MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy

• Published in: PloS one Vol. 8; no. 4; p. e61384
• Main Authors: Dössinger, Georg, Bunse, Mario, Bet, Jeannette, Albrecht, Julia, Paszkiewicz, Paulina J, Weißbrich, Bianca, Schiedewitz, Isabell, Henkel, Lynette, Schiemann, Matthias, Neuenhahn, Michael, Uckert, Wolfgang, Busch, Dirk H
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 26.04.2013; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Animals; Antigen (tumor-associated); Antigens; Antigens, Neoplasm - immunology; Binding sites; Biology; CD3 antigen; Cell culture; Cell Culture Techniques; Cloning; Cooperation; Cytomegalovirus; Cytomegalovirus - immunology; Epitopes; ErbB-2 protein; Gene expression; Gene Transfer Techniques; HEK293 Cells; Histocompatibility Antigens - chemistry; Histocompatibility Antigens - metabolism; Humans; Hygiene; Immunology; Immunotherapy; Infections; Jurkat Cells; Leukemia; Lymphocytes; Lymphocytes T; Major histocompatibility complex; Medicine; Mice; Molecular Sequence Data; Multiple sclerosis; Polymerase Chain Reaction; Process controls; Protein Multimerization; Receptors; Receptors, Antigen, T-Cell - chemistry; Receptors, Antigen, T-Cell - immunology; Receptors, Antigen, T-Cell - isolation & purification; Receptors, Antigen, T-Cell - therapeutic use; Receptors, Antigen, T-Cell, alpha-beta; Sequence Analysis, Protein; Single-Cell Analysis; Species Specificity; T cell receptors; T-cell receptor; Transgenes; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0061384

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.