Studies On Specific Antigen Efficiently Activated Cytotoxic T Lymphocyte

Following surgery, radiotherapy and chemotherapy, with the rapid development of the technology of gene engineering and cell engineering. biological anti-tumor therapy is a new type of anti-tumor therapy.Biological anti-tumor therapy has rapidly developed and been widely used in clinical treatment with notable efficacy.Adoptive cellular immunotherapy is a kind of therapy which focuses on infusion of the anti-tumor immunocompetent cells to tumor patients in order to directly or indirectly kill tumor cells. Currently, the main immunocompetent cells for adoptive immunotherapy include lymphokine-activated killer cells (LAK), tumor-infiltrating lymphocytes (TIL), anti-CD₃ monoclonal antibody activated killer cells (CD₃AK) ,cytokine activated killer cells (CIK), cytotoxic T lymphocyte (CTL) natural killer cells (NK)and so on.Some of them have entered the stage of clinical application.The others still are ongoing pre-clinical studies.According to the theory of two-signal activation of T cells,our studies simulated the in vivo two-signal activation pathway of T cells, combined rhHSP70 with the antigen peptide in vitro under certain conditions. HSP70-PC were transmitted to the major histocompatibility antigen complex (MHC) molecules and triggered specific cytotoxic T lymphocytes (CTL) response. Or stimulated specific cellular immune response mediated by T-cells through direct activation of T cells. Under certain circumstance,mistletoe extracts can be used as biological response modifier which can almost stimulate all cells in immune system. We named our cells SAA-CTL(specific antigen activated cytotoxic T lymphocyte).The purpose of our studies were to carry out the following tasks :â‘ To prove rhHSP70-peptide complex,which is the complex of rhHSP70 combining with two synthesized antigenic peptides,has immune activity on human peripheral blood and can activate immune cells' cytotoxicity;â‘¡To establish the fermentation process of mistletoe using Lactobacillus plantarum and to validate the effect of. the fermentation extracts of mistletoe on co-stimulator;â‘¢To establish a new cell culture system which is suitable for large-scale cultivation of SAA-CTL;â‘£To prove that the in vitro SAA-CTL has strong and specific ability to kill tumor cells.â… . Study on specific immune response induced by rhHSP70-peptide complexes1. Proliferation of lymphocytes activated by rhHSP70-peptide complexesCombined synthetic peptides, the HER2/neu protein T cell epitope P_106-114 and P369-377, with purified rhHSP70.Stimulated human peripheral blood lymphocytes using six groups of stimulator: 1) the control group, 2) rhHSP70 group; 3) P_106-114 Group; 4)P_369-377 group; 5)rhHSP70-P_106-114 group; 6) rhHSP70_-P369-377 group. The sample groups was added 0.05ml samples while the control group was added to the same volume 1640 medium with 10% human AB serum. 48 hours later, put up MTT assay. The result showed that lymphocytes stimulated by rhHSP70,P_106-114 and P_369-377 had no significant proliferation (P＞0.05) compared with unstimulated lymphocytes,.Lymphocytes stimulated by rhHSP70-P_106-114 and rhHSP70-P_369-377 had significant proliferation (P＜0.001).This activation was dose-dependent.When rhHSP70-peptide complexes were diluted for 5 times and 25 times, they still had activation effect on lymphocyte.2. Cytotoxic activity of lymphocytes activated by rhHSP70-peptide complexes on different tumor cell lines in vitroRespectively cultivated tumor cells to the logarithmic growth phase.Those cells,including mouse tumor cell line D₂F₂ and human tumorl cell line Hela,Eca-109, HCC-9724, and SKOV₃, as target cells, were seeded in 96-well plates and put into incubator for 24 hours.The growth of cancer cells would be fully adherent on the plates.Then,stimulated peripheral blood respectively using rhHSP70, P_106-114, P3_69-377, rhHSP70-P_106-114 or rhHSP70-P_369-377, and cultured for 7 days to collect the cells as effector cells.Next,added effector cells to the target cells which were adherent to the 96-well plates and made the ratio of effector cells and tumor cells be 10: 1,20:1 and 40:1.Non-stimulated lymphocytes were as control.After 24～72 hours' cultivatation, MTT test was made to assay cytotoxic activity. The results were showed as the following :Compared with the control group, when the ratio of target cells and effect cells were 10:1, 20:1and 40:1, lymphocytes stimulated by rhHSP70, P_106-114, P_369-377, had no significant cytotoxic effect on various tumor cells (P＞0.05).When the lymphocytes were simulated by rhHSP70-P_106-114 and rhHSP70-P_369-377.No show significant cytotoxic effect on Hela and Eca-109 cells was showed(P＞0.05). But D₂F₂, HCC-9724, SKOV₃ were killed significantly. (P＜0.05 or P＜0.01).â…¡. Studies on the fermentation process of mistletoe and on the immune effect of mistletoe fermentation extracts.1. Research on the mistletoe fermentation.In our studies, Lactobacillus plantarum were used to ferment mistletoe. The fermentation process were studied and focused on the fermentation medium, pH, initial biomass and other factors which could influence the process of fermentation. Those studies identified the appropriate conditions for mistletoe fermentation were MRS medium with peptone, glucose, The appropriate concentration of MgSO₄ and MnSO₄ is 0.2% and 0.05% respectively.The best age of seed was 12h.The best initial mass were 8～10%.Under the condition of 37℃and pH 5.5～6.5.2. Immune effect of mistletoe fermentation extractsDetected the promotion of CD₂₈⁺ activated by mistletoe fermentation extracts using flow cytometry. Compared with the negative control, among the lymphocytes stimulated by mistletoe fermentation extracts, the percentage of CD₂₈⁺ and CD₄⁺CD₂₈⁺ cells increased significantly (P＜0.01). â…¢. Establishment and identification of large-scale SAA-CTL cultivation system.1. Establishment of SAA-CTL cultivation systemCoated flask (bottom plot 225cm²)with anti-CD₃ monoclonal antibody (final concentration of 1μg·mL^-1) for 18 h. Harvested PBMC and modified the cell density to 1×10⁹·L^-1 with RPMI-1640. Then,added rhHSP 70-peptide complexes and mistletoe extract; After that,half changed RPMI 1640 medium with rIL-2 500U·ml^-1,and counted the cell's quantity and regulated the cell's concentration to 1×10⁹ L^-1 every 3 days. Ended the cultivation at the 28th day.2. Observation of morphologyObserved the morphology and the dynamic growth of SAA-CTL using biological difference inverted microscope. Minority cells began its coloning growth 3 days later after being activated. Cell volume increased. The shape of cells changed. 14 days later, the cell volume increased and the shape of cells was single and obviously polymorphous. 28 days later, large-scale exuberant proliferation and significant coloning growth of cells could be seen. Observed through Transmission Electron Microscope, the integrity of membrane could be seen. The shape of cells were irregular with a little protuberance. There was no mycoplasma in the culture system.Intact Organelles, a small amount of lipid droplets , well-organized mitochondria, dense secretory granules of membrane vesicles ,lysosomes and lots of free ribosomes existed in cytoplasma.All of the above structure confirmed that the cultured cells had the features of normal T lymphocytes.There were particle substance relevant to cytotoxic activity which were structural and material basis of cytotoxic activity of SAA-CTL.3. Karyotype analysisAfter colchicine treatment,hypotonic treatment and centrifugation, fixed and re-fixed.Then,produced and stained the film of cells. Microscopic examination showed the appropriate choice of scattered-split cell through low-fold raster.Next,observed and counted the chromosome using the high-fold raster.The results showed they were normal human with Karyotype of 23 pairs of chromosome,â…©â…©.4. Compare of proliferation kinetics with LAK and CD3AK cellsIn the first week of being activated, SAA-CTL cells proliferated slowly.Then, they went into the rapid proliferation phase in the 2weeks. On the 28day ,which is logarithmic growth phase, cells increased 517.6±20.28The proliferation of LAK cells was slow. The number of LAK cells merely increased 112.73±9.76 times on the 28day.The number of CD₃AK cells increased 376.94±14.81times.On different day, the proliferation multiple of SAA-CTL cells were significantly higher than CD₃AK and LAK at the same period (P＜0.05, P＜0.01orP＜0.001).5. Compare of immune phenotype identification with LAK and CD₃AK cellsFCM result showed that CD₃⁺ cells of activated SAA-CTL cells increased from 50.9±9.41% to 95.6±10.41% (P＜0.001). CD₃⁺ CD₄⁺cells increased from 19.6±2.98% to 51.4±4.68% (P＜0.01).CD₃⁺ CD₈⁺ cells increased from 27.5±1.97% to 53.4±3.72% (P＜0.01).6. Test of IFN-γ,TNF-αexcreted by different cells.Results indicated that the level of IFN-γand TNF-αinduced by these three immunoreactive cells increased significantly with the growing of culture days.In the different days of culture,the level of IFN-γand TNF-αexisted in SAA-CTL culture supernatant were significantly higher than that in LAK, CD₃AK (P＜0.05, P＜0.01 or P＜0.001). But in the third week of cultivation,IFN-γand TNF-αinduced by LAK, CD₃AK had no significant increase.While IFN-γand TNF-αinduced by SAA-CTL still had significant increase.7. Observation of cytotoxic activityCompared the cytotoxic activity of SAA-CTL in different cultivation days.The results showed that cytotoxic activity in the 7,10,13,16 ,19 and 22day were significantly powerful than in the 4 day(P＜0.05, P＜0.01 or P＜0.01),which indicated by SAA-CTL from 7 to 19 days was the best option for anti-tumor therapy in clinical application.In summary, our research demonstrated for the first time that synthesized rhHSP70-peptide complexes ,which were the in vitro combination of rhHSP70 expressed in Pichia pastoris and tumor peptide, had the same immune activity with the natural hHSP70- peptide complexes.This kind of rhHSP 70-peptide complexes could activate the proliferation the specific immune response of human peripheral blood lymphocyte. Firstly discovered the fermentation extracts of Northeast Mistletoe had the role of stimulating costimulatory molecules CD₂₈ on CD₄⁺ T cell surface to increase . Firstly established a large-scale in vitro culture system of SAA-CTL and confirmed its high proliferative ability, and strong cytotoxicity advantages. SAA-CTL is expected to become the most promising anti-tumor effect cells. Our study laid the groundwork of immune mechanism for tumor and the development of the clinical utilization of SAA-CTL.