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Muhammed Ali, Zsofia Foldvari, Eirini Giannakopoulou, Maxi-Lu Böschen, Erlend Strønen, Weiwen Yang, Mireille Toebes, Benjamin Schubert, Oliver Kohlbacher, Ton N Schumacher, and Johanna Olweus (accepted)

Induction Of Neoantigen Reactive T Cells From Healthy Donors

Nat Protocols.

Identification of immunogenic neoantigens and their cognate T cells represent 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-engineered T cells. Recent advances in deep sequencing technologies and in silico prediction algorithms enable rapid identification of candidate neoepitopes. However, large scale validation of putative neoepitopes and isolation of reactive T cells is challenging due to limited availablity of patient material and low frequencies of neoepitope-specific T cells. Here, we describe a standardized protocol for 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 transfected with mRNA encoding candidate neoepitopes are utilized to prime autologous naïve CD8+ T cells. Antigen-specific T cells recognizing endogenously processed and presented epitopes are detected using peptide-MHC (pMHC) multimers. Single multimer-positive T cells are sorted for identification of TCR sequences, preceded by an optional step that includes clonal expansion and functional characterization. The time required to identify neoepitope-specific T cells is 15 days, with an additional two to four 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 data sets generated by this technology should be of value to improve algorithms to predict immunogenic neoantigens.