Fig 2: More potent specific lysis to the DAC treated tumor cell lines by the TCR-T cells in vitro. (A) LDH assay for the cytotoxicity. The TCR-T cells were used as effector cells (E) and SW480 cells treated without or with 0.1 µM, 1 µM DAC as target cells (T). Various E/T ratios were tested as indicated. LDH spontaneous release of the target cells was less than 20%. Results shown were mean ± SD of quadruplicate wells. (B) ELISPOT assay for IFN-? release by the TCR-T cells. The target cells in the cytotoxicity assay were used as stimulators (S). SW480 cells were treated without or with 0.1 µM, 1 µM DAC. Various E/S ratios were tested as indicated. A375 melanoma cell line was selected to be the positive stimulator cells for the high-expressed NY-ESO-1. The IFN-? spot-forming cells (SFCs) were counted and analyzed by CTL-ImmunoSpot S6 Ultimate Micro Analyzer. ns: no significance. *P<0.05; **P<0.01.

Fig 3: Construction of lentiviral vector and preparation of NY-ESO-1 TCR transduced T lymphocytes. (A) The construction of lentiviral vector encoding the NY-ESO-1 TCR gene. (B) T cells were analyzed by FACS with FITC-conjugated anti-CD3 antibody and PE-conjugated anti-TCR Vß13.1 antibody that recognized the ß chain of the 1G4-a95:LY. (C) T cells were analyzed by FACS with APC-conjugated HLA-A2 tetramer containing the NY-ESO-1157–165 (SLLMWITQC). The percentage of cells in each quadrant was indicated. Representative of three independent experiments.

Fig 4: Induction of antigen expression by DAC on CRC cell lines with MSI or MSS. (A) After exposed at various lengths of time and at various concentrations of DAC, NY-ESO-1 transcript expression in the indicated CRC cell lines, including LOVO and HCT116 with MSI-H, and SW480 and HT29 with MSS, was assayed by RT-qPCR. The results were normalized to GAPDH and the baseline of untreated groups (controls) was the mean value of one experiment performed in technical triplicate. The results of fold changes were analyzed in comparison to the DAC untreated group as control. *P<0.05, **P<0.01, ***P<0.001. (B) NY-ESO-1 protein expression in the above cell lines was detected by Western blot assay. The proteins on the cell membranes were captured by the anti-NY-ESO-1 mAb (E976). GAPDH was used as a loading control.

Fig 5: In vitro and in vivo specific cytotoxicity of the NY-ESO-1 TCR-T cells. (A) IFN-? ELISPOT assay: T cells transduced with mock gene were used as ctrl-T cells, and the NY-ESO-1 TCR-T cells as effectors. T cells were co-cultured with T2 cells, which were used as stimulators, and loaded with different concentrations of cognate antigens, respectively. The unloaded T2 cells alone and T cells alone served as background controls, while the T cells+PHA served as a positive control. ESO10/50/100 indicated the concentration of antigens at 10 ng/ml, 50 ng/ml, 100 ng/ml NY-ESO-1 peptides (p157-165) respectively, and OVA100 indicated 100 ng/ml irrelevant OVA peptides (p257-264). The effector-stimulator ratio was 20:1 in this experiment. Spots of IFN-? were counted and analyzed. PHA: Phytohaemagglutinin, a selective T cell mitogen for T cell activation. *P<0.05. **P<0.01. (B) IFN-? ELISPOT assay: ctrl-T cells or the NY-ESO-1 TCR-T cells were co-cultured with tumor cells expressed different genotypes of HLA-A2 and NY-ESO-1, which served as stimulators. The T cells alone and tumor cells alone (not shown) served as background controls, while the T cells+PHA served as a positive control. The effector-stimulator ratio was 20:1 in this experiment. The spots of IFN-? were counted and analyzed. (C) LDH assay: ctrl-T cells (empty column) and the NY-ESO-1 TCR-T cells (full column) were used as effectors and the tumor cells with different genotypes of HLA-A2 and NY-ESO-1 were used as targets. The effectors(E) and targets(T) were co-cultured at different E/T ratios as indicated. Spontaneous release of target cells was less than 20%. Results shown were mean ± SD of quadruplicate wells. ns: no significance. **P<0.01; ***P<0.001. (D) Antitumor effects of the TCR-T cells on A375 xenograft nude mice. The PBS, ctrl T cells or the TCR-T cells were infused three times weekly via tail vein. The tumor size was measured every three days. Bars, mean ± SD. ***P<0.001. (E) Kaplan-Meier survival plot. The survival curve was analyzed by log-rank test. **P= 0.0054.