Fig. 4: TCRs specifically react to NEJ-derived neoantigens.

a, Pipeline overview for identifying T cell populations reactive to NEJ-derived neoantigen through IVS of CD8⁺ T cells derived from PBMCs from healthy donors against APC-presented neopeptides. b, IFNγ ELISA of reactive CD8⁺ T cell populations (n = 3) following IVS with neoantigen. c, 10× V(D)J sequencing shows IFNG signatures of highly proliferated TCR clonotypes cultured with T2 cells pulsed with neoantigen (coloured), control peptide (light grey) or no peptide (dark grey). Specific TCR clonotypes are highlighted for NeoA_RPL22 and NeoA_GNAS reactivity in donors 3 (left) and 4 (centre and right). d, Clonotype frequency analysis of TCR clones in CD8⁺ T cells from donors 3 (left) and 4 (centre and right) following IVS with NeoA_RPL22 or NeoA_GNAS. Neoantigen-reactive TCR clones are denoted by text. e, NeoA_GNAS-specific (top) and NeoA_RPL22-specific (bottom) TCR-transduced PBMC-derived CD8⁺ T cells were activated against neoantigen-pulsed T2 cells in a dose-dependent manner. TCR-transduced cells were also co-cultured with control-peptide-pulsed T2 cells at the highest dose concentration (1 μM). PBMC-derived CD8⁺ T cells were stained with CD107a and CD137 antibodies, and surface expression of the TCR coactivation markers was analysed by flow cytometry. The percentages of activated (CD107a and CD137 antibody-stained) CD8⁺ T cells detected in flow analysis are indicated by the numbers within the box. f, IFNγ ELISA (n = 3) of NeoA_GNAS-reactive (top) and NeoA_RPL22-reactive (bottom) TCR-transduced CD8⁺ T cells co-cultured with dose-dependent neoantigen (neo)-pulsed (left) and control-peptide-pulsed T2 cells (right). g, NeoA_GNAS-specific (top) and NeoA_RPL22-specific (bottom) TCR-transduced triple-reporter Jurkat76 cells were co-cultured with non-pulsed T2 cells (left), 0.1 μM neoantigen-pulsed T2 cells (centre) or 0.1 μM neoantigen-pulsed T2 cells treated with pan-HLA class I blocking antibody (right). Cells were stained with CD3 antibody, and TCR activation was evaluated by NFAT–GFP activity. The percentages of CD3⁺ and NFAT-GFP⁺ TR Jurkat76 cells detected in flow analysis are indicated by the numbers within the box. h, NeoA_GNAS-dextramer staining of bulk CD8⁺ T cells derived from an HLA-A*02:01 healthy donor (left) and patients with glioma (right) following two cycles of NeoA_GNAS IVS. Further statistical details are provided in Supplementary Table 3. a, Created in BioRender (credit: D.W.K., https://BioRender.com/z79j394; 2024).