Purpose: Triggering of CD40 on antigen-presenting cells via its ligand CD154 is an important event in the initial phase of an immune response against cancer cells. In this study, we investigated the effects of adenoviral CD154 immunomodulatory gene therapy on the activation of human dendritic cells (DCs) in a well-defined in vitro system. Experimental design: Human bladder cancer cell lines and tumor cells from patients with renal cell carcinoma (RCC) were transduced with Ad-CD154 vectors or control vectors. Activation of human in vitro generated DCs after coculture with transduced tumor cells was analyzed. Therapeutic efficacy and cytotoxic T-lymphocyte (CTL) activity were assessed in a subcutaneous (s.c.) murine bladder cancer model. Results: Human bladder cancer cell lines expressing CD154 showed a decreased growth rate, increased apoptosis, and modulated expression of molecules important for recognition by cytotoxic lymphocytes. Further, CD154-expressing allogeneic bladder tumor cell lines and autologous tumor cells from patients with renal cell cancer induced maturation of DCs and stimulated IFN-γ production from lymphocytes cocultured with mature DCs. In vivo studies showed that CD154 gene therapy was highly effective in wild-type mice but only minimally effective in nude mice. Consequently, strong tumor-specific CTL activity was detected in mice vaccinated with tumor cells expressing CD154. Conclusions: Using tumor cell lines as well as patient-derived material, we could show that tumor cells expressing CD154 efficiently induce maturation and activation of DCs as well as activation of lymphocytes. Our murine in vivo studies demonstrate that lymphocytes contribute to the observed antitumor effect in a s.c. bladder tumor model. These studies should stimulate CD154 gene therapy approaches for the treatment of urologic malignancies.
At least for some cancers it is now generally accepted that despite tumor growth the host immune system is capable of recognizing antigens present in tumor cells. The problem is, however, that because of various mechanisms (tumor escape, immunosuppression, etc.) the immune response is not sufficient to eliminate the cancer.1 One current strategy of biologic therapy of cancer is based on the concept that tumor cells should be genetically modified to act as antigen-presenting cells2 or as immunostimulating cells3 to activate tumor-specific cells, e.g., CD4+ helper T lymphocytes and CD8+ cytotoxic T lymphocytes (CTLs). Key players in the activation of CTLs are the dendritic cells (DCs). These cells are superior APCs because of their high expression of MHC and because they supply important adhesion and costimulatory molecules to lymphocytes.4,5 Immature DCs are efficient antigen-capturing cells patrolling the body. Upon activation, they differentiate into a mature stage, their ability to take up antigen decreases, and they gain several properties to process and present captured antigens with high efficacy. The maturation of immature DCs seems to be a crucial step for potent antitumor CTL responses.6 This event is triggered by interaction of CD40 on the DCs with its ligand CD154 on CD4+ T lymphocytes.7 It was also shown that this signaling between DCs and CD4+ T lymphocytes could be replaced by direct CD40 stimulation.8,9 The possibility of manipulating the activation and function of DCs has resulted in many potent immunotherapy protocols in animal studies,10,11 and some are now facing clinical trials.12,13
The potential of CD154 gene therapy has been demonstrated in some experimental animal models including bladder cancer.14,15,16,17 Notwithstanding these observations, the immunologic basis of this therapeutic approach remains poorly defined. A possible scenario may be that interactions between the Ad-CD154–transduced tumor cells and immature DCs in the tumor area take place and that such interactions evoke potent antitumor responses. Therefore, we hypothesized that transduction of tumor cells with the gene for CD154 would result in activation of immature DCs as a starting signal for an effective immune response. Hence, in the current study we directly assessed the immunological consequences of the interaction of CD154-expressing tumor cells and DCs. In addition, the transduction with Ad-CD154 itself may modulate the phenotype and biology of tumor cells. For example, in a B-cell chronic lymphocytic leukemia model, transduction of the cells with Ad-CD154 vectors enhanced the expression of not only costimulatory molecules on the tumor cell surface, but also the expression of the death-signaling receptor CD95.18,19 These up-regulations were also seen in non–small cell lung cancer cells when trimeric fusion protein complexes of CD154 were added to the tumor cells.20 In different tumor cell lines it has been shown that triggering of CD40 on the tumor cell with recombinant CD154 or agonistic antibodies leads to growth inhibition and apoptosis20,21,22,23,24 and also to increased susceptibility to specific lysis by CTLs.21
In the present investigation, we have scrutinized the effects of adenoviral CD154 transduction on cultures of human urinary bladder carcinoma cells and analyzed the interaction of CD154-transduced tumor cells with DCs in a well-defined in vitro system. Because renal cancer is a urologic malignancy with currently insufficient treatment options, we extended our investigations to material from patients with renal cell carcinoma (RCC).
Materials and methods
The human transitional cell carcinoma cell lines J82 and T2425 and the murine bladder tumor cell line MB49 (kind gift of Dr K Esuvaranathan, University of Singapore) were cultured in RPMI containing 10% FBS, 1% L-glutamine, and 1% penicillin–streptomycin. Medium and supplements were purchased from Life Technologies, Paisley, UK.
Tumor material and blood samples for in vitro generation of DCs were obtained from patients with RCC undergoing radical tumor nephrectomy. Tumor tissue was minced using a scalpel and sieve, washed, and short-term cultures were established in DMEM containing 10% FBS, 1% L-glutamine, and 1% penicillin–streptomycin. Written informed consent was obtained from the patients before use of the material.
Replication-incompetent E1+E3 deleted adenoviral serotype 5 vectors were used. Both Ad-CD154 (murine) and an empty control vector, Ad-dl70-3 were kind gifts from Dr Gert Maass, Roche, Penzberg, Germany.18
The human cell lines and cultures of tumor tissue were transduced by pulsing them with adenoviral vectors (10–200 pfu/cell) for 1 hour in a serum-free cell culture medium (200 μl). After this first incubation, 2 mL culture medium was added and the vector pulse was continued. Cells were analyzed for expression of CD154 transgene and various surface molecules at different time points post transduction by flow cytometry.
Cell growth and apoptosis assays
Three days post transduction with either Ad-CD154 or Ad-dl70.3, bladder tumor cells were seeded into 96-well plates at high density of 10,000 cells per well. After 24 and 48 hours cell growth assays were performed with Alamar Blue™ assay, which incorporates a colorimetric growth indicator, based on detection of metabolic activity.26 At the same time points cells were harvested and an apoptosis assay was performed using Annexin V kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer's recommendations.
Stimulation of DCs with transduced tumor cells
Peripheral blood mononuclear cells (PBMCs) from heparinized blood of healthy donors and patients with RCC were obtained by Ficoll-Paque centrifugation. Monocytes and lymphocytes were separated by counterflow elutriation. Monocytes were cultured in RPMI 1640 supplemented with 10% FBS, 1% L-glutamine, 1% penicillin–streptomycin, GM-CSF (500 U/mL), and IL-4 (500 U/mL) for 7 days to generate immature DCs. Medium was exchanged every 2–3 days. Immature DCs were cocultured with transduced tumor cells (ratio 10:1) for 2 to 3 days±25 μg α-CD154 antibodies (PharMingen International, Hamburg, Germany). Screening of the cell surface markers and detection of transgene expression was performed by flow cytometry.
Coculture of lymphocytes and activated DCs
Lymphocytes were obtained from the monocyte-negative fraction during counterflow elutriation of PBMCs and cocultured with DCs for 4 days at a DC/lymphocyte ratio of 1:10. Thereafter, culture supernatant was harvested and the concentrations of IFN-γ and IL-4 were determined by sandwich ELISA (Becton Dickinson, Heidelberg, Germany).
Expression of cell surface molecules was analyzed by flow cytometry (FACSCalibur, Becton Dickinson) using α-human mAbs conjugated to PE or FITC: α-HLA-DR (BD Becton Dickinson), α-CD54 (Immunotech, Krefeld, Germany), α-CD1a, α-CD83, α-(murine) CD154 (PharMingen International), α-HLA-ABC class I, α-CD14, α-CD40, α-CD80, α-CD86, α-CD95, α-CD120a (Serotec, Oxford, UK). Single-cell suspensions of 1×105 cells were incubated with mAbs for 10 minutes at room temperature, washed in 2 mL PBS, centrifuged at 200×g and the supernatant was decanted. Cells were resuspended in 200 μl 1% paraformaldehyde in PBS before analysis.
Subcutaneous bladder tumor model
Male C57BL/6J or nude mice (B&K, Stockholm, Sweden) received a single dose of 10,000 murine MB49 bladder tumor cells subcutaneously (s.c.) into the hind leg. Two days before injection the tumor cells were transduced with either Ad-CD154 or Ad-dl70-3. Tumor growth was monitored daily and palpable tumors greater than 10 mm3 were considered positive. Animal studies were approved by the local Animal Ethics Committee (Dnr:C67/0).
CTL assay with vaccinated mice
Male C57BL/6J mice received four weekly s.c. injections of MB49-Ad-CD154 followed by one challenge with parental MB49 cells. Two to three weeks after challenge mice were euthanized and CD8-positive cells were purified from splenocytes using the MACS system (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer's recommendations. CD8-positive cells from vaccinated and naïve control mice were restimulated with irradiated MB49 in a 5:1 ratio for 5 days in RPMI-based medium, supplemented with 25 U/mL IL-2. Cytotoxicity against MB49, LLC1, and YAC-1 target cells was determined in a standard radioactive chromium-release assay.
Phenotype of transduced human tumor cell lines
Ad-CD154 transduction of human bladder cancer cell lines up-regulated immunomodulatory molecules and receptors important for cytotoxic killing. The J82 cell line was highly MHC class I, CD40, and CD54 (ICAM-I) positive but there was no or very little expression of CD80 (B7.1) and CD86 (B7.2) or the death receptors CD95 (Fas) and CD120a (TNFR) in the different experiments. Upon Ad-CD154 transduction, CD86, CD95, and CD120a were slightly up-regulated whereas CD40 was down-regulated compared with Ad-dl70-3 (empty control) vector-transduced cells (Fig 1, A and B). The cell line T24 was positive for MHC class I, CD40, CD54, CD80, CD86, and CD95, whereas CD120a expression was low. After Ad-CD154 transduction of the T24 cell line, the molecules mentioned above were up-regulated on a proportion of tumor cells except for CD40, which was down-regulated (Fig 1, C and D). Untransduced cells showed the same pattern as did control-vector–transduced cells.
CD154 expression slows down growth rate and induces apoptosis
CD154 expression by the tumor cells did not affect the growth rate unless the cells were grown at high density. In the latter case, the tumor cells clustered and the growth rate decreased (Fig 2A). The viability (propidium iodide staining) of Ad-CD154–transduced tumor cells was also reduced (data not shown) and apoptosis assays using Annexin V showed that the percentage of apoptotic cells was increased compared to Ad-dl70-3–transduced cells. Whereas the percentage of Annexin V–positive cells was negligible in J82 and T24 cultures transduced with control vector, 38% (T24) and 16.5% (J82) of tumor cells showed signs of early apoptosis in cultures expressing transgenic CD154 (Fig 2B). Nontransduced cells were similar to control-vector–transduced cells regarding growth rate, viability, and percentage of apoptotic cells (data not shown).
Ad-CD154–transduced tumor cells induce maturation of DCs
Immature DCs were cocultured with J82 or T24 cells transduced with Ad-CD154 or control vector. After 3 days, Ad-CD154–transduced tumor cell lines had stimulated the transition of allogeneic immature DCs to a mature phenotype with strong expression of CD86 and CD83. Tumor cells transduced with mock vector did not induce expression of either surface molecule on DCs (Fig 3A). To prove the involvement of CD40–CD154 interaction in the activation of DCs, transduced tumor cells and DCs were cocultured in the presence of α-CD154 inhibitory mAb. As depicted in Figure 3B the addition of the inhibitory antibody completely blocked the expression of CD83 and CD86 on a proportion of DCs, which remained in the immature state. In a further set of experiments, we tested the capacity of activated DCs to induce lymphocyte responses. DCs activated by CD154 expressing tumor cells strongly induced IFN-γ production by autologous lymphocytes. IFN-γ production was drastically reduced when an inhibitory mAb to CD154 was added to the coculture of DCs and tumor cells. Minimal IFN-γ production was detected in cocultures of lymphocytes and DCs stimulated with J82 Ad-dl70-3 (Fig 4) and in cultures of DCs in the absence of lymphocytes (data not shown). The production of IL-4 by lymphocytes stimulated with activated or nonactivated DCs was assessed in parallel but was found to be below the detection limit of a sandwich ELISA (data not shown).
CD154-expressing tumor cells efficiently activate autologous DCs from patients with RCC
Because of its bad prognosis and the lack of effective therapy, RCC has become a target for innovative immunotherapy.27 Consequently, we also examined the effects of CD154-expressing RCC cells on DCs. To strengthen the clinical relevance of our study, we used tumor cells and autologous DCs from patients with renal cancer. In three patients tested, we consistently observed that RCC transduced with Ad-CD154 up-regulated the expression of the maturation markers CD86 and CD83 on cocultured autologous DCs (Fig 5). In line with this observation, we also found an increased secretion of IFN-γ by lymphocytes cocultured with activated but not with nonactivated DCs (Fig 6). These findings were of special interest to us because for the first time we have analyzed the immunologic effects of CD154 gene transfer of freshly isolated solid tumors from patients.
Activity of T lymphocytes is required for effective Ad-CD154 gene therapy in a s.c. model of murine bladder cancer
To obtain information on the relevance of activation of T lymphocytes for the antitumor effect of Ad-CD154 immunomodulatory gene therapy in the in vivo situation, we performed additional animal studies. When injected into immunocompetent wild-type mice transduction of MB49 bladder tumor cells with Ad-CD154 totally inhibited tumor outgrowth compared to the control group receiving MB49 transduced with empty vector. In contrast, in nude mice we observed only a minimal antitumor effect of Ad-CD154 transduction (Fig 7). These data indicate a requirement for T lymphocytes during CD154 gene therapy in this model. To further assess the role of cytotoxic lymphocytes we performed cytotoxicity assays with splenocytes from mice vaccinated with MB49 expressing CD154. Based on previous studies,17 mice were injected four times in weekly intervals with MB49 expressing CD154 to obtain full protection against a subsequent challenge with parental MB49. Splenocytes from vaccinated mice that rejected a challenge with parental tumor and splenocytes from naïve control mice were isolated, and purified CD8 cells were restimulated with irradiated parental MB49. As indicated in Figure 8A, CTLs from mice vaccinated with MB49-CD154 effectively killed parental MB49 targets but not syngeneic LLC1 tumors or NK cell sensitive YAC-1. No cytotoxic activity was observed using CTLs from nonvaccinated control mice (Fig 8B).
Gene therapy of cancer, involving transfer of CD154 into tumor cells, has been applied in several animal models and has produced protective antitumor immunity.14,15,16,17 Clinical trials are now ongoing for hematologic malignancies with encouraging results.19 The cellular and immunological effects of the CD154 transduction into tumor cells are under investigation. It has been shown that triggering of CD40 leads to growth inhibition, apoptosis, and enhanced susceptibility for CTL lysis of the tumor cell.20,21,22,23,24 Here we show for the first time that CD154 transduced human solid tumor material from both cell culture and freshly isolated tumors can induce differentiation of immature DCs toward a mature phenotype. Investigated cell lines also exhibited a changed phenotype, retarded growth, and enhanced apoptosis.
The proliferation rate was reduced if the cells were cultured at high density, implying that interactions between CD40 and CD154 are taking place. Annexin V assays showed that a higher number of cells transduced with the Ad-CD154 vector were apoptotic compared with tumor cells transduced with an empty control vector. The J82 cells were not as sensitive, probably due to their lower expression of CD40. It has been shown earlier that DCs efficiently take up apoptotic cells28 and present the processed exogenous antigens on MHC class I to CD8+ CTLs.29 Initial experiments performed in our laboratory suggest that Ad-CD154–transduced tumor cells are taken up by DCs and that this increased uptake might be due to the increased rate of apoptosis in CD154-positive tumor cell cultures (data not shown).
The Ad-CD154–transduced tumor cells exhibited a changed phenotype with up-regulation of several cell surface molecules important for CTL recognition, whereas their expression of CD40 was down-regulated. Similar results have been shown in B-cell chronic lymphocytic leukemia.18,19 These cells are, however, APCs by nature and it was interesting to observe similar findings in urinary bladder carcinoma. The up-regulation of receptors for CTL recognition (MHC class I), attachment (CD54), and killing (CD95, CD120a) likely renders the J82 and T24 cells more sensitive for CTL-specific lysis, although this remains to be formally demonstrated.
Interaction of CD154 with its receptor CD40 leads to reciprocal activation. During this process, expression of CD154 can be down-regulated by receptor-mediated endocytosis, proteolytic cleavage, or reduced mRNA production.30 In our studies, we occasionally observed a reduced expression of CD40 in tumor cell cultures transduced with CD154. At least to our knowledge CD154-mediated modulation of the expression of CD40 has not been thoroughly investigated yet. It might be that at least under certain circumstances the interaction of CD154 and CD40 leads to reciprocal modulation of the expression of both the receptor (CD40) and its ligand (CD154).
Whereas CD154 gene therapy was successfully applied in some animal models of solid tumors including bladder cancer,16,17 the immunological mechanisms of this therapy remain poorly defined. It has been shown that triggering of CD40 on DCs induces expression of costimulatory signals (CD80, CD86) and adhesion molecules (CD54) and leads to efficient cross-priming of CTLs.30 Furthermore, ligation of CD40 results in increased DC viability, maturation, and IL-12 secretion by these cells.31 Chiodoni et al showed in a murine colon carcinoma model that CD154 and GM-CSF cotransduced tumor cells injected s.c. were less tumorigenic and that growing tumors were infiltrated by DCs. These DCs were also shown to stimulate IFN-γ production by antigen-specific CTLs in vitro.15 Based on the animal studies mentioned above we wanted to explore the direct interaction of tumor cells expressing CD154 with DCs utilizing human tumor cells and human DCs from healthy donors and from tumor patients. We showed in well-defined human allogeneic and autologous in vitro systems that Ad-CD154–transduced tumor cells cocultured with immature DCs induced a mature phenotype of the DCs with high expression of CD86 and the maturation marker CD83. DCs cocultured with control-vector–transduced tumor cells were ineffective in this regard. To further determine the immunostimulatory potential of the DCs we cocultured them with autologous T lymphocytes. T lymphocytes exhibited dramatically enhanced IFN-γ production when cocultured with the mature DCs. The maturation of DCs and the lymphocytic IFN-γ response could be partially blocked by adding α-CD154 antibodies to the culture, suggesting that the CD40/CD154 interaction is crucial for DC activation. The up-regulation of other important molecules on the Ad-CD154–transduced tumor cells may also be responsible for the differentiation of the DCs, which may explain why adding α-CD154 antibodies only partially blocked the maturation.
In our in vitro studies we observed an increased rate of apoptosis and reduced growth in tumor cultures transduced with Ad-CD154. At the same time CD154-expressing tumor cells induced DC maturation and subsequent activation of lymphocytes. We performed in vivo experiments using a murine s.c. bladder tumor model to dissect to what extent T lymphocytes and increased apoptosis each contribute to the antitumor effect of CD154 gene therapy. Whereas we observed a dramatic antitumor effect of CD154 gene therapy in immunocompetent wild-type mice, tumor outgrowth was only marginally affected in nude mice. As we also demonstrated cytotoxic activity of CTLs isolated from mice vaccinated with MB49-Ad-CD154, our data collectively suggest that activity of T lymphocytes is essential for CD154 gene therapy in this model. T-lymphocyte–independent effects (one of which might be increased apoptosis of tumor cells expressing CD154) seem to have only marginal antitumor effects in this experimental setting.
In conclusion, CD154 transduction of both freshly isolated patient tumor cells and tumor cell lines engender tumor cells capable of inducing differentiation of immature DCs and further IFN-γ production by lymphocytes cocultured with mature DCs. Experiments performed with cell lines showed an altered phenotype of CD154 transduced cells, a reduced growth rate, and enhanced apoptosis. In vivo experiments employing nude mice suggest that for eradication of s.c. bladder tumors the activity of T lymphocytes is of prime importance, whereas increased apoptosis seems to be of minor importance for the observed antitumor effect in vivo. Together with previously published animal studies,17 our experiments provide a rationale for the use of adenoviral CD154 gene therapy in the treatment of urologic malignancies such as bladder cancer and RCC.
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This study was supported by The Swedish Cancer Society, the Lions' Cancer Fund at Uppsala University Hospital, and Deutsche Forschungsgemeinschaft SFB 367. The authors thank Gabriele Bentien (Research Center Borstel) for excellent technical assistance and the colleagues from the Department of Urology at the Medical University of Lübeck for providing tumor tissue and peripheral blood of patients with RCC.
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Loskog, A., Tötterman, T., Böhle, A. et al. In vitro activation of cancer patient–derived dendritic cells by tumor cells genetically modified to express CD154. Cancer Gene Ther 9, 846–853 (2002). https://doi.org/10.1038/sj.cgt.7700507
- gene therapy
- adenoviral vector
- bladder cancer
- renal cell carcinoma
- dendritic cell
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