Prediction And Identification Of Glioma-Associated Antigen MAGE-D4 T Cell Antigen Peptide

Objective:To predict the glioma-associated antigen MAGE-D4 HLA-A2 restricted T cell antigen peptide,and to analyze whether these antigen peptides can induce T cell with tumor killing effect after DC loading,and provide MAGE-D4 glioma immunotherapy Experimental basis.Methods:(1)Prediction of MAGE-D4 epitope peptide: HLA-A*0201-restricted MAGE-D4 T cell antigen peptide was analyzed using SYFPEITHI and BIMAS websites to screen for high-scoring candidate peptides;(2)Peptide-T2/HLA-A2 complex binding stability assay: synthesizing the selected antigen peptide,incubating the antigen peptide with T2 cells,and analyzing the affinity of the antigen peptide peptide to HLA-A2;(3)Screening and identification of HLA-A*0201 phenotype: DNA of glioma U87 cells was extracted and identified by HLA-A*0201 characteristic primers;peripheral blood mononuclear cells(PBMC)of healthy volunteers were isolated and flowed.The cytometer screened HLA-A2 healthy volunteers.(4)In vitro induction culture and identification of dendritic cells(DC):Peripheral blood PBMC of HLA-A* 0201 healthy volunteers were isolated,CD14+ cells were isolated from PBMC by magnetic bead sorting,and cytokines were used(rhGM-CSF,rhIL-4 and rTNF)were incubated with CD14+ cells to induce mature DCs and detected by flow cytometry by morphological observation,cell surface markers(CD83,CD80,CD1 a and HLA-DR)Identification.(5)Preparation of MAGE-D4 antigen peptide-loaded DC: DCs induced by cytokine induction to day 3 and day 5 were stimulated with MAGE-D4 antigen peptide,and culture was continued until day 8,and MAGE-D4 antigen peptide-loaded DC was collected;(6)Preparation of effector T cells: T cell magnetic bead sorting kit was used to isolate T cells in HLA-A* 0201 healthy volunteer PBMC,and T cells were loaded with MAGE-D4 epitope peptide DC(T cells: DC Incubate for 10:1),culture until day 4,incubate with MAGE-D4 antigen peptide-loaded DCs in the same ratio,and continue to culture for 4 days with IL-2,then,CCK-8 and ELISPOT were used to detect the proliferation of T cells and the frequency of secretion of T cells.(7)Effector T cell killing experiment: 10: 1,20: 1 effector cells: target cells were incubated,Lactate dehydrogenase(LDH)release assay was used to determine the killing effect of effector T cells on U87 cells.Results:(1)Six HLA-A*0201 restriction candidate peptides were predicted by the website: P1(SLLLVILGV),P2(LLQERANK),P3(CLPPPNVIL),P4(SLGPGLRIL),P5(ILSNEPWEL),P6(RLSLLLVIL).(2)Except for P1,the other five peptides have good affinity with HLA-A*0201 molecule(FI is greater than 1).(3)U87 cells were identified as HLA-A* 0201 phenotype by PCR,andHLA-A2 healthy volunteers were screened by flow cytometry.(4)CD14+ cells,after induction by cytokines,exhibit typical DC morphology and high expression of CD1 a,CD83,CD80 and HLA-DR molecules.(5)After incubation of T cells with MAGE-D4 antigen peptide-loaded DCs,CCK-8 assay showed T cell proliferation,but there was no statistical difference between the groups.IFN-? ELISPOT assay showed a significant increase in T cells secreting IFN-?,which was statistically different from the control group(P< 0.05).(6)LDH test results showed that when the effective target ratio was 20:1,the effector T of P2-DC+TC group,P3-DC+TC group,P4-DC+TC group and P5-DC+TC group was loaded.The killing efficiency of cells against U87 cells was significantly higher than that of unloaded peptides in DC+TC group(P<0.05).The killing effect of P3-DC +TC group,P4-DC+TC group and P5-DC+TC group was effective.The target ratio increased and increased(P<0.05).Conclusion:The dendritic cell vaccine sensitized with MAGE-D4 polypeptide(LLQERANK,CLPPPNVIL,ILSNEPWEL,RLSLLLVIL)can effectively induce CTL in vitro,resulting in strong anti-glioma cytotoxicity,suggesting that MAGE-D4 peptide vaccine has the potential for glioma immunotherapy.