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

PLoS One. 2013 Apr 26;8(4):e61384. doi: 10.1371/journal.pone.0061384. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antigens, Neoplasm / immunology
  • Cell Culture Techniques
  • Cytomegalovirus / immunology
  • Epitopes
  • Gene Transfer Techniques
  • HEK293 Cells
  • Histocompatibility Antigens / chemistry*
  • Histocompatibility Antigens / metabolism
  • Humans
  • Immunotherapy*
  • Jurkat Cells
  • Mice
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Protein Multimerization*
  • 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
  • Transgenes

Substances

  • Antigens, Neoplasm
  • Epitopes
  • Histocompatibility Antigens
  • Receptors, Antigen, T-Cell
  • Receptors, Antigen, T-Cell, alpha-beta

Grant support

This work was supported by the SFB (Sonderforschungsbereich/Collaborative Research Centre) TR36 (TP-B10/13, TP-Z1) and the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Immunotherapy of Cancer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.