title: MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy.
abstract: Adoptive therapy using T cells redirected to target tumor- or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs), we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN). Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25%) and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3(+) T cells) without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes.
authors: Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.
authors: Georg
authors: G
authors: Dössinger
authors: Mario
authors: M
authors: Bunse
authors: Jeannette
authors: J
authors: Bet
authors: Julia
authors: J
authors: Albrecht
authors: Paulina J
authors: PJ
authors: Paszkiewicz
authors: Bianca
authors: B
authors: Weißbrich
authors: Isabell
authors: I
authors: Schiedewitz
authors: Lynette
authors: L
authors: Henkel
authors: Matthias
authors: M
authors: Schiemann
authors: Michael
authors: M
authors: Neuenhahn
authors: Wolfgang
authors: W
authors: Uckert
authors: Dirk H
authors: DH
authors: Busch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology; D018929:Cell Culture Techniques; D003587:Cytomegalovirus / Q000276:immunology; D000939:Epitopes; D018014:Gene Transfer Techniques; D057809:HEK293 Cells; D006649:Histocompatibility Antigens / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007167:Immunotherapy; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000627:therapeutic use*; D016693:Receptors, Antigen, T-Cell, alpha-beta; D020539:Sequence Analysis, Protein; D059010:Single-Cell Analysis; D013045:Species Specificity; D019076:Transgenes
chemical_list: D000951:Antigens, Neoplasm; D000939:Epitopes; D006649:Histocompatibility Antigens; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta
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