IL-4 myeloma cells secreting cytokine interleukin-4 (IL-4) and immature DCs (DCIMAT ) or relative mature DCs (DCRMAT ). DCRMAT displayed an up-regulated expression of immune molecules (Iad , CD40, CD54, CD80 and CD86) and certain cytokines/chemokines, and enhanced ability of allogeneic T cell stimulation when compared to DCIMAT . These DCs were fused with myeloma cells by polyethylene glycol (PEG). The fusion efficiency was approximately 20%. Our data showed that immunization of C57BL/6 mice with DCRMAT /J558 hybrids induced protective immunity against a high dose of J558 tumor challenge (1 × 106 cells) in 3 out of 10 immunized mice, compared with no protection seen in mice immunized with DCIMAT /J558 hybrids. Furthermore, immunization of mice with engineered DCRMAT /J558-IL-4 hybrids elicited stronger J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro and induced more efficient protective immunity (10/10 mice; tumor free) against J558 tumor challenge in vivo than DCRMAT /J558 hybrid vaccines. The results demonstrate the importance of DC maturation in DC-tumor hybrid vaccines and indicate that the engineered fusion hybrid vaccines which combine gene-modified tumor and DC vaccines may be an attractive strategy for cancer immunotherapy. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Dendritic cells; Engineered tumor cells; IL-4; Cell fusion; Antitumor immunity

1. Introduction Cytotoxic T lymphocytes (CTLs) play a major role in rejection of immunogenic tumors [1]. Classically, CTLs target tumors through recognition of a ligand consisting of the self major histocompatibility complex (MHC) class I molecule and peptide antigen generally derived from tumor antigens synthesized within the tumor cells [2,3]. However, effective immune responses against tumor antigens arising Abbreviations: APC, antigen presenting cell; BM, bone marrow; cpm, counts per minute; CTL, cytotoxic T lymphocyte; DC, dendritic cell; DMEM, Dulbecco’s modified medium; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GM-CSF, granulocyte macrophage colony-stimulation factor; ICAM, intercellular adhesion molecule; IL-4, interleukin-4; MIP, macrophage inflammatory protein; MHC, major histocompatibility complex; MLR, mixed lymphocyte reaction; PEG, polyethylene glycol; TRITC, tetramethyl rhodamine; TNF, tumor necrosis factor ∗ Corresponding author. Tel.: +1-306-655-2917; fax: +1-306-655-2635. E-mail address: [email protected] (J. Xiang).

during transformation are rarely observed. A number of mechanisms have been proposed to explain the failure to develop effective endogenous immunity against tumor. These include the generation of antigen-loss tumor variants, loss of MHC expression, and down-regulation of antigen processing machinery [4,5]. To augment the host immune response against tumors, gene-modified tumor vaccine using tumor cells engineered to express immunogenes have been conducted to enhance their immunogenicity and potential antigen-presenting ability and thus. These strategies include introduction of genes coding for MHC antigens [6], co-stimulatory molecules [4], cytokines [7] and chemokines [8] into tumor cells. The vaccine strategies using these engineered tumor cells have shown to be capable of stimulating T cell-mediated antitumor immune responses in animal tumor models. However, recent studies demonstrated that the in vivo generation of immune responses against tumor cells generally occurred through cross-priming, with tumor antigen presentation being dependent on bone marrow-derived

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(BM-derived) antigen presenting cells (APCs) of the host [9]. The mechanism by which tumor antigens are taken up and presented by host APCs remains unclear. In addition, the clinical use of these strategies may be limited by unknown defect(s) of tumor cells derived from each cancer patient. Dendritic cells (DCs) which are one of the most potent APCs have been used as DC vaccines of cancer. DCs pulsed with synthetic tumor-derived MHC class I-restricted peptides or tumor lysates have been shown to induce significant CTL-dependent antitumor immune responses in vitro as well as in mice in vivo [10–12]. Recently, Labeur et al. have demonstrated that the induction of antitumor immunity by DC vaccines is correlated with the maturation stages of DCs [13]. However, these DC vaccine strategies are currently limited by their dependence on in vitro antigen loading and the availability of appropriate, defined tumor antigens. Fusion of two different kinds of cells can generate hybrid cells that presumably have phenotypic characteristics of both of the progenitor cells. In a novel approach to tumor cell-based immunization, Guo et al. have originally shown that the fusion of activated B cells to tumor cells produced a potent immunogen, capable of inducing tumor-specific immunity [14]. The advantage of this novel approach include its ability to (i) correct defects in co-stimulatory signaling, (ii) provide both MHC class I and II epitopes and (iii) not require the identification of tumor antigens. Recently, Gong et al. have further demonstrated the induction of antitumor activity by immunization with fusions of granulocyte macrophage colony-stimulation factor/interleukin-4 (GM-CSF/IL-4) stimulated, peripheral blood mononuclear cell (PBMC)-derived DCs and carcinoma cells [15,16]. More recently, Kugler et al. have shown that 41% of the patients responded to hybrid cell vaccine with four complete remissions of metastatic renal cell carcinoma [17]. However, the maturation of GM-CSF/IL-4 stimulated, PBMC-derived DCs is not stable and needs to be stabilized in macrophage-conditioned medium [18]. Sometimes, these PBMC-derived DCs also display immature phenotypes in a medium containing GM-CSF/IL-4 [19]. So far, the potential effect of DC maturation stages on the antitumor efficiency of hybrid vaccines has not been studied. The induction of stronger CTL responses has become a major goal of current cancer vaccine strategies. In this study, we investigated the antitumor immunity derived from vaccination of hybrids between myeloma cells and BM-derived DCs at two different stages of differentiation, immature versus relatively mature DCs cultivated in medium containing GM-CSF alone and GM-CSF plus IL-4, respectively [20,21]. To further enhance the potential vaccine efficiency of fusion hybrids, we proposed a novel approach of engineered fusion hybrids to combine the above two strategies: gene-modified myeloma and DC vaccines. Therefore, in this study, we also investigated the antitumor immunity derived from vaccination of hybrids between DCs and engineered J558-IL-4 myeloma cells secreting cytokine IL-4 [7].

2. Materials and methods 2.1. Cell lines, antibodies, chemokines, peptides and animals J558 and A20 are poorly immunogenic myeloma and B cell lymphoma cell lines of BALB/c (H-2Kd ) origin, respectively. These two cell lines were maintained in Dulbecco’s modified medium (DMEM) (GIBCO, Gaithersburg, MD) plus 10% fetal calf serum (FCS). A tumor cell line J558-IL-4 [7] engineered to secrete interleukin-4 (IL-4) was obtained from Dr. Hook, Harvard Medical School, Boston, MA. Monoclonal antibodies including the anti-mouse H-2Kd , Iad , CD11b, CD11c, CD40, CD80, CD86, and ICAM-I (CD54) antibodies were obtained from PharMingen (San Diego, CA), respectively. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG antibody was obtained from Bio/Can Scientific (Missisasauga, Ont., Canada). The recombinant mouse IL-4 and GM-CSF were purchased from Endogene, Woburn, MA. Female BALB/c (H-2Kd ) and C57BL/6 (H-2Kb ) mice were obtained from the animal resources center of the University of Saskatchewan and housed in the animal facility of the Saskatoon Cancer Center. 2.2. DC culture Two procedures were used for generation of immature DCs (DCIMAT ) and relative mature DCs (DCRMAT ) from BM cell culture, respectively [20,21]. Briefly, BM cells prepared from femora and tibias of normal BALB/c mice were depleted of red blood cells with ammonium chloride and plated in DMEM plus 10% FCS and GM-CSF (low dose, 2 ng/ml) alone for generation of DCIMAT , and in GM-CSF (high dose, 10 ng/ml) with conjunction of IL-4 (10 ng/ml) for generation of DCRMAT on day 1. On day 3, nonadherent granulocytes, T and B cells were gently removed and the respective fresh media were added. On day 5, loosely adherent proliferating DC aggregates were dislodged and re-plated in the respective fresh media. On day 7 of culture, released, nonadherent DCIMAT and DCRMAT were harvested. The CD11c-positive DCs account for more than 95% of the harvested population as measured by flow cytometry. The yield of these DCs was 6 × 106 to 8 × 106 cells per mouse. These DCs were then subject to (i) phenotypic analysis by flow cytometry and using RNase protection assays, (ii) functional analysis using phagocytosis and mixed lymphocyte reaction (MLR) assays, and (iii) fusion with engineered tumor cells in vitro. 2.3. Immunofluorescence analysis For phenotypic analysis, DCIMAT and DCRMAT derived from the BM culture were stained with a panel of antibodies and then quantified by flow cytometry, respectively. These antibodies are rat anti-mouse H-2Kd , Iad , CD11b, CD11c, CD40, CD80, CD86 and ICAM-1 (CD54) antibodies. Briefly, DCs were incubated with each of the above

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antibodies (5 ␮g/ml) on ice for 30 min. After three washes with PBS, cells were then incubated with FITC-conjugated goat anti-rat IgG antibody (1:60) on ice for another 30 min. After three washes with PBS, cells were then analyzed by flow cytometry. Isotype-matched monoclonal antibodies were used as controls. 2.4. RNase protection assay We used RNase protection assays (RiboQuant Multi-Probe kit; Pharmingen, San Diego, CA) to examine the expression of mRNA for multiple cellular markers. RNA was extracted from the cells using a commercial kit and 32 P-UTP (Amersham Canada Ltd., Oakville, Ontario, Canada)-labeled probes were generated by in vitro transcription of cytokine/chemokine-related multi-probe template sets (Pharmingen) using T7 RNA polymerase. The labeled probes were purified by phenol–chloroform extraction and ethanol precipitation and adjusted to 3 × 105 cpm/␮l, then hybridized to the RNA samples (5 ␮g each). The reactions were subsequently digested with RNase, followed by Proteinase K treatment and phenol–chloroform extraction. After ethanol precipitation with 4 M ammonium K acetate, the protected samples were resuspended in 1 × loading buffer and realized on 5.7% acrylamide–bisacrylamide urea gels. The gels were absrobed to filter paper, dried under vacuum, and exposed to Kodak X-AR film with intensifying screens at −80 ◦ C. The relative expression of cytokine, chemokine and chemokine receptor encoding mRNA were assessed by scanning densitometry (Molecular Dynamics, Sunnyvale, CA), using only autoradiograph signals within the linear signal density range of the film, and then normalized using the housekeeping gene value (GAPDH). 2.5. DC phagocytosis The ability of DC phagocytosis was assessed using FITCconjugated dextran (10 kDa, molecular weight) (Molecular Probes, Eugene, OR), respectively. Briefly, 20 ␮l of dextran (0.05 mM) was incubated with 5 × 105 DCs in DMEM at 37 ◦ C. After 2 h of incubation, cells were harvested and resuspended in medium with the addition of trypan blue, which quenches the fluorescence of extracellular particles. Next, DCs were washed, resuspended in PBS and analyzed by flow cytometry. 2.6. Allogenic mixed lymphocyte reactions T cells were purified from C57BL/6 mouse splenocytes as nylon wool non-adherent cells [22]. The primary MLRs were performed as previously described [23]. Briefly, graded doses of irradiated DCs (3000 rad) were co-cultured in 96-well plates with a constant number (2 × 105 ) of allogeneic T cells from C57BL/6 mice. After 3 days, T cell proliferation was measured using an overnight 3 H-thymidine (1 mCi/ml, Amersham) uptake assay (1 ␮Ci per well). The

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levels of 3 H-thymidine incorporation into cellular DNA were determined by liquid scintillation counting. 2.7. Fusion of DCs with J558-IL-4 DCs derived from BM culture were fused with tumor cells at a 6:1 (DC: tumor) ratio using polyethylene glycol (PEG, molecular weight 1450)/DMSO solution (Sigma, St. Louis, MO). Briefly, 6 × 106 DCs were mixed with 1 × 106 tumor cells and washed with serum-free DMEM. After removing the medium, 1 ml of PEG was added to the cell pellet while resuspending the cells by stirring for 2 min. An additional 10 ml of DMEM was added to the cell suspension over the next 3 min with constant stirring. The cells were centrifuged at 400 × g for 5 min to remove the PEG. The cells were further washed with PBS for 3 times, and then resuspended in PBS for immunization of mice. Fusion preparation of DCs with J558-IL-4 or J558 cells were termed DC/J558-IL-4 and DC/J558 fusion hybrids, respectively. The method for labeling tumor cells with tetramethyl rhodamine (TRITC) (Sigma) was similar as previously described [24]. Briefly, tumor cells were resuspended in DMEM at 1 × 106 cells/ml and incubated with TRITC (0.5 ␮g/ml) at 37 ◦ C for 45 min. The labeled cells were washed with PBS for 3 times. To evaluate the fusion efficiency, DCs were fused with these TRITC-labeled tumor cells according to the protocol described above. Fusion preparations were further stained with the rat anti-mouse CD11c antibody, and then the FITC-conjugated goat anti-rat IgG antibody as described in Section 2.6. The cell suspensions were checked by fluorescence micorscopy. Single tumor cell and DC showed red fluorescence TRITC in cytoplasm and green fluorescence FITC on cell surface, respectively, whereas, the fused hybrid cell simultaneously displayed red and green fluorescence in its cytoplasm and on its cell surface membrane. After randomly picking up 12 high power fields (×400) for counting fused versus unfused cells in three independent experiments, we observed a fusion efficiency of 20 ± 2.3% for both DCIMAT and DCRMAT populations. 2.8. Cytotoxicity assay In general, CTL activation becomes maximal 9–12 days after immunization [25]. Ten days after mice were vaccinated with DCIMAT /J558, DCRMAT /J558-IL-4 and DCRMAT / J558 cells, their spleens were removed for preparation of single cell suspensions by pressing against fine nylon mesh. Red cells were lysed by using 0.84% ammonium chloride. Spleen lymphocytes were co-cultured with irradiated J558 cells (6000 rad) at 25:1, 5 × 106 lymphocytes and 2 × 105 irradiated J558 cells in 2 ml of DMEM plus 10% FCS in each well of a 24-well plate, respectively. Four days later, T cells were harvested and purified from the cultures using Ficoll–Paque density gradient centrifugation. These T cells were used as effector cells in a chromium-release assay

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against J558 and irrelevant A20 target cells. The target cells (104 per well) were incubated for 8 h in triplicate culture with effector cells at various effector/target ratios. Percentage of specific lysis was calculated as: 100 × [(experimental cpm − spontaneous cpm)/(maximal cpm − spontaneous cpm)]. The spontaneous cpm released in the absence of effector cells was less than 10% of specific lysis; the maximal cpm release was effected by adding 1% Triton X-100 to the cells. 2.9. Animal studies For evaluation of tumor immunity, mice were s.c. vaccinated with 1.4 × 106 irradiated DCRMAT /J558-IL-4, DCRMAT /J558 and DCIMAT /J558 fusion hybrid cells (8000 rad). For controls, mice were vaccinated with 1.4×106 irradiated mixed cells (1.2 × 106 DCs and 0.2 × 106 J558-IL-4 cells) or 0.2 × 106 irradiated J558-IL-4 or J558 cells or PBS. Ten days subsequent to the vaccination, mice were s.c. injected with 0.25 × 106 (low dose) and 1 × 106 (high dose) J558 tumor cells parallelly. Animal mortality and tumor growth were monitored daily for up to 10 weeks; for humanitarian reasons, all mice with tumors that achieved a size of 1.5 cm in diameter were sacrificed.

3. Results 3.1. Phenotypic and functional characteristics of DCs DCs used in this study were derived from mouse BM cells cultivated in the complete medium containing GM-CSF (2 ng/ml) alone for generation of DCIMAT or GM-CSF (10 ng/ml) in conjunction with IL-4 (10 ng/ml) for generation of DCRMAT . We first conducted experiments to analyze the phenotypic characteristics of these DCs. The DCRMAT enhanced the expression of (i) cell surface immune molecules such as the MHC class II Iad antigen, the adhesion molecule ICAM-1 (CD54) and the co-stimulatory molecules (CD40, CD80 and CD86) (Fig. 1), and (ii)

Fig. 2. Analysis of cytokine and chemokine expression of DCIMAT and DCRMAT by RNase protection assays. (A) RNase protection assay of DCIMAT and DCRMAT . (B) Relative expression of cytokine and chemokine mRNA of DCIMAT and DCRMAT .

inflammatory cytokines and chemokines such as IL-1␤, IL-6, RANTES, MIP-1␣, MIP-1␤ and MIP-2 (Fig. 2) than the DCIMAT . In addition, a low level of expression of cytokines (TNF-␣ and GM-CSF) was also seen in DCRMAT , but not in DCIMAT . However, the expression of MHC class I antigen, CD11b and CD11c remained unchanged on these DCs (data not shown). We then conducted experiments to analyze the functional characteristics of these DCs. We first conducted DC phagocytosis assay. We found that DCRMAT exhibited less efficient phagocytosis of FITC-conjugated dextran beads than the DCIMAT in three independent experiments (data not shown). Since DCs are potent stimulators of primary MLRs and are able to induce the proliferation of allogeneic CD8+ T cells in vitro [26], we next compared the abilities of DCs to stimulate primary MLRs against allogeneic CD8+ T cells. We found that the DCRMAT

Fig. 1. Phenotypic analysis of DCIMAT and DCRMAT by flow cytometry. The expression of Iad , CD40, CD54, CD80 and CD86 on DCs (solid lines) were analyzed using respective FITC-conjugated antibodies by flow cytometry. Isotype-matched monoclonal antibodies (dotted lines) were used as controls. One representative experiment of two is shown.

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Fig. 3. DCRMAT induce stronger MLRs than DCIMAT . Irradiate DCRMAT /(black circles) and DCIMAT (open triangles), 1 × 104 cells per well in 96-well plates and reciprocal dilutions thereof, were co-cultured for 3 days with 1 × 105 allogeneic C57BL/6 T cells. The overnight [3 H]-thymidine uptake seen on day 4 is expressed as the mean of three determinations. The S.D. of each point is less than 5% of the mean value. Background proliferation of DCs or T cells alone was always below 4000 cpm. One representative experiment of two is shown.

significantly induced strong T cell proliferative responses, while the DCIMAT was unable to stimulate allogeneic T cell proliferation (P < 0.01) (Fig. 3).

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Fig. 4. Cytotoxicity assay. Spleen lymphocytes were harvested from mice vaccinated with DCRMAT /J558-IL-4, DCRMAT /J558 and DCIMAT /J558. T cells were subsequently generated by co-cultivation of spleen lymphocytes of these immunized mice with irradiated J558 cells (6000 rad) for 4 days. T cells derived from these mice immunized with DCRMAT /J558-IL-4 (䊊), DCRMAT /J558 (䊉) and DCIMAT /J558 () were used as effector cells in a chromium release assay, in which 51 Cr-labeled J558 tumor cells were used as target cells. To confirm that T cell cytotoxicity was J558 tumor specific, we also included A20 cells (䉱) as a target control. Each point represents the mean of triplicates and the standard error. One representative experiment of two is shown.

3.2. Enhanced in vitro CTL responses induced by DCRMAT /J558-IL-4 vaccination Next, we addressed the specific antitumor effector functions induced by vaccination of the mice with fusion hybrids (DCRMAT /J558-IL-4, DCIMAT /J558-IL-4 and DCRMAT / J558), assessing the CTL activities against 51 Cr-labelled J558 target cells of splenocytes from the vaccinated animals. As shown in Fig. 4, T cells from mice vaccinated with DCRMAT /J558-IL-4 displayed substantially enhanced CTL activity (46% specific killing; E:T ratio, 50) compared to that of mice vaccinated with DCRMAT /J558 (30% specific killing, E:T ratio, 50) (P < 0.05) and DCIMAT /J558 (8% specific killing; E:T ratio, 50) (P < 0.01). This CTL activity was immunologically specific, in as much as none of these populations showed cytotoxic activities against the irrelevant A20 tumor cells, and T cells from naive mice had no activity against the J558 cells (data not shown). 3.3. Enhanced in vivo antitumor immunity induced by DCRMAT /J558-IL-4 vaccination To examine whether the maturation stage of DCs used for fusion hybrids may affect its antitumor immunity in vivo, we vaccinated mice with DCRMAT /J558 and DCIMAT /J558, and 10 days later challenged the animals with J558 tumor cells. Both low (0.25 × 106 ) (assay I in experiment I of Table 1) and high (1×106 ) (assay II in experiment I of Table 1) doses of J558 tumor challenges were invariably lethal for the PBS and J558 tumor vaccination control mice. Vaccination of mice with DCRMAT /J558 displayed enhanced immune protection (low dose, 100%; high dose, 30%) compared to mice

Table 1 Tumor growth of J558 cells in vaccinated mice Vaccination

Tumor incidence Experiment I

Experiment II

Assay I: (0.25 × 106 J558 cells)a DCRMAT /J558 0/10 DCIMAT /J558 8/10 0/10 DCRMAT /J558-IL-4 DCRMAT and J558-IL-4 3/10 J558-IL-4 6/10 J558 10/10 PBS 10/10

0/8 7/8 0/8 3/8 4/8 8/8 8/8

Assay II: (1 × 106 J558 cells)b DCRMAT /J558 DCIMAT /J558 DCRMAT /J558-IL-4 DCRMAT and J558-IL-4 J558-IL-4 J558 PBS

6/8 8/8 0/8 7/8 8/8 8/8 8/8

7/10 10/10 0/10 10/10 10/10 10/10 10/10

a In assay I, mice were s.c. vaccinated with 1.4 × 106 irradiated DCRMAT /J558-IL-4, DCRMAT /J558 and DCIMAT /J558 fusion hybrid cells. For controls, mice were vaccinated with 1.4 × 106 irradiated mixed cells (1.2 × 106 DCs and 0.2 × 106 J558-IL-4 cells) or 0.2 × 106 irradiated J558-IL-4 or J558 cells or PBS. Ten days after vaccination, mice were s.c. injected with 0.25 × 106 J558 tumor cells. b In assay II, mice were s.c. vaccinated with 1.4 × 106 irradiated DCRMAT /J558-IL-4, DCRMAT /J558 and DCIMAT /J558 fusion hybrid cells. For controls, mice were vaccinated with 1.4 × 106 irradiated mixed cells (1.2 × 106 DCs and 0.2 × 106 J558-IL-4 cells) or 0.2 × 106 irradiated J558-IL-4 or J558 cells or PBS. Ten days after vaccination, mice were s.c. injected with 1 × 106 J558 tumor cells.

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vaccinated with DCIMAT /J558 (low dose, 20%; high dose, 0%). To examine whether engineered fusion hybrid DCRMAT / J558-IL-4 is capable of induction of enhanced antitumor immunity in vivo, we vaccinated mice with various fusion hybrids including DCRMAT /J558-IL-4, DCIMAT /J558 and respective controls, and 10 days later challenged the animals with J558 tumor cells. Our data demonstrated that vaccination of mice with DCRMAT /J558-IL-4 resulted in more efficient protection against the second challenge of J558 tumor cells (low dose, 100%; high dose, 100%) than DCRMAT /J558 (low dose, 100%; high dose, 30%), a mixture of DCRMAT and J558-IL-4 (low dose, 70%; high dose, 0%), and J558-IL-4 alone (low dose, 40%; high dose, 0%). The results seen in experiment II are similar to those in experiment I (Table 1).

4. Discussion and conclusion DCs are the most potent stimulators of primary immune responses known thus far [10], and have been recognized as potential tools for immunotherapy and vaccine strategies, especially for the therapy of tumors [10–12]. Recently, it has been shown that the induction of antitumor immunity by DC vaccines is correlated with the maturation stages of DCs [13]. The degree of differentiation or maturation of a population of DCs largely determines their functional capabilities. In general, immature DCs expressing less MHC and costimulatory molecules migrate from the BM into various organs, where they usually reside in an nascent state [27]. During this time, these immature DCs are efficient in phagocytosis and antigen-processing [28]. Upon activation, they initiate a maturation process that results in decreased antigen-processing capacity, but enhanced expression of MHC and co-stimulatory molecules. These more mature DCs then migrate to lymphoid organs to interact/activate naive T cells [29,30]. IL-4 is a cytokine that stimulates the differentiation and maturation of DCs [21,31]. In this study, we generated DCs at two maturation stages, the immature DCs (DCIMAT ) and the relative mature DCs (DCRMAT ) from BM cell culture in the absence and presence of IL-4, respectively. Our data showed that DCRMAT displayed more expression of cell-surface molecules (Iad , CD40, CD54, CD80 and CD86), inflammatory cytokines (IL-1␤ and IL-6) and chemokines (RANTES, MIP-1␣, MIP-1␤ and MIP-2) as well as stronger stimulation on T cell proliferation, but less phagocytic activity than DCIMAT . Therefore, these two types of DCs became good candidates for studying the effect of DC maturation on the antitumor efficiency of DC hybrid vaccines. Our data showed that vaccination of mice with DCRMAT /J558 displayed enhanced immune protection (low dose, 100%; high dose, 30%) compared to mice vaccinated with DCIMAT /J558 (low dose, 20%; high dose, 0%). This indicates that the efficiency of DC hybrid vaccines is related to the maturation stage of the DCs; the latter correlating

with the expression of important immune molecules for presentation of tumor antigen to T cells and further stimulation of antitumor T cell responses. Our recent study showed that phagocytosis of apoptotic tumor cells is able to induce DC maturation leading to enhanced antitumor immunity [32]. However, the fusion hybrid between immature DCs and tumor cells did not induce efficient antitumor immunity in this study. This may be because the physical fusion of DCs with tumor cells differs from the phagocytosis and may not be able to induce any DC maturation which is critical in induction of antitumor immunity.In addition to stimulating DC maturation, IL-4 prevents the blockade of DC differentiation by tumor cells [33] and plays a key role as a mediator in T cell responses and IL-12p70 induction [34,35]. Vaccines using tumor cells engineered to secrete IL-4 enhanced DC infiltration and their indirect antigen presentation; this has been shown to enhance the antitumor immunity mediated by CD8+ T cells [36,37]. These have prompted us to investigate the vaccine efficiency of IL-4 gene-modified tumor-DC hybrids. In this study, we demonstrated that vaccination of mice with DCRMAT /J558-IL-4 induced stronger J558 tumor-specific CTL cytotoxicity in vitro and resulted in more efficient immune protection in vivo against the second challenge of J558 tumor cells (low dose, 100%; high dose, 100%) than DCRMAT/J558 (low dose, 100%; high dose, 30%), a mixture of DCRMAT and J558-IL-4 (low dose, 70%; high dose, 0%), and J558-IL-4 alone (low dose, 40%; high dose, 0%). Our data thus indicate that engineered fusion hybrid vaccine of IL-4-gene-modified myeloma and relative mature DCs significantly enhances the antitumor immunity. Taken together, our study demonstrated the importance of DC maturation stages in DC-tumor hybrid vaccines and indicated that the engineered fusion hybrid vaccines which combine gene-modified tumor and DC vaccines may be an attractive strategy for cancer immunotherapy.

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