Figure 3.

Frequencies of neoantigen-specific T cells in TIL and assessment of their ability to recognize autologous tumor cells. HLA-A2/mutated peptide dextramers were produced for peptides found to activate TIL and/or found to be presented on tumor cell MHC-I. The dextramers were used to stain tumor-infiltrating lymphocytes (A), and anti-PE beads were used to enrich for stained cells followed by a rapid-expansion protocol. Thereafter, recognition of KADA (B) and ANRU (C) tumor cells by the sorted TIL was assessed by IFN-γ ELISA with or without IFN-γ pretreatment of the tumor. Only the specificities that could be significantly enriched by the sorting are shown. Unsorted cells were used as a control. The tumor cells were analyzed for neoepitope expression by MS and presented as LC-DIAMS Poisson detection plots for mutant ETV6 in untreated (D) and IFN-γ-treated ANRU tumor cells (E) with the arrow in the latter indicating detection. The total TIL population (F. ANRU) was co-cultured with untreated or IFN-γ pretreated autologous tumor cells, or with the different neoepitope peptides, analyzed by dextramer staining for the same epitope and for cell surface CD107a. As negative control, TIL alone were used and stained as described above. Dot plots are gated on lymphocytes/singlets/live cells and frequency indicates % dextramer⁺ out of CD8⁺ cells (A) and gated in the same way plus on CD8⁺ and frequency indicates % dextramer⁺CD07a⁻ or dextramer⁺CD107a⁺ of CD8⁺ cells (F).