title: Identification of peptide-specific TCR genes by in vitro peptide stimulation and CDR3 length polymorphism analysis.
abstract: Identification of TCR genes specific for tumor-associated antigens (TAAs) is necessary for TCR gene modification of T cells, which is applied in anti-tumor adoptive T cell therapy (ACT). The usual identification methods are based on isolating single peptide-responding T cells and cloning the TCR gene by in vitro expansion or by single-cell RT-PCR. However, the long and exacting in vitro culture period and demanding operational requirements restrict the application of these methods. Immunoscope is an effective tool that profiles a repertoire of TCRs and identifies significantly expanded clones through CDR3 length analysis. In this study, a survivin-derived mutant peptide optimized for HLA-A2 binding was selected to load DCs and activate T cells. The monoclonal expansion of TCRA and TCRB genes was separately identified by Immunoscope analysis and following sequence identification, the properly paired TCR genes were transferred into T cells. Peptide recognition and cytotoxicity assays indicated that TCR-modified PBMCs could respond to both the mutant and wild type peptides and lyse target cells. These results show that combining Immunoscope with in vitro peptide stimulation provides an alternative and superior method for identifying specific TCR genes, which represents a significant advance for the application of TCR gene-modified T cells.
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Shao
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China.
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Lin
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Southern Medical University, Guangzhou, China.
authors.forename: Teng
authors.initials: T
authors.lastname: Wang
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China.
authors.forename: Yusheng
authors.initials: Y
authors.lastname: Ou
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China.
authors.forename: Han
authors.initials: H
authors.lastname: Shen
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China.
authors.forename: Changli
authors.initials: C
authors.lastname: Tao
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China.
authors.forename: Fenglin
authors.initials: F
authors.lastname: Wu
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China.
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Zhang
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China.
authors.forename: Huaben
authors.initials: H
authors.lastname: Bo
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China; Guangdong Province Key Laboratory for Biotechnology Drug Candidate, Guangzhou, China; Southern Medical University, Guangzhou, China. Electronic address: shulhuang@sina.com.
authors.forename: Shulin
authors.initials: S
authors.lastname: Huang
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D002999:Clone Cells; D018920:Coculture Techniques; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D019672:Genes, T-Cell Receptor / Q000276:immunology*; D057809:HEK293 Cells; D015789:HLA-A2 Antigen / Q000276:immunology / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009369:Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D010455:Peptides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011110:Polymorphism, Genetic; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D000077022:Survivin; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014162:Transfection
chemical_list: D000951:Antigens, Neoplasm; C107489:BIRC5 protein, human; D022801:Complementarity Determining Regions; D015789:HLA-A2 Antigen; D051034:Inhibitor of Apoptosis Proteins; D010455:Peptides; D000077022:Survivin
keywords: Immunoscope; In vitro stimulation; Monoclonal expansion; Mutated peptide; TCR