The Immunotherapy Of Prostate Cancer In Mice Vaccination With Dendritic Cells Pulsed With Recombinant Human PSA/Tumor Cell Lysate

Prostate cancer is main cause of cancer-related death among men in our station. Vaccine strategies represent a novel therapeutic approach. One potential target for a prostate cancer vaccine is prostate-specific antigen, owing to its restricted expression in prostate cancer and normal prostatic epithelial cells. T cell-based immunotherapy via the in vitro or in vivo expansion of prostate tumor associa- ted antigen (TAA)-specific T lymphocytes is one of the most promising thera- peutic approaches to treat prostate cancer.Strategies employed in animal and clinical trials consist of dendritic cell vaccines, recombinant protein and recom- binant DNA vaccines, as well as viral vector delivery of vaccines. New approaches incorporating a combination of a vaccine with traditional treatments for prostate cancer are now on the way.Subjective.The aims of this study were to investigate: A) To construct pcDNA3.1/His-Myc -B PSA recombinant eukaryotic expression plasmid and investigate its PSA expression in eukaryotic cells. Recombinant Human PSA protein was purified; B) Study focused on maturation/ co- stimulation and antigen presentation func- tion of bone merrow derived DC puLsed by PSA, Lysate, OVA and PBS; Its ability of priming Tcells by PSA, Lysate, OVA and PBS puLsed dendritic cells and the generation of PSA/lysate-specific CD4+T cells; Its ability of inducing PSA/ lysate -specific CTL response in vitro; DTH-positive reaction was elicited by PSA/ lysate puLsed dendritic cells were also examined;C) The CTL induced in vitro by PSA/ ly- sate puLsed dendritic cells were adoptive transfered in experiment nude model and lysate puLsed dendritic cells as vaccine were also tested in RM-1 mice model. The survival time of mice and vlolume of tumor size were valuated by such vaccine theraphy.The role of CD4+, CD8+Tcells and NK cells were identified in RM-1 mice model.Methods.1.pcDNA3.1/His-Myc(-)BPSA recombinant eukaryotic expression plasmid were constructed, The coding sequence of GDNF was amplified from human prostate carcinoma sample cells by reverse transcription PCR (RT-PCR) and cloned into pcDNA3.1/His-Myc-B eukaryotic expression vector. The recombinant pcDNA3.1/His-Myc-BPSA plasmid was then transfected into CHO cells mediated by using Lipofectamine 2000 method. Analysis by restricting enzyme digestion and DNA sequencing were carried out to demonstrate the sequence of the plasmid. PSA protein and its activity were then determined using pcDNA3.1/His-Myc-BPSA plasmid transfected in CHO cells cells. The positive cell clones were selected by G418. PSA positive cloning was expressed into CHO cells by large-scale culture.2.mDC were derived from bone marrow (BM) cell, culture in the presence of GM-CSF plus IL-4 for 5 days. 7 d-DC were generated by stimulating with rTNF-α(500 U/ml) for 2 days. 7 d-DC pulsed with PSA, Lysate, OVA and PBS were designated as PSA-DC, Lysate-DC, OVA-DC, and non-DC, respectively. These DCs were examined the expression of Iab, CD40, B7.2, CD25, and CD11c by FACS. The secretion of IL-12 (p70) and IL-1βof DCs were assayed by ELISA. OVA-specific proliferation of MF2.2D9 cell line was used to examine the Ag-presenting function of mDCs cultured with OVA by IL-2 levels assay secreted by MF2.2D9 cell line.3.CD4+ T cells were purified by autoMACS. For MLR assay, CD4+T cells (2×105/well) from C57BL/6 mice (H-2b) were cocultured with PSA-DC, Lysate-DC, OVA-DC, and non-DC at various ratios, and thymidine incur- poration was determined using a liquid scintillation counter, respectively. MLR superna- tants were collected for cytokine detection of IFN-γ, IL-2, IL-4 and IL-10 levels.4.DTH assay was performed with modifications. C57BL/6 mice (H-2b) were immunized on the dorsal flank by s.c. inoculation of PSA-DC, Lysate-DC, OVA-DC, and non-DC cells (2×10~6 cells/animal) isolated from homogeneic mice on day 0 and 14, and challenged on day 21 at the right hind footpad by PSA in complete adjuvant injecting (10μg/animal). Footpad thickness was then measured on day 22 with a calipers-type engineer's micrometer.5.CTL Cytotoxicity assay. Recipient C57BL/6 mice were sacrificed 7 days after inoculated with PSA-DC, Lysate-DC, OVA-DC, and non-DC cells of C57BL/6 mice. Effectors were prepared by harvesting nylon wool-eluted splenic T cells. Effectors were plated at the appropriate ratios in quadruplicate. The LINCaP cell line (HLA A2 PSA specific) was used as syngeneic target and the E.G7 cell line (H-2b) was used as OVA-specific target cells. A standard 4 hrs cytotoxicity assay was performed with LINCaP cell or E.G7 cell labeled with 51Cr as target cells.6.Nude mice were immunized on the dorsal flank by s.c. inoculation of LNCaP tumor cells to established prostate nude mice model. Peptide-pulsed dendritic cells (10μg/mL for 2 hours) were irradiated (4,200 rad) and cocultured with purified spleen T cells from C57BL/6 mice in 24-well round-bottomed culture plates meanwhile rmIL-2 was added. One week later, the spleen T cells were restimulated with peptide-pulsed irradiated homogenetic DCs and 2 days later, T cells were collected used as CTL for adoptive transfer to nude mice loaded with LNCaP tumor cells. The survival of nude mice loaded with tumor cells and the size of tumor were calculated.7.C57BL/6 mice were immunized on the dorsal flank by s.c. inoculation of Lysate-DC, OVA-DC, and non-DC on day -7. On day 0, 2×10~6 cells of RM-1 tumor cells (H-2b) were injected s.c. in C57BL/6 mice pre-treated by s.c. inoculation of DC, correspondingly. In some test, for the determination of which immune cells were required for antitumor activity, mice were immunodepleted of CD4, CD8, or natural killer (NK) NK1.1 cells with the corresponding monoclonal antibodies.The survival of nude mice loaded with tumor cells and the size of tumor were calculated.Results. 1. The coding sequence of GDNF was amplified from human prostate carcino- ma sample cells by reverse transcription PCR (RT-PCR) and cloned into pcDNA 3.1/His-Myc-B eukaryotic expression vector. The recombinant pcDNA3.1/His- Myc(-)B/PSA plasmid was then transfected into CHO cells mediated by using Lipofectamine 2000 method. Analysis by restricting enzyme digestion and DNA sequencing were carried out to demonstrate the sequence of the plasmid. PSA protein and its activity were then determined using pcDNA3.1/His-Myc-B PSA plasmid transfected in CHO cells cells. The positive cell clones were selected by G418. RT-PCR product is 700bp specific segment. Analysis by restricting enzyme digestion and DNA sequencing of pcDNA3.1/His-Myc-BPSA recombinant showed results from restricting enzyme were 640 bp and 300 bp segments respectively. RT-PCR eletrophoretic analysis and Western blot method revealed that PSA cloning was successful. The recombine- ant plasmid can express active PSA protein in CHO cells. PSA positive cloning was expressed into CHO cells by large-scale culture. The max cell density could be approved to 3～9×10 cells/ml. The output of recombination protein was to 200～800mg/ L/d. The expressed protein was purified by affinity resin Ni-NTA and analyzed by SDS-PAGE and the purity of PSA was reached over 90%.2. We showed bone-merrow derived mDC cultured from C57BL/6 mice expre- ssed high levels of MHC class II, CD40, and CD86 upon TNF-αor by PSA, Lysate and OVA antigen stimulation. In addition, mDC secreted high levels of IL-12 (p70) and IL-1β. The phenotype and cytokine of DCs indicated those DCs were at mature state. OVA-presenting mDC may enhance secretion level of IL-2 of MF2.2D9 cell line by stimulation.3. The stimulatory activities and secretion of cytokines of mDC in different group in allogeneic MLR were compared. Compared with PSA-DC, Lysate- DC, , OVA-DC and non-DC was less able to stimulate the proliferation of CD4+T cells in 3-day classic MLR ( p<0.05). The cytokine profiles of CD4+T cells were analyzed after stimulation with DC in MLR supernatants. High levels of IFN-γand IL-2 ( p<0.05)were detected in the supernatants of MLR stimulated by PSA-DC, Lysate- DC, whereas the level of IL-10 and IL-4 was very low. These results indicate that both PSA-DC and Lysate- DC possess the ability to polarize CD4+T cells to the Th1 cell subset and can induce the generation of IFN-γ-producing T cells. Moreover, PSA-DC and Lysate-DC enhanced DTH responses of C57BL/6 mice to PSA antigen immunization. These results suggest that s.c. innoculation of PSA-DC and Lysate-DC potently elicited high DTH response and can induce T cell high sensitive response by pre-sensitization of C57BL/6 mice to PSA antigen in vivo.4. To observe the influence of administration of PSA-DC and Lysate-DC on inducing anti-PSA CTL reactions in vitro, splenic T cells isolated from C57BL /6 mice were isolated and restimulated in secondary MLR with PSA-DC, Lysate- DC, OVA-DC and non-DC. One part of the restimulated T cells after the 72-hr culture was used as an effector in the 4-hr CTL assays.The LINCaP cell line (PSA specific) was used as syngeneic target and the E.G7 cell line (H-2b) was used as OVA-specific target cells. We found that enhanced CTL activity of T cells to LINCaP cells were from PSA-DC, Lysate- DC( p<0.05), to E.G7 cells were from OVA-DC( p<0.05). These data indicate that PSA-DC, Lysate-DC can induce PSA specific cellular immunity in Vitro.5. Compared with control group, CTL T cells induced in vitro by PSA-DC, Lysate -DC were adoptive transferred to nude mice loaded with LNCaP tumor cells, can significant prolonged the survival of nude mice( p<0.01) and obviously repressed the size of tumor( p<0.001). Likewise in RM-1 mice prostate cancer model, immunized with Lysate-DC, compared with OVA-DC and non-DC, the pre-infection vaccine resulted in 100% clearance of primary tumors, whereas On day 0 of injection vaccine cleared 40-60% of primary tumors. In vivo depletion of NK cells resulted in a 40-60% reduction in growth suppression within the primary tumor, depletion of CD4+ cells resulted in a 20% reduction in growth suppression. These data indicate vaccine generated protection of anti-tumor immunity required an important cooperativity between NK cells, CD4+, and CD8+ T cells. Conclusion:DC-based multi-epitope immunotherapy with hormone-refractory prostate carcinoma yielded protective immunity, generates efficient cellular antitumor responses. Thereby providing further preclinical support for feasible immuno- therapy approaches for prostate cancer.