and Bcl-2
Departments of
* Pathology,
Surgery, and
Immunology, University of Pittsburgh Medical Center and University of Pittsburgh Cancer Institute, and
Department of Molecular Biology and Biochemistry, University of Pittsburgh, Pennsylvania
Correspondence: Galina V. Shurin, Ph.D., Clinical Immunopathology, University of Pittsburgh Medical Center, 5725 CHP MT, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail: shuringv{at}msx.upmc.edu
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chain mRNA. In summary, these results suggest that transduction of DC with the IL-15 gene markedly stimulates DC function and protects them from tumor-induced apoptosis.
Key Words: apoptosis • immunosuppression • prostate cancer
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subunit shared with IL-2R, IL-4R, IL-7R, IL-9R, and a specific IL-15R subunit . Mast cells respond to IL-15 through a type 2 receptor system that does not share elements with IL-2R but uses a novel 60- to 65-kDa IL-15RX subunit. Based on the fact that dendritic cells (DC) express the common subunit [5
], it is likely that DC express a type 1 IL-15R complex. In fact, it has been recently demonstrated that murine DC express IL-15R [6
]. Although IL-15 shares activities with IL-2 and uses the common ß and subunits of the IL-2R, it became evident that these cytokines exert differential effects on many cell populations.
IL-15 acts on various cells of the immune system, including T and B lymphocytes, NK cells, monocytes, eosinophils, and circulating neutrophils. IL-15 stimulates NK cells, T cells, and B cells to proliferate, secrete cytokines, and exhibit increased cytolytic activity or produce antibody. It also stimulates phagocytes, maintains mast cells, and regulates migration of activated/memory T cells. IL-15 is involved in protection against different viral and bacterial infections regulating not only innate immunity but also adaptive immune responses. There is increasing evidence to suggest that IL-15 plays a pivotal role in protective immune responses, allograft rejection, and the pathogenesis of various autoimmune and chronic inflammatory diseases [7
]. IL-15 is also a potent inhibitor of several apoptosis pathways: It delays apoptosis of neutrophils more efficiently than IL-2 [8
] and inhibits cytokine deprivation-induced apoptosis in activated T cells, tumor necrosis factor -mediated apoptosis in fibroblasts, and apoptosis induced by anti-Fas, anti-CD3, dexamethasone, and/or anti-immunoglobulin (Ig)M in activated T and B cells [9
, 10
]. We have recently reported that IL-15 also protects DC from tumor-induced apoptosis [11
]. Thus, IL-15 is likely to be a general protector from apoptosis for different cell types in vitro and in vivo. Furthermore, using murine tumor models, it has been shown that IL-15 exhibits a strong therapeutic potential that is superior to IL-2 [12
]. For instance, the daily administration of IL-15 has been reported to mediate antitumor effects after the cyclophosphamide injection in tumor-bearing mice and enhance adoptive immunotherapy by activating NK cells [13
]. IL-15 also displays a stronger target-specific accumulation and more rapid clearance from the circulation than IL-2 [14
]. Thus, IL-15 might be considered a novel, potential therapeutic agent with strong immunomodulating properties.
Although it is well-documented that IL-15 plays an important role in the regulation of macrophage and NK cell differentiation and T cell chemotaxis and proliferation [15
16
17
], the role of IL-15 in the regulation of the DC system is not completely resolved. It has been reported that DC produce IL-15 upon stimulation with bacterial products, interferon-, or CD40 ligand, and this effect was correlated with an enhanced stimulatory ability of DC [18
, 19
]. It has also been demonstrated that DC coincubated with IL-15 express high levels of the Bcl-2 family of proteins and perform an increased resistance to tumor-induced apoptotic death [11
]. The aim of this study was to evaluate the functional activity and tumor resistance of human DC transduced with adenovector encoding the human IL-15 gene. We have demonstrated that genetic modification of peripheral blood mononuclear cell (PBMC)-derived DC with the IL-15 gene activates DC, enhances their function, and protects them from prostate cancer-induced apoptosis by up-regulating the expression of IL-15R and Bcl-2.
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DC were generated from PBMC of healthy donors. After gradient separation on Histopaque-1077 (Sigma Chemical Co., St. Louis, MO), PBMC were resuspended in CM (5x106 cells/ml) and incubated for 1 h at 37°C. Nonadherent cells were removed, and adherent DC precursors were cultured in CM with 1000 U/ml recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) and 1000 U/ml rhIL-4 (Schering-Plough Research Institute, Kenilworth, NJ).
Day 4 nonadherent DC culture cells were transduced with the IL-15 gene using an adenoviral system. Enhanced green fluorescent protein (EGFP) encoding adenovector was used for evaluation of DC transduction efficacy, whereas 
5 adenovirus was used as a control for all other experiments. Twenty-four hours later, control and transduced DC (106 cells/well) were incubated with LNCaP cells (5x105 cells/well), separated by membrane inserts with 0.4-µm pore size for 48 h.
On day 7, nonadherent DC were harvested, and their phenotype and function were analyzed. For morphological evaluation, DC were placed on microscope slides using a Cytospin centrifuge (100 g, 5 min; Shandon Lipshaw, Pittsburgh, PA) and were stained with LeukoStat stain kit (Fisher Scientific, Pittsburgh, PA). The DC viability in cultures was assessed using the trypan blue exclusion protocol (0.2%; Gibco). Trypan blue-negative cells were considered alive, and their percentage among the total cell number was calculated. For the survival experiments, transduced and nontransduced 7-day-old DC treated with LNCaP cells and nontreated cells were set (based on the number of live cells) at 1 x 106 DC/ml in 96-well plates in a total volume of 200 µl. During the next 7 days, DC were cultured without the addition of growth factors or cytokines, and the number and percentage of live cells were counted at third, fourth, fifth and seventh days.
Adenoviral vectors and DC transduction
Adenovector encoding human IL-15 was constructed, propagated, and titered according to the standard protocol previously described by Hardy et al. [20
]. IL-15 cDNA (kindly provided by Immunex, Seattle, WA) was amplified by polymerase chain reaction (PCR) using a set of primers to create a SalI site at the 5' end and a NotI site at the 3' end. Then, plasmid was digested by SalI and NotI restriction enzymes to release IL-15 cDNA, which was subcloned into the SalI-NotI site of the adenovirus shuttle plasmid (pAdlox) to get pAdlox/hIL-15. The plasmid was digested with SfiI and cotransfected with Ad5-derived, E1- and E3-deleted adenoviral backbone into 293 cells by a calcium phosphate precipitation. The inserted cDNA sequence is expressed under the transcriptional control of the cytomegalovirus promoter. Recombinant adenoviruses were isolated from a single plaque, expended in CRE8 cells, and purified three times by consecutive cesium chloride gradient ultracentrifugation. Purified virus was extensively dialyzed against 10 mM Tris/1 mM MgCl2 sterile buffer at 4°C and stored in aliquots at -80°C. Recombinant adenovirus was titered on CRE8 cells for plaque-forming units. Control adenoviral vectors encoding the EGFP gene and 5 gene (Ad5) were constructed as described previously [21
]. For the transduction, day 4-cultured DC were collected, washed in Hanks’ balanced saline solution (HBSS), counted, incubated with vectors (50 µl per 5–10 x 106 cells; 200 multiplicity of infection) for 2 h at 37°C, and further cultured in CM (0.25x106 cells/ml) with 1000 U/ml rhGM-CSF and 1000 U/ml rhIL-4.
Flow cytometry analysis
Harvested cells were washed in the fluorescein activated cell sorter (FACS) medium (HBSS containing 0.1% bovine serum albumin and 0.1% NaN3) and were stained with appropriately diluted antibodies directly conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE), according to the standard procedure followed by fixation in 2% paraformaldehyde. The following antibodies were used for the FACS staining: FITC-labeled anti-human CD1a, human leukocyte antigen (HLA)-DR, CD86, CD80, CD40, and PE-labeled CD83 (PharMingen, San Diego, CA). Fluorescence was measured using a FACScan flow cytometer, and data analysis was performed using the Cell Quest software (Becton-Dickinson, San Diego, CA).
For an Annexin V assay, harvested DC were washed in FACS medium and were double-stained with FITC-conjugated Annexin V (PharMingen) and propidium iodide (PI; Sigma Chemical Co.). The percentage of cells undergoing early apoptosis in a quantitative manner was determined as the percentage of Annexin V-positive PI-negative cells.
Mixed leukocyte reaction (MLR)
Functional activity of DC was determined in the primary allogeneic MLR assay using T lymphocytes as responder cells. Allogeneic T cells were obtained from the peripheral blood of healthy donors by passage through nylon wool columns after the lysing of red blood cells. The MLR assays were carried out in triplicates in round-bottom 96-well plates to ensure efficient DC/T cell contact and were mixed at various ratios (1:810, 1:270, 1:90, 1:30, 1:10, 1:3, and 1:1) in a total volume of 200 µl CM. Proliferation of T cells was measured by uptake of 3H-thymidine (1 µCi/well, 5 Ci/mmol; DuPont-NEN, Boston, MA), pulsed for the last 16–18 h after 3 days in culture. The cultures were harvested onto GF/C glass fiber filter paper (Whatman Intl. Ltd., Maidstone, UK) using a MACH III microwell harvester (Tomtec, Hamden, CT). Incorporation of 3H-thymidine was determined on a MicroBeta TRILUX liquid scintillation counter (Wallac, Gaithersburg, MD). Index of stimulation (IS) was expressed as a ratio of DC-stimulated to nonstimulated T cell proliferation determined as counts per minute (cpm).
Cytokine assays
IL-12 and IL-15 protein production by control and genetically engineered DC was assessed in cell-free culture supernatants (300 g, 30 min) by enzyme-linked immunosorbent assay (ELISA; Quantikine, R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. To evaluate IL-12 production, DC (5x105 cells/ml/24 h) were stimulated with 10 µl/ml inactivated Staphylococcus aureus (Sigma Chemical Co.) on day 6, and 24 h later, the levels of IL-12 protein in cell-free supernatants were measured by ELISA (IL-12p70). IL-15 protein production by control and IL-15-transduced DC (5x105 cells/ml/48 h) was measured in 48 h after transduction.
Western blot
The protein expression of IL-15, IL-15R, and antiapoptotic protein Bcl-2 in transduced and control DC was assessed using a Western blot technique. Cells were collected, washed in HBSS, and lysed in 100 µl extraction buffer [1% Triton X-100, 0.1% sodium dodecyl sulfate, 50 mM Tris, 150 mM NaCl, 0.6 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin] for 30 min on ice. After centrifugation (9000 g, 15 min, at 4°C), the protein concentration was determined in the supernatants by the Bradford method using the BioRad protein kit (BioRad Laboratories, Hercules, CA). Total proteins (50 µg) were dissolved in electrophoresis sample buffer, separated by 4–12% polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane (Novex, San Diego, CA). The membrane was blocked with 0.5% nonfat milk and 0.1% Tween-20 (Fisher, Fairlawn, NJ) in 20 mM Tris-HCl buffer (pH 7.2). IL-15 and IL-15R were detected using mouse monoclonal antibodies (mAb) against human IL-15 (IgG1, HuIL15-M112) or human IL-15R chain (HuIL15R-M161; a gift from Genmab, Utrecht, The Netherlands) at a final concentration of 100 µg/ml and goat anti-mouse secondary mAb [IgG (H+L), 1/150,000 dilution; Pierce, Rockford, IL]. Bcl-2 was detected with mouse anti-human mAb (IgG1, 1/500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Expression of ß-actin was evaluated as a housekeeper control protein using mouse anti-human mAb (IgG1, 1/5000 dilution; Sigma Chemical Co.). Secondary antibodies were the same as for IL-15 detection. The membrane was processed and treated with chemiluminescent reagents (Pierce), and the bands were visualized on Kodak film (Eastman Kodak, Rochester, NY).
Immunocytochemistry
DC were cytospinned (100 g, 5 min), air-dried, and fixed in ice-cold acetone. Slides were washed in phosphate-buffered saline with 0.05% saponin (Sigma Chemical Co.) and were incubated for 1 h at room temperature with the mouse anti-IL-15 mAb (20 µg/ml), anti-IL-15R mAb (20 µg/ml), or mouse IgG1 (20 µg/ml; PharMingen) as an isotype control. Biotinylated horse anti-mouse IgG (H+L) was used as secondary Ab (1/500 dilution; Vector Laboratories, Burlingame, CA) and was applied for 45 min. After developing with the peroxidase-based chromogen AEC kit (3-amino-9-ethylcarbazol; Biomega, Foster City, CA), counterstaining was performed with hematoxylin. The presence of IL-15 or IL-15R was evident as a red-brown reaction product.
Reverse transcriptase (RT)-PCR
Expression of IL-15R chain mRNA in transduced and nontransduced DC was assessed by RT-PCR. Total RNA was extracted from cells using the Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH). cDNA was synthesized from 2 µg RNA in 20 µl reactions using random primers (Boehringer Mannheim, Manheim, Germany) and avian myloblastosis virus RT (Gibco-BRL, Gaithersburg, MD). cDNA (2 µl) was amplified using Taq polymerase in a 25-µl reaction volume. Amplification of each cDNA sample within the linear range was achieved by 30 or 39 cycles using a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) under optimized conditions for each set of primers: for IL-15R, 39 cycles of denaturation at 94°C for 60 s, annealing at 60°C for 60 s, and extension at 72°C for 60 s; for human glyceraldehydes 3-phosphate dehydrogenase (GAPDH), which was used as a housekeeping control for the PCR amplification, 30 cycles of denaturation at 94°C for 60 s, annealing at 60°C for 60 s, and extension at 72°C for 60 s. For detection of IL-15R, the following primers were used: IL-15R sense, 5'-GTCAAGAGCTACAGCTTGTAC-3'; antisense, 5'-GGTGAGCTTGCTCCTGGAG-3'. IL-15R cDNA amplified with these primers was expected to yield a 779-bp product. The sequence of sense and antisense primers for GAPDH was 5'-CATCAAGAAGGTGGTGAAGCA-3' and 5'-TCTACATGGCAACTGTGAGGA-3'. Amplification with these primers was expected to yield a 363-bp product. PCR products were separated on 1% agarose gels containing 1 mg/ml ethidium bromide together with an appropriate marker ladder.
Statistical analysis
Statistical significance of differences between two groups was determined using the Student’s t-test or the nonparametric Mann-Whitney test after evaluation for normality. ANOVA was used for the comparison among more than two groups. For all statistical analysis, the level of significance was set at a probability of 0.05 to be considered significant. Data are presented as the mean ± SEM.
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5-transduced or nontransduced DC (Fig. 1
). These data demonstrated the elevation of intracellular IL-15 protein expression by DC transduction with Ad5IL-15 vector and suggested a high transduction efficacy of the method used here.
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Figure 1. Transduction of human DC with the IL-15 gene results in a high level of protein expression. Human DC were generated from PBMC precursors and transduced with the IL-15 (C) or 5 (B) genes using an adenovector system as described in Materials and Methods. Nontransduced DC (A) served as a control. Immunocytochemical analysis of IL-15 expression was performed on saponin-treated cells after acetone fixation. Positive cells appear dark red. The results of one representative out of three independent experiments are shown.
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5-transduced, and nontransduced DC revealed a significant increase in expression [assessed as mean fluorescence intensity (MFI)] of CD83 (30.4 vs. 41.8 vs. 58.8), CD86 (68.8 vs. 97.4 vs. 127.2), and CD40 (61.4 vs. 76.2 vs. 89.1) on DC after IL-15 transduction. In addition, transduction with the IL-15 gene slightly enlarged expression of CD80, CD1a, and HLA-DR molecules (Fig. 2A
). Although we have observed a slight elevation of surface-marker expression on DC transduced with Ad5, this effect was significantly lower than the effect in IL-15-transduced DC (P<0.05). Furthermore, addition of neutralizing mAb against human IL-15 after transduction significantly blocked the elevation of CD83, CD1a, and CD40 expression on IL-15-transduced DC (P<0.01), although it did not change the phenotype of 5-transduced DC (Fig. 2B)
. These results suggest that increased expression of surface molecules on DC transduced with the IL-15 gene is specific for IL-15 effects.
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Figure 2. Transduction of DC with the IL-15 gene results in increased expression of different DC-related surface markers. DC were generated and transduced with the IL-15 or 5 genes as described in Materials and Methods. Nontransduced DC served as a control. FACScan analysis was performed using staining with anti-human FITC-labeled CD1a, CD86, CD40, and HLA-DR, and PE-labeled CD83 mAb. (A) Expression levels of the CD83, CD86, CD80, CD40, CD1a, and HLA-DR molecules are shown by filled histograms; open histograms show background staining with isotype-matched control antibody. Data are represented as MFI. (B) The anti-IL-15 Ab were added to cultured DC after transduction (shaded columns), and 24 h later, DC were harvested, washed, and stained with anti-human PE-labeled CD83 and FITC-labeled CD40, CD1a, and CD86 mAb. The alteration of expression of DC markers was counted as a difference between the expression on transduced DC and the expression on nontransduced DC, accepted as 100%: *, P < 0.01 - anti-IL-15 Ab-treated IL-15-transduced DC group versus nontreated IL-15-transduced DC group. Data represent mean ± SEM from three independent experiments.
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5-transduced and nontransduced DC cultures, respectively (P<0.01; Fig. 3A
). This suggests that transduction of DC with the IL-15 gene resulted in a significant increase in induced IL-12 production by DC.
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Figure 3. IL-15 transduction of DC increases S. aureus-induced IL-12 production by DC and their ability to induce T cell proliferation. DC were generated from PBMC precursors and transduced with the IL-15 (dark-gray column) or 5 (light-gray column) gene using an adenovector system as described in Materials and Methods. Nontransduced DC (solid column) served as a control. (A) Human DC (5x105 cells/ml) were treated with inactivated S. aureus for 24 h, and the levels of IL-12 protein were measured in cell-free supernatants using ELISA (IL-12p70). Data represent the mean ± SEM from three independent experiments. *, P < 0.01. (B) Primary MLR assay was performed as described in Materials and Methods. DC were used as stimulators and allogeneic T lymphocytes as responders. DC/T cells were mixed at ratios 1:810, 1:270, 1:90, 1:30, 1:10, 1:3, and 1:1. The counts were presented as IS. Spontaneous proliferation of T cells varied from 400 to 600 cpm. The optimal counts for the control group in a MLR assay was observed at a DC:T cell ratio from 1:30 to 1:10, which corresponds to  10,000 cpm range. Data represent the mean ± SEM of triplicate measurements from three independent experiments. P < 0.05.
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5-transduced or nontransduced DC at effector:target ratios 1:270, 1:90, 1:3, and 1:1 (P<0.05; Fig. 3B
). For instance, at ratio 1:1, IS was 4.4 ± 0.3 for nontransduced DC, 7.3 ± 0.2 for 5-transduced DC, and 10.3 ± 0.7 for IL-15-transduced DC. Taken together, these results suggest that transduction of DC with IL-15 increases their functional activity in allogeneic MLR.
Overexpression of IL-15 in DC increased the expression of IL-15R
To determine whether the elevation of IL-15 protein production by IL-15-transduced DC is associated with increased synthesis of IL-15 receptors, we have measured the expression of IL-15R in DC using immunocytochemistry, Western blot, and RT-PCR. The analysis of Western blot data revealed that the level of IL-15R expression in DC transduced with the IL-15 gene was significantly higher than the level of expression in Ad55-transduced and nontransduced DC (Fig. 4
). For instance, index of relative receptors expression (total pixel IL-15R/total pixel ß-actin ratio) measured by densitometry was 0.612 ± 0.093 in IL-15-transduced DC compared with 0.198 ± 0.058 and 0.255 ± 0.074 in -transduced and nontransduced DC, respectively. Next, we have applied an immunocytochemical technique to determine whether IL-15R is expressed on cell surface or localized intracellularly. Immunocytochemical analysis of DC with and without permeabilization of cellular membranes has shown an intensive intracellular staining for IL-15R in DC transduced with the IL-15 gene when compared with 5-transduced and nontransduced DC (Fig. 5
). No significant difference in extracellular membrane staining in different groups was detected, suggesting that most of IL-15R chains stay within the cytoplasmic compartments, shed, or were transported to the cell surface, bound with secreted IL-15, and quickly internalized back into the intracellular compartment.
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Figure 4. Overexpression of IL-15 in DC increases the expression of IL-15R protein as determined by Western blot. DC were generated from PBMC precursors and transduced with the IL-15 (lane 2) or 5 (lane 3) genes using an adenovector system as described in Materials and Methods. Nontransduced DC (lane 1) served as a control. The anti-IL-15 Ab were added to nontransduced (lane 4), IL-15-transduced (lane 5), or 5-transduced (lane 6) DC for 24 h. ß-Actin served as a housekeeping control protein. The results of one representative out of three independent experiments are shown.
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Figure 5. Overexpression of IL-15 in DC increases the expression of intracellular IL-15R as determined by immunocytochemistry. DC were generated from PBMC precursors and transduced with the IL-15 (C) or 5 (B) gene using an adenovector system as described in Materials and Methods. Nontransduced DC (A) served as a control. Immunocytochemical analysis of IL-15R expression was performed on the cytospinned (100 g, 5 min) cells, air-dried, fixed in ice-cold acetone, and permeabilized with saponin. Positive staining appears dark red. The representative results of one out of three independent experiments are shown.
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chain is synthesized de novo in IL-15-transduced DC, we have assessed the expression of IL-15R mRNA in different DC populations. As shown in Figure 6
, a significantly higher level of chain mRNA was detected in DC transduced with the IL-15 gene.
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Figure 6. Transduction of DC with the IL-15 gene increases the expression of IL-15R mRNA. The level of IL-15R mRNA expression on DC was determined by RT-PCR. Total RNA was extracted using the Tri Reagent, and cDNA was synthesized from IL-15-transduced (lane 3), 5-transduced (lane 2), and nontransduced (lane 1) DC as described in Materials and Methods. GAPDH primers were used as an internal control for PCR amplification. Amplification of each cDNA sample was achieved under optimized conditions for each set of primers. cDNA from human lymphocytes (lane 4) and muscle cells (lane 5) was used for additional controls. The results of one out of three independent experiments are shown.
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, suggesting the specificity of the effect and its mediation by overproduction by IL-15 itself. It also suggests that this effect was mediated by extracellular soluble or membrane-bound IL-15 and its interaction with primary IL-15R on DC surface. Thus, it is likely that IL-15 produced by DC increases expression of IL-15R in a paracrine manner.
Transduction of DC with the IL-15 gene increased their resistance to prostate cancer-induced apoptosis
To examine how overexpression of IL-15 influences the sensitivity of DC to prostate cancer-induced apoptosis, we have determined the survival of DC in the tumor microenviroment. For this purpose, cultured DC were coincubated with LNCaP cells separated by a membrane, and 48 h later, the level of DC apoptosis was measured using Annexin V binding assay (Fig. 7
). In addition, the kinetics of DC viability in cultures was also assessed 1–7 days later (Fig. 8
). The percentage of Annexin V-positive, i.e., apoptotic, cells among IL-15-transfected DC was significantly lower compared with 5-transduced and nontransduced DC (27±4% vs. 35±4% and 46±5%, respectively). For the evaluation of DC survival, transduced and nontransduced, 7-day-old DC treated with LNCaP cells and control, nontreated cells were set at 1 x 106 DC/ml in 96-well plates, 200 µl/well. During the next 7 days, DC were grown without the addition of any growth factors, and live cells were counted. The analysis of cell viability demonstrated that DC grown in the absence of tumor cells had a better survival rate than DC grown in the presence of the tumor, and transduction of DC with the IL-15 gene markedly prolonged their survival in tumor-treated and untreated groups when compared with nontransduced or 5-transduced DC. For DC coincubated with LNCaP cells starting from day 4, the percentage of live cells in IL-15-transduced DC cultures was significantly higher compared with nontransduced and 5-transduced DC (82±6% vs. 60±8 and 62±7%, respectively). Finally, on day 7, the survival of DC transduced with the IL-15 gene was also significantly higher (up to 30%) when compared with other groups (P<0.01). Thus, these data suggest that transduction of DC with the IL-15 gene increases their survival in cultures coincubated with tumor cells.
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Figure 7. Genetic modification of DC with the IL-15 gene is accompanied by decreased levels of tumor-induced apoptosis in DC. DC were generated and transduced with the IL-15 or 5 genes as described in Materials and Methods. Nontransduced DC served as a control. Cultured DC (1x106 cells/well) were coincubated with LNCaP prostate adenocarcinoma cells (5x105 cells/well) separated by membrane inserts with 0.4-µm pore size for 48 h. The level of DC apoptosis was measured using an Annexin V binding assay. The Annexin-positive PI-negative (apoptotic) staining is shown by filled histograms, and open histograms show nonstained cells. Data are represented as relative fluorescence intensity on the x-axis and relative cell numbers on the y-axis. The results of one representative out of three independent experiments are shown.
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Figure 8. Transduction of DC with the IL-15 gene prolongs their survival in the tumor microenviroment. DC were generated, transduced with the IL-15 or 5 genes, and incubated with LNCaP cells as described in the legend to Figure 7
. DC viability was assessed using the trypan blue exclusion protocol as described in Materials and Methods. Percentages of live cells among nontransduced (•, treated with LNCaP;  , nontreated), 5-transduced ( , treated with LNCaP;  , nontreated), and IL-15-transduced ( , treated with LNCaP;  , nontreated) DC were calculated. Data represent the mean ± SEM of triplicate measurements from three independent experiments.
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5-transduced and nontransduced DC but did not influence Bcl-2 protein expression in IL-15-transduced DC (Fig. 9
). Thus, these data suggest that one of the mechanisms of protection of IL-15-transduced DC from prostate cancer-induced apoptosis is the blockade of tumor-induced inhibition of Bcl-2 expression in DC.
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Figure 9. Transduction of DC with the IL-15 gene protects them from tumor-induced inhibition of Bcl-2 expression. DC were generated, transduced with the IL-15 or 5 genes, and incubated with LNCaP cells as described in the legend to Figure 7
. Bcl-2 protein expression in IL-15-transduced (lanes 2A and 2B), 5-transduced (lanes 3A and 3B), and nontransduced (lanes 1A and 1B) DC before (A) and after (B) coincubation with LNCaP cells was measured using Western blot as described in Materials and Methods. The results of one out of two independent experiments are shown.
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5 induced a slight activation of DC in comparison with a significant activation of DC after transduction with Ad5IL-15 vector.
The transduction efficacy, determined as expression of EGFP protein on DC varied between 60 and 80%. DC transduced with Ad5IL-15 expressed significantly higher levels of IL-15 as compared with 5-transduced and nontransduced DC, suggesting an active synthesis of the cytokine in DC. However, concentrations of the secreted form of IL-15 in cell-free supernatants were low (up to 15 pg/ml/5x105 cells/48 h), but they were still significantly higher than nondetectable levels of IL-15 in 5-transduced or nontransduced DC cultures (P<0.01). We have also shown that the transduction of DC with the IL-15 gene results in an elevation of their function, including expression of costimulatory molecules, IL-12 production, and the ability to induce proliferation of T cells in the primary MLR assay. The addition of anti-IL-15 Ab blocked the increase of surface-marker expression of IL-15-transduced DC. As it did not change the phenotype of 5-transduced DC, it suggests that this effect was mediated via produced IL-15, whereas a minor activation of 5-transduced DC was not related to IL-15.
The mechanisms of IL-15-induced activation of IL-15-transduced DC are not yet clear. One of the likely pathways might involve the up-regulation of IL-15R. As monocytes have been shown to express the IL/15Rß, , and the specific IL-15R, it allowed suggesting the existence of the same receptor complex on DC [33
]. Using a Western blot and immunocytochemical analysis with mAb against the human IL-15R chain (HuIL15R-M161), we have detected the expression of IL-15R in DC and its up-regulation in DC transduced with the IL-15 gene. IL-15-transduced DC expressed a higher level of IL-15R mRNA as well. The addition of antibodies against IL-15 blocked the expression of IL-15R protein, suggesting that IL-15 produced by DC might increase the expression of IL-15R by extracellularly released IL-15 and its interaction with IL-15R on DC surface. As immunocytochemical analysis of IL-15R localization revealed that most of the expressed IL-15R chain was localized intracellularly, most likely IL-15R were transported to the cell surface, bound with secreted IL-15, and internalized back into the intracellular compartment. Taken together, these results suggest that overexpressed IL-15 mediates its effects on DC activity through the interaction with the extracellular IL-15R, which could be quickly internalized after IL-15 ligation.
In addition to the functional activation of IL-15-transduced DC, transduction of DC with the IL-15 gene increased their resistance to prostate cancer-induced apoptosis. It has been recently reported that IL-15 is a potent inhibitor of several apoptosis pathways [9 ]. It has also been demonstrated that stimulation of DC for 24 h with IL-15 or IL-12 prior to their coincubation with prostate cancer cells results in expressed high levels of the Bcl-2 family of proteins and performs an increased resistance to tumor-induced apoptotic death [11 ]. We have shown here that the transduction of DC with the IL-15 gene prolongs DC survival in cultures and significantly increases their resistance to tumor-induced apoptosis. One of the mechanisms of protection of IL-15-transduced DC from prostate cancer-induced apoptosis was related to the blockade of tumor-induced inhibition of Bcl-2 expression in DC. Thus, together with our previous results, these data demonstrate that endogenous and exogenous IL-15 protects DC from tumor-induced cell death by regulating the expression of antiapoptotic proteins from the Bcl-2 family. Furthermore, the transduction of DC with the IL-15 gene resulted in increased IL-12 production. As IL-12 has been reported to enhance the survival of DC in tumor microenvironment, it is possible that IL-15-induced IL-12 release might serve as an additional mechanism of protection of IL-15-transduced DC from tumor-induced apoptotic death. Determination of further mechanisms, which allow IL-15-transduced DC to escape from tumor-induced suppression and apoptosis, is in progress.
In summary, we have demonstrated that transduction of human DC with the IL-15 gene results in a significant activation of their functions, increased resistance to prostate cancer-induced apoptosis, and prolonged survival in the tumor microenvironment. These effects are likely to be mediated by IL-15-induced up-regulation of IL-15R and Bcl-2 expression. This approach of DC modification might be used to study the mechanisms involved in tumor-induced inhibition of DC and may further improve the efficacy of DC-based therapies in preclinical and clinical trials.
Received November 14, 2001; revised August 9, 2002; accepted August 12, 2002.
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