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Induction of neoantigen-reactive T cells from healthy donors

Nature Protocols volume 14, pages 1926–1943 (2019)Cite this article

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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.

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Fig. 1: Schematic summary outlining the identification of neoepitope-reactive T cells in healthy donors.

Science Shaped

Fig. 2: Optimizing transfection of DCs and minigene design.
Fig. 3: Priming and expansion of antigen-specific T cells.
Fig. 4: Multiplexed functional analysis of T cell clones.

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Acknowledgements

We thank the Oslo University Hospital (OUH) flow cytometry core facility for excellent technical assistance. This work was supported by Stiftelsen Kristian Gerhard Jebsen (J.O. and T.N.S.), South-Eastern Regional Health Authority Norway, the Research Council of Norway, the Norwegian Cancer Society, the University of Oslo, Oslo University Hospital (all J.O.) and the Queen Wilhelmina Cancer Research Award (T.N.S.).

Author information

Author notes

  1. These authors contributed equally: Muhammad Ali, Zsofia Foldvari, Eirini Giannakopoulou.

Authors and Affiliations

  1. Department of Cancer Immunology, Oslo University Hospital Radiumhospitalet, Oslo, Norway

    Muhammad Ali, Zsofia Foldvari, Eirini Giannakopoulou, Maxi-Lu Böschen, Erlend Strønen, Weiwen Yang & Johanna Olweus

  2. K.G. Jebsen Center for Cancer Immunotherapy, Institute for Clinical Medicine, University of Oslo, Oslo, Norway

    Muhammad Ali, Zsofia Foldvari, Eirini Giannakopoulou, Maxi-Lu Böschen, Erlend Strønen, Weiwen Yang & Johanna Olweus

  3. Division of Molecular Oncology & Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, the Netherlands

    Mireille Toebes & Ton N. Schumacher

  4. Institute for Biomedical Informatics, University of Tübingen, Tübingen, Germany

    Benjamin Schubert & Oliver Kohlbacher

  5. Applied Bioinformatics, Department of Computer Science, University of Tübingen, Tübingen, Germany

    Benjamin Schubert & Oliver Kohlbacher

  6. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    Benjamin Schubert

  7. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Benjamin Schubert

  8. cBio Center, Dana-Farber Cancer Institute, Boston, MA, USA

    Benjamin Schubert

  9. Biomolecular Interactions, Max Planck Institute for Developmental Biology, Tübingen, Germany

    Oliver Kohlbacher

  10. Translational Bioinformatics, University Medical Center Tübingen, Tübingen, Germany

    Oliver Kohlbacher

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Contributions

M.A., Z.F., E.G., M.-L.B. and J.O. conceived and designed the experiments. M.A., Z.F. and E.G. performed the experiments and analyzed the data. B.S., O.K., W.Y. and M.T. designed and synthesized mRNA minigenes and pMHC multimers. M.A., Z.F., E.G., E.S., T.N.S. and J.O. wrote the manuscript. All authors reviewed and commented on the manuscript.

Corresponding author

Correspondence to Johanna Olweus.

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Competing interests

J.O. has a collaboration with Kite Pharma and is a member of the Scientific Advisory Board of Intellia Therapeutics. J.O is the inventor on the patent WO2015071763 (CTL peptide epitopes and antigen-specific T cells, methods for their discovery, and uses thereof). T.N.S. is a consultant for Adaptive Biotechnologies, AIMM Therapeutics, Allogene Therapeutics, Amgen, Merus, Neon Therapeutics and Scenic Biotech; is a recipient of grant/research support from MSD, Bristol-Myers Squibb and Merck KGaA; is a stockholder in AIMM Therapeutics, Allogene Therapeutics, Merus, Neogene Therapeutics and Neon Therapeutics; and is a venture partner at Third Rock Ventures.

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Journal peer review information: Nature Protocols thanks Timothy Chan and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Strønen, E. et al. Science 352, 1337–1341 (2016): http://science.sciencemag.org/content/352/6291/1337

Kumari, S. et al. Proc. Natl. Acad. Sci. USA 111, 403–408 (2014): https://www.pnas.org/content/111/1/403

Integrated supplementary information

Supplementary Figure 1

Flow cytometric characterization of the purity of naive and memory CD8+ T cells isolated from the same donor.

Supplementary Figure 2 Different ratios of DCs to naive CD8+ T cells.

M2-minigene-electroporated DCs were cocultured with T cells at DC:T cell ratios of 1:8, 1:4, 1:2 and 1:1 (n = 4 donors, 3 cultures/donor), and cultures were analyzed for the presence of neo-1, -2, -3, -4 and -5 pMHC-multimer-reactive populations on day 12. Each dot represents the percentage of pMHC-multimer-positive cells among CD8+ cells identified by flow cytometry.

Supplementary Figure 3 Comparison of the ability of M2 and M3 minigenes to induce T cell responses.

Blood from 20 different donors was used to initiate cultures stimulated with either M2 or M3 minigene-electroporated DCs to compare the induction of T cell responses against neo-1, -2, -3, -4 and -5 epitopes (n = 10 donors/minigene, 3 cultures/donor). Circles designate data points from M2-induced cultures, and triangles designate data points from M3-induced cultures. Data plotted as mean ± s.e.m. *P < 0.05.

Supplementary Figure 4 Multiplexed functional analysis of T cell clones.

Clones were labeled as described in the legend of Fig. 4. To the right, the CD107a/b degranulation response of 16 individual clones against target cells pulsed with 10 nM neo-3 peptide is shown. Numbers in the lower left corner of plots correspond to individual clones in the dot plot to the left.

Supplementary Figure 5 Gating strategy for the identification of pMHC-multimer-positive CD8+ T cells.

Lymphocytes were identified, and doublets and dead cells were excluded with the help of forward (FSC) and side scatter (SSC) gates and live/dead fixable near-IR dead cell staining. From the live cell gate, CD8+ T cells were gated and pMHC-multimer-reactive cells were identified as double positive for PE- and APC-conjugated pMHC multimers.

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Supplementary Information

Supplementary Figures 1–5, Supplementary Note, Supplementary Methods and Supplementary Table 1

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Ali, M., Foldvari, Z., Giannakopoulou, E. et al. Induction of neoantigen-reactive T cells from healthy donors. Nat Protoc 14, 1926–1943 (2019). https://doi.org/10.1038/s41596-019-0170-6

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