| The studies demonstrated the occure and development of the cancer was related with the functional defection of T lymphocyte, the further studies found the reason of which was the down-regulation of the mature dendritic cells (DC) by the body. In vitro, many experiments verified that the lymphocyte isolated from the advance stage cancer patient's peripheral blood still maintained very good reaction activity to the outside stimulation. So through culturing the DC in vitro and inducing the specific and high biology acitvity cytotoxic T lymphocyte (CTL), then injected back into the patient, which maybe becoming excellent therapy for the cancer. Therefore, if we know the target antigen for the caner, how can we get the good biology acitvity and large amount specfic CTL?DC is the most effective Antigen Presenting Cell at activating naive T Cells, which can activate antigen specific CTL with the antigen successful loading. At present, granulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin-4(IL-4) can promote the generation of large numbers of Dendritic Cells from peripheral blood monocytes. However, the focus point of the protocal in pulsing DC and inducing CTL in vitro is how to loading the antigen effectively?The studies showed: with different antigen loading technic, the DC activating efficiency is different. The CTL activity is also different. There are several ways to load the antigen, the popular strategy are: the antigen peptide transducing DC and virus vector carrying the antigen gene transfecting the DC.Transducing DC with antigen peptide has advantages such as antigen peptide is specific and which can immunize times, but which also has one main problem is the half-life of this kind of antigen peptide usually us very short, so only after several times of immunization can we get CTL with good activity. Moreover, the transduction efficiency is low.Transfection DC with virus vector carring antigen gene can make the DC produce a constant and large amount of antige peptide, which can resolve the problem of antigen peptide transducing DC directly. At present, the common virus vectors for the DC transfection are: retrovirs (Retrov), adenovirus (Ad) and adeno-associated virus (AAV). Among three of the virus vector, AAV is much more suitabl for the DC therapy in cancer. The reasons are: AAV is non-pathogenic (no disease is known), which can not pack by itself without the help virus such as adenovirus, so it is very safe for the gene therapy; AAV can transduce by speicfic the nineteenth chromosomal integration. This results in a very stable, long term transducti, which results in very long term transgene expression and very few chance of carcinomatous mutation. No known problems due to this unlike retroviruses; AAV is about 5kb, which is low immune response Unlike adenovius; AAV can infect many kinds of target cell, not just dividing cell but also non-dividing cell. Moreover, the transfection efficiency is high. Our pre-study demonstrated the effciency of AAV transfecting DC was about 90%. And AAV can up-regulate the surface marker of DC such as CD80, CD83 and CD86, which results in up-regulation of the activity of DC. Generating the CTL by gene trasfecting DC using the AAV as vector is very rapid and effective. Only one time of DC pusling can we generate the high activity CTL within two weeks; AAV is a tough virus, which is very easy to stores and transport.Two parts of gene therapy study using AAV as vector were designed on the base of pre-studis. The first part is: Generating the CTL by BA46 gene trasfecting DC to treat breast cancer. The second part is: Generating the CTL by prostate special antigen (PSA) gene trasfecting DC to treat prostate cancer. Through them to study the possibility of cancer antigen gene transducing DC generating the CTL in cancer treatment. Furthermore, we will optimize the protocol and make it as the experiment base for the future clinical trial in some kinds of cancer with clear antigen. The first part: Study on Generating the CTL by BA46 gene trasfecting DC using AAV as vector to treat breast cancer.1 Methods1.1 Generation of AAV/BA46 virus stockTotal RNA was isolated from Hs 578T breast cancer cells. The mRNA was separated. The primer pair was designed as: 5' CCGCAGCATGCCGCGCCC 3' and 5' CACTAACAGCCCAGCAGC 3', BA46 cDNA was generated by RT-PCR anaplification. The BA46 cDNA product was then gel-purified. The BA46 cDNA was sequenced and determined to be identical to the published sequence, then ligated into basic gutted AAV vector d16-95, constructed the plasmid of AAV/BA46. Adenovirus-free AAV/BA46 virus stocks was generated using the complementor plasmid pSH3. The virus stock was purified by heparin-agarose column and titered by extracting viral DNA and carrying out a comparative dot blot hybridization.1.2 AAV/BA46 trsansfecting the DC and generating the CTLPeripheral blood was derived from healthy donors. Ficoll gradient-purified PBMCs were inoculated into sixwell culture plates for 5 hours, and the adherent cells were selected following three gentle washes. Immediately after the removal of the nonadherent cells, the adherent cells were divided into 2 groups, one was gene transfer group, the other was control group, the gene transfer group infected with AAV/BA46 virus. After 5 hours of incubation, the medium/virus solution was removed, the flesh AIM-V medium added. The control group pulsed with 293 cells lysate for 5 hours. GM-CSF at a final concentration of 800 IU/ml was included in the medium in both groups throughout the culture. Human IL-4 at 1000 IU/ml was added at day 3, TNF-αat 100 IU/ml was added at day 5. On day 6, non-adherent PBMCs from the same healthy donors were washed and added to AAV/BA46-loaded or mock-treated DCs (ratio of 20:1, responders:dendritics) in six-well culture plates. The cultures were supplemented with recombinant human GM-CSF (800 U/ml), recombinant human IL-2 (10 U/ml), and IL-7 (20 ng/ml). After 7 days of coculture (day 12), the cells were collected for the CTL activity assay. 1.3 The BA46 mRNA test, the detection of viral integration, the DC surface marker analysis.On day 6, collected the suspend cells (Mature DC), observed the morphologic features of DC with light microscope. BA46 mRNA expression was detected in transduced DCs using RT-PCR amplification, The resulting BA46 RT-PCR products were Southern blotted and probed with 32P-labeled BA46 sequences to observe expression.Chromosomal integration of the AAV/BA46 genome was undertaken by vector-chromosome junction PCR amplification and Southern blot analysis.Cell surface marker analysis of DCs was by flow cytometry. For the characterization of DCs, a panel of surface marker was used: CD40, CD80, CD86.1.4 The immunity activity testing of BA46-specific CTLsCell surface marker analysis of stimulated T cells: The primed T-cell populations were analyzed for surface markers on day 13. A panel of mAbs recognizing the following antigens was used: PE-anti-CD4, FITC-anti-CD8, and PE-anti-CD56. Thus, the ratio of CD8/CD4 and CD8/CD56 was analysed with the flow cytometric.Analysis of CTL for intracellular cytokines: The mixed T-cell population was tested on day 13. The cells were harvested, washed and fixed with 2% paraformaldehyde in PBS for 20 minutes at room temperature. The cells were washed and permeabilized with PBS/1% BSA/0.5% saponin for 10 minutes at room temperature. Activated and control cells were stained with FITC-anti-IFN-r and PE-anti-IL-4 and analyzed by flow cytometric.Analysis of CTL for cytotoxicity assays and MHCâ… limitation in a 51Cr-release assay: Target cells: The primary breast cancer cell line Hs578T (HLA-A1 haplotype) was used as one BA46-positive target. The HLA haplotypes of all donors were compatible with this cell line. The autologous donor EBV-transformed lymphoblastoid cell lines (LCLs) were generated according to the routine method and were derived from the six healthy donors. The LCLs were infected with AAV/BA46 at a multiplicity of infection and became BA46 positive tested by the the flow cytometric. The cells were used for cytotoxicity assays in a 6-hour 51Cr-release assay. Analysis of CTL for MHCâ… limitation cytotoxicity in a 51Cr-release assay: Where indicated, target cells were preincubated with anti-Classâ… blocking antibody at 1/1000. After 7 days of coculture, the cells were used for cytotoxicity assays in a 6-hour 51Cr-release assay, as previously described.2 ResultConstucted the AAV/BA46 plasmid and prepared the high tite of AAV/BA46 virus stock, AAV/BA46 load DC successfully. AAV/BA46-1oading of DCs resulted in: (1) BA46 expression in DCs, (2) chromosomal integration of the AAV/BA46 vector within DC, (3) high CD80 and CD86 expression in DCs, (4) high CD8:CD4 and CD8:CD56 T cell ratios s, (5) T-cell populations with significant interferon-g (IFN-r) expression but low IL-4 expression, and (6) trong, rapid BA46-specific, MHC classâ… -restricted CTLs in only 1 week3 ConclusionThese data suggest that rAAV-loading of DCs may be useful for immunotherapeutic protocols against self-antigens and that the BA46 antigen is a potentially appropriate target for cell-mediated immunotherapeutic treatment for the ductal breast cancer.The second part: Study on Generating the CTL by PSA gene trasfecting DC using AAV as vector to treat prostate cancer.1 Methods1.1 Generation of AAV/PSA virus stockTotal RNA was isolated from LNCaP (prostate cancer cells) which was PSA positive. The mRNA was separated. The primer pair was designed as: 5'-TCGCGCCGAGATGTGGAAT-3' and 5'-TGTTCTTCTAGGTCTCCACC-3', PSA cDNA was generated by RT-PCR amplification. The PSA cDNA product was then gel-purified. The PSA cDNA was sequenced and determined to be identical to the published sequence, then ligated into basic gutted AAV vector d16-95, constructed the plasmid of AAV/PSA. Adenovirus-free AAV/PSA virus stocks was generated using the complementor plasmid pSH3. The virus stock was purified by heparin-agarose column and titered by extracting viral DNA and carrying out a comparative dot blot hybridization.1.2 AAV/PSA trsansfecting the DC and generating the CTLPeripheral blood was derived from healthy donors. Ficoll gradient-purified PBMCs were inoculated into sixwell culture plates for 5 hours, and the adherent cells were selected following three gentle washes. Immediately after the removal of the nonadherent cells, the adherent cells were divided into 3 groups, one was gene transfer group, the other was PSA protein lipoinfection group, the third was control group pulsed by 293 cell lysate. The gene transfer group infected with AAV/PSA virus. After 5h of incubation, the medium/virus solution was removed, the fresh AIM-V medium added. The PSA protein group was lipoinfected with PSA protein (200ng/ml) overnight. The control group pulsed with 293 cells lysate overnight. GM-CSF at a final concentration of 800 IU/ml was included in the medium in both groups throughout the culture. Human IL-4 at 1000 IU/ml was added at day 3, TNF-αat 100 IU/ml was added at day 5. On day 6, non-adherent PBMCs from the same healthy donors were washed and added to AAV/PSA-loaded or mock-treated DCs (ratio of 20:1, responders: dendritics) in six-well culture plates. The cultures were supplemented with recombinant human GM-CSF (800 U/ml), recombinant human IL-2 (10 U/ml), and IL-7 (20 ng/ml). After 7 days of coculture (day 12), the cells were collected for the CTL activity assay.1.3 Analysis of AAV/PSA Expression and efficient infection, the detection of viral integration, the DC surface marker analysis.On day 6, collected the suspend cells (Mature DC), observed the morphologic features of DC with light microscope. After three groups of suspension cells (DC) were harvested, the cells were fixed and permeabilized. The cells were incubated with mouse anti-PSA monocional antibody, and were subsequently stained with FITC-conjugated goat anti-mouse monoclonal antibody. A FACSCalibur flow cytometer was used for data acquisitions. The AAV/PSA transfecting DCs were smeared after centrifugation, observed with fluorescence microscope to determine the expression of PSA protein. Chromosomal integration of the AAV/PSA genome was undertaken by vector-chromosome junction PCR amplification and Southern blot analysisCell surface marker of DCs was analysed by flow cytometry. For the characterization of DCs, a panel of surface marker was used: HLA-DR,CD1a,CD14,CD83,CD80,CD86,CD40.1.4 The immunity activity testing of PSA-specific CTLs activating by DCCell surface marker analysis of stimulated T cells: The primed T-cell populations were analyzed for surface markers on day 13. A panel of mAbs recognizing the following antigens was used: PE-anti-CD4, FITC-anti-CD8, PE-anti-CD56, FITC-anti-CD25 and FITC-anti-CD69. Thus, the ratio of CD8/CD4, CD8/CD56 and the expression of CD25 and CD69 were analysed with the flow cytometric.Analysis of CTL for intracellular cytokines: The mixed T-cell population was tested on day 13. The cells were harvested, washed and fixed with 2% paraformaldehyde in PBS for 20 minutes at room temperature. The cells were washed and permeabilized with PBS/1% BSA/0.5% saponin for 10 minutes at room temperature. Activated and control cells were stained with FITC-anti-IFN-r and PE-anti-IL-4 and analyzed by flow cytometric.Analysis of CTL for cytotoxicity assays in a 51Cr-release assay: Target cells: The primary prostate cancer cell line LNCaP (HLA-A1 haplotype) was used as one PSA-positive target. The HLA haplotypes of all donors were compatible with this cell line. The autologous donor EBV-transformed lymphoblastoid cell lines (LCLs) were generated according to the routine method and were derived from the six healthy donors. The LCLs were infected with AAV/PSA at a multiplicity of infection and became PSA positive tested by the the flow cytometric. The cells were used for cytotoxicity assays in a 6-hour 51Cr-release assay. Addition to the analysis of the antigen specificity, we designed different dose virus transfecting the DC, inducing the CTL and analysed the killing, the dose effecting on the CTL killing was analysed through which.Analysis of CTL for MHCâ… limitation cytotoxicity in a 51Cr-release assay: Where indicated, target cells were preincubated with anti-Classâ… blocking antibody at 1/1000. The anti-Classâ…¡blocking antibody was used for negtive control, After 7 days of coculture, the cells were used for cytotoxicity assays in a 6-hour 51Cr-release assay to analyse the MHCâ… limitation of the CTL killing, as previously described.2 ResultConstuctcd the AAV/PSA plasmid and prepared the high tite of AAV/PSA virus stock, AAV/PSA load DC successfully. AAV/PSA-loading of DCs resulted in: (1) The PSA expression in DCs, (2) chromosomal integration of the AAV/PSA vector within DC, (3) high HLA-DR,CD1a,CD83,CD80,CD40 and CD86 expression in DCs, (4) higher CD8:CD4 and CD8:CD56 T cell ratios than the PSA protein pulsing, (5) T-cell populations with significant higher CD69 and interferon-r (IFN-r) expression but lower CD25 and IL-4 expression than the PSA protein pulsing, and (6) trong, rapid PSA-specific, MHC classâ… -restricted CTLs in only 1 week3 ConclusionThese datas suggested again that rAAV-loading of DCs may be useful for immunotherapeutic protocols against self-antigens and that the PSA antigen is potentially appropriate for cell-mediated immunotherapeutic protocols prostate cancer. |
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