Mammaglobin A Epitope Long Peptide-loaded Dendritic Cells Induce Specific Cytotoxic T Lymphocytes Against Breast Cancer Cells In Vitro

Objective: Breast cancer is one of common female malignant tumor types,accounting for the second cancer death in women.Surgery,radiotherapy,chemotherapy and endocrine therapy and so on are common treatment methods of breast cancer.Although early screening and treatment of breast cancer patients have been improved,but a part of patients are resistant to radiation and chemotherapy and can't tolerate the side effects of radiation and chemotherapy,which can lead to failure of treatment.Tumor immunotherapy takes advantage of the host's immune system to specifically recognize and kill tumor cells,which has the very good application prospect.Dendritic cells(DC)is the most powerful antigen presenting cells in the body and the complex of antigen epitope peptide(8-10 amino acids)and major histocompatibility complex(MHC)is presented to the cell surface.Mature DCs activate the initial T cells and induce cytotoxic T lymphocyte(CTL)to kill tumor cells.Tumor long peptide vaccine has the properties of chemical stability,simple operation,high efficiency and so on.Long peptide vaccines overcome the drawbacks of short peptide vaccines including quick degradation and immune tolerance.Mammaglobin A(MGBA)specifically express in breast cancer,which make it a potential therapeutic target.Therefore,this study predict and synthetic MGBA epitope long peptide to verify the immune activity in vitro and provide a new insights for peptide vaccine.Methods: MGBA-derived HLA-A2 positive epitopes were predicted by the online database.According to the epitope concentration sequence,long peptide was designed and synthesized by Fmoc.Peripheral blood of HLA A2 + healthy volunteers was used to separate peripheral blood mononuclear cells(PBMCs).The granulocyte macrophage colony stimulating factor(GM-CSF)and interleukin 4(IL-4)were used to induce DC and tumor necrosis factor alpha(TNF-?)to promote DC maturation.The long peptide sensitized DC(experimental group,no peptide sensitized DC as control group)induce specific CTL.Molecular markers(CD83,CD80,CD86 and HLA-DR)of DC surface were detected using flow cytometry and IL-10,IL-12,IFN-?secretion level were detected using enzyme-linked immunosorbent(ELISA).At the same time,the killing effect of DC-CTL and MGBA-derived long peptide DC-CTL on UACC-812 and MCF-7 were detected by flow cytometry.Results: MGBA P69-92 was predicted by bioinformatics prediction method.The long peptide-loading DC and control group both induced mature DC with typical morphology.Compared with the control group,The expression level of IL-10 and IL-12 of experimental group separately was 161.90±17.90 vs 196.15±17.18 and259.48±22.45 vs 212.08±21.50(P < 0.05).For secretion levels of IFN-?,MGBA P69-92 CTLs with UACC-812 group was 636.64 ±61.09,which was significantly higher than other group(P < 0.05).According to the result of Annexin V-FITC/PI apoptosis detection,the killing activity of MGBA P69-92 DC-CTL to UACC-812(HLA-A2+,MGBA+)breast cancer cell at 20?1 ratio was significantly higher than DC-CTL to UACC-812(P < 0.05).At the same time,DC-CTL and MGBA P69-92DC-CTL has not obvious killing activity to MCF-7(HLA-A2+,MGBA-)cells without significant difference in the two groups(P > 0.05).Conclusion: MGBA P69-92 long peptide can be effectively presented to DC.MGBA P69-92 CTLs has specifically killing activity to HLA-A2 and MGBA positive breast cancer cells,high levels of IFN-?,no killing effect on MGBA-negative breast cancer cells.On the basis of this study,the further study need be certified immune activity of long peptide in vivo.This result provide a theoretic base for the design of the vaccine.