Chapter 3. Results
3.5 Generation of DC/TGF-β-expressed cell fusion vaccines using
To generate DC/TGF-β-expressed cell fusion vaccines, BALB/3T3 cells were fused with DCs. For detection of the fusion efficiency, DCs and BALB/3T3 were labeled with DiO and DiI lipophilic fluorescent dyes, respectively. The fusion efficiencies were determined by the percent of double-stained cells using flow cytometer. The dual fluorescent dot plot showed that LPPC could not significantly affect the abilities of PEG to fuse cells (Fig.
10a). After calculation of data from 3 independent experiments, the results revealed that PEG treatment without or with LPPC both caused about 50% of double-stained cells (Fig. 10b). There was no significant difference in fusion efficiency between PEG treatment and LPPC/DNA with PEG treatments, which
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coincided with the results in the BALB/3T3_pAsRed and B16-F10_pLEGFP fusion experiments. Sequentially, the results showed LPPC could also simultaneously deliver IL-6 gene into the DCs and BALB/3T3 cells during fusion process (Fig. 11; 743.6 ± 179.7 pg). While the PEG treatment only induced the expression of IL-6 (33.0 ± 2.4 pg) and it was as similar as the amounts in the cultured medium (27.4 ± 9.9 pg; Fig. 11). Sequentially, whether the simultenous transfection of IL-6 gene by LPPC could affect the expression of TGF-β was verified. Figure 12 showed that the simultaneous transfection of IL-6 gene by LPPC could not affect the secretion of TGF-β comparing to PEG treatment without LPPC addition.
3.6 Evaluation of the efficacy of co-transfection-fusion DC vaccine
To evaluate the efficacies of co-transfection-fusion DC vaccines for initiating the host immune system, each kind of vaccines were respectively administered to mice by i.p. on day 0 and the splenocytes from naïve mice or immunized mice were harvested at 7th day after immunization to monitor the cytokines profiles and cell proliferation with stimulation of antigens derived from BALB/3T3 cells. The results showed that there were no significantly differences between PEG, LPPC/IL-6 with PEG and naïve groups among the productions of IFN-γ, IL-2, IL-4 and IL-10 (Fig. 13), which might be due to the period of immunization was too short to trigger enough immune response.
However, the LPPC/IL-6 with PEG group induced a significant proliferative response comparing to the naïve group with the stimulation of antigen (p < 0.05) (Fig. 14). In contrast, PEG alone group could not induce any significant proliferative response comparing to the naïve group with the stimulation of
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antigen. Besides, without the stimulation of antigens, there were no significant differences among all groups, which indicated that the LPPC/IL-6 with PEG group could trigger an antigen-specific immune response during a short period of immunization than PEG group.
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Chapter 4. Discussion
In this study, we firstly proved that the co-transfection-fusion strategy of using LPPC as a transfection reagent to deliver a transgene simultaneously during the PEG fusion process is workable. According to this strategy, the LPPC/IL-6/PEG DC vaccines were made by this co-transfection-fusion protocol, they could produce interleukin (IL)-6, a pleiotropic cytokine that was known to increase host immune responses and antagonize immunosuppressive effects of TGF-β (Hsiao, Liao et al. 2004). They seemingly had the abilities to initiate the specific host immunity faster than the PEG DC vaccine in mice.
DC-based vaccines represent a promising approach to stimulating specific-antitumor immunity (Banchereau, Palucka et al. 2001; Reichardt, Brossart et al. 2004). Thus, a large number of strategies have been developed to deliver Ags to DC including the defined peptides, specific tumor-associated Ags (TAA) or whole tumor cell material by using viral or non-viral technique in recent years (Mayordomo, Zorina et al. 1995; Boczkowski, Nair et al. 1996;
Paglia, Chiodoni et al. 1996; Schmidt, Ziske et al. 2003; Tian, Wang et al. 2008).
However, strategies in which single TAA is loaded onto DCs are limited by few defined TAAs or the possibilities of immunologic escape through down-regulation of the target Ag in tumor cells (Wang and Rosenberg 1996). In contrast, strategies that DCs loaded with whole tumor cells or tumor lysate have the advantage to induce multiple antitumor immunities against a broad tumor Ags, even unidentified tumor Ags (Trefzer, Herberth et al. 2000; Walden 2000;
Tian, Wang et al. 2008). Furthermore, the comparison between the efficacies of
26
DC/tumor fusion vaccines and tumor lysate-pulsed DC vaccines demonstrated that DC/fusion vaccines had superior efficacy, because DC/tumor fusion vaccines have the longer interaction with tumor antigens in vivo (Kao, Zhang et al. 2005). However, in a clinical trial for patients with metastatic breast carcinoma, immunizations with DC/tumor hybrids induced antitumor immunity in a majority of patients while tumors regressed on only a subset of patients (Avigan, Vasir et al. 2004). The possibilities for the clinical failures may result from the weakness of patients to have a compromised immune system, or the maturation state or subsets of DCs to cause immune tolerance or Treg expansion (de Vries, Lesterhuis et al. 2003; McIlroy and Gregoire 2003; Tuyaerts, Aerts et al. 2007). Moreover, soluble immunosuppressive factors (TGF-β, VEGF, IL-10, PGE-E2) secreted by malignant tumors are present in the microenvironment and interfere with effective T-cell function (Kuppner, Sawamura et al. 1990;
Torre-Amione, Beauchamp et al. 1990; Bomstein, Ophir et al. 1993)
Consistently, the inhibitory effect of tumor-derived TGF-β on the efficacy of DC/tumor fusion vaccine has been demonstrated in mouse model (Kao, Gong et al. 2003). TGF-β is a potent immunosuppressive cytokine that often secreted by tumor cells (Pasche 2001). It has been reported that TGF-β could inhibit CTL generation and allow tumors to escape immune surveillance (Kehrl, Wakefield et al. 1986; Rook, Kehrl et al. 1986). Moreover, TGF-β has a direct effect on DC which would interfere with the abilities of DCs to present antigen, stimulate the IFN- expression of tumor-specific CTL, and migrate to draining lymph nodes (Kobie, Wu et al. 2003). Similarly, in our study, most tumor cell lines did secrete higher amounts of TGF-β (Fig. 8), and the immortalized cells-derived TGF-β had the inhibitory effect on the Ag presentation abilities of DCs (Fig. 9).
Therefore, several TGF-β neutralizing strategies to boosting antitumor immunity
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have been reported to resolve the inhibitory effect of TGF-β. Transgenic expression of a dominant-negative TGF-β receptor (TGF-β-R) in DCs can enhance the ability of host immune response against tumors (Muraoka, Dumont et al. 2002; Shah, Tabayoyong et al. 2002). In addition, neutralization of TGF-β by using an adenovirus encoding TGF-β-R to infect tumor cells and then fusing with DCs has been proven to enhance the efficacy of DC fusion vaccine (Zhang, Berndt et al. 2008). On the other hand, IL-6 has the ability to antagonize the immunosuppressive effects of TGF-β (Ohta, Yamagami et al. 2000; Hsiao, Liao et al. 2004), which implied that the IL-6’s anti-TGF-β activities might be used in gene-modified DC fusion vaccine as an alternative approach.
We found that LPPC could not only deliver IL-6 gene into the cell mixture but also has no effect on the fusion efficiencies of vaccine during fusion process (Fig. 10). As expect, the LPPC/IL-6/PEG DC vaccine showed a superior efficacy in triggering antigen-specific immunogenic effects than the PEG DC vaccine (Fig. 14), which may result from the strong antagonism of IL-6 against TGF-β in the microenvironment.
To elevate the efficacy of DC fusion vaccine, several types of gene-modified DC fusion vaccines have been established. In addition to the TGF-β-R-secreting DC/tumor fusion vaccines (Zhang, Berndt et al. 2008), Suzuki et al. showed IL-12 gene-transgeic murine colon-26 adenocarcinoma cells were fused with DCs markedly enhanced antitumor effect in vivo therapeutic model (Suzuki, Fukuhara et al. 2005). Iinuma et al. also reported that IL-12-modified DCs and IL-18-modified murine neuroblastoma cells were fused together to induce protective and therapeutic effects for liver-metastasis neuroblastoma (Iinuma, Okinaga et al. 2006). The essential need for previous strategies is the tumor cells have to be cultured. However, the clinical isolated
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tumor cells may not be cultured and maintained until completion of transfection and fusion procedural. Therefore, the co-transfection/fusion strategy may be a more suitable choice to prepare the DC/autologous tumor cell vaccine in the clinical setting.
Our strategy showed that immuno-modulated gene transfection can be simultaneous with fusion protocol. We proposed that IL-6 gene should be delivered into BALB/3T3 cells, DCs or hybrids. If the IL-6 gene was delivered to DCs or hybrids, the immuno-stimulating effects of IL-6 may affect directly on DCs or hybrids as other gene-modified DC fusion cells (Suzuki, Fukuhara et al.
2005; Iinuma, Okinaga et al. 2006; Zhang, Berndt et al. 2008). Alternatively, if the IL-6 gene was delivered to BALB/3T3 cells, there have been reported that the use of genetically-modified tumor cells as antitumor vaccines could elicit potent immunogenic effects (Hsieh, Chen et al. 2000; Antonia, Seigne et al.
2002; Berzofsky, Terabe et al. 2004; Frankenberger, Regn et al. 2005; Naruishi, Timme et al. 2006). Based on these ideas, the preparation of gene-modified DC vaccine has no needs to be performed sequentially. Besides, the use of LPPC as the transfection reagent in the co-transfect-fusion strategy may be replaced with other viral vectors to perform higher transfection efficiency during the PEG fusion process.
Furthermore, effective adjuvants for enhancing the abilities of DC/tumor cell fusion vaccines have also been reported, including exogenous IL-12, IL-18, or CpG oligodeoxynucleotides (CpG ODNs) to activate tumor-specific T cells (Gong, Koido et al. 2002; Vasir, Wu et al. 2008). In addition, pre-treatment of DCs and tumor cells with TLR agonist penicillin-killed Streptococcus pyogense (OK-432) and heat shock respectively induce stronger induction of antigen-specific CTLs (Koido, Hara et al. 2007). Moreover, we speculate that
29
the addition of these adjuvants in combination with the co-transfection-fusion strategy may further enhance the effectiveness of DC fusion vaccines. Besides, the combination of other immuno-stimulating genes, such as IL-15 with IL-6 may activate NK cell cytotoxicity in presence of tumor-derived TGF-β (Lin, Chuang et al. 2008). Thus, the co-transfection of IL-6 and IL-15 into DC/tumor fusion cells by our strategy may result in extremely stronger antitumor immunity.
In conclusion, our study demonstrated that the co-transfection-fusion strategy is feasible. We generated IL-6-secreting DC fusion vaccines made by LPPC/IL-6/PEG and proved this vaccine could elicit superior immunogenic effects in the short period of immunization, which proposed the gene transfection and cell fusion can be performed simultaneously. This alternative approach is simple and has the potential to be a promising strategy for gene-modified DC fusion vaccine in the clinical use.
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Figures
Fig. 1 Micrograph of Lipo-PEI-PEG Complex (LPPC) nanoparticles
The SEM photo of LPPC; the sizes of LPPC nanoparticles were ranged from 200 nm to 300 nm. Length of scale bar = 1 μm.
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Fig. 2 DNA retarding assay for LPPC-DNA complexes
LPPC at different amounts were incubated with a fixed amount (18 μg) of DNA for 30 min at room temperature, and the different DNA complexes were run on an agarose gel. Lane 1 was DNA marker and lane 2 was naked DNA, lane 3 to lane 5 were 100μg LPPC, 200μg LPPC and 300μg LPPC respectively. The replacement of DNA from complexes by competition of heparin was shown on lane 6 to lane 8. These DNA complexes were run in a 0.8% agarose gel.
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a.
b.
Fig. 3 Particle size and zeta-potential analysis of LPPC-DNA complexes 100μg, 300μg and 900μg LPPC were mixed with fixed amount (18 μg) of DNA and gave rise to DNA complexes at different N/P ratios. a. and b. The particle sizes and zeta-potential of LPPC, DNA and LPPC-DNA complexes at different N/P ratios from 7,22 and 66 measured by DLS and ZetaPlus Z-potential analyzer respectively.
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Fig. 4 In vitro transfection efficiency of LPPC/DNA complexes
3 μg DNA were complexed with different amounts of LPPC and then transfected into BALB/3T3 cells. After 48 hours, the transfection efficiencies of each group were measured by flow cytometer. NC: cells alone; PEI: 5mM PEI, 18 μl; data represents the mean ± S.D. of three independent experiments. * represents p <
0.01, ** represents p < 0.001 v.s. PEI.
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a.
b.
35
Fig. 5 Addition of LPPC has no effect on fusion efficiency caused by PEG BALB/3T3_pAsRed cells and B16-F10_pLEGFP cells were fused at 1:1 ratio by PEG treatment alone, LPPC+PEG treatments or LPPC treatment alone. The fusion efficiencies of each treatment were examined by FACS analysis. a. The characteristic diagram of one experiment for measuring the fusion efficiency; b.
Statistical analysis of fusion efficiencies of each treatment. Data represents the mean ± S.D. of three independent experiments. * represents p < 0.05 v.s. LPPC alone.
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Fig. 6 Cytokine production by fusion cells after the co-transfection-fusion protocol
18 g IL-6 plasmid was complexed with 300 g LPPC firstly and then added into cells during the PEG fusion process. After 48 hours, the supernatants of fused cells made by LPPC/DNA complex and PEG treatments or LPPC/DNA treatment alone were collected and examined by ELISA to measure the expression of IL-6 protein level. NC: cells mixture alone; data represents the mean ± S.D. of two independent experiments.* represents p < 0.05, **
represents p < 0.01 v.s. NC.
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a. b.
c.
CD11c MHCI MHCII CD86 Fig. 7 Generation of bone marrow-derived dendritic cells
Bone marrow derived dendritic cells were generated using medium containing 200 U/ml GM-CSF; iBMDC and mBMDC were harvested at Day 8 and 10. a.
Phase contrast microscope revealed the morphology of iBMDC. b. Phase contrast microscope revealed the morphology of mBMDC. Original magnification, × 320. c. Surface phenotypes were determined using FACS cytometer . The antibodies CD11c, MHCI, MHCII and CD86 were used to stain the iBMDC (the upper raw) and mBMDC (the lower raw), respectively. Data represents the mean ± S.D. of three independent experiments.
mBMDC iBMDC
209.0±60.13 47.6±29.24 129.6±45.00 13.2±3.80
194.6±60.52 40.3±2.38 575.2±41.16 259.3±37.44
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Fig. 8 TGF-β secretion of different cell lines
Different cell lines (1 × 105 cells/ml) were grown in 2% culture medium.
Supernatants of different cell lines were collect at 12 hours, 24 hours and 36 hours after seeding, respectively. Supernatants and medium alone were each assessed for TGF-β by ELISA. All cell lines tested expressed TGF-β. Data represents the mean ± S.D. in duplicate of three independent experiments (n = 6).
+ represents p < 0.01 v.s. 3T3 of 12 hr; # represents p < 0.01 v.s. 3T3 of 24 hr; * represents p < 0.01 v.s. 3T3 of 36 hr.
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Fig. 9 Effect of TGF-β derived from immortalized cells on BMDC’s phenotypes
BMDCs were incubated with the BALB/3T3 conditioned medium containing TGF-β or recombinant TGF-β (10 ng/ml) for 6 days. Cytokines were replenished every 2 days and then DCs were matured with LPS (0.5 μg/ml) for 48 hours.
DCs of each treatment were stained with anti-MHCII or CD86 antibodies and analyzed by flow cytometer. The results are the mean ± S.D. of two independent experiments. * represents p < 0.05 v.s. DC without any treatments; ** represents p < 0.01 v.s. DC without any treatments.
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a.
b.
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Fig. 10 Fusion efficiencies of LPPC/IL-6/PEG DC vaccine and PEG DC vaccine
DiI-labeled BALB/3T3 cells were fused with DiO-labeled DCs at 1:2 ratio by LPPC/IL-6/PEG treatments or PEG treatment alone, and fusion efficiencies were examined by FACS analysis. a. The characteristic diagram of one experiment for measuring the fusion efficiency in upper right panel; b.
Statistical analysis of fusion efficiencies of each treatment. Data represents the mean ± S.D. of two independent experiments.
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Fig. 11 Cytokine production by LPPC/IL-6/PEG DC fusion cells and PEG DC fusion cells
LPPC/IL-6/PEG DC fusion cells and PEG DC fusion cells have been generated.
After 48 hours, the supernatants of fused cells were collected and examined by ELISA to measure the expression of IL-6 protein level. NC: cells mixture alone;
data represents the mean ± S.D. of two independent experiments.* represents p
< 0.05 v.s. NC.
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a.
b.
Fig. 12 TGF-β secretion by DC fusion cells
DC/BALB/3T3 fusion cells continue to secrete bioactive TGF-β. The supernatants of LPPC/IL-6/PEG DC fusion cells and PEG DC fusion cells were collected 48 hours after the experiment. ELISA was performed on supernatants in acidified or nonacidified procedures to detect total or active forms of cells-derived TGF-β. NC: medium alone; data represents the mean ± S.D. of two or three independent experiments.
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Fig. 13 Cytokines profiles produced by splenocytes from mice immunized with LPPC/IL-6/PEG DC vaccine or PEG DC vaccine
Mice were immunized with LPPC/IL-6/PEG DC vaccine or PEG DC vaccine (1
× 106 cells/injection) on day 0. After a week, immunized mice were sacrificed and the splenocytes were isolated (1 × 106 cells/well) to incubate with
BALB/3T3 lysate (3.3 × 105 cells/well). After 48 hours, the supernatants were collected and detected a broad array of the protein levels by ELISA. There were no significantly difference between LPPC/IL-6/PEG group and PEG group among the cytokines which are related to Th1 or Th2 response.
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Fig. 14 The cell proliferation of Ag-specific splenocytes from LPPC/IL-6/PEG DC vaccine-immunized mice
Splenocytes from immunized mice of each group were isolated and seeded in 96-well plate (2.5 × 105 cells/well) in the absence or presence of BALB/3T3 lysate (2.5 × 104 cells/well). MTT assay have been performed after 3 days incubation. The result showed that splenocytes from mice vaccinated with LPPC/IL-6/PEG DC vaccine could stimulate Ag-specific immune response (n=2,
* represents p < 0.05 v.s. naïve group).
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Table
Appearance Milky, turbid solution
Components DOPC, DLPC, PEI and PEG
Size (nm) 218.35±12.94
Zeta potential (mV) 40.25±11.24
Character Could be easily purified by
centrifugation
Table 1. Physical properties of Lipo-PEI-PEG Complex (LPPC)
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