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