| ObjectiveDCs are highly potent initiators of the immune response, characterized by their ability to engulf, process, and present antigens to T lymphocytes. In recent years, DC-based anti-tumor vaccines have emerged as promising strategies for cancer immunotherapy. But high levels of TGF-βproduced by cancer cells inhibit the ability of DCs to present antigen, stimulate tumor-sensitized T lymphocytes. C57BL/6 murine bone marrow DCs were pulsed with freeze–thawed TRAMP-C2 tumor lysate and induced prostate cancer specific DCs. We successfully constructed a retrovirus containing dominant-negative TGF-βtype II receptor (TβRIIDN). The tumor lysate-pulsed DCs were rendered TGF-βinsensitive by infecting with a retrovirus containing TβRIIDN, leading to the blockade of TGF-βsignals to members of the Smad family. After transfection we detected surface antigen, cell proliferation, secretion and evaluated the immunotherapy of TGF-β insensitive DC vaccines to TRAMP-C2 tumor-bearing C57BL/6 mice. Materials and methods1. To construct a retrovirus containing dominant-negative TGF-βtype II receptor (TβRIIDN): Pantropic GP293 retroviral packaging cells were seeded at a density of 2.5×106 cells in T-25 collagen I-coated flasks 24 h before plasmid transfection in antibiotic-free 10% Dulbecco's Modified Eagle Medium. A mixture of 2μg retroviral plasmid and 2μg vesicular stomatitis virus envelope G protein (VSV-G) envelope plasmid was cotransfected in serum-free DMEM using LipofectAMINE-Plus, according to the manufacturer's protocols. Briefly, cells were transfected for 12 h followed by the addition of an equivalent volume of 10%DMEM and incubation for an additional 12 h. Afterward, the supernatant was aspirated, the cells were rinsed gently in PBS, and 3 ml of fresh 10%DMEM was added each flask. 24 h later, virus-containing supernatant was collected and used to infect target cells.2. Isolation, cultivation and identification of tumor lysate-pulsed DC: Erythrocyte-depleted murine bone marrow cells were obtained from the femurs and tibiae of C57BL/6 mice under aseptic conditions and cultured at 1×106 cells/ml in CM supplemented with 1000 U/ml recombinant murine granulocyte-macrophage-colony-stimulating factor (rmGM-CSF) and 1000 U/ml recombinant murine interleukin-4 (rmIL-4). The medium was replaced on day 2 with additional recombinant cytokines. On day 6, nonadherent DCs were harvested by gradient centrifugation and were further purified with MACS CD11c beads. Immature DCs were pulsed with freeze–thawed tumor lysate to obtain tumor lysate-pulsed DC. Tumor lysate-pulsed DCs were identified under light microscope. Immatured DCs, tumor lysate-pulsed DCs, GFP-transduced DCs and tumor lysate-pulsed TGF-β-insensitive DCs, which were cultured for an additional 18 h, were analyzed by flow cytometry, using a panel of Abs specific for MHC class II, CD40, CD11c, CD80, and CD86.3. Infection of DCs with Retrovirus: Tumor lysate-pulsed DCs were infected with the retrovirus containing TβRIIDN or GFP vector. The cells were rinsed gently in 5%CO2, 37℃for 48 hours to get TβRIIDN DCs. The immunophenotype of TβRIIDN DCs were analyzed by flow cytometry.4. Experiment of TβRIIDN DCs in vitro: Different DCs were cultured with TGF-β. The surface co-stimulatory molecules (CD80/CD86) were analyzed by flow cytometry. The antiproliferative effects of TGF-βwere observed by thymidine incorporation assay. Smad-2 and phosphorylated Smad-2 were detected by Western blot.5. Antitumor Analyses in Vivo: TRAMP-C2 tumors were established in mice. 40 C57BL/6 tumor-bearing mice were divided into four groups randomly and inoculated s.c. with nontransduced DCs, tumor lysate-pulsed TGF-β-insensitive DCs, GFP-transduced DCs, or PBS. The vaccination was repeated on day 15. Serum levels of IFN-γand IL-12 were determined by enzyme-linked immunoabsorbant assay (ELISA). Tumor growth and mouse survival were monitored daily post-inoculation. 6. Cytotoxicity T Lymphocyte Assays: Splenic cells were obtained from the tumor-bearing mice 5 days after the final vaccination and cocultured with X-ray (40 Gy)-irradiated TRAMP-C2 cells (2×105) in 24-well plates for 4 days. The activated T-cells were harvested and used as effector cells against 51Cr-loaded TRAMP-C2 target cells. An irrelevant cancer cell line, mouse melanoma cell line, B16-F1 was used as a nonspecific control.Results:1. Erythrocyte-depleted murine bone marrow cells were freshly isolated from the femurs and tibiae of C57BL/6 mice. The purity of bone marrow–derived DC, determined through analysis on CD11c staining by flow cytometry, was 90.8%. Tumor lysate-pulsed DCs were infected with the TβRIIDN-containing or GFP control retrovirus. The infection efficiency was determined by flow cytometry analysis. They were 92.7% and 90.6%, respectively, for the T?RIIDN- containing and the GFP control retroviruses.2. DC surface molecules MHC class II, CD40, CD11c, CD80 and CD86, were analyzed and compared among immatured DCs, tumor lysate-pulsed DCs, GFP-transduced DCs and TβRIIDN-transduced DCs by flow cytometry. The expression of CD86, CD80, MHC class II and CD40 on tumor lysate-pulsed DCs, GFP-transduced DCs and TβRIIDN-transduced DCs was higher than those on immatured DCs (P<0.01). No obvious differences were observed among tumor lysate-pulsed DCs, GFP-transduced DCs and TβRIIDN-transduced DCs (P>0.05), indicating that the transduction of TβRIIDN did not affect the immunophenotype of DCs.3. Smad-2 and phosphorylated Smad-2 were detected by Western blot analysis after the TβRIIDN-transduced or GFP-transduced DCs were treated with 5 ng/ml TGF-β1. The presence of Smad-2 was detected in all DC groups. But, phosphorylated Smad-2 was only detected in nontransduced and GFP-transduced DCs in response to TGF-β1; absence of phosphorylated Smad-2 in TβRIIDN- transduced DCs confirmed that TGF-βsignal transduction was blocked by the presence of the TβRIIDN.4. The inhibitory rate of TGF-βon thymidine uptake was compared among the TβRIIDN-transduced DCs, GFP-transduced, and nontransduced DCs after the addition of TGF-β1 for 72 h. TGF-β1 showed a dramatic antiproliferative effect on the established nontransduced and GFP-transduced DCs, inhibiting uptake by a mean of 62.5% and 65.5% respectively. Whereas the mean inhibitory rate of thymidine uptake by TβRIIDN-transduced DCs was 15%, the resistance to the antiproliferative effects of TβRIIDN-transduced DCs was statistically significant when compared with the other groups (P<0.05). Importantly, when cells were maintained under normal growth conditions in the presence of TGF-β1, the nontransduced and GFP-transduced DCs failed to proliferate and died within 15 days. TβRIIDN-transduced DCs, however, continued to proliferate and grow normally, showing significant resistance to the antiproliferative effects of TGF-β1.5. Nontransduced DCs, GFP-transduced DCs and TβRIIDN- transduced DCs were cultured with freeze–thawed tumor lysate and 10 ng/mL TGF-β1 for 7 days. The surface co-stimulatory molecules CD80 and CD86, were analyzed by flow cytometry. As expected, expression of CD86 and CD80 was higher on TβRIIDN-transduced DCs than on nontransduced DCs and GFP-transduced DCs (P<0.01) in the presence of TGF-β1.6. To assess the antitumor effect of the TβRIIDN-transduced DC vaccine in vivo, TRAMP-C2 tumors were established in C57BL/6 mice. A suspension of nontransduced DCs, GFP-transduced DCs, or TβRIIDN-transduced DCs was injected into tumor-bearing mice (n = 10/group). PBS was used as a negative control. These experimental groups were designed to evaluate whether blocking TGF-βsignaling alters the efficacy of DC vaccine in inducing anti-tumor immune responses and mortality of the tumor-bearing mice. Immunization with TβRIIDN-transduced DCs, GFP-transduced DCs or nontransduced DCs significantly suppressed the growth of the tumor (P<0.01, P<0.05 and P<0.05, vs. control, respectively), with the TβRIIDN-transduced DCs showing the more significant inhibitory effect. Complete tumor regression occurred in 20% of TRAMP-C2-tumor-bearing mice that were treated with TβRIIDN-transduced DCs. These results indicated that the tumor lysate-pulsed TGF-β-insensitive DC vaccine had the strongest anti-tumor effect in all the vaccination groups. Another four groups of ten mice were used to evaluate the survival rate after 50 days. Results showed that survival rate of the untreated, GFP-vector control and nontransduced DCs treated mice was 0%, 20%and 30% respectively, while the survival of the TβRIIDN-transduced DCs treated cohort was 80%. Statistical analysis by using the Mantel-Haenszel log-rank test indicated a significant difference between the TGF-β-insensitive-DC and the other two control groups (P<0.01). The result demonstrate that the TGF-β-insensitive DC vaccine was effective in improving the survival rate in mice bearing TGF-β-secreting tumors.7. In animals injected with PBS, there was a basal level of IFN-γand IL-12. In animals received nontransduced DCs or GFP-transduced DCs, there was a significant increase in levels of both cytokines. A further increase in serum IL-12 and IFN-γwas observed when these cells were rendered insensitive to TGF-? (the T?RIIDN-transduced DCs group), suggesting the increase of activated immune cells in these hosts.8. The ability of CTLs to lyse TRAMP-C2 cells was assayed in vitro using a standard 51Cr release assay. Compared with the PBS-vaccinated group, mice treated with TβRIIDN-transduced DCs, GFP-transduced DCs, or non-transduced DCs all showed a higher cytotoxicity against the TRAMP-C2 cells. The most potent TRAMP-C2-specific splenic CTL response was induced by the TGF-?-insensitive DCs in the tumor bearer (85% killing activity at an effector:target cell ratio of 100:1). No apparent lysis was observed against irrelevant B16-F1 cells. This result indicates that tumor-specific cytolysis is generated by blocking TGF-βsignaling, which enhances the efficacy of DC vaccines. Conclusion1. 1. We successfully constructed a retrovirus containing dominant-negative TGF-βtype II receptor (TβRIIDN).2. C57BL/6 murine bone marrow DCs were pulsed with freeze–thawed TRAMP-C2 tumor lysate and induced prostate cancer specific DCs.3. The tumor lysate-pulsed DCs were rendered TGF-βinsensitive by infecting with a retrovirus containing dominant-negative TGF-βtype II receptor (TβRIIDN), leading to the blockade of TGF-βsignals to members of the Smad family.4. Expression of TβRIIDN did not affect the phenotype of transduced DCs. Expression of CD86 and CD80 was higher on TβRIIDN-transduced DCs than on nontransduced DCs and GFP-transduced DCs (P<0.01) in the presence of TGF-β1.5. TβRIIDN-transduced DCs suppressed tumor growth and increased survival rate of TRAMP-C2 tumor-bearing mice. Furthermore, complete tumor regression occurred in 2 vaccinated mice. TβRIIDN-transduced DCs induced higher IFN-γand IL-12 level in vivo.6. The most potent TRAMP-C2-specific splenic CTL response was induced by the TGF-?-insensitive DCs in the tumor bearer (85% killing activity at an effector:target cell ratio of 100:1). No apparent lysis was observed against irrelevant B16-F1 cells.
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