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Dendritic cells transduced with a PSMA-encoding adenovirus and cocultured with autologous cytokine-induced lymphocytes induce a specific and strong immune response against prostate cancer cells
Urologic Oncology: Seminars and Original Investigations 27 (2009) 26 –32
Original article
Dendritic cells transduced with a PSMA-encoding adenovirus and cocultured with autologous cytokine-induced lymphocytes induce a specific and strong immune response against prostate cancer cells夡 Kebing Wang, Ph.D., Xin Gao, Ph.D.*, Jun Pang, Ph.D., Xiaopeng Liu, Ph.D., Yubin Cai, Ph.D., Yan Zhang, Ph.D., Jianhua Zhou, Ph.D., Hailun Zhan, Ph.D. Department of Urology, Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China Received 19 July 2007; received in revised form 25 September 2007; accepted 28 September 2007
Abstract Objective: The lack of curative therapies for advanced prostate cancer (PCa) has prompted a search for novel treatments such as immunotherapy. In this study, we analyzed whether dendritic cells (DCs) from healthy donors transduced with a PSMA-encoding adenovirus (Ad-PSMA) and cocultured with autologous cytokine-induced killer cells (CIKs) can induce a strong specific immune response against PCa cells in vitro. Materials and Methods: Ad-PSMA was constructed by DNA recombination. DCs and CIKs were prepared by cytokines induction from peripheral blood mononuclear cells, and flow cytometry was used to measure the phenotypes of DCs and CIKs. DCs were transduced with Ad-PSMA and then cocultured with autologous CIKs. The cytotoxicity of the cocultured cells against specific target LNCaP cells and control targets DU145 and PC3 cells was analyzed by a 4-h LDH release assay. Results: DCs were transduced with Ad-PSMA with transfection efficiency of 70% and the transduction did not alter typical morphology of mature DCs. The PSMA protein was effectively expressed in DCs, which were transfected with Ad-PSMA. Ad-PSMA-transduced DCs stimulated CIKs strongly to lyse about 75% of PSMA-expressing PCa cells. Furthermore, the cocultivation of Ad-PSMA-transduced DCs with CIKs could significantly increase the production of interferon-␥ after restimulated with PSMA peptide mixtures. Conclusions: The data demonstrate that DCs, which were transduced with a PSMA-expressing adenovirus and cocultured with autologous CIKs, induce a PSMA-specific, strong immune response against PCa cells. Therefore, this approach may have a potential for an adoptive immunotherapy for patients with advanced PCa. © 2009 Elsevier Inc. All rights reserved. Keywords: Dendritic cell; Cytokine-induced killer cell; Adenovirus; Prostate specific membrane antigen; Prostate cancer; Immunotherapy
1. Introduction Prostate cancer (PCa) is the most frequently diagnosed cancer in old men and also the second leading cause of male cancer death in the Western countries [1]. Radical prostatectomy, which is the standard and curative form of treatment, is suitable only for a small percentage of patients because the tumor has often extended beyond the confinement of the prostate capsule at the time of diagnosis. Advanced, recurrent, and metastatic tumors treated by andro夡 This work was supported by the National Natural Science Foundation of China (no. 30471730). * Corresponding author. Tel.: ⫹(86)20-85516867-2052; fax: ⫹(86)2087536401; and ⫹(86)20-87533706. E-mail address: [email protected] (X. Gao).
1078-1439/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2007.09.003
gen deprivation often develop into hormone-refractory prostate cancer (HRPC) within a few years. In patients with HRPC, systemic treatment combined with effective palliative chemotherapy improves only median survival time from an average of about 12 months to 17–18 months [2]. Therefore, effective novel therapeutic approaches such as immunotherapy are needed. Prostate specific membrane antigen (PSMA) is a welldefined tumor-associated antigen (TAA), and its expression is maintained highly in progressed stages of PCa and in HRPC [3,4]. Patricia et al. have shown that breaking immune-tolerance towards PSMA is possible [5]. Dendritic cells (DCs) are professional antigen-presenting cells that can be loaded ex vivo with TAAs to induce a strong immune response against these antigens [6,7]. DC-based im-
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munotherapy has been shown to be effective for TAAs as CA19-9 in pancreatic carcinoma [8] or MART-1 and MAGE-3 in melanoma [9]. However, primary experiments against PCa showed only limited success [5]. Cytokineinduced killer cells (CIKs) can be developed in large numbers from peripheral lymphocytes, and they are able to lyse tumor cells in a non-MHC-restricted manner [10]. Besides the MHC-independent way, CIKs cultures can be primed by antigen-loaded DCs [10]. Recent studies have demonstrated that DCs pulsed with tumor antigens and coincubated with CIKs boosted cytotoxic activity toward leukemia [11], multiple myeloma [12], osteosarcoma [13], and hepatocellular carcinoma [14]. Therefore, the use of DC and CIK cell cultures could increase the potential of immune response against PCa. Although many epitopes of TAAs are identified, studies have shown that the endogenous processing and presentation of TAA might be more efficient for DCs than the exogenous loading of synthetic TAA peptides. Transduction of DCs with a TAA-encoding adenovirus offers potential advantages over peptide-pulsing. In this paper, it was our aim to evaluate the probability of an adoptive and/or a DC-based immune therapy in patients with PCa. DCs from healthy donors were transduced with a PSMA-expressing adenovirus at high efficiency and cocultured with autologous CIKs. With this approach, we demonstrated that DCs are able to induce a strong and specific immune response against PCa cells in vitro.
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2.3. Construction of adenoviral plasmids pAdTrackCMV-GFP and pAdTrackCMV-GFP-PSMA were recombined with pAdEasy-1 by homologous recombination in Escherichia coli, according to the manufacturer’s instructions [15], to generate adenoviral plasmids pAd and pAd-PSMA. 2.4. Production of virus stocks Adenoviral plasmids were cleaved with PacI. Afterwards, 293A cells were transfected with the plasmids and Lipofectamine2000 (Invitrogen, Carlsbad, CA). Viruses (Ad and Ad-PSMA) were collected 7 to 10 days after transfection and used to generate high-titer viral stocks [16]. Plaque assays were performed as described by Graham and Prevec [17]. 2.5. Generation of DCs Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat from healthy donors by Ficoll density gradient centrifugation (Lymphoprep, Nypacon, Norway). All individuals gave informed consent, and blood was drawn according to the instruction of our local ethics committee. The cells were allowed to adhere for 2 h at 37°C in RPMI 1640 (GIBCO) with 10% human AB serum (the Blood Center of Guangzhou, China). The nonadherent cells were collected to generate CIKs. Adherent cells were cultured in RPMI 1640 with 10% human AB serum, 1000 U/ml GM-CSF and 1000 U/ml IL-4 (PeproTech, Rocky Hill, NJ). On day 7, 500 U/ml TNF-␣ (PeproTech) was added.
2. Materials and methods 2.6. Adenoviral transduction of DCs 2.1. Cell lines 293A Cells (ATCC) were used to propagate E1-deleted adenoviruses. The human PSMA-positive PCa cell line LNCaP, the human PSMA-negative PCa cell lines DU145, and PC-3 (ATCC) were used as targets for cytotoxicity assays. LNCaP cells were maintained in F12 (GIBCO, Grand Island, NY) and others were maintained in high glucose DMEM (GIBCO) with 10% fetal bovine serum (GIBCO) at 37°C in a 5% CO2 atmosphere.
On day 6 of the culture, transduction of DCs with virus was conducted in 6-well plates with 1 ⫻ 106 cells/well in 300 ul serum-free medium. Virus was added to the wells at a multiplicity of infection (MOI) 200. Transduction was allowed to proceed for 2 h at 37% in 5% CO2. Then fresh RPMI 1640 containing 10% AB serum, GM-CSF, and IL-4 was added to the culture up to 3 mL per well. After 48 h of incubation, cell viability was assessed by trypan blue exclusion (Sigma) and the transduction efficiency of DCs was checked by GFP expression with fluorescence microscopy and flow cytometry.
2.2. Plasmids 2.7. PSMA expression in adenovirus-transduced DCs Adenoviral backbone-vector pAdEasy-1 and transfervector pAdTrackCMV-GFP (Stratagene, La Jolla, CA) were used to generate replication-deficient adenovirus. The PSMA-encoding plasmid pET-30a-PSMA (PSMA cDNA was extracted from fresh PCa tissues of one HLA-A24⫹ patient) was kindly provided by Dr. Huangjiang, Sun YatSen University, China. The PSMA gene was cut out and cloned into pAdTrackCMV-GFP to generate pAdTrackCMV-GFP-PSMA.
Forty-eight hours after transduction, 5 ⫻ 106 adenovirusinfected DCs were collected and their proteins were extracted by chemical reagents (Geme, Shanghai, China). For Western blot analysis, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) through an 8% polyacrylamide gel and were then transferred onto a nitrocellulose membrane. The membrane was incubated with 5% non-fat milk in PBS and later with
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mouse anti-PSMA mAb (Bioscience, Buckinghamshire, United Kingdom) for 2 h at room temperature. After washing, the membrane was incubated with an alkaline phosphatase-conjugated goat anti-mouse IgG antibody (Bioscience) for 1 h at room temperature. Immunoreactive bands were detected by the ECL Western blot analysis system (Bioscience). 2.8. Surface marker analysis of DCs On day 8, non-transduced and adenovirus-transduced DCs were collected and resuspended in cold FACS buffer (phosphate-buffered saline with 0.2% BSA and 0.09% sodium azide). Cells were immunostained with fluorescein isothio-cyanate (FITC) conjugated mouse anti-human CD14, CD40, CD80, CD83 mAbs, and phycoerythrin (PE) conjugated mouse anti-human CD86 mAb, together with irrelevant isotype-matched FITC- or PE-conjugated control mAbs (all mAbs were obtained from BD Pharmingen [San Jose, CA]). Cells were incubated with antibodies on ice for 15 min, washed twice with PBS, resuspended, and phenotyped on flow cytometric analysis using a FACS Calibur instrument (IMK-Lymphocyte; BD Biosciences, San Jose, CA). 2.9. Generation of CIKs Nonadherent human PMBCs were prepared and grown in culture flasks coated with 30 ug/ml human anti-CD3 mAb (Yes Biotech Lab, Ontario, Canada) at a density of 5 ⫻ 106 cells/ml in 5ml complete medium, consisting of RPMI 1640, 10% human AB serum, 1000 U/ml IL-2 (Boehringer, Mannheim, Germany), and 2-mM glutamine. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. On day 8, CIKs were cocultured for another 7 d with adenovirus-transduced mature DCs at a stimulator to responder ratio of 1:50. Then CIKs were tested for their activities. 2.10. Analysis of IFN-␥-secreting lymphocytes IFN-␥-secreting cells were detected on day 7 of the coculture. Briefly, pure CIKs and the cocultured cells of CIKs with Ad- and Ad-PSMA-infected DCs were restimulated respectively for 24 h at 37°C with 20 ug/ml PSMA peptide mixtures including the PSMA 624 – 632 peptide (HLA-A24⫹, TYSVSFDSL) and the PSMA 178 –186 peptide (HLA-A24⫹, NYARTEDFF) (Institute of Huada Gene, Beijing, China). In addition, 10 ug/ml intracellular transport inhibitor brefeldin A (BFA) was added. Cells were labeled with antibodies against CD3 (PE-Cy5) and CD8 (allophycocyanine, APC) for 15 min; then they were permeabilized with FACS permeabilizing solution for another 10 min at room temperature. These cells were washed and incubated with antibody against IFN-␥ (FITC) for 30 min at room temperature in the dark (All mAbs were obtained from BD Pharmingen). Unbound antibodies were removed by two washes. Finally, cells were analyzed by flow cytometry.
2.11. Cytotoxicity assay A CytoTox 96® nonradioactive cytotoxicity assay-lactate dehydrogenase (LDH) release (Promega, Madison, WI) was used to measure cytotoxic activity according to the manufacturer’s instruction. In brief, target cells [10,000] including LNCaP, PC3 and DU145 cells were plated in triplicate in a U-bottomed 96-well plate and incubated for 4 h with different effector cells, including CIKs, Ad-DC-CIKs (CIKs cocultured with Ad-transduced DCs), and Ad-PSMA-DC-CIKs (CIKs cocultured with Ad-PSMA-transduced DCs) in various effector-to-target ratios. Maximal release of LDH was performed by completely lysing target cells. Target cells without effector cells were used as negative controls (spontaneous release). Cytotoxicity was calculated as percentage cytotoxicity ⫽ (experimental absorbance minus spontaneous release of effector cells) minus spontaneous release of target cells/(maximal release minus spontaneous release of target cells). 2.12. Statistical analysis Data were presented as mean ⫾ standard deviation. Statistical differences were considered to be significant at a P-value less than 0.05 as determined by a paired Student’s t-test using SPSSv11.5 (SPSS Inc., Chicago, IL).
3. Results 3.1. Construction of recombinant adenoviruses and transduction of DCs with adenovirus The PSMA cDNA orientation in pAdTrackCMV-GFPPSMA was confirmed by sequencing (data not shown). After digestion with PacI, both pAd and pAd-PSMA formed 2 fragments of about 30 kb and 3.0 or 4.5 kb, which indicated the success of their structures (data not shown). Adenoviral constructs were cleaved with PacI, and then encapsulated with 293A cells to produce adenovirus: Ad (without transgene) and Ad-PSMA (encoding PSMA). Final yields were about 1011plaque-forming units (pfu). DCs were transduced with adenovirus at MOI 200 to analyze the transduction efficiency. About 70% of DCs were positive for GFP expression by fluorescence microscopy (Fig. 1A) and flow cytometry (Fig. 1B). With regard to cell viability, MOI 200 was considered optimal for gene transduction because cell viability was ⬎95% (Fig. 1B). Forty-eight hours after transduction, the PSMA expression in DCs was evaluated by Western blot. As seen in Fig. 1C, the PSMA protein was expressed in Ad-PSMA-transduced DCs and LNCaP cells, but not in Ad-transduced DCs. 3.2. Phenotype of DCs and of Ad-PSMA-transduced DCs On day 8 of cell culture, mature DCs were harvested. These cells were loosely suspended, exhibited irregular
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Fig. 1. Transduction of dendritic cells (DCs) with adenovirus. (A) Ad-PSMA-transduced DCs under inverted fluorescence microscope (200⫻). (B) Transduction efficiency in DCs. GFP expression was evaluated by flow cytometry 48 h after gene transduction (M1, % of GFP-positive cells). The percentage of viable cells was evaluated by trypan blue staining. (C) Western blot analysis of PSMA expression. The specific immunoreactive single band can be seen in Ad-PSMA-transduced DCs and LNCaP cells, but not in Ad-transduced DCs. (Color version of figure is available online.)
shapes, and displayed many fine processes at their edges. The phenotype of transduced and untransduced mature DCs was analyzed by FACS. The results showed that the expression of CD40, CD80, CD86, CD14, and CD83 was 96.78 ⫾ 2.51%, 66.25 ⫾ 2.78%, 97.24 ⫾ 2.15%, 1.37 ⫾ 0.21%, and 33.52 ⫾ 2.81%, respectively, in untreated DCs, and 95.10 ⫾ 2.60%, 68.12 ⫾ 2.45%, 96.97 ⫾ 2.73%, 1.35 ⫾ 0.25%, and 35.42 ⫾ 2.15%, respectively, in Ad-PSMA-transduced DCs, which suggested insignificant changes in the expression profile of DC surface markers between adenoviraltransduced DCs and non-transduced DCs (P ⬎ 0.05). 3.3. Phenotype of CIKs CIKs were generated from nonadherent PBMCs with IL-2 in culture flask immobilized anti-CD3 mAb. On day 4 of the cocultivation, the expression of CD3, CD4, CD8, and CD56 was checked by flow cytometry. CIKs cocultured with Ad-PSMA-pulsed DCs increased significantly the rate of CD3⫹CD4⫹, CD3⫹CD8⫹, and CD3⫹CD56⫹ (Table1). Furthermore, we found an enhanced proliferation of CIKs after contact with DCs. Between day 7 and day 15, the proliferative power was 800-fold for Ad-PSMA-DC-CIKs, 560-fold for Ad-DC-CIKs, and 220-fold for CIKs. 3.4. PSMA-specific activation of CIKs through CD8 population PSMA-specific activation of lymphocytes was examined using an IFN-␥-secretion assay after restimulation with PSMA peptide mixtures. When lymphocytes were cocultured with Ad-PSMA- transduced DCs, lymphocytes double
positive for CD8 and IFN-␥-secretion amounted to 4.35%. In contrast, only 2.15% lymphocytes were CD8⫹ and IFN␥⫹, when cocultured with Ad-transduced DCs, and 1.99% in CIKs (P ⬍ 0.01) (Fig. 2). 3.5. Induction of strong and specific cytotoxic activity of Ad-PSMA-transduced DCs cocultured with autologous CIKs The cytotoxicity of CIKs was investigated on LNCaP cells which expressed the PSMA protein, DU145 and PC3 cells, which were negative for the PSMA expression [18]. Lysis of PSMA-positive LNCaP cells was significantly higher (P ⬍ 0.01), when CIKs were cocultured with AdPSMA-transduced DCs than with Ad-transduced DCs at an appropriate rate of 20:1 (effector cells:target cells) that was optimized for our experiments. CIKs without coculture showed only moderate cytotoxicity. In contrast to LNCaP cells, DCs pulsed with Ad-PSMA did not induce higher cytotoxicity against PSMA-negative DU145 and PC3 cells, demonstrating a PSMA specificity of the immune response of CIKs (Fig. 3).
Table 1 The phenotype changes of CIKs cocultured with adenovirus-transfected DC
CIKs Ad-DC-CIKs Ad-PSMA-DC-CIKs
D3⫹CD4⫹ (%)
CD3⫹CD8⫹ (%)
CD3⫹CD56⫹ (%)
8.76 ⫾ 1.10 10.25 ⫾ 1.34 28.60 ⫾ 1.56
68.41 ⫾ 3.21 76.32 ⫾ 3.52 85.27 ⫾ 3.40
18.21 ⫾ 2.70 20.81 ⫾ 2.15 27.23 ⫾ 2.33
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Fig. 2. IFN-␥-assay. Gates were set on CD3⫹ T lymphocytes. After restimulation with PSMA peptide mixtures, Ad-PSMA-DC-CIKs (CIKs cocultured with Ad-PSMA-transduced DCs) increased significantly the production of CD8⫹ (IFN-␥⫹) T cells compared with Ad-DC-CIKs (CIKs cocultured with Ad-transduced DCs) and CIKs. Representative data from 3 independent experiments are shown.
4. Discussion DC-based immunotherapy holds promises but needs enhancing immunogenicity of DCs, and CIKs exhibit nonspecific cytotoxicity against tumor targets but lack antitumor specificity. The cocultivation of tumor antigen-loaded DCs with autologous CIKs appear to make up for their individual deficiencies and boost their antitumor immune effects. In this report, we demonstrated that DCs from healthy donors, which were efficiently transduced with a PSMA-expressing adenovirus and cocultured with autologous CIKs, are able to induce a strong and specific immune response against PSMA-positive PCa cells. Therefore, this approach may have a potential for an adoptive immunotherapy for patients with PCa. PSMA is a well-defined TAA and has been correlated with aggressive disease [19]. It is a transmembrane-carboxypeptidase [20], which is up-regulated 10-fold or more in PCa, from androgen-sensitive to androgen-independent PCa, and in metastatic deposits. Furthermore, PSMA is an internalized membrane protein and it has been shown to serve as a tissue-specific target for adenoviral vectors, which may be applicable for gene therapeutical treatment of prostate cancer [21]. DNA-based immunization could break immunologic tolerance towards TAA and did not induce autoimmune effects in mice. However, the potential of this approach was only efficient for the prevention of tumor growth in mice [22]. Recently, several studies have demonstrated that DCs pulsed with PSMA peptides stimulated CTL responses in advanced cancer patients, but achieved only limited success [5,23]. Recent studies have shown that the immunity of patients with PCa is depressed and could have negative effects on the stimulation of DCs or the capability of lymphocytes to lyse tumor cells [24]. The PCa microenvironment not only kills mature DCs, but also inhibits their generation and maturation [24]. This leads to deficiencies in number and function of DCs. In this study, we used specific cytokines to expand healthy donors-derived DCs to numbers sufficient for the induction of an efficient immune response against
PCa cells. Several mechanisms contribute to the low immunogenicity of PCa. One of the most important ones is the down-regulation of MHC class I expression and blocking of the recognition of tumor cells through T cells. As effector cells, CIKs cultures are able to kill in a MHC independent manner but could be primed by antigen-loaded DCs [10]. Furthermore, CIKs have a high proliferation rate and can be efficiently stimulated by DCs and induce a strong lytic activity in comparison with other immunologic effector cells [25]. Moreover, it has recently been shown that CIKs possess antitumor activity. In accordance with our results, interactions between DCs and CIKs lead to an activation of both populations and augmentation of cytotoxicity of CIKs against tumor cells. This may be an additional advantage to obtain a strong immune response against PCa. Despite their high cytotoxic activity against tumor cells in all clinical
Fig. 3. Strong and specific cytotoxicity of Ad-PSMA-DC-CIKS (CIKs cocultured with Ad-PSMA-transduced DCs). CIKs, Ad-DC-CIKs (CIKs cocultured with Ad-transduced DCs) and Ad-PSMA-DC-CIKs were used as effector cells to test the cytotoxic activities against specific target LNCaP cells and unspecific targets DU145 and PC3 cells. Cytotoxic activities at an effector-to-target ratio of 20:1 were determined by a 4-h LDH release assay. Data are representative of 5 separate experiments (*P ⬍ 0.01 vs. CIKs; #P ⬍ 0.01 vs. Ad-DC-CIKs).
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trials that had used CIKs against different tumors, no autoimmune disease had been observed [26]. TAA loading of DCs can be accomplished by a variety of techniques. Nonviral gene delivery transfer such as lipofection or electroporation can achieve only small transfection efficiencies [27]. The use of purified and defined PSMA peptides to load DCs has been shown for PCa. However, their use is limited to already known MHC-restricted peptides, which can be recognized by human T cells only in the context of MHC class I. Moreover, peptides are stable for a short time and their immunogenic potential is limited [5]. The adenoviral vector is a highly efficient and reproducible method of gene transfer. Several studies have shown that successful adenoviral gene transfer into human DCs resulted in induction of a T-cell response against the tumors [28]. In the present study, adenoviral gene transfer was performed to load DCs efficiently. With adenoviral transduction, we achieved a high PSMA expression. According to Gonzalez-Carmona [14], the high and long-term PSMA expression in adenovirus-transduced DCs seems to be a crucial parameter for induction of a potent immune response against a TAA, which can pulse large numbers of functional DCs in the course of DCs maturation and increase strongly expansion and stimulation of PSMA-specific effector cells against PSMA-expressing PCa cells. Despite the efficient adenoviral transduction and high PSMA expression were achieved in DCs, the phenotype of DCs did not change, which are similar to the results of Gonzalez-Carmona [14]. Other advantages of adenoviral transduction of full-length PSMA gene include the presentation of both MHC class I and II epitopes and previously unknown antigenic epitopes of PSMA [29]. The induction of a specific TAA immune response is a crucial factor for the design of immunotherapeutic strategies against cancer. The use of tumor lysates, dendritomas formed by fusion of DCs with allogenic carcinoma cells, or irradiated carcinoma cells have been included in recent protocols against carcinoma. However, these induce an immune response against unknown antigens, which could produce severe autoimmune diseases. In this report, we demonstrated a PSMA-specific immune response by an IFN-␥secretion assay. Antigen-specific interferon-␥ secreted by the cocultured cells of CIKs with Ad-PSMA-transduced DCs was significantly increased after restimulation with PSMA peptide mixtures, which indicated that the immune response was PSMA specific. In fact, such IFN-␥ can also inhibit tumor cell growth and promote cytotoxic effects [30]. Further research in vivo and its effects on humoral response are still to be done.
5. Conclusion Our data indicate that DCs from healthy donors infected with a PSMA-expressing adenovirus at a high transduction efficiency and cocultured with autologous CIKs can induce
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a PSMA-specific and strong immune response against PCa cells in vitro. This approach may induce regression of PCa and thus represents a novel promising adoptive immunotherapeutic approach to be tested in clinical trials for advanced PCa patients.
Acknowledgments The authors thank Mr. Yue He and Mr. Xinbo Xie from Cellkine Technology Co. Ltd. of Guangzhou for their excellent technical assistance.
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Original article
Dendritic cells transduced with a PSMA-encoding adenovirus and cocultured with autologous cytokine-induced lymphocytes induce a specific and strong immune response against prostate cancer cells夡 Kebing Wang, Ph.D., Xin Gao, Ph.D.*, Jun Pang, Ph.D., Xiaopeng Liu, Ph.D., Yubin Cai, Ph.D., Yan Zhang, Ph.D., Jianhua Zhou, Ph.D., Hailun Zhan, Ph.D. Department of Urology, Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China Received 19 July 2007; received in revised form 25 September 2007; accepted 28 September 2007
Abstract Objective: The lack of curative therapies for advanced prostate cancer (PCa) has prompted a search for novel treatments such as immunotherapy. In this study, we analyzed whether dendritic cells (DCs) from healthy donors transduced with a PSMA-encoding adenovirus (Ad-PSMA) and cocultured with autologous cytokine-induced killer cells (CIKs) can induce a strong specific immune response against PCa cells in vitro. Materials and Methods: Ad-PSMA was constructed by DNA recombination. DCs and CIKs were prepared by cytokines induction from peripheral blood mononuclear cells, and flow cytometry was used to measure the phenotypes of DCs and CIKs. DCs were transduced with Ad-PSMA and then cocultured with autologous CIKs. The cytotoxicity of the cocultured cells against specific target LNCaP cells and control targets DU145 and PC3 cells was analyzed by a 4-h LDH release assay. Results: DCs were transduced with Ad-PSMA with transfection efficiency of 70% and the transduction did not alter typical morphology of mature DCs. The PSMA protein was effectively expressed in DCs, which were transfected with Ad-PSMA. Ad-PSMA-transduced DCs stimulated CIKs strongly to lyse about 75% of PSMA-expressing PCa cells. Furthermore, the cocultivation of Ad-PSMA-transduced DCs with CIKs could significantly increase the production of interferon-␥ after restimulated with PSMA peptide mixtures. Conclusions: The data demonstrate that DCs, which were transduced with a PSMA-expressing adenovirus and cocultured with autologous CIKs, induce a PSMA-specific, strong immune response against PCa cells. Therefore, this approach may have a potential for an adoptive immunotherapy for patients with advanced PCa. © 2009 Elsevier Inc. All rights reserved. Keywords: Dendritic cell; Cytokine-induced killer cell; Adenovirus; Prostate specific membrane antigen; Prostate cancer; Immunotherapy
1. Introduction Prostate cancer (PCa) is the most frequently diagnosed cancer in old men and also the second leading cause of male cancer death in the Western countries [1]. Radical prostatectomy, which is the standard and curative form of treatment, is suitable only for a small percentage of patients because the tumor has often extended beyond the confinement of the prostate capsule at the time of diagnosis. Advanced, recurrent, and metastatic tumors treated by andro夡 This work was supported by the National Natural Science Foundation of China (no. 30471730). * Corresponding author. Tel.: ⫹(86)20-85516867-2052; fax: ⫹(86)2087536401; and ⫹(86)20-87533706. E-mail address: [email protected] (X. Gao).
1078-1439/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2007.09.003
gen deprivation often develop into hormone-refractory prostate cancer (HRPC) within a few years. In patients with HRPC, systemic treatment combined with effective palliative chemotherapy improves only median survival time from an average of about 12 months to 17–18 months [2]. Therefore, effective novel therapeutic approaches such as immunotherapy are needed. Prostate specific membrane antigen (PSMA) is a welldefined tumor-associated antigen (TAA), and its expression is maintained highly in progressed stages of PCa and in HRPC [3,4]. Patricia et al. have shown that breaking immune-tolerance towards PSMA is possible [5]. Dendritic cells (DCs) are professional antigen-presenting cells that can be loaded ex vivo with TAAs to induce a strong immune response against these antigens [6,7]. DC-based im-
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munotherapy has been shown to be effective for TAAs as CA19-9 in pancreatic carcinoma [8] or MART-1 and MAGE-3 in melanoma [9]. However, primary experiments against PCa showed only limited success [5]. Cytokineinduced killer cells (CIKs) can be developed in large numbers from peripheral lymphocytes, and they are able to lyse tumor cells in a non-MHC-restricted manner [10]. Besides the MHC-independent way, CIKs cultures can be primed by antigen-loaded DCs [10]. Recent studies have demonstrated that DCs pulsed with tumor antigens and coincubated with CIKs boosted cytotoxic activity toward leukemia [11], multiple myeloma [12], osteosarcoma [13], and hepatocellular carcinoma [14]. Therefore, the use of DC and CIK cell cultures could increase the potential of immune response against PCa. Although many epitopes of TAAs are identified, studies have shown that the endogenous processing and presentation of TAA might be more efficient for DCs than the exogenous loading of synthetic TAA peptides. Transduction of DCs with a TAA-encoding adenovirus offers potential advantages over peptide-pulsing. In this paper, it was our aim to evaluate the probability of an adoptive and/or a DC-based immune therapy in patients with PCa. DCs from healthy donors were transduced with a PSMA-expressing adenovirus at high efficiency and cocultured with autologous CIKs. With this approach, we demonstrated that DCs are able to induce a strong and specific immune response against PCa cells in vitro.
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2.3. Construction of adenoviral plasmids pAdTrackCMV-GFP and pAdTrackCMV-GFP-PSMA were recombined with pAdEasy-1 by homologous recombination in Escherichia coli, according to the manufacturer’s instructions [15], to generate adenoviral plasmids pAd and pAd-PSMA. 2.4. Production of virus stocks Adenoviral plasmids were cleaved with PacI. Afterwards, 293A cells were transfected with the plasmids and Lipofectamine2000 (Invitrogen, Carlsbad, CA). Viruses (Ad and Ad-PSMA) were collected 7 to 10 days after transfection and used to generate high-titer viral stocks [16]. Plaque assays were performed as described by Graham and Prevec [17]. 2.5. Generation of DCs Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat from healthy donors by Ficoll density gradient centrifugation (Lymphoprep, Nypacon, Norway). All individuals gave informed consent, and blood was drawn according to the instruction of our local ethics committee. The cells were allowed to adhere for 2 h at 37°C in RPMI 1640 (GIBCO) with 10% human AB serum (the Blood Center of Guangzhou, China). The nonadherent cells were collected to generate CIKs. Adherent cells were cultured in RPMI 1640 with 10% human AB serum, 1000 U/ml GM-CSF and 1000 U/ml IL-4 (PeproTech, Rocky Hill, NJ). On day 7, 500 U/ml TNF-␣ (PeproTech) was added.
2. Materials and methods 2.6. Adenoviral transduction of DCs 2.1. Cell lines 293A Cells (ATCC) were used to propagate E1-deleted adenoviruses. The human PSMA-positive PCa cell line LNCaP, the human PSMA-negative PCa cell lines DU145, and PC-3 (ATCC) were used as targets for cytotoxicity assays. LNCaP cells were maintained in F12 (GIBCO, Grand Island, NY) and others were maintained in high glucose DMEM (GIBCO) with 10% fetal bovine serum (GIBCO) at 37°C in a 5% CO2 atmosphere.
On day 6 of the culture, transduction of DCs with virus was conducted in 6-well plates with 1 ⫻ 106 cells/well in 300 ul serum-free medium. Virus was added to the wells at a multiplicity of infection (MOI) 200. Transduction was allowed to proceed for 2 h at 37% in 5% CO2. Then fresh RPMI 1640 containing 10% AB serum, GM-CSF, and IL-4 was added to the culture up to 3 mL per well. After 48 h of incubation, cell viability was assessed by trypan blue exclusion (Sigma) and the transduction efficiency of DCs was checked by GFP expression with fluorescence microscopy and flow cytometry.
2.2. Plasmids 2.7. PSMA expression in adenovirus-transduced DCs Adenoviral backbone-vector pAdEasy-1 and transfervector pAdTrackCMV-GFP (Stratagene, La Jolla, CA) were used to generate replication-deficient adenovirus. The PSMA-encoding plasmid pET-30a-PSMA (PSMA cDNA was extracted from fresh PCa tissues of one HLA-A24⫹ patient) was kindly provided by Dr. Huangjiang, Sun YatSen University, China. The PSMA gene was cut out and cloned into pAdTrackCMV-GFP to generate pAdTrackCMV-GFP-PSMA.
Forty-eight hours after transduction, 5 ⫻ 106 adenovirusinfected DCs were collected and their proteins were extracted by chemical reagents (Geme, Shanghai, China). For Western blot analysis, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) through an 8% polyacrylamide gel and were then transferred onto a nitrocellulose membrane. The membrane was incubated with 5% non-fat milk in PBS and later with
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mouse anti-PSMA mAb (Bioscience, Buckinghamshire, United Kingdom) for 2 h at room temperature. After washing, the membrane was incubated with an alkaline phosphatase-conjugated goat anti-mouse IgG antibody (Bioscience) for 1 h at room temperature. Immunoreactive bands were detected by the ECL Western blot analysis system (Bioscience). 2.8. Surface marker analysis of DCs On day 8, non-transduced and adenovirus-transduced DCs were collected and resuspended in cold FACS buffer (phosphate-buffered saline with 0.2% BSA and 0.09% sodium azide). Cells were immunostained with fluorescein isothio-cyanate (FITC) conjugated mouse anti-human CD14, CD40, CD80, CD83 mAbs, and phycoerythrin (PE) conjugated mouse anti-human CD86 mAb, together with irrelevant isotype-matched FITC- or PE-conjugated control mAbs (all mAbs were obtained from BD Pharmingen [San Jose, CA]). Cells were incubated with antibodies on ice for 15 min, washed twice with PBS, resuspended, and phenotyped on flow cytometric analysis using a FACS Calibur instrument (IMK-Lymphocyte; BD Biosciences, San Jose, CA). 2.9. Generation of CIKs Nonadherent human PMBCs were prepared and grown in culture flasks coated with 30 ug/ml human anti-CD3 mAb (Yes Biotech Lab, Ontario, Canada) at a density of 5 ⫻ 106 cells/ml in 5ml complete medium, consisting of RPMI 1640, 10% human AB serum, 1000 U/ml IL-2 (Boehringer, Mannheim, Germany), and 2-mM glutamine. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. On day 8, CIKs were cocultured for another 7 d with adenovirus-transduced mature DCs at a stimulator to responder ratio of 1:50. Then CIKs were tested for their activities. 2.10. Analysis of IFN-␥-secreting lymphocytes IFN-␥-secreting cells were detected on day 7 of the coculture. Briefly, pure CIKs and the cocultured cells of CIKs with Ad- and Ad-PSMA-infected DCs were restimulated respectively for 24 h at 37°C with 20 ug/ml PSMA peptide mixtures including the PSMA 624 – 632 peptide (HLA-A24⫹, TYSVSFDSL) and the PSMA 178 –186 peptide (HLA-A24⫹, NYARTEDFF) (Institute of Huada Gene, Beijing, China). In addition, 10 ug/ml intracellular transport inhibitor brefeldin A (BFA) was added. Cells were labeled with antibodies against CD3 (PE-Cy5) and CD8 (allophycocyanine, APC) for 15 min; then they were permeabilized with FACS permeabilizing solution for another 10 min at room temperature. These cells were washed and incubated with antibody against IFN-␥ (FITC) for 30 min at room temperature in the dark (All mAbs were obtained from BD Pharmingen). Unbound antibodies were removed by two washes. Finally, cells were analyzed by flow cytometry.
2.11. Cytotoxicity assay A CytoTox 96® nonradioactive cytotoxicity assay-lactate dehydrogenase (LDH) release (Promega, Madison, WI) was used to measure cytotoxic activity according to the manufacturer’s instruction. In brief, target cells [10,000] including LNCaP, PC3 and DU145 cells were plated in triplicate in a U-bottomed 96-well plate and incubated for 4 h with different effector cells, including CIKs, Ad-DC-CIKs (CIKs cocultured with Ad-transduced DCs), and Ad-PSMA-DC-CIKs (CIKs cocultured with Ad-PSMA-transduced DCs) in various effector-to-target ratios. Maximal release of LDH was performed by completely lysing target cells. Target cells without effector cells were used as negative controls (spontaneous release). Cytotoxicity was calculated as percentage cytotoxicity ⫽ (experimental absorbance minus spontaneous release of effector cells) minus spontaneous release of target cells/(maximal release minus spontaneous release of target cells). 2.12. Statistical analysis Data were presented as mean ⫾ standard deviation. Statistical differences were considered to be significant at a P-value less than 0.05 as determined by a paired Student’s t-test using SPSSv11.5 (SPSS Inc., Chicago, IL).
3. Results 3.1. Construction of recombinant adenoviruses and transduction of DCs with adenovirus The PSMA cDNA orientation in pAdTrackCMV-GFPPSMA was confirmed by sequencing (data not shown). After digestion with PacI, both pAd and pAd-PSMA formed 2 fragments of about 30 kb and 3.0 or 4.5 kb, which indicated the success of their structures (data not shown). Adenoviral constructs were cleaved with PacI, and then encapsulated with 293A cells to produce adenovirus: Ad (without transgene) and Ad-PSMA (encoding PSMA). Final yields were about 1011plaque-forming units (pfu). DCs were transduced with adenovirus at MOI 200 to analyze the transduction efficiency. About 70% of DCs were positive for GFP expression by fluorescence microscopy (Fig. 1A) and flow cytometry (Fig. 1B). With regard to cell viability, MOI 200 was considered optimal for gene transduction because cell viability was ⬎95% (Fig. 1B). Forty-eight hours after transduction, the PSMA expression in DCs was evaluated by Western blot. As seen in Fig. 1C, the PSMA protein was expressed in Ad-PSMA-transduced DCs and LNCaP cells, but not in Ad-transduced DCs. 3.2. Phenotype of DCs and of Ad-PSMA-transduced DCs On day 8 of cell culture, mature DCs were harvested. These cells were loosely suspended, exhibited irregular
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Fig. 1. Transduction of dendritic cells (DCs) with adenovirus. (A) Ad-PSMA-transduced DCs under inverted fluorescence microscope (200⫻). (B) Transduction efficiency in DCs. GFP expression was evaluated by flow cytometry 48 h after gene transduction (M1, % of GFP-positive cells). The percentage of viable cells was evaluated by trypan blue staining. (C) Western blot analysis of PSMA expression. The specific immunoreactive single band can be seen in Ad-PSMA-transduced DCs and LNCaP cells, but not in Ad-transduced DCs. (Color version of figure is available online.)
shapes, and displayed many fine processes at their edges. The phenotype of transduced and untransduced mature DCs was analyzed by FACS. The results showed that the expression of CD40, CD80, CD86, CD14, and CD83 was 96.78 ⫾ 2.51%, 66.25 ⫾ 2.78%, 97.24 ⫾ 2.15%, 1.37 ⫾ 0.21%, and 33.52 ⫾ 2.81%, respectively, in untreated DCs, and 95.10 ⫾ 2.60%, 68.12 ⫾ 2.45%, 96.97 ⫾ 2.73%, 1.35 ⫾ 0.25%, and 35.42 ⫾ 2.15%, respectively, in Ad-PSMA-transduced DCs, which suggested insignificant changes in the expression profile of DC surface markers between adenoviraltransduced DCs and non-transduced DCs (P ⬎ 0.05). 3.3. Phenotype of CIKs CIKs were generated from nonadherent PBMCs with IL-2 in culture flask immobilized anti-CD3 mAb. On day 4 of the cocultivation, the expression of CD3, CD4, CD8, and CD56 was checked by flow cytometry. CIKs cocultured with Ad-PSMA-pulsed DCs increased significantly the rate of CD3⫹CD4⫹, CD3⫹CD8⫹, and CD3⫹CD56⫹ (Table1). Furthermore, we found an enhanced proliferation of CIKs after contact with DCs. Between day 7 and day 15, the proliferative power was 800-fold for Ad-PSMA-DC-CIKs, 560-fold for Ad-DC-CIKs, and 220-fold for CIKs. 3.4. PSMA-specific activation of CIKs through CD8 population PSMA-specific activation of lymphocytes was examined using an IFN-␥-secretion assay after restimulation with PSMA peptide mixtures. When lymphocytes were cocultured with Ad-PSMA- transduced DCs, lymphocytes double
positive for CD8 and IFN-␥-secretion amounted to 4.35%. In contrast, only 2.15% lymphocytes were CD8⫹ and IFN␥⫹, when cocultured with Ad-transduced DCs, and 1.99% in CIKs (P ⬍ 0.01) (Fig. 2). 3.5. Induction of strong and specific cytotoxic activity of Ad-PSMA-transduced DCs cocultured with autologous CIKs The cytotoxicity of CIKs was investigated on LNCaP cells which expressed the PSMA protein, DU145 and PC3 cells, which were negative for the PSMA expression [18]. Lysis of PSMA-positive LNCaP cells was significantly higher (P ⬍ 0.01), when CIKs were cocultured with AdPSMA-transduced DCs than with Ad-transduced DCs at an appropriate rate of 20:1 (effector cells:target cells) that was optimized for our experiments. CIKs without coculture showed only moderate cytotoxicity. In contrast to LNCaP cells, DCs pulsed with Ad-PSMA did not induce higher cytotoxicity against PSMA-negative DU145 and PC3 cells, demonstrating a PSMA specificity of the immune response of CIKs (Fig. 3).
Table 1 The phenotype changes of CIKs cocultured with adenovirus-transfected DC
CIKs Ad-DC-CIKs Ad-PSMA-DC-CIKs
D3⫹CD4⫹ (%)
CD3⫹CD8⫹ (%)
CD3⫹CD56⫹ (%)
8.76 ⫾ 1.10 10.25 ⫾ 1.34 28.60 ⫾ 1.56
68.41 ⫾ 3.21 76.32 ⫾ 3.52 85.27 ⫾ 3.40
18.21 ⫾ 2.70 20.81 ⫾ 2.15 27.23 ⫾ 2.33
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Fig. 2. IFN-␥-assay. Gates were set on CD3⫹ T lymphocytes. After restimulation with PSMA peptide mixtures, Ad-PSMA-DC-CIKs (CIKs cocultured with Ad-PSMA-transduced DCs) increased significantly the production of CD8⫹ (IFN-␥⫹) T cells compared with Ad-DC-CIKs (CIKs cocultured with Ad-transduced DCs) and CIKs. Representative data from 3 independent experiments are shown.
4. Discussion DC-based immunotherapy holds promises but needs enhancing immunogenicity of DCs, and CIKs exhibit nonspecific cytotoxicity against tumor targets but lack antitumor specificity. The cocultivation of tumor antigen-loaded DCs with autologous CIKs appear to make up for their individual deficiencies and boost their antitumor immune effects. In this report, we demonstrated that DCs from healthy donors, which were efficiently transduced with a PSMA-expressing adenovirus and cocultured with autologous CIKs, are able to induce a strong and specific immune response against PSMA-positive PCa cells. Therefore, this approach may have a potential for an adoptive immunotherapy for patients with PCa. PSMA is a well-defined TAA and has been correlated with aggressive disease [19]. It is a transmembrane-carboxypeptidase [20], which is up-regulated 10-fold or more in PCa, from androgen-sensitive to androgen-independent PCa, and in metastatic deposits. Furthermore, PSMA is an internalized membrane protein and it has been shown to serve as a tissue-specific target for adenoviral vectors, which may be applicable for gene therapeutical treatment of prostate cancer [21]. DNA-based immunization could break immunologic tolerance towards TAA and did not induce autoimmune effects in mice. However, the potential of this approach was only efficient for the prevention of tumor growth in mice [22]. Recently, several studies have demonstrated that DCs pulsed with PSMA peptides stimulated CTL responses in advanced cancer patients, but achieved only limited success [5,23]. Recent studies have shown that the immunity of patients with PCa is depressed and could have negative effects on the stimulation of DCs or the capability of lymphocytes to lyse tumor cells [24]. The PCa microenvironment not only kills mature DCs, but also inhibits their generation and maturation [24]. This leads to deficiencies in number and function of DCs. In this study, we used specific cytokines to expand healthy donors-derived DCs to numbers sufficient for the induction of an efficient immune response against
PCa cells. Several mechanisms contribute to the low immunogenicity of PCa. One of the most important ones is the down-regulation of MHC class I expression and blocking of the recognition of tumor cells through T cells. As effector cells, CIKs cultures are able to kill in a MHC independent manner but could be primed by antigen-loaded DCs [10]. Furthermore, CIKs have a high proliferation rate and can be efficiently stimulated by DCs and induce a strong lytic activity in comparison with other immunologic effector cells [25]. Moreover, it has recently been shown that CIKs possess antitumor activity. In accordance with our results, interactions between DCs and CIKs lead to an activation of both populations and augmentation of cytotoxicity of CIKs against tumor cells. This may be an additional advantage to obtain a strong immune response against PCa. Despite their high cytotoxic activity against tumor cells in all clinical
Fig. 3. Strong and specific cytotoxicity of Ad-PSMA-DC-CIKS (CIKs cocultured with Ad-PSMA-transduced DCs). CIKs, Ad-DC-CIKs (CIKs cocultured with Ad-transduced DCs) and Ad-PSMA-DC-CIKs were used as effector cells to test the cytotoxic activities against specific target LNCaP cells and unspecific targets DU145 and PC3 cells. Cytotoxic activities at an effector-to-target ratio of 20:1 were determined by a 4-h LDH release assay. Data are representative of 5 separate experiments (*P ⬍ 0.01 vs. CIKs; #P ⬍ 0.01 vs. Ad-DC-CIKs).
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trials that had used CIKs against different tumors, no autoimmune disease had been observed [26]. TAA loading of DCs can be accomplished by a variety of techniques. Nonviral gene delivery transfer such as lipofection or electroporation can achieve only small transfection efficiencies [27]. The use of purified and defined PSMA peptides to load DCs has been shown for PCa. However, their use is limited to already known MHC-restricted peptides, which can be recognized by human T cells only in the context of MHC class I. Moreover, peptides are stable for a short time and their immunogenic potential is limited [5]. The adenoviral vector is a highly efficient and reproducible method of gene transfer. Several studies have shown that successful adenoviral gene transfer into human DCs resulted in induction of a T-cell response against the tumors [28]. In the present study, adenoviral gene transfer was performed to load DCs efficiently. With adenoviral transduction, we achieved a high PSMA expression. According to Gonzalez-Carmona [14], the high and long-term PSMA expression in adenovirus-transduced DCs seems to be a crucial parameter for induction of a potent immune response against a TAA, which can pulse large numbers of functional DCs in the course of DCs maturation and increase strongly expansion and stimulation of PSMA-specific effector cells against PSMA-expressing PCa cells. Despite the efficient adenoviral transduction and high PSMA expression were achieved in DCs, the phenotype of DCs did not change, which are similar to the results of Gonzalez-Carmona [14]. Other advantages of adenoviral transduction of full-length PSMA gene include the presentation of both MHC class I and II epitopes and previously unknown antigenic epitopes of PSMA [29]. The induction of a specific TAA immune response is a crucial factor for the design of immunotherapeutic strategies against cancer. The use of tumor lysates, dendritomas formed by fusion of DCs with allogenic carcinoma cells, or irradiated carcinoma cells have been included in recent protocols against carcinoma. However, these induce an immune response against unknown antigens, which could produce severe autoimmune diseases. In this report, we demonstrated a PSMA-specific immune response by an IFN-␥secretion assay. Antigen-specific interferon-␥ secreted by the cocultured cells of CIKs with Ad-PSMA-transduced DCs was significantly increased after restimulation with PSMA peptide mixtures, which indicated that the immune response was PSMA specific. In fact, such IFN-␥ can also inhibit tumor cell growth and promote cytotoxic effects [30]. Further research in vivo and its effects on humoral response are still to be done.
5. Conclusion Our data indicate that DCs from healthy donors infected with a PSMA-expressing adenovirus at a high transduction efficiency and cocultured with autologous CIKs can induce
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a PSMA-specific and strong immune response against PCa cells in vitro. This approach may induce regression of PCa and thus represents a novel promising adoptive immunotherapeutic approach to be tested in clinical trials for advanced PCa patients.
Acknowledgments The authors thank Mr. Yue He and Mr. Xinbo Xie from Cellkine Technology Co. Ltd. of Guangzhou for their excellent technical assistance.
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