Identification Of Point-mutated Survivin Antigen Peptide-specific TCR Gene With HLA-A2Restricted And Verification Of Its Anti-tumor Function

Objective: To screen the TCR gene which specifically recognizeHLA-A2-restricted survivin point-mutated peptide by stimulate T lymphocytes withHLA-A2-restricted. Then the TCR gene dual expression vector was constructed andrecombinant chimeric adenovirus were packaged for TCR-transfection into Tlymphocytes.to research the antitumor function of transgenic T cells. This study mayprovide foundation for new method of point-mutated tumor associated antigen T celladoptive tumor treatment and new targets of gene therapeutic agents.Method:1.PBMC were isolated from peripheral blood of HLA-A2+healthyindividuals and were induced into DC in vitro. Mature DC cells were loaded withHLA-A2-restricted survivin point-mutated peptide and co-cultured with Tlymphocytes. Control group DC didn’t load with HLA-A2-restricted survivinpoint-mutated peptide but co-cultured with T lymphocytes.2. Analysis of the T cell antigen receptor (TCR) Vα and Vβ subfamilyexpression pattern in the experimental group and the control group were detected byflow cytometry (FCM) and GeXP Genetic Analysis System.3. The total RNA was extracted from the experimental group. The preferredmonoclonal/oligoclonal TCRVα and Vβ genes were cloned and the correctsequences were subcloned into the vector to construct shuttle plasmidp315-TCRVα-IRES-Vβ.4.The adenovirus backbone plasmid and shuttle plasmid p315-TCRVα-IRES-Vβwere cotransfered into HEK293cells with Lipofectamine2000,and recombinant TCR Vα and Vβ adenovirus was generated. The exogenous targetgene was analyzed by PCR using viral genomic DNA as template and the expressionof target protein in PBMC was detected by flow cytometry. The recombinantadenovirus was propagated in HEK293cells,and isolated by sonication. The titter ofviruses were detected by TCID50method.5. PBMC was infected by recombinant TCR Vα and Vβ adenovirus andcocultured with different tumor cells (T2cell loaded with Sur79M2-HLA-A2+、Survivin+, HepG2-HLA-A2+、 Survivin+, Mcf-7-HLA-A2+、 Survivin+,7402-HLA-A2+、Survivin-, NCI-H1299-HLA-A2-、Survivin-) for36h afterinfection. The killing effects and the secretion of IFN-γ on tumor cells were separatelyanalyzed by MTT and enzyme linked immunosorbent assay(Elisa) after12,24and48hours.Results: The FCM results showed that TCRVβ7.1of PBMC that were stimulatedby Sur79M2were significantly higher than that in control group. The Gexp resultshowed a monoclonal/oligoclonal proliferation in TCR Vα4and Vβ7genes in theexperimental group. The sequencing results and analysis of CDR3sequence showedthat TCR Vα4and Vβ7genes appeared the clonal proliferation phenomenon. ThePCR and flow cytometry result showed that TCR Vα4and Vβ7target genes weresuccessfully integrated into the virus genome and could expressed in host cells. Thekilling assay showed that the lysis efficiency of PBMC infected with recombinantTCR adenovirus against different tumor cells: T2cell loaded with Sur79M2（HLA-A2+、Survivin+）> HepG2（HLA-A2+、Survivin+）> Mcf-7（HLA-A2+、Survivin+）>7402（HLA-A2-、Survivin+）> NCI-H1299（HLA-A2-、Survivin-）,The results of IFN-γ secretion almost were consistent with the MTT lysis efficiency.Conclusion:Through the methods of T lymphocytes were stimulated with cellsloaded with HLA-A2-restricted survivin point-mutated peptide in vitro, wesuccessfully screened out the HLA-A2-restricted survivin point-mutated peptidespecificic TCRVα4and Vβ7genes. The TCRVα4and Vβ7genes were cloned andconstructed into recombinant adenovirus. Afer transfection of PBMC, killing assays confirmed that the TCRVα4and Vβ7genes can effectively recognize and kill T2cellsloaded with HLA-A2-restricted survivin point-mutated peptides. The TCRgene-modified PBMC also could have a certain activation kill function against tumorcell lines expressed wild-type survivin antigens.