Induction of neoantigen-reactive T cells from healthy donors

Nat Protoc. 2019 Jun;14(6):1926-1943. doi: 10.1038/s41596-019-0170-6. Epub 2019 May 17.


The identification of immunogenic neoantigens and their cognate T cells represents the most crucial and rate-limiting steps in the development of personalized cancer immunotherapies that are based on vaccination or on infusion of T cell receptor (TCR)-engineered T cells. Recent advances in deep-sequencing technologies and in silico prediction algorithms have allowed rapid identification of candidate neoepitopes. However, large-scale validation of putative neoepitopes and the isolation of reactive T cells are challenging because of the limited availablity of patient material and the low frequencies of neoepitope-specific T cells. Here we describe a standardized protocol for the induction of neoepitope-reactive T cells from healthy donor T cell repertoires, unaffected by the potentially immunosuppressive environment of the tumor-bearing host. Monocyte-derived dendritic cells (DCs) transfected with mRNA encoding candidate neoepitopes are used to prime autologous naive CD8+ T cells. Antigen-specific T cells that recognize endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for the identification of TCR sequences, after an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 d, with an additional 2-4 weeks required for clonal expansion and downstream functional characterization. Identified neoepitopes and corresponding TCRs provide candidates for use in vaccination and TCR-based cancer immunotherapies, and datasets generated by this technology should be useful for improving algorithms to predict immunogenic neoantigens.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD8-Positive T-Lymphocytes / immunology*
  • Cells, Cultured
  • Dendritic Cells / immunology*
  • Dendritic Cells / metabolism
  • Electroporation / methods
  • Epitopes / genetics
  • Epitopes / immunology*
  • Humans
  • Immunotherapy / methods
  • Neoplasms / immunology*
  • Neoplasms / therapy
  • RNA, Messenger / genetics
  • Receptors, Antigen, T-Cell / analysis
  • Receptors, Antigen, T-Cell / immunology
  • Transfection / methods


  • Epitopes
  • RNA, Messenger
  • Receptors, Antigen, T-Cell