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

Induction of neoantigen-reactive T cells from healthy donors.

Author information

1
Department of Cancer Immunology, Oslo University Hospital Radiumhospitalet, Oslo, Norway.
2
K.G. Jebsen Center for Cancer Immunotherapy, Institute for Clinical Medicine, University of Oslo, Oslo, Norway.
3
Division of Molecular Oncology & Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, the Netherlands.
4
Institute for Biomedical Informatics, University of Tübingen, Tübingen, Germany.
5
Applied Bioinformatics, Department of Computer Science, University of Tübingen, Tübingen, Germany.
6
Department of Systems Biology, Harvard Medical School, Boston, MA, USA.
7
Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
8
cBio Center, Dana-Farber Cancer Institute, Boston, MA, USA.
9
Biomolecular Interactions, Max Planck Institute for Developmental Biology, Tübingen, Germany.
10
Translational Bioinformatics, University Medical Center Tübingen, Tübingen, Germany.
11
Department of Cancer Immunology, Oslo University Hospital Radiumhospitalet, Oslo, Norway. johanna.olweus@medisin.uio.no.
12
K.G. Jebsen Center for Cancer Immunotherapy, Institute for Clinical Medicine, University of Oslo, Oslo, Norway. johanna.olweus@medisin.uio.no.

Abstract

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.

PMID:
31101906
DOI:
10.1038/s41596-019-0170-6
[Indexed for MEDLINE]

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