title: Functional TCR retrieval from single antigen-specific human T cells reveals multiple novel epitopes.
abstract: The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-β chains directly from single antigen-specific CD8(+) or CD4(+) T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro-transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8(+) and CD4(+) T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes.
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Mainz, Germany. Translational Oncology at the University Medical Center, Johannes Gutenberg University, Mainz gGmbH, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Simon
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Mainz, Germany. Translational Oncology at the University Medical Center, Johannes Gutenberg University, Mainz gGmbH, Germany.
authors.forename: Tana A
authors.initials: TA
authors.lastname: Omokoko
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Mainz, Germany. Translational Oncology at the University Medical Center, Johannes Gutenberg University, Mainz gGmbH, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Breitkreuz
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Mainz, Germany. Translational Oncology at the University Medical Center, Johannes Gutenberg University, Mainz gGmbH, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Hebich
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Mainz, Germany. Translational Oncology at the University Medical Center, Johannes Gutenberg University, Mainz gGmbH, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kreiter
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Mainz, Germany. Translational Oncology at the University Medical Center, Johannes Gutenberg University, Mainz gGmbH, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Attig
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Mainz, Germany.
authors.forename: Abdo
authors.initials: A
authors.lastname: Konur
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Mainz, Germany.
authors.forename: Cedrik M
authors.initials: CM
authors.lastname: Britten
authors.affiliation: Translational Oncology at the University Medical Center, Johannes Gutenberg University, Mainz gGmbH, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Paret
authors.affiliation: Department of Pathology, Algemeen Stedelijk Ziekenhuis Aalst, Aalst, Belgium.
authors.forename: Karl
authors.initials: K
authors.lastname: Dhaene
authors.affiliation: Ganymed Pharmaceuticals AG, An der Goldgrube 12, Mainz, Germany.
authors.forename: Özlem
authors.initials: Ö
authors.lastname: Türeci
authors.affiliation: Division of Translational and Experimental Oncology, Department of Medicine III, Johannes Gutenberg University, Mainz, Germany. Translational Oncology at the University Medical Center, Johannes Gutenberg University, Mainz gGmbH, Germany. front-office@tron-mainz.de.
authors.forename: Ugur
authors.initials: U
authors.lastname: Sahin
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000276:immunology / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology; D051379:Mice; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism*; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000276:immunology
chemical_list: D000951:Antigens, Neoplasm; C104948:CTAG1B protein, human; D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D008565:Membrane Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; C446603:TPTE protein, human; D051059:PTEN Phosphohydrolase