Prediction Of HLA-A~*0201 Restricted Cytotoxic T Lymphocyte Epitope From Tumor-associated Antigen MUC4 And Experimental Study For The Identification Of Epitope In Vitro

Background Adopted as one of the therapeutic methods for malignant tumors, tumor vaccines are now playing an important role in this field,especially for the epitope-based peptide/DNA vaccines.The primary step for the design of specific tumor peptide vaccine is to predict and identify the antigen-specific CTL epitope.Dendritic cell(DC)is the most powerful antigen presenting cells (APC)of human kind,The effective cellular immune response induced by tumor antigen modified DC provide the enormous application perspective for many kinds of malignant tumors.MUC4 is one potential oncogene located at 3q29;its expression product mucin4 has essential biological functions for the development,metastases and invasion of many common tumors such as breast cancer,lung cancer,colon cancer & pancreatic cancer.Screening of active antigen epitope of MUC4 and divise of relative peptide or DC vaccine would be of great help for the study of tumor vaccine in MUC4 positive neoplasms.Objective To predict and synthesize the epitope peptide of tumor associated antigen MUC4 HLA-A^* 0201 restricted cytotoxic T lymphocyte(CTL).To establish the successful technique of culturing the mature DC originated from peripheral blood mononuclear cells(PBMCs).To explore the immune response to MUC4-induced specific CTL epitope-pulsed DC and validate the possible epitopes of MUC4.Methods(1)Combination of two epitope prediction databases(SYFPEITHI and ProPred-â… ),the possible epitopes were predicted,artificially synthesized & purified.The predicted peptides were examined for their affinity to HLA-A^*0201 molecule using T2 cells.(2)DCs were induced & cultured from the HLA-A^*0201 positive healthy volunteers' PBMCs combined with cytokines of GM-CSF,IL-4 and TNF-α.DCs' phenotype was identified by flow cytometry.(3)CD8⁺ T cells were isolated and purified through MACS (magnetic activated cell sorting).Mature DCs were pulsed with each of the five synthesized peptides & positive control peptide CML28_173-181.Autologous CD8⁺ T cells from the HLA-A^*0201 donors were stimulated with the peptide-pulsed DCs to produce CTLs.CTL activity was assessed by LDH release assay & standard ⁵¹Cr-releasing assay.The IFN-γreleased was detected by enzyme-linked immuno spot assay(ELISPOT).Results(1)With comprehensive analysis,5 candidate HLA-A^*0201 restricted epitopes were predicted and synthesized:P01202(¹¹²⁵LLLGVGTFV¹¹³³), P01203(⁴⁹⁸WLLEPHDAI⁵⁰⁶),P01204(¹¹²⁶LLGVGTFVV¹¹³⁴),P01205 (¹¹¹⁹GALGGLLLL¹¹²⁷)å’ŒP01206(⁹⁴⁴GTLDMRAFL⁹⁵²).The peptide-MHC complex affinity was evaluated using T2 cells.The results indicated that affinity of P01202,P01204 to the MHC was stronger than other 3 predicted peptides.(2) Mature DCs could be induced from healthy donors' PBMC with combination use of cytokines including GM-CSF,IL-4 and TNF-α.(3)CTL induced by P01204 pulsed DC presented the strongest cytotoxic effect on HCT-116(colon cancer cell,MUC4⁺,HLA-A2⁺)and T2 cells pulsed with peptide P01204 at various E:T ratios of 5:1,10:1,20:1 than the other 4 peptide-induced CTLs.The P01204-induced CTL had significant specific lysis(%)on HCT-116(MUC4⁺, HLA-A2⁺)than that of MCF-7(breast cancer cell,HLA-A2⁺,MUC4)and BXPC-3(pancreatic cancer cell,HLA-A2^-,MUC4⁺).The P01204-induced CTL had significant effect on HCT-116 cells with anti-HLA-A24 antibody pretreated than on anti-HLA-A^*0201 pretreated HCT- 116 cells.Conclusion(1)The results suggest that bioinformatics methods could predict the MUC4 HLA-A^*0201 restricted CTL epitopes effectively.MHC-peptide binding assay could provide the information on the identification of the possible epitopes.(2)Sufficient mature DC could be induced & cultured in vitro from the collected PBMCs with combined use of cytokines GM-CSF,IL-4 & TNF-α.(3) The specific CTLs induced by MUC4 antigen epitope P01204 (¹¹²⁶LLGVGTFVV¹¹³⁴)definitely would generate the cytotoxic function on tumor cells espressed MUC4 and HLA-A^*0201.The peptide P01204 (¹¹²⁶LLGVGTFVV¹¹³⁴)is thought be one of the suitable epitope of MUC4 antigen.