Extended Data Fig. 10: The expression of ETV7 is correlated with the expression of memory genes and exhaustion marker genes and generation of anti-MSLN CAR T-shETV7 therapeutic system.
a-c, Related to Fig. 7f and g. Mouse naive CD8+ T cells were stimulated with anti-mouse CD3/CD28 antibodies for 24 hours and then transfected with lentivirus for overexpression of ETV7 or vector control (Ctrl). The cells were then stimulated 3 times every two days to induce exhaustion. The mRNA levels (b) and protein (percentage of CD8+ T cells and MFI) (a, c) expression of TCF1, CTLA4, TOX, CXCR4, EOMES, CXCR5, CXCL13, LAG3, TIM3, and TNFRSF9 in CD8+ T cells were determined by RT-PCR and FACS analysis, respectively (n = 4 biological independent replicates). d, Pearson correlation coefficient between expression level of ETV7 and memory, exhaustion genes across different cancer types. e, Schematic illustration of the anti-MSLN CAR T-shCtrl, anti-MSLN CAR T-shETV7 constructs. f, Anti-MSLN CAR T cells stably overexpressing ETV7 shRNA (shRTV7) or vector control (shCtrl) were lysed for RT-PCR (n = 3 biological independent replicates) and WB. The experiment was independently repeated three times with similar results. g, Total number of cells at the time of transduction with anti-MSLN CAR T-shCtrl and anti-MSLN CAR T-shETV7 (n = 3 biological independent replicates). h, Calcein release assay was used for in vitro cytotoxicity testing at 3 different effectors: target ratios on K562 overexpressing human MSLN stability cell lines as indicated (n = 3 biological independent replicates). i-k, Related to Fig. 8g–k. Schematic of the mouse model experiment (i). OVCAR8 cells were subcutaneously implanted into the flanks of NCG mice to establish a xenograft tumor model. The mice were then treated with 3×106 anti-MSLN CAR T-shCtrl cells, 3×106 anti-MSLN CAR T-shETV7 or T cells per mouse on day 8 after tumor cell injection. Tumor burden was monitored twice weekly using Vernier calipers (j) (n = 5–7 mice per group) Kaplan-Meier survival curves of OVCAR8 xenograft mice treated with anti-MSLN CAR T-shCtrl cells, anti-MSLN CAR T-shETV7 or T cells (k) (n = 7 mice per group). All data are the mean ± SD. P-values are indicated. Significance was calculated by two-sided unpaired Student’s t-test.
