Fig. 5

TCR-T production and functional assay. a Framework of TCR-T production, activation, and expansion. T cells were activated on day 0, transduced with asTCR lentivirus on day 1, and expanded for two weeks. (+) sign indicates days for evaluating and quantifying essential T cell markers, and (*) asterisks indicate days for performing functional assays. b IFNγ was detected after co-culturing TCR-Ts with (NY-ESO-1(_157–165)) and other peptides-enriched T2 cells to validate TCR-T function and specificity. c TCR-Ts activation and potential antitumor function were evaluated by quantifying IFNγ release after an overnight co-culturing of TCR-Ts with ECA109(NY-ESO-1⁻/HLA-A*02:01⁻), ECA109(NY-ESO-1⁺/HLA-A*02:01⁺) and OE19(NY-ESO-1⁺/HLA-A*02:01⁺) EC cells. d T cell activation before performing functional assay was evaluated using a late T cell activation marker (CD25) by selecting sampling two different TCR-Ts. e T cell resting phase was verified before performing functional assay using CD137 marker using two different sampled TCR-Ts. f–h TCR-T function and specificity of two selected TCR-Ts (TCRT4 and TCRT5) were validated by quantifying their IFNγ release or killing effect (cytotoxicity) against multiple cancer cells with activated native T cell as negative control (f), T2 cells expressing different HLA allotype pulsed with relevant peptide (NY-ESO-1_(157–165)) (g), and (NY-ESO-1_(157–165)) or other peptides-enriched T2 cells (h)