In-depth profiling of IDH2^R140Q-specific neo-T cells. (A) Deconvolution of the IDH2^R140Q response to identify the stimulatory 15mer neopeptide. Schematic showing the 5 overlapping 15mers spanning the IDH2^R140Q mutation (left). IFN-γ ELISpot responses toward each 15mer for 2 representative donors (center), and summary results (right, n = 8). Vertical lines for the box indicate the median, 20th, and 80th percentiles. (B) Intracellular cytokine staining analysis to identify reactive CD8 and CD4 subsets recognizing the immunogenic 15-SPN neopeptide based on IFN-γ production. The gating strategy for CD8⁺ and CD4⁺ T cells is shown on the left; the percentage of reactive CD8 and/or CD4 cells for individual responders is shown on the right (percentage obtained by subtracting response from unstimulated control). (C) Schematic of 10mers overlapping by 9 AAs that encompass the 15-SPN neopeptide used for minimal epitope mapping (left). Representative example of minimal epitope mapping profiled for neo-T cells from 2 donors (right) and (D) summary of the 8 responding donors, with each colored square indicating the minimal epitope recognized. (E) Analogous IDH2 wild-type peptide sequences generated for each of the IDH2 minimal epitope (top). IFN-γ ELISpot response of neo-T cells after stimulation with the mutant or wild-type peptides for the 10-SPN (left), 10-TIQ (middle), and 10-IQN (right) neoepitopes.