Enhancing dendritic cell vaccine potency by combining a BAK/BAX siRNA-mediated antiapoptotic strategy to prolong dendritic cell life with an intracellular strategy to target antigen to lysosomal compartments

Authors

  • Tae Heung Kang,

    1. Laboratory of Infection and Immunology, Graduate School of Medicine, Korea University, Ansan, Gyeonggi, Korea
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  • Jin Hyup Lee,

    1. Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD
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  • Kyung Hee Noh,

    1. Laboratory of Infection and Immunology, Graduate School of Medicine, Korea University, Ansan, Gyeonggi, Korea
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  • Hee Dong Han,

    1. Department of Advanced Materials, Korea Research Institute of Chemical Technology, Yuseong, Daejeon, Korea
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  • Byung Cheol Shin,

    1. Department of Advanced Materials, Korea Research Institute of Chemical Technology, Yuseong, Daejeon, Korea
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  • Eun Young Choi,

    1. Graduate Program of Immunology, Seoul National University College of Medicine, Seoul, Korea
    2. Center for Animal Resource Development, Seoul National University College of Medicine, Seoul, Korea
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  • Shiwen Peng,

    1. Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD
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  • Chien-Fu Hung,

    1. Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD
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  • T.-C. Wu,

    1. Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD
    2. Department of Obstetrics and Gynecology, Johns Hopkins Medical Institutions, Baltimore, MD
    3. Department of Oncology, Johns Hopkins Medical Institutions, Baltimore, MD
    4. Department of Molecular Microbiology and Immunology, Johns Hopkins Medical Institutions, Baltimore, MD
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  • Tae Woo Kim

    Corresponding author
    1. Laboratory of Infection and Immunology, Graduate School of Medicine, Korea University, Ansan, Gyeonggi, Korea
    2. Research Center for Women' s Diseases, Sookmyung Women' s University, Seoul, Korea
    • Laboratory of Infection and Immunology, Graduate School of Medicine, Korea University, 516 Gojan-1 Dong, Ansan-Si, Gyeonggi-Do 425-707, South Korea
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    • Fax: +82-31-412-6718.


Abstract

Dendritic cell (DC)-based vaccines have become important in immunotherapeutics as a measure for generating antitumor immune responses. We have previously demonstrated that linkage of the antigen gene to a lysosomal targeting signal, a sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhances the potency of DC-based vaccines. DCs have a limited life span, hindering their long-term ability to prime antigen-specific T cells. In this study, we attempted to further improve the potency of a DC vaccine that targets human papilloma virus 16 (HPV16) E7 to a lysosomal compartment (DC-Sig/E7/LAMP-1) by combining a strategy to prolong DC life. We show that small interfering RNA-targeting Bak and Bax proteins can be used to allow transfected DCs to resist being killed by T cells. This is done by downregulating these proapoptotic proteins, which have been known as so-called gate keepers in mitochondria-mediated apoptosis. DCs expressing intact E7 or Sig/E7/LAMP-1 became resistant to attack by CD8+ T cells after transfection with BAK/BAX siRNA, leading to enhanced E7-specific T cell activation in vitro and in vivo. More importantly, vaccination with E7-presenting DCs transfected with BAK/BAX siRNA generated a strong therapeutic effect against an E7-expressing tumor in vaccinated mice, compared with DCs transfected with control siRNA. Our data indicate that a combination of strategies to enhance intracellular Ag processing and to prolong DC life may offer a promising strategy for improving DC vaccine potency. © 2007 Wiley-Liss, Inc.

Antigen presentation by dendritic cells (DCs) is a critical element in the induction of the cellular immune responses necessary for tumor immunotherapy. DCs have an intrinsic ability to prime immune responses.1 Because of this, there has been a great deal of interest in the use of these cells for cancer therapy.2, 3, 4, 5, 6 However, clinical results have not been very promising. One of the limitations in the use of DC vaccines for clinical applications is their low potency. In particular, the generation of cellular immunity using a DC vaccine against low immunogenic tumor-specific antigens, such as human papillomavirus (HPV) E7, has been difficult. This problem presents a major hurdle in the goal of controlling cervical cancer. In a previous study, we reported that only 60% of mice that received intramuscular immunization with DC-E7 survived following a challenge with a low number of E7-expressing tumor cells (TC-1) (1 × 104/mouse).7 The development of strategies for improvement of DC vaccine potency is indispensable if we are to use DC-mediated cancer immunotherapy. To compensate for the weak immune response generated by DCs expressing wild-type E7 antigen, we have developed intracellular targeting strategies that increase MHC class I and class II presentation of E7 antigen by DCs.8, 9, 10, 11 Recently, we found that linking the sorting signal of the lysosome-associated membrane protein 1 (Sig/LAMP-1)-targeted E7 to endosomal and lysosomal compartments enhanced MHC class I presentation to CD8+ T cells, as well as MHC class II presentation of E7 to CD4+ T cells.12 More importantly, immunization of mice with DC-Sig/E7/LAMP-1 led to more effective antitumor protection and treatment against a TC-1 cervical tumor model than did DC-E7. Despite these efforts, the potency of DC-base vaccines still needs to be improved to treat a large tumor. After administration, the life span of antigen-presenting DCs is limited in various ways, which hinders their ability to prime the immune response.13 A principal contributor to the short life of these DCs is cytotoxic cell-induced apoptosis.14, 15 After activation by DCs, cytotoxic T lymphocytes (CTLs) recognize antigens and kill the cells that express them via apoptosis.16 Because DCs express MHC-I:antigen peptide complexes on their surface, newly primed CTLs can kill the very DCs that activated them.17 From these observations, we reasoned that an intracellular targeting strategy, employing an approach to inhibit apoptosis and prolong the survival of antigen-expressing DCs in vivo, could work better. In previous reports, we also found that a variety of antiapoptotic factors can enhance DC survival and the antigen-specific CD8+ T cell immune responses induced by various DNA vaccines.15, 17 Since antiapoptotic proteins, such as Bcl-xL, raise significant concerns related to oncogenicity, there are practical limitations in introducing them to DC-based vaccines for clinical trials. To overcome this problem, we have tried to introduce RNA interference (RNAi) technology to DC vaccine systems, using small interference RNA (siRNA) for targeting and silencing key pro-apoptotic proteins, such as Bax and Bak. They are members of the Bcl-2 family and play key gatekeeping roles in the mitochondria-mediated intrinsic apoptotic pathway.18, 19, 20, 21 By using siRNA, concerns related to biohazards are alleviated since RNA-based strategies avoid problems of integration and permanent genetic change. In this study, we show that the delivery of BAK and BAX siRNA to antigen-expressing DCs prolongs the lives of transfected DCs and that DCs-Sig/E7/LAMP-1 transfected with BAK and BAX siRNA are capable of generating strong antigen-specific CD8+ T cell immune responses and antitumor effects in vaccinated mice.

Abbreviations:

BM-DC, bone marrow-derived DC; CFSE, 5- (and 6-) carboxy fluorescein diacetate succinimidyl ester; CTL, cytotoxic T lymphocyte; DC, dendritic cell; FITC, fluorescein isothiocyanate; HPV16, human papilloma virus 16; LAMP-1, lysosome-associated membrane protein type 1; MHC, major histocompatibility complex; PE, phycoerythrin; RNAi, RNA interference; siRNA, small interference RNA.

Material and methods

Preparation of siRNAs and transfection

siRNAs were synthesized using 2′-O-ACE-RNA phosphoramides (Dharmacon, Lafayette, CO). The sense and antisense strands of siRNA were: Bak, beginning at nt 310, 5′-P-UGCCUACGAACUCUUCACCdTdT-3′ (sense), 5′-P-GGUGAAGAGUUCGUAGGCAdTdT-3′ (antisense); Bax, beginning at nt 217, 5′-P-UAUGGAGCUGCAGAGGAUGdTdT-3′ (sense), 5′-P-CAUCCUCUGCAGCUCCAUAdTdT-3′ (antisense); P represents 5′ phosphate. RNAs were deprotected and annealed according to the manufacturer's instruction. Nonspecific Control siRNA (Target: 5′-NNATTGTATGCGATCGCAGAC-3′) was also acquired from Dharmacon. Two hundred thousand recombinant DCs on a 6-well vessel were transfected with 300 pmol of the synthesized siRNAs using Oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The transfected cells were used for subsequent experiments 3 days later. We used FITC-labeled siRNA to document transfection efficiency of the DCs, using flow cytometry analysis. Virtually 100% of the DCs were successfully transfected with siRNA (data not shown).

Construction of the DC vaccines

The immortalized DC line, kindly provided by Dr. Kenneth Rock (University of Massachusetts, Worcester, MA), was genetically manipulated using a retroviral system.22 Briefly, Bone marrow cells flushed from the femurs and tibias of C57BL/6 mice were infected with a retrovirus encoding murine GM-CSF, myc and raf oncogene. This DC line was used for construction of the DC vaccines expressing no insert, E7, or Sig/E7/LAMP-1 as described previously.12 For this, phoenix (ϕNX) packaging cells were transfected with a retroviral vector plasmid (pMSCV, pMSCV-E7, or pMSCV-Sig/E7/LAMP-1) using Lipofectamine 2000. Retroviral supernatant from the transfected phoenix cells was incubated with 50% confluent DC in the presence of polybrene (8 μg/ml; Sigma). Following transduction, the retroviral supernatants were removed, and DCs were grown in a culture medium containing 7.5 μg/ml of puromycin for selection. The expression of E7 antigens was confirmed by Western blot analysis.12

Western blot analysis

The expression of E7 and Sig/E7/LAMP-1 proteins in the DCs and the expression of Bak and Bax pro-apoptotic proteins in DC cells transfected with BAK and BAX siRNA was characterized by Western blot analysis as described previously.23 Cells were lysed with protein extraction reagent (Pierce, Rockford, IL). Equal amounts of protein (50 μg) were loaded and separated by SDS-PAGE using a 15% polyacrylamide gel. The gels were electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). Blots were then blocked for 2 hr at room temperature with phosphate buffered saline (PBS)/0.05% Tween 20 (TTBS) containing 5% nonfat milk. Membranes were probed with E7-specific Ab (Zymed, San Francisco, CA) and anti-BAK or BAX mouse monoclonal antibody (Cell Signaling Technology, Beverly, MA), at 1:1,000 dilution in TTBS for 2 hr. They were washed 4 times with TTBS, then incubated with goat antimouse IgG conjugated with horseradish peroxidase (Zymed, San Francisco, CA) in a 1:1,000 dilution in TTBS containing 5% nonfat milk. Membranes were washed 4 times with TTBS and developed using Hyperfilm-enhanced chemiluminescence (Amersham, Piscataway, NJ).

Determination of apoptotic cells after CTL assay

DCs expressing no insert, E7, or Sig/E7/LAMP-1 transfected with control siRNA or BAK/BAX siRNA, were incubated for 4 or 18 hr with an E7-specific CD8+ T cell line at different E:T ratios (12.5:1, 2.5:1, 0.5:1 or 0.1:1).7 We used FITC-conjugated anti-CD8 antibody to stain for CD8+ E7-specific T cells. We gated CD8 cells (DCs) for activated caspase-3 analysis in order to characterize the percentage of apoptotic dendritic cells. Detection of apoptotic cells in the DCs was accomplished using PE-conjugated rabbit antiactive Caspase-3 antibody (BD Bioscience, San Diego, CA) according to the manufacturer's instructions. The percentage of apoptotic cells was analyzed using flow cytometry.

In vitro activation of E7-specific CD8+ T cells by DCs

For assessing whether the recombinant DCs transfected with the siRNAs were capable of presenting the E7 antigen to CD8+ T cells, 5 × 104 DCs were incubated for 18 hr with 5 × 105 of the E7-specific CD8+ T cell line.7, 24 Activated IFN-γ-secreting E7-specific CD8+ T cells were identified by staining for both surface CD8+ and intracellular IFN-γ, and analyzed by flow cytometry analysis as described earlier.

Immunization with DCs

Six- to eight-week-old female C57BL/6 mice were purchased from Daehan Biolink (Chungbuk, Korea). All animal procedures were performed according to approved protocols and in accordance with recommendations for the proper use and care of laboratory animals. For the obvious downregulation of Bax and Bak expression, DCs transfected with BAK and BAX siRNA were used 3 days after transfection as described previously.25 After 2 washes in phosphate-buffered saline, 1 × 106 of the retrovirus-transduced DCs in 0.1 ml of phosphate-buffered saline were injected intramuscularly into mice twice, with 1 week interval between the injections as described previously.7

Intracellular cytokine staining and flow cytometry analysis

Splenocytes were harvested from mice (3 mice per group) 1 week after the last vaccination. Prior to intracellular cytokine staining, 4 × 106 pooled splenocytes from each vaccination group were incubated overnight with 1 μg/ml of E7 (RAHYNIVTF) peptide containing an MHC class I epitope (aa 49-57) for detection of E7-specific CD8+ T-cell precursors.25 IFN-γ staining and flow cytometry analysis were performed as described previously. Analysis was performed on a Becton-Dickinson FACScan with CELLQuest software (Becton Dickinson Immunocytometry System, Mountain View, CA). For the determination of the avidity of E7-specific CD8+ T cells in mice vaccinated with DC-Sig/E7/LAMP-1 transfected with the siRNAs, the pooled splenocytes were incubated overnight with different concentrations of E7 peptide (aa 49-57; 1, 10−1, 10−2, 10−3, 10−4, 10−5, 10−6, 10−7, or 10−8 μg/ml). The number of E7-specific IFN-γ-secreting CD8+ T cells was determined using intracellular cytokine staining and FACScan analysis as described earlier.

In vivo tumor treatment

The HPV-16 E7-expressing murine tumor model, TC-1, has been described previously.11 In brief, HPV-16 E6, E7 and ras oncogene were used to transform primary C57BL/6 mouse lung epithelial cells to generate TC-1. Mice (5 per group) were challenged by intravenous injection of 5 × l05 TC-1 tumor cells/mouse into the tail, in order to simulate hematogenous spread.11 Mice were treated with DCs 3 days after the tumor challenge. Mice were monitored twice a week and killed on day 42 after the last vaccination. The mean number of pulmonary nodules in each mouse was evaluated by experimenters blinded as to sample identity. To study the subsets of lymphocytes that are important for the antitumor effects, a tumor protection experiment was performed, coupled with in vivo antibody depletion that used a protocol similar to one previously described.11 Briefly, antibody depletion was initiated 1 week after the second vaccination and continued until the animals were killed. mAb GK1.5 was used for CD4 depletion, mAb 2.43 for CD8 depletion, and mAb PK136 for NK depletion. In vivo tumor treatment and antibody depletion experiments were performed at least twice to generate reproducible data.

In vivo clearance of DCs in the primed mouse

C57BL/6 mice (5 per group) were first primed with DC-no insert or DC-Sig/E7/LAMP-1. Seven days later, the mice were boosted with carboxyfluorescein (CFSE)-labeled DC-Sig/E7/LAMP-1 transfected with control siRNA or BAK/BAX siRNA. To create CFSE positive DCs transfected with different siRNA, DCs were labeled with 5 μM CFSE for 10 min. Unlabeled DCs transfected with BAK/BAX siRNA were used as a negative control. Two days after boosting with the DCs, popliteal lymph nodes were harvested from the vaccinated mice. Isolated lymphocytes were analyzed by forward and side scatter and gated around a population of CD80+ cells with the size and granular characteristics of DCs. The percentage of CFSE+ cells among the gated CD80+ cells was analyzed using a protocol described previously.26

Statistical analysis

All data, expressed as means ± standard deviation (SD), are representative of at least 2 different experiments. Data for intracellular cytokine staining with flow cytometry analysis and tumor treatment experiments were evaluated by analysis of variance (ANOVA). Comparisons between individual data points were made using Student's t-test. In the tumor protection experiment, the principal outcome of interest was time to tumor development. The event time distributions for different mice were compared using the method of Kaplan and Meier and the log-rank statistic. All p values < 0.05 were considered significant.

Results

Transfection of DC cell lines with BAK/BAX siRNA silences the expression of Bak and Bax proteins

To examine if E7 proteins were translated in DCs, Western blot analysis was performed with an E7-specific antibody. In DC-E7 and DC-Sig/E7/LAMP-1, the bands of E7 and Sig/E7/LAMP-1 at their corresponding molecular weight were detected. In contrast, there was no band in the DC-no insert. This result is the same as that previously described.12 We also performed Western blot analysis to determine whether transfection of recombinant DCs expressing no insert, E7 or Sig/E7/LAMP-1 with BAK/BAX siRNA would down-regulate the expression of Bak and Bax proteins in transfected cells. As shown in Figure 1, the expression of Bak and Bax proteins was abolished 3 days after transfection in lysates from the DCs transfected with BAK/BAX siRNA. No expression of Bak or Bax was identified up to 7 days after transfection. Expression of Bak and Bax was detected at below-normal levels by day 9, and this expression returned to normal levels by day 11 after transfection as our previous report.25 In contrast, expression of Bak and Bax proteins was detected in the DCs after transfection with control siRNA, and the levels of expression were similar to the expression of Bak and Bax proteins by non-siRNA-transfected DC cells (data not shown). We also analyzed β-actin expression in transfected DCs in order to demonstrate that equal amounts of cell lysates were loaded for Western blot analysis. These results indicate that BAK/BAX siRNA abolish Bak and Bax protein expression during the period of immune priming by the transfected DCs.

Figure 1.

Western blot analysis to detect expression of Bak and Bax protein in DCs transfected with the various siRNA constructs. Various DCs were transfected with control or BAK/BAX siRNAs. Equal amounts of protein (50 μg) were loaded and separated by SDS-PAGE using a 15% polyacrylamide gel. Western blot analysis was performed with 50 μg of the cell lysate and anti-BAK or BAX mouse monoclonal antibody 3 days after transfection. β-actin was used as a control to indicate that equal amounts of cell lysates were loaded.

DCs transfected with BAK/BAX siRNA are more resistant to CTL killing than those with control siRNA

To determine whether the DCs expressing E7 or Sig/E7/LAMP-1 transfected with BAK and BAX siRNA could resist a CTL-induced apoptosis, we incubated the DCs transfected with the siRNAs with an E7-specific CD8+ T cell line and determined the percentages of apoptotic cells in DC populations 4 or 18 hr after incubation. DC-no insert was used as a negative control. As shown in Figure 2, the significant difference in apoptotic cell percentages between the E7-presenting DCs transfected with control siRNA and those with BAK/BAX siRNA was observed 4 hr after incubation. The difference was greater at 18 hr. Although 80–90% of DC-E7 and DC-Sig/E7/LAMP-1 cells transfected with control siRNA were apoptotic, the DCs transfected with BAK/BAX siRNA generated less than 15% apoptotic cells 18 hr after incubation with the CD8+ T cell line, particularly at low E:T ratios (0.1:1). These data suggest that transfection of DCs with BAK/BAX siRNA leads to down-regulation of Bak and Bax protein expression, resulting in resistance to apoptosis induced by antigen-specific CD8+ T cells.

Figure 2.

In vitro resistance of the DCs transfected with the siRNAs to CD8+ T cell-mediated CTL-killing. DCs were incubated with an E7-specific CD8+ T cell line at different E:T ratios (12.5:1, 2.5:1, 0.5:1 or 0.1:1) for 4 (a) or 18 hr (b). FITC-conjugated anti-CD8 antibody was used to stain for a CD8+ E7-specific T cell line, and then CD8 cells (DCs) were gated to identify the percentage of apoptotic dendritic cells. Detection of apoptotic cells in the DC cells was performed using PE-conjugated rabbit antiactive caspase-3 antibody (BD Bioscience). The percent of apoptotic cells was analyzed using flow cytometry.

E7-expressing DCs transfected with BAK/BAX siRNA increase the number of activated E7-specific IFN-γ+ CD8+ T cell lines in vitro

We determined whether the transfection of E7-expressing DCs with the siRNAs influenced the ability of the DCs to activate an E7-specific CD8+ T cell line in vitro. For this, the transfected DCs were co-cultured with an MHC class I-restricted E7-specific T cell line at a DC:T-cell ratio of 1:10 in vitro.24 As shown in Figure 3, the DC-no insert transfected with control or BAK/BAX siRNAs failed to induce significant IFN-γ production in E7-specific CD8+ T cells. After 18 hr, IFN-γ-secreting CD8+ T cells were counted using FACS analysis. Figures 3a and 3b demonstrate a more than 10 fold increase in the number of activated T cells following co-incubation of the E7-specific T cell line with the DCs transfected with BAK/BAX siRNA, when compared with DCs transfected with control siRNA. Taken together, these data suggest that the treatment of BAK/BAX siRNA may influence the ability of DCs to activate antigen-specific CD8+ T cells by prolonging DC life span during in vitro activation of an E7-specific IFN-γ+ CD8+ T cell line.

Figure 3.

Intracellular cytokine staining and flow cytometry analysis to demonstrate the in vitro activation of E7-specific T cells by DCs transfected with siRNAs. DCs expressing E7 or Sig/E7/LAMP-1, transfected with control or BAK/BAX siRNAs, were incubated with an E7-specific CD8+ T cell line at a 1:10 mixture of DC:T cell ratios for 18 hr. Intercellular cytokine staining assay was performed to count activated IFN-γ-secreting CD8+ T cells using flow cytometry. The data presented in this figure are from one representative experiment of the 3 performed (a). Bar graph depicting the % of E7 specific CD8+ T cell (b).

Vaccination with the E7-expressing DCs transfected with BAK/BAX siRNA leads to a significant increase in the number and avidity of E7-specific CD8+ T cells in the immune response

To determine whether vaccination with E7-expressing DCs transfected with the siRNAs could enhance the generation of E7-specific IFN-γ+ CD8+ T-cell precursors in vaccinated mice, we performed an intracellular cytokine staining and flow-cytometry analysis using splenocytes from mice vaccinated with the various DCs. As shown in Figure 4a, mice vaccinated with DC-Sig/E7/LAMP-1 cells transfected with BAK/BAX siRNA exhibited an approximately 5 fold increase in the number of E7-specific IFN-γ+ CD8+ T cells, compared with mice vaccinated with DCs transfected with control siRNA. DC-E7 cells transfected with BAK/BAX siRNA also generated more E7-specific T-cell precursors than those with control siRNA. In contrast, the DC-no insert transfected with control or BAK/BAX siRNAs failed to induce significant IFN-γ production in E7-specific CD8+ T cells. Our results demonstrate that immunization with E7-expressing DCs transfected with BAK/BAX siRNA can significantly increase the number of E7-specific IFN-γ+ CD8+ T cells generated in vaccinated mice. In previous studies, the duration of dendritic cell and T-cell interaction has been felt to be important for the generation of high avidity T cells.27, 28 In addition, we have shown that high-avidity CTLs provide better protection against a tumor challenge than low-avidity CTLs.27 Therefore, we performed a functional avidity assay to determine the avidity of E7-specific CD8+ T cells generated by vaccination with DC-Sig/E7/LAMP-1 transfected with control or BAK/BAX siRNAs. We defined the number of IFN-γ-secreting CD8+ T cells stimulated by 1 μg/ml of E7 peptide (aa 49-57) as a maximum response and compared the functional avidity of T cells from mice vaccinated with DC-Sig/E7/LAMP-1 transfected with control or BAK/BAX siRNAs at 50% of the maximum. We found that the concentration of E7 peptide required to achieve 50% of the maximum IFN-γ+ CD8+ T-cell response was about 8 × 10−3 μg/ml for mice vaccinated with the DCs transfected with BAK/BAX siRNA, and about 9 × 10−2 μg/ml for mice vaccinated with DCs transfected with control siRNA (Fig. 4b). Transfection of DC-Sig/E7/LAMP-1 with BAK/BAX siRNA generates higher avidity E7-specific CD8+ T cells in vaccinated mice than transfection of DC-Sig/E7/LAMP-1 with control siRNA.

Figure 4.

Intracellular cytokine staining and flow cytometry analysis to determine the number (a) and functional avidity (b) of IFN-γ-producing E7-specific CD8+ T cells in mice after immunization with E7-expressing DCs transfected with various siRNA constructs. Mice (5 per group) were vaccinated twice with E7-expressing DCs transfected with BAK/BAX siRNA or control siRNA. There was a 1-week interval between injections. Splenocytes were harvested 1 week after the last vaccination, stained for CD8+ and IFN-γ, and analyzed by flow cytometry to detect activated E7-specific CD8+ T cells. The bar graph depicts the number of IFN-γ-expressing E7-specific CD8+ T cells per 3 × 105 splenocytes from vaccinated mice (mean ± SD) (a). For the determination of the avidity of E7-specific CD8+ T cells, pooled splenocytes were incubated overnight with different concentrations of E7 peptide. The number of E7-specific IFN-γ-secreting CD8+ T cells was determined using intracellular cytokine staining and FACScan analysis as described earlier. We defined the number of IFN-γ-expressing CD8+ T cells stimulated with E7 peptide (amino acids 49-57; 100 μg/ml) as the maximal response. The horizontal line allows comparison of E7 peptide concentrations needed for 50% of maximal E7-specific CD8+ T-cell response in mice vaccinated using the 2 regimens. The data presented in this figure are from 1 representative experiment of the 2 performed (b).

Vaccination with DC-Sig/E7/LAMP-1 cells transfected with (BAK/BAX) siRNA generates better antitumor effects than controls

To determine whether the observed increase in the number and functional avidity of E7-specific CD8+ T-cell precursors could translate into a better E7-specific antitumor effect, we performed an in vivo tumor treatment experiment using a previously characterized E7-expressing tumor model, TC-1.11 DC-Sig/E7/LAMP-1 cells, which exhibited better immunogenicity than DC-E7 cells in Figure 4, were selected for this experiment. To compare the antitumor effects generated by vaccination with DC-Sig/E7/LAMP-1 transfected with BAK/BAX siRNA or control siRNA, we performed an in vivo tumor treatment experiment in a lung tumor metastasis model at a high lethal dose. In this experiment, 5 × 105 TC-1 cells were injected via the tail vein. This number was 50 times more TC-1 cells than that commonly used in our studies.7, 10, 11, 12, 15, 17, 29 Mice were first challenged with the tumor cells, followed 3 days after tumor challenge by treatment with DC-Sig/E7/LAMP-1 cells transfected with control or BAK/BAX siRNAs. Mice were killed 28 days after challenge, and the number of pulmonary tumor nodules was counted. As shown in Figure 5a, mice treated with the DCs transfected with BAK/BAX siRNA demonstrated the lowest number of pulmonary nodules, compared with mice treated with the DCs transfected with control siRNA (p < 0.007), or the naïve control group. These results show that vaccination with DCs transfected with BAK/BAX siRNA generates a significantly better therapeutic antitumor effect than vaccination with DCs transfected with control siRNA.

Figure 5.

In vivo tumor treatment experiments in mice vaccinated with DCs expressing E7 or Sig/E7/LAMP-1. An in vivo tumor treatment experiment was performed using a hematogenous spread lung model. Mice were inoculated with 5 × l05 TC-1 tumor cells via tail vein injection and then treated with DC-Sig/E7/LAMP-1 cells transfected with control or BAK/BAX siRNAs 3 days after inoculation. No treatment served as a negative control (a). In vivo antibody depletion experiments to determine the contribution of subsets of lymphocytes to the observed protective antitumor effect (b). Mice were challenged and vaccinated as described in panel (a). CD4, CD8 or NK1.1 depletion was initiated 1 week after the second vaccination. Mice were killed 28 days after tumor challenge to examine the growth of pulmonary nodules. Data are expressed as the mean number of lung nodules. The data presented in this figure are from 1 representative experiment of the 2 performed.

We also performed a tumor treatment experiment with antibody depletion to determine the subset of T lymphocytes responsible for the antitumor response. Mice were challenged with TC-1 and subsequently vaccinated with the DCs as described earlier. Antibody depletion was initiated 1 week after the booster vaccination and continued for 28 days following the challenge. Mice depleted of CD8+ T cells displayed nearly the same degree of tumor growth as naïve mice, and mice depleted of CD4+ T cells displayed slightly increased tumor growth when compared with nondepleted mice. Mice depleted of NK cells did not generate a significantly different number of tumor nodules when compared with mice with no depletion (Fig. 5b). These data suggest that CD8+ T cells are essential for the antitumor effect and that CD4+ T cells may also contribute to the observed antitumor effect, though not as strongly as CD8+ T cells.

E7 peptide-loaded DCs transfected with BAK/BAX siRNA survive longer in vivo than E7 peptide-loaded DCs transfected with control siRNA

To determine whether transfection with BAK/BAX siRNA improves the survival of DC-Sig/E7/LAMP-1 during in vivo conditions, mice were first primed with DC-no insert or DC-Sig/E7/LAMP-1. One week later, the mice were boosted with CFSE-labeled DC-Sig/E7/LAMP-1 transfected with control siRNA or BAK/BAX siRNA. Unlabeled DCs transfected with BAK/BAX siRNA were used as a negative control. Two days after boosting with the DCs, popliteal lymph nodes were harvested from the vaccinated mice. Isolated lymphocytes were further analyzed using flow cytometry. The percentage of CFSE+ cells among the gated CD80+ monocyte-like cells with size and granular characteristics of DC was measured, using a protocol described previously.26 As shown in Figures 6a and 6b, at 2 days after vaccination with DC-no insert, there was no significant difference between the percentage of CFSE+ cells among the gated CD80+ monocyte-like cells in mice given BAK/BAX siRNA from mice given control siRNA. In comparison, in mice primed with DC-Sig/E7/LAMP-1, we detected a significant decrease in the percentage of CFSE+ CD80+ DCs in mice that received cells with control siRNA, compared with that of CFSE+ CD80+ DCs in mice that received cells with BAK/BAX siRNA. More than 90% of CFSE+ CD80+ DCs were caspase-3 negative, indicating that these cells were not apoptotic (data not shown). Our data suggest that transfection of E7-expressing DCs with BAK/BAX siRNA may protect DCs from being killed by E7-specific immunity, thus prolonging DC life in vivo.

Figure 6.

Flow cytometry analysis to determine the survival of DC-Sig/E7/LAMP-1 transfected with control siRNA or BAK/BAX siRNA in draining lymph nodes. C57BL/6 mice (5 per group) were first primed with DC-no insert or DC-Sig/E7/LAMP-1. Seven days later, the mice received CFSE-labeled DC-Sig/E7/LAMP-1 transfected with control siRNA or BAK/BAX siRNA. Unlabeled DCs transfected with BAK/BAX siRNA were used as a negative control. Two days after boosting with the DCs, popliteal lymph nodes were harvested and isolated lymphocytes were analyzed by flow cytometry. The monocyte-like cells with size and granular characteristics of DCs were gated. The percentage of CFSE+ cells among the gated CD80+ cells was analyzed. The data presented in this figure are from 1 representative experiment of the 2 performed (a). Bar graph depicting percentages of CFSE-expressing cells out of total CD80+ cells (b).

Discussion

In this study, we have shown that vaccination with HPV16 E7-expressing DCs transfected with siRNA targeting BAK and BAX increased E7-specific antitumor immune responses. These siRNA transfected DCs were highly resistant to apoptotic cell death mediated by E7-specific CD8+ T cells, leading to prolonged DC survival. This resulted in a further increase in DC-mediated vaccine potency. In our previous studies, we demonstrated that retrovirally-transduced DCs endogenously expressing Sig/E7/LAMP-1 (the linkage of a Sig/LAMP-1 molecule to E7) increased the presentation of antigenic E7 peptides to E7-specific T cells in the context of MHC class I and class II, and enhanced the potency of DC vaccines.12 This enhancement of dendritic cell-based vaccine potency might be explained by qualitative changes in antigen-expressing DCs that lead to enhanced activation of E7-specific T cells. DCs, however, have a limited life span that hinders their long-term ability to prime antigen-specific T cells. DCs that present the relevant MHC-peptide complexes qualify as potential targets and are at risk of being eliminated by the CTLs they have activated. This would seriously limit the capacity of DCs to prime CTL immunity.14, 25 During CTL-killing process, as shown in Figures 2 and 6, highly immunogenic DCs having more antigenic peptides on their surface such as DC-Sig/E7/LAMP-1 might be better targets for E7-specific CD8+ T cells, compared with less immunogenic DCs such as DC-E7. Thus, modulation of apoptosis in DCs using siRNA technology is necessary to prolong DC survival and further enhance the potency of DC-based vaccines.

The BAK and BAX siRNA technology can be extended to the preparation of antigen-specific T cells ex vivo. We have shown that E7-expressing DCs transfected with BAK/BAX siRNA were capable of resisting being killed by E7-specific CD8+ T cells, compared with DC cells transfected with control siRNA, leading to an increase in the number of activated CD8+ T cells (Fig. 3). It would be possible that the significant increase in activated CD8+ T cells might be due to change in the expression of molecules important for antigen presentation in DCs, such as CD11c, CD40, CD80, CD86, MHC I and MHC II and pro-inflammatory cytokines, such as IFN-β, IFN-α and TNF-α.30, 31 We therefore performed flow cytometry to determine the expression levels of them important for antigen presentation in DC-Sig/E7/LAMP-1 transfected with BAK/BAX siRNA or control siRNA in nontransfected DC cell line. There were no significant changes in the expression of the tested molecules among the DC cell lines. In our previous report, we observed a similar result in an E7 peptide-loaded DC cell line transfected with BAK/BAX siRNA or control siRNA and in nontransfected DC cell line.26 Recently, it has been reported that synthetic siRNAs complexed with liposomes can be potent inducers of pro-inflammatory cytokines.30, 31 To confirm the induction of the IFN-β, IFN-α, and TNF-α, we performed RT-PCR to determine the mRNA expression levels of them in these DC cell lines transfected with or without the BAK/BAX siRNA or control siRNA/Oligofectamine complexes. Unexpectedly, we found that there were no significant changes in the mRNA levels of the cytokines among these DC cell lines (data not shown). One of plausible explanations about this discrepancy may be a point of time to assess the level of cytokines. Most literatures have shown the induction of cytokines within 1 or 2 days after transfection. Notably, no expression of Bak or Bax was detected in the siRNA-treated DCs we used at days 3 after transfection. This silence of Bak and Bax was lasted by day 9, and the expression of them returned to normal levels by day 11 after transfection.25 From this reason, in this study, the siRNA-treated DCs were used for in vitro and in vivo experiments 3 days after transfection. Similarly, the levels of the immune-modulating molecules were also determined 3 days after transfection of the DCs with the siRNAs. It would be possible that the pro-inflammatory molecules were induced within 1 day after transfection. Subsequently, the levels of the molecules could return to normal levels 3 days after treatment of siRNAs. We could not rule out, however, other possibility that transfection of DCs with siRNA/liposome complexes may affect immune-priming capacity of DCs through other cytokines we did not test such as IL-12, IL-6, IL-10 and INF-γ or other factors such as chemokines that influence DC homing to the draining lymph nodes. Despite a bundle of possible factors to affect DC capacity of priming an immune response, most important thing is that the only difference between control siRNA and BAK/BAX siRNA is a sequence. In some literatures, base sequence motifs including 5′-GUCCUUCAA-3′, which can induce inflammatory cytokines, were identified.31, 32 It is worth to take a notice that the siRNAs we used do not contain these sequence motifs. Thus, the enhanced E7-specific T-cell-mediated immune response may not be due to changes in the expression of the molecules important for antigen presentation in DCs and the pro-inflammatory cytokines in the DC cell lines transfected with BAK/BAX siRNA or control siRNA. Therefore, the increase in activated CD8+ T cells might be due to enhanced survival of dendritic cells mediated by BAK and BAX siRNA. Considering the difficulty in gathering blood from patients for ex vivo T-cell preparation, the application of BAK/BAX siRNA technology might be promising for tumor immunotherapy using adoptive transfer of T cells.

We have shown that DC survival can be prolonged by their transfection with DNA encoding antiapoptotic proteins.17 Among them, the antiapoptotic Bcl-2 family, such BCL-xL and BCL-2, were most effective in increasing the life span of DCs tranfected with a gene gun via an intradermal route. However, since these molecules have been implicated as contributors to oncogenic transformation, utilization of the antiapoptotic DNA encoding Bcl-2 family for clinical applications might be limited.33 We have recently demonstrated that peptide-pulsed BM-DCs transfected with BAK/BAX siRNA were capable of resisting being killed by antigen-specific CD8+ T cells in vivo.29 RNA interference (RNAi) using siRNA targeting proapoptotic proteins provided similar effects, while alleviating oncogenic concerns associated with the use of DNA encoding antiapoptotic proteins.

The discovery of defined tumor antigens and their application in therapeutic cancer vaccines has not yet resulted in a successful therapy for cancer patients. One of the reasons is most of tumor antigens are self ones. There are simple evidences in mouse and human that most cancers, similar to normal somatic cells, do not directly prime self-tumor-antigen-specific T cells very efficiently.34, 35, 36 Like this, self tolerance that normally exists to prevent autoimmune disease may preclude the development of an adequate antitumor response and thwart the development of effective immune responses against tumors.37 Thus, breaking tolerance in tumor-bearing hosts has been seen as a primary requirement for cancer immunotherapy. In this aspect, it is worth to notify that DCs expressing the baculoviral caspase inhibitor, p35, display defective apoptosis, resulting in their accumulation and, in turn, chronic lymphocyte activation and systemic autoimmune manifestations.38 Considering the number of well characterized self-tumor antigens, it will be an interesting challenge to check whether antiapoptotic DCs that present a self tumor antigen break self-tolerance and induce a therapeutic immune response against various tumors.

In this study, we further increased the potency of a Sig/E7/LAMP-1 expressing DC vaccine by prolonging DC life with BAK/BAX siRNA. The DCs transfected with BAK/BAX siRNA enhanced E7-specific CD8+ T-cell activation in vitro. They elicited stronger antitumor effects in vivo, compared with DCs transfected with control siRNA. Thus, a DC-based vaccine strategy incorporating Sig/E7/LAMP-1 DCs transfected with BAK/BAX siRNA may be a promising strategy for tumor immunotherapy.

Ancillary