title: Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries.
abstract: The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.
authors.affiliation: Translational Research Program, Benaroya Research Institute, Seattle, Washington, United States of America.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hondowicz
authors.forename: Katharine V
authors.initials: KV
authors.lastname: Schwedhelm
authors.forename: Arnold
authors.initials: A
authors.lastname: Kas
authors.forename: Michael A
authors.initials: MA
authors.lastname: Tasch
authors.forename: Crystal
authors.initials: C
authors.lastname: Rawlings
authors.forename: Nirasha
authors.initials: N
authors.lastname: Ramchurren
authors.forename: Martin
authors.initials: M
authors.lastname: McIntosh
authors.forename: Leonard A
authors.initials: LA
authors.lastname: D'Amico
authors.forename: Srinath
authors.initials: S
authors.lastname: Sanda
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Standifer
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
authors.forename: Brad
authors.initials: B
authors.lastname: Stone
mesh_terms: D000595:Amino Acid Sequence; D000941:Antigens / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D016022:Case-Control Studies; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000276:immunology; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D058501:Enzyme-Linked Immunospot Assay; D000071858:Epithelial Cell Adhesion Molecule; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D015723:Gene Library; D006680:HLA Antigens / Q000276:immunology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000941:Antigens; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D000939:Epitopes; C094538:GLIPR1 protein, human; D006680:HLA Antigens; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins