Functional TCR retrieval from single antigen-specific human T cells reveals multiple novel epitopes

Cancer Immunol Res. 2014 Dec;2(12):1230-44. doi: 10.1158/2326-6066.CIR-14-0108. Epub 2014 Sep 22.


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.

MeSH terms

  • Animals
  • Antigens, Neoplasm / immunology
  • Cell Line
  • Cloning, Molecular
  • Epitopes, T-Lymphocyte / immunology*
  • Histocompatibility Antigens Class I / immunology
  • Histocompatibility Antigens Class I / metabolism
  • Histocompatibility Antigens Class II / immunology
  • Histocompatibility Antigens Class II / metabolism
  • Humans
  • Membrane Proteins / genetics
  • Membrane Proteins / immunology
  • Mice
  • PTEN Phosphohydrolase / genetics
  • PTEN Phosphohydrolase / immunology
  • Phosphoproteins / immunology
  • Protein Binding
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / metabolism*
  • T-Lymphocyte Subsets / immunology*
  • T-Lymphocyte Subsets / metabolism*
  • Viral Matrix Proteins / immunology


  • Antigens, Neoplasm
  • CTAG1B protein, human
  • Epitopes, T-Lymphocyte
  • Histocompatibility Antigens Class I
  • Histocompatibility Antigens Class II
  • Membrane Proteins
  • Phosphoproteins
  • Receptors, Antigen, T-Cell
  • Viral Matrix Proteins
  • cytomegalovirus matrix protein 65kDa
  • TPTE protein, human
  • PTEN Phosphohydrolase