As a new generation of treatment, tumor immunotherapy targeting tumor-associated antigens (TAA) has attracted widespread attention. The survivin antigen belongs to TAA. It is a key inhibitor of apoptosis and a key regulator of cell cycle progression; furthermore, it may be a candidate target for tumor therapy. In addition, studies have confirmed that granulocyte-macrophage colony-stimulating factor (GM-CSF) and CCL17 significantly affect local anti-tumor immunity in the tumor microenvironment. The mouse survivin gene was screened by BIMAS and SYFPEITHI to obtain the highest scored mouse survivin epitope peptide, which was synthesized into a peptide vaccine to immunize normal mice. Subsequently, spleen lymphocytes were isolated to induce survivin-specific cytotoxic T lymphocytes (CTL). Next, genetic engineering was used to construct the B16F10 cell line that stably expressed CCL17 and GM-CSF genes. A mouse melanoma model was used to observe the effects of the combination of the three on tumor volume and tumor weight. In-vitro survivin-specific CTL combined with CCL17 gene had a stronger inhibitory effect on B16F10 cells, while combined GM-CSF gene did not enhance the inhibitory effect of CTL on B16F10 cells. In-vivo experiments demonstrated that survivin-specific CTL combined with GM-CSF and CCL17 genes can inhibit the growth of mouse melanoma. HE staining and immunohistochemistry showed that the tumor had more necrotic cells and more infiltrating lymphocytes. The results showed that survivin-specific CTL combined with CCL17 and GM-CSF genes could inhibit tumor growth better.
The aim of the present study was to use The Cancer Genome Atlas (TCGA) database to identify tumor neoantigens, combined with a bioinformatics analysis to design and analyze antigen epitope peptides. Epitopes were screened using immunogenicity tests to identify the ideal epitope peptides to target tumor neoantigens, which can specifically activate the immune response of T cells. The high-frequency mutation loci (top 10) of colorectal, lung and liver cancer genes were screened using TCGA database. The antigenic epitope peptides with high affinity for major histocompatibility complex molecules were selected and synthesized using computer prediction algorithms, and were subsequently detected using flow cytometry. The cytotoxicity of specific cytotoxic T lymphocytes (CTLs) on peptide-loaded T2 cells was initially verified using interferon IFN-γ detection and a calcein-acetoxymethyl (Cal-AM) release assay. Tumor cell lines expressing point mutations in KRAS, TP53 and CTNNB1 genes were constructed respectively, and the cytotoxicity of peptide-induced specific CTLs on wild-type and mutant tumor cells was verified using a Cal-AM release assay and carboxyfluorescein succinimidyl ester-propidium iodide staining. The high-frequency gene mutation loci of KRAS proto-oncogene (KRAS) G12V, tumor protein p53 (TP53) R158L and catenin β1 (CTNNB1) K335I were identified in TCGA database. A total of 3 groups of wild-type and mutant peptides were screened using a peptide prediction algorithm. The CTNNB1 group had a strong affinity for the human leukocyte antigen-A2 molecule, as determined using flow cytometry. The IFN-γ secretion of specific CTLs in the CTNNB1 group was the highest, followed by the TP53 and the KRAS groups. The killing rate of mutant peptide-induced specific CTLs on peptide-loaded T2 cells in the CTNNB1 group was higher compared with that observed in the other groups. The killing rate of specific CTLs induced by mutant peptides present on tumor cells was higher compared with that induced by wild-type peptides. However, when compared with the TP53 and KRAS groups, specific CTLs induced by mutant peptides in the CTNNB1 group had more potent cytotoxicity towards mutant and wild-type tumor cells. In conclusion, point mutant tumor neoantigens screened in the three groups improved the cytotoxicity of specific T cells, and the mutant peptides in the CTNNB1 group were more prominent, indicating that they may activate the cellular immune response more readily.
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