Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries

• Published in: PloS one 2012-01, Vol.7 (1), p.e29949-e29949
• Main Authors: Hondowicz, Brian D, Schwedhelm, Katharine V, Kas, Arnold, Tasch, Michael A, Rawlings, Crystal, Ramchurren, Nirasha, McIntosh, Martin, D'Amico, Leonard A, Sanda, Srinath, Standifer, Nathan E, Shendure, Jay, Stone, Brad
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antigen-presenting cells; Antigens; Antigens - immunology; Antigens, Neoplasm - chemistry; Antigens, Neoplasm - immunology; Autoantigens; Autografts; Autoimmune diseases; Autoimmunity; B cells; Biology; Cancer; Case-Control Studies; CD8 antigen; CD8-Positive T-Lymphocytes - immunology; Cell Adhesion Molecules - chemistry; Cell Adhesion Molecules - immunology; Consortia; Deoxyribonucleic acid; Diabetes; Diabetes mellitus; Diabetes Mellitus, Type 1 - immunology; DNA; Enzyme-linked immunosorbent assay; Enzyme-Linked Immunospot Assay; Epithelial Cell Adhesion Molecule; Epitopes - chemistry; Epitopes - immunology; Error correction & detection; Gene Library; Genes; Genomes; High-throughput screening; High-Throughput Screening Assays - methods; HLA Antigens - immunology; Human subjects; Humans; Immune system; Immunology; Infectious diseases; Libraries; Lymphocytes; Lymphocytes T; Medical research; Medicine; Molecular Sequence Data; mRNA; Neoplasm Proteins - chemistry; Neoplasm Proteins - immunology; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - immunology; Optimization; Pancreas; Peptides; Peripheral blood; Population; Protein Binding; Researchers; RNA; Screening; T cell receptors; T cells; T-Lymphocytes - immunology; Transgenic animals; Vaccines
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0029949

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.