Extended Data Fig. 10: Activation, cytokine secretion, and proliferation induced by neoantigen-specific TCRs from patients without response to anti-PD-1 upon co-culture with the autologous cell lines.

Healthy donor T cells genetically engineered to express the captured neoTCRs from patient9 (a), 10 (b-d) and 11 (e-f) were co-cultured with the autologous (M488, M485 and M486 respectively) or a mismatched cell line (M202). a, 4-1BB upregulation in the CD8⁺ neoTCR⁺ T cells from patient 9 after co-culture. Melanoma cell lines were pre-treated with regular media or media with IFNγ 24 h prior co-culture with T cells (n = 3). b, 4-1BB, OX-40, and CD107a upregulation in CD8⁺ neoTCR⁺ T cells from patient 10 after co-culture. Melanoma cell lines were pre-treated with regular media or media with IFNγ 24 h prior co-culture with T cells (n = 3). c, Cytokine release at 24 h after co-culture (n = 3). d, Proliferation of CD8⁺ neoTCR⁺ T cells from patient 10 measured by Ki67 mean fluorescence intensity upon 24, 48 and 72 h co-culture with autologous melanoma cell line (M485, top panel) or a mismatched cell line (M202, bottom panel) (n = 3). e, 4-1BB and OX-40 upregulation in CD8⁺ neoTCR⁺ T cells from patient 11 after co-culture. Melanoma cell lines were pre-treated with regular media or media with IFNγ 24 h prior co-culture with T cells (n = 3). f, Cytokine release at 24 h after co-culture (n = 3). * p < 0.05, ** p < 0.005, ***p < 0.0005, ****p < 0.0001 vs Neo12, two-tailed unpaired t test with Holm-Sidak adjustment for multiple comparisons in figure a, b, c, e, and f. * p < 0.05, ** p < 0.005, ***p < 0.0005, ****p < 0.0001 vs M202, two-tailed unpaired t test with Holm-Sidak adjustment for multiple comparisons in figure d. Exact p-values provided in Supplementary Information. (n) indicates the number of biological replicates. Mean ± SD and individual values are shown. All T-cell products contain CD8⁺ and CD4⁺ gene-edited T cells.