Extended Data Fig. 9: Identification of T cells reactive to tryptophan-derived aberrant peptides.

a, Flow cytometry analysis of CD8⁺ T cells reactive to BV421 and PE-labelled pMHC multimers complexed with the ZNF513-derived aberrant peptide in co-cultures of naïve CD8⁺ T cells and autologous monocyte-derived dendritic cells pulsed with peptide (right) or DMSO vehicle (left). Cells were from an HLA-C*07:02^pos healthy donor. b, Boolean gating strategy for analysis of combinatorial pMHC multimer staining limits false positive signals arising due to background fluorescence when combining in one staining multiple different pMHC multimers labelled with different fluorochromes. In total, four different fluorochromes were used in pMHC multimer preparation and combined into four dual-colour pMHC multimer pairs, each pair complexed with a different peptide. For simplicity, gating for only one dual-colour pMHC multimer population is shown. Gating strategy: (1) Live CD8⁺ T cell singlets were identified with the use of FSC and SSC gates, live/dead fixable near-IR dead cell and CD8 staining. (2) Separate gates were used to define positive events in each pMHC channel. (3) NOT gates were used to select CD8⁺/pMHC multimer⁻ events for each pMHC channel. (4) AND gates for two pMHC multimer⁺ populations and NOT gates for each of the remaining pMHC channels were used to select for cells that are positive only in two channels. Events staining positively for one or more than two pMHC channels were gated out. (5) AND gate was used to combine all pMHC NOT gates. (6) OR gate was made with gates from step 4 and 5. The final plot shows only CD8⁺ T cells that are positive for two pMHC channels and excludes cells positive for only one or positive for more than two channels. c, For analysis of T cells upregulating the activation marker CD137, viable CD8⁺ T cell singlets were identified by FSC and SSC gates, live/dead fixable near-IR dead cell staining and CD8 staining. Subsequently, activated CD8⁺ T cells were identified as CD137⁺ events, in which cut-off was set based on staining of T cells incubated without target cells. Plots depicting CD137⁺ events show the KCNK6 T cell clone 1 incubated with target cells pulsed with the KCNK6 aberrant peptide (1 μg ml⁻¹, top), or not (bottom).