In vivo immunogenic characteristics of cytotoxic T lymphocyte epitopes from pancreatic tumor antigen MUC4 / 中国病理生理杂志

AIM:Peptide vaccines have been conceived as promising therapies for tumor-inflicted patients due to their easy production and capability of inducing specific immune response required for defending the tumor.During our previous research,4 HLA-A2-restricted peptides had been identified as immunogenic in vivo.In this study, we aimed to testify the in vivo immunogenicity of the 4 peptides.METHODS: BALB/c mice were vaccinated with HLA-A2 restricted peptides emulsified in incomplete Freund's adjuvant(IFA)subcutaneously in combination with the epitope at the adjacent location.After the 3rd peptide vaccination for 10 d,the peptide-specific immune response was evaluated by ELISPOT and ELISA.The ability to induce T cell response was investigated by using cytotoxicity assay in vivo and the presence of pep-tide-specific CD8+T cells capable of recognizing the MHC-peptide was detected by flow cytometry.RESULTS: Among the 4 candidate HLA-A2 restricted peptides,the immune response elicited by P2004-1Y9V was superior to that of the other 3 peptides.The CTLs induced by P2004 and P2004-1Y9V lysed CAPAN-2 cells.P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell lines of CAPAN-2 than the native peptide-specific CTLs.Intracellu-lar cytokine staining assay indicated the presence of P 2004-1Y9V specific CD8 +T cells in the P2004-1Y9V vaccinated mice.CONCLUSION:P2004-1Y9V is the most immunogenic peptide in vivo, and can be explored as potential tumor peptide vaccine in the future clinical study.

HLA-A2-restricted point-mutated Survivin peptide elicited specific cytotoxic T lymphocyte reaction against hepatocarcinoma / 中华微生物学和免疫学杂志

Objective To investigate the anti-hepatocarcinoma(anti-HCC) function of HLA-A2-restricted point-mutated Survivin peptide induced CTLs.Methods The HLA-A2-restricted Survivin nonapeptides were evaluated using bioinformatics software.The binding affinity of Survivin peptide to HLA-A2 molecular was determined with flow cytometry analysis.After peptide-induced CTLs were generated in vitro,flow cytometry and ELISA were performed to detect the levels of IFN-γ,which were secreted by reactive CTLs.Peptide-induced CTLs were co-cultured with hepatoma cell lines HepG2 and BEL-7402.The rates of tumor cells lysis were assayed using CytoTox 96(R) and the morphological changes of tumor cells were observed with inverted phase contrast microscope.Results Point-mutated Survivin nonapeptide Sur79M2 (KMSSGCAFL) was filtered out,which was shown higher scores compared with the wild-type peptide Sur79.Consistent with the results of software analysis,Sur79M2 showed higher binding ability in T2 binding assays.At the same time,Sur79M2-induced CTLs could release a large number of IFN-γ after incubated with target cells rather than Sur79.When co-cultured with HCC cell lines HepG2 and BEL-7402,Sur79M2-induced CTLs effectively lysis HepG2 on HLA-A2-restricted manner without killing effect on BEL-7402 that do not express HLA-A2 molecules.Conclusion Sur79M2 could elicited specific cytotoxic T lymphocytes in vitro,which were able to specifically kill HCC cell lines on HLA-A2-restricted manner.

Selection and identification of HLA-A2-restricted CTL epitopes within heparanase / 中华微生物学和免疫学杂志

Objective To select and identify human leukocyte antigen(HLA)-A2-restricted T-cell epitope within heparanase(Hpa),and to provide aeademic bases for peptide-based immunotherapy with malignant tumor.Methods The Hpa sequence was scanned for prediction of the immunogenic peptides-based CTL epitopes using the HLA-binding prediction software SYFPEITHI and BIMAS.Ten the affinity of candidate epitopes to HLA-A2 was evaluated by T2 binding test.Acfivation of T cell was assessed by ELIS-POT and cytotoxictiy by lactate dehydrogenase(LDH)release assay.Results of the six predicted nona-peptides,Hpa(310-318,FLNPDVLDI)showed high affinity of binding to HLA-A2 and led to IFN-γ secre-tion in vitro.Furthermore,Hpa(310-318) FLNPDVILDI could induce PBMC from a HLA-A2 positive healthy donor to lyse specifically HCC-LM6 and SW-480 cells which expressing both Hpa and HLA-A2.Conclusion The nona-peptide Hpa(310-318)FLNPDVLDI may be all HLA-A2-restricted CTL epitope.which is capable of inducing Hpa-specific CTL in vitro.