The Immunological Fuction Of Dendritic Cells From Human Peripheral Blood Into Which Her-2/neu Gene Was Delivered By An Adeno-assoiciated Virus Vector

ObjectivesThis researth will discuss dendritic cells from human peripheral blood into which Her-2/neu gene was delivered by an adeno-assoiciated virus vector.Check the surface markers and Her-2 expression of DC, T lymphocte subsets,the mixed lymphocyte reaction, and the specific killing activety. Methods and Material 1.MaterialRPMI1640, PBS (PH7.4) , 10%AB human serum , rhGM-CSF, IL-4, TNF-α rAAV-Her-2/neu, MMC, Mouse mAb to human FITC-CD1 a , FITC-CD83, PE -CD40, PE -CD80, FITC -CD86, FITC-HLA-DR, PE-CD8, FITC-CD4,PE-CD3, APC-Her-2 , PE-IgG1, FITC-IgG1, ~3H-TdR, FPS, MTT, SK-BR-3 cell line, MCF7 cell line, Carbon dioxide incubation box, Invered phase contrast microscope, Flow cytometry, etc. 2. Methods(1) Mononuclear cells in healthy person's were isolated by Ficol-Hypaque density gradient separation.Using granulocyte/macrophage colony-stimulating factor(GM-CSF) ,interleukin 4 (IL-4),tumor necrosis factor a(TNF- FITC-CD83^ PE-CD4(K PE -CD80> FITC-CD86> FITC-HLA-DR> APC-Her-2, PE-IgGK FITC-lgGl.(4) Using granulocyte/macrophage colony-stimulating factor (GM-CSF) ,interleukin 2 (IL-2), lymphoctes were initially cultivated in RPMI1640 supplemented with 10%AB human serum for seven days.The lymphoctes were divided into two groups and mixed with two groups of DC treated with MMC for 96h. Checked the T lymphocte subsets of CD4+T, CD8+T and CD3+T cells by flow cytometry.(5) Separated pure T lymphoctes by nylon wool column. T lymphoctes were divided into two groups and mixed with two groups of DC treated with MMC according to DC/T 115. 1 I 10, II 20, II 40 for 96h. Checked the mixed lymphocyte reaction by 3H-TdR.(6) Generated SK-BR-3 and MCF7 cell lines.Make growth cures of SK-BR-3 and MCF7 by MTT.(7) Checked SK-BR-3 's HLA-A by PCR-SPP using primer alleles: 002A2 ? 118A1K 148A24> 023A33> 015A11. Checked HLA-A of twelve healthy volunteers too,and selected the volunteers of HLA-A2 and HLA-A11.(8) Mononuclear cells in HLA-A2 and HLA-A 11 volunteers 's were isolated by Ficol-Hypaque density gradient separation. Generated DC and T lymphoctes. Bothvolunteers' DC were divided into two groups:one was added rAAV-Her-2/neu, the other was none. T lymphoctes were divided into two groups and mixed with two groups of DC treated with MMC according to DC/T 1 : 20 for 96h.(9) SK-BR-3 cells mixed with two groups of CTLs of HLA-A11 volunteers "s according to E/ T 80 : 1, 40 : 1, 20 : 1 , 10 : 1 for 12h.MCF7 cells mixed with two groups of CTLs of HLA-A2 volunteers 's according to E/ T 80 : 1, 40 I 1, 20 : 1 , 10 I 1 for 12h. Checked the specific killing activety by MTT.(lO)Statistics: SPSS10.0 software.3. Results(1) Both groups of cells had the same process of growing.DC shows dramatic changes in cell morphology during culture with in seven days.Many dendritic appeared at the cells gradually observed by light microscope.(2) Both groups of DC highly expressed CD1 a ,CD86,CD83 and HLA-DR. CD1a ,CD86,CD83 and HLA-DR were not obvious different among the two groups.CD40,CD80 of the DC added virus were higher than the other. Her-2 expression of the DC added virus was 88.97%, the other was nearly none.(3) CD3+T cells equaled to the summation of CD4+T cells and CD8+T cells in all groups. T cells mixed with DC had more CD8+ T cells. CD8+ T cells mixed with DC added virus were more than the other. CD8+/CD4+were inverse in both groups of T cells mixed with DC. CD8+/CD4+ of T cells mixed were DC added virus was higher than the other group.(4) After both groups of DC mixed with T cells, T cells got together to DC. After 24h ,T cells conjected and composed of cluster. After 78h, T cells proliferated dramatically .Both groups of DC stimulated strong T-cell proferative resposes.(5) SK-BR-3 and MCF7 grew attaching on Castaneda bottles.Growth cures of SK-BR-3 and MCF7 showed: both SK-BR-3 and MCF7's seeding time were 24h.(6) Checked SK-BR-3's HLA-A by PCR-SPP showed: HLA-A2.(7) Volunteers B, C> D were HLA-A2,Volunteer K was HLA-A11.(8) After CTL and tumor cells mixed 4h, the CTL induced by DC added virus got together to SK-BR-3. After 12h, lots of SK-BR-3 mixed CTL induced by DC added virus were killed.The other three groups weren't seen obviously killing. The killing rates of CTL induced by DC added virus to SK-BR-3 were higher with higher E/T.When E/T were 80 : 1, 40 : 1, 20 ! 1,CTL induced by DC added virus to SK-BR-3 were obviously higher than CTL induced by DC no virus. The killing rates of the other three groups were not obvious different. The DC added virus specifically lysed tumor targets.4. Conclusions(1) Mature DC with typical appearance could be obtained in both groups.(2) Her-2 expression of the DC added virus was 88.97%, suggested that rAAV-Her-2/neu can transfer Her-2/neu gene into DC. Both groups had mass and mature DC. Infective DC had more ability in stimulating immunostimulatory effect.(3) Both groups of DC stimulated CD8+Tcells proliferation. DC added virus were more efficient than the other group.(4) It was detected that both infective DC and uninfective DC could stimulated T lymphocyte proliferation in mixed lymphocyte reaction,but there was no difference between them.(5) The killing to breast cancer cell line with Her-2 over expression was more efficient with infective DC priming autologus T cells to generate CTL than with uninfective DC. CTL activity induced by infective DC was stronger than by uninfective DC,but lower elicited efficiency to breast cancer cell line without Her-2 expression.The immunological fuction of DC in human peripheral blood was enhanced by Her-2/neu gene deliveried into by AAV.