Design Of Broad-Spectrum Multiple Antigen Peptides Based On CTL Epitopes Of Human MPGES1 And Anti-HCC Activity In Vitro

Background and ObjectiveMicrosomal prostaglandin E synthase 1(m PGES1)is one of the two key rate-limiting enzymes in the synthesis of prostaglandin E2(PGE2).It plays an crucial role in inflammation,moreover,in the development,metastasis and recurrence of hepatocellular carcinoma(HCC).Because of its high expression on the surface of various tumor cells and no expression or very low expression on the surface of normal tissue cells,m PGES1 is considered as a tumor-associated antigen(TAA),which could be used as an effective molecular target for the prevention and treatment of HCC.As a new generation of tumor vaccines,cytotoxic T lymphocyte(CTL)epitope vaccine is regarded as a promising treatment method in the novel immunotherapy strategies for tumor,especially dendritic cell(DC)vaccines loaded with CTL epitopes.In order to design and synthesize broad-spectrum multiple antigen peptide(MAP)based on CTL epitopes of human m PGES1 and detect the anti-HCC activity in vitro,HLA-A2/A3-restricted CTL epitopes of m PGES1 are screened and used for tree-like MAPs design and synthesis with polylysine as cores and four branches.After isolation and culture DCs from murine bone marrow,vaccines are synthesized by loading MAPs on mature DCs and their effect of killing liver cancer cells is observed in vitro.Methods1 HLA-A2/A3-restricted CTL epitopes of m PGES1 were predicted online through SYFPEITHI,and nonapeptides were screened for their high predictive value with both HLA-A2 and HLA-A3 genotypes.MHCPred prediction tool was used to analyze the ability of nonapeptides binding to HLA,and which scored greater than 5.0 was considered to have structural affinity.2 Standard Solid Phase Peptide Synthesis(SPPS)was used to synthesize three MAPs with polylysine as their cores to form tight and orderly four-branch tree-like structure,namely,(VSRIPARLK)4,(LRVQGTIIA)4,(VTHAVRGVL)4(hereinafter referred to as M1,M2,M3).An ordinary MAP with single-chain structure was also synthesized,namely VSRIPARLK(hereinafter referred to as M5).3 The purity of MAPs was analyzed by Reversed Phase High Pressure Liquid Chromatograph(RP-HPLC).Molecular weight of MAPs was determined by Liquid Chromatography/Mass Spectrometry(LC/MS).4 After isolation and culture DCs derived from murine bone marrow until mature,the cell morphology was observed under inverted fluorescence microscope and surface structure was photographed by scanning electronic microscope.Flow cytometer was used to identify cell surface molecular markers like CD83,CD86 and CD11 c.5 After isolation and culture lymphocytes of murine spleen,the co-incubation mature murine DCs loaded MAPs and murine lymphocytes induced m PGES1-specific CTLs proliferation,which were served as effector cells.Human liver cancer cell lines including Hep G2,Huh-7,MHCC97 h and SMMC7721 were cultured and m PGES1 expression was detected by Western Blot.Three strains with high expression,medium expression and low expression of m PGES1 were selected as target cells.6 The effector cells(E)were co-cultured with the target cells(T)at the E : T ratios of 1 : 3,1 : 1,3 : 1,10 : 1,25 : 1 and 50 : 1.Killing experiments were divided into six groups:(1)murine DCs loaded with M1 and lymphocytes were recorded as M1 group;(2)murine DCs loaded with M2 and lymphocytes were recorded as M2 group;(3)murine DCs loaded with M3 and lymphocytes were recorded as M3 group;(4)murine DCs loaded with M1,M2 and M3 at the same time and lymphocytes were marked as M4 group;(5)murine DCs loaded with M5 and lymphocytes were recorded as M5 group;(6)murine DCs and lymphocytes without MAP load were recorded as M0 group.In addition,human liver cancer cells were set as the control groups without any immune cells.7 The cytotoxicity of effector cells were detected by Cell Counting Kit-8(CCK-8)assay.At the E : T ratio of 10 : 1,both flow cytometer and Annexin V-FITC/PI staining kit were used to measure the apoptosis of target cells caused by effector cells.At the E : T ratio of 25 : 1,the secretion level of cytokines IFN-? in the process of effector cells killing target cells was estimated by Enzyme Linked Immunosorbent Assay(ELISA).Results1 HLA-A2/A3-restricted CTL epitopes of m PGES1 were obtained.Three four-branch MAPs with tree-like structure and an single-chain MAP were synthesized.The purity of all MAPs was above 95%,reaching the international standard of testing peptides.Their molecular weight were 4495.00 k Da,4215.60 k Da,4140.00 k Da and 1040.20 k Da respectively,and their differences from the theoretical value were non-significant.2 DCs derived from murine bone marrow were obtained and cultured until mature.Under inverted fluorescence microscope,special dendritic shape of DCs was observed.Scanning electronic microscope showed typical DCs surface structure with yarn-like protuberances.Mature murine DCs expressed high levels of CD83,CD86 and CD11 c,up to 89.61%,89.74% and 95.16%,conformed to the phenotypic characteristics of DC.3 Lymphocytes of murine spleen were isolated and cultured,and then effective cells proliferation was induced by the co-incubation of murine DCs loaded MAPs and lymphocytes.MHCC97 h with high expression of m PGES1,Huh-7 with medium expression and Hep G2 with low expression were cultured as target cells.4 At the E:T ratio of 1 : 3,1 : 1,3 : 1,10 : 1,25 : 1 and 50 : 1,CCK-8 assay results showed that with the higher concentration of effector cells or E : T ratio,the effector cells in each group had stronger toxic effects on target cells.When the same target cells were killed and the E : T ratio was same,the toxic effect of effector cells in the M1 group,M2 group,M3 group and M4 group on target cells were significantly enhanced compared with those in the M5 group and M0 group(p<0.05).When the same effector cells were used for killing,the toxic effect of effector cells on target cells was the strongest in MHCC97 h,followed by Huh-7,Hep G2(p<0.05).5 At the E : T ratio of 10 : 1,Annexin V-FITC/PI apoptosis kit and flow cytometer revealed that when killing the same target cells,apoptosis effect in M3 or M4 group was significantly obvious,followed by M1 or M2 group,M5 or M0 groups(p<0.05).However,the differences between M3 group and M4 group,between M1 group and M2 group,between M5 group and M0 group were non-significant(p>0.05).When the same effector cells were used for killing,the apoptosis effect was most obvious in MHCC97 h,followed by Huh-7,Hep G2(p<0.05).6 At the E : T ratio of 25 : 1,the results of ELISA displayed that when killing the same target cells,the secretion level of murine IFN-? in M3 or M4 group were significantly obvious,followed by M1 or M2 group,M5 or M0 groups(p<0.05).However,the differences between M3 group and M4 group,between M1 group and M2 group,between M5 group and M0 group were non-significant(p>0.05).When the same effector cells were used for killing,the secretion of murine IFN-? was most obvious in MHCC97 h,followed by Huh-7,Hep G2(p<0.05).Conclusions1 Broad-spectrum MAPs based on CTL epitopes of human m PGES1 are synthesized successfully.2 DCs derived from murine bone marrow are successfully obtained and cultured until mature,which conform to the characteristics of mature DCs.3 Murine DC vaccine loaded with four-branch MAPs of human m PGES1 CTL epitopes has specific killing effect on human liver cancer cells by secreting IFN-?,and its killing efficiency is positively correlated with the m PGES1 expression level of liver cancer cells.The killing effect of murine DC vaccine loaded with four-branch MAPs is more obvious than single-chain MAP.In addition,the killing effect is relevant to CTL dominant epitope(VTHAVRGVL)rather than the number of CTL epitopes.