Induction Of Specific Anti-tumor Immune Responses Based On Mouse Survivin Antigen MHC Class Ⅰ And MHC Class Ⅱ Epitope Peptides

Objective: Induction of Survivin antigen MHC class-Ⅰ and MHC class-ⅠI antigen epitope peptides in lymphocytes and investigation of the functional characteristics of MHC class-Ⅰ and MHC class-ⅠI epitope peptide-induced lymphocytes in the anti-tumor immune response.Methods: 1.The 15 amino acid MHC-Ⅱ class epitopes of mouse Survivin antigen were screened using Net MHCIIpan 4.0 and SYFPEITHI,two online prediction systems for predicting T-cell epitopes.The screened MHC-Ⅱ class epitope peptides were synthesized to make a peptide vaccine and immunized in C57BL/6 wild-type mice,whose spleen lymphocytes were isolated.T lymphocytes were induced with Survivin antigen MHC class-Ⅰ and MHC class-ⅠI antigen epitope peptides.Detection of Survivin antigen MHC class-Ⅰ and MHC-Ⅱ antigen epitope peptide induced T lymphocytes on mouse melanoma B16F10 cells using Flow cytometry and Calcein assay.Expression of granzyme B and perforin in Survivin antigen MHC class-Ⅰ and MHC class-ⅠI antigen epitope peptide induced lymphocytes co-cultured with B16F10 cells was detected using Western Blot.The secretion of IFN-γ and IL-10 in Survivin antigen MHC class-Ⅰ and MHC class-ⅠI antigenic epitope peptides-induced lymphocytes was detected by ELISA.2.Epitope cassettes targeting the MHC class-Ⅰ and MHC class-ⅠI epitope genes of the mouse Survivin antigen were designed.Mouse Survivin antigen epitope gene vaccines containing MHC class-Ⅰ and MHC class-ⅠI epitopes of Survivin antigen were constructed by genetic engineering method,respectively.3.Mice were rerouted with B16F10 melanoma cells to establish a rerouted mouse model.At different time points,gene vaccines with MHC class-Ⅰ and MHC class-ⅠI epitopes of Survivin antigen were wrapped in liposomes and injected into the mice through the tail vein.Tumor growth changes were observed,tumor volume size and weight were measured,local lymphocyte infiltration of tumor was detected by HE staining and IHC,and the degree of depletion of local infiltrating CD8+ T cells was detected by QPCR.Results:In vitro cytological assay,Survivin antigen MHC class-Ⅰ and MHC class-ⅠI epitope peptides induced a killing effect of T lymphocytes on mouse melanoma B16F10 cell line and increased expression of granzyme B and perforin compared to the control group.Killing is more effective when Survivin antigen MHC class-Ⅰ and MHC class-ⅠI epitope peptide induced T lymphocytes act together.The high expression of IFN-γ and low expression of IL-10 in the MHC-Ⅱ class epitope peptide induced group indicated that Survivin antigen MHC-Ⅱ class epitope peptide induced T lymphocytes mainly in Th1 class cells.In vivo animal studies,Survivin antigen MHC class I and MHC-Ⅱ epitope gene vaccines are effective in inhibiting tumor growth.Compared to the early and late treatment groups,the tumor growth in the tumor vaccine prevention group was considerably slower and the treatment effect was more significant.Tumor growth was effectively controlled regardless of whether early or late treatment was administered.The results of HE staining and IHC showed that there were more localized necrotic cells and infiltration of lymphocytes in the experimental group.The expression of the surface inhibitory receptors PD-1,Tim-3,and 2B4 on CD8+ T cells with local tumor infiltration was decreased during the combined action of MHC class-Ⅰ and MHC class-ⅠI epitope gene vaccines.This suggests that the depletion of tumor locally infiltrating T cells may be reduced when MHC class-Ⅰ and MHC class-ⅠI epitope antigens act in combination.Conclusions: Mouse Survivin antigen MHC class-Ⅰ and MHC-Ⅱ epitope peptide-induced T lymphocytes have a certain killing function against B16F10 cells in vitro.The gene vaccine based on mouse Survivin antigen MHC class-Ⅰ and MHC-Ⅱ epitopes can activate antigen-specific T cell responses,thus exerting anti-tumor effects and effectively controlling tumorigenesis in mice.