title: SING: a novel strategy for identifying tumor-specific, CTL-recognized tumor antigens.
abstract: Traditional methods for identifying T cell-recognized tumor antigens (Ags) are laborious and time-consuming. In an attempt to simplify the procedure, a novel strategy, SING (SIgnal transduction molecule-mediated, NFAT-controlled, GFP expression) was established as a direct approach for cloning T cell-recognized tumor Ags. In the SING system, a mouse T cell line (BW5147) was transduced with a chimeric H-2Kb construct containing T cell-signaling domains and a green fluorescent protein (GFP) reporter gene under the transcriptional control of nuclear factor of activated T cells (NFAT). The resultant BW5147 cells were named BS cells. This cell line could "sense" TCR stimulation through the T cell-signaling domains after coculture with Ag-specific T cells and then become fluorescent (expressing green fluorescence protein, GFP+) in the presence of Ag peptides. The interaction between BS cells and Ag-specific T cells could be enhanced by addition of costimulatory signals. Currently, BS cells have been optimized to "sense" TCR stimulation after being pulsed with the relevant peptides at concentrations as low as 10(-9) M. Endogenous Ag-expressing BS cells could also become fluorescent after coculture with Ag-specific T cells. Our results provide a proof of principle for using the SING system to directly isolate Ag-expressing BS cells from BS cell repertoires, which are retrovirally transduced with tumor-derived cDNA libraries. Once tumor Ag-marked BS cells are identified, the sequences encoding tumor Ags could be easily retrieved by PCR amplification of the genomic DNA using vector-specific primers.
authors.affiliation: Gene Therapy Group, Department of Microbiology and Immunology, University of Arizona, Tucson, Arizona 85721, USA.
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
authors.forename: Xianghui
authors.initials: X
authors.lastname: He
authors.forename: Tom C
authors.initials: TC
authors.lastname: Tsang
authors.forename: David T
authors.initials: DT
authors.lastname: Harris
mesh_terms: D016475:3T3 Cells; D000818:Animals; D017951:Antigen Presentation / Q000187:drug effects; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods*; D018920:Coculture Techniques; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004527:Egg Proteins / Q000276:immunology; D005089:Exocytosis / Q000187:drug effects; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006183:H-2 Antigens / Q000276:immunology; D006825:Hybridomas / Q000473:pathology; D015759:Ionomycin / Q000494:pharmacology; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D016399:Lymphoma, T-Cell / Q000473:pathology; D018942:Macrolides / Q000494:pharmacology; D051379:Mice; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010047:Ovalbumin / Q000276:immunology; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014161:Transduction, Genetic
chemical_list: D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D004527:Egg Proteins; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D008164:Luminescent Proteins; D018942:Macrolides; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; C110366:OVA-8; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D015759:Ionomycin; C036978:concanamycin A; D010047:Ovalbumin; D013755:Tetradecanoylphorbol Acetate