Extended Data Fig. 7: ETV7 drives CD8+ T cell exhaustion program in MC38 OVA tumor model and effect of CD4+ T cells and identification in bone marrow chimera experiment.
a-c, C57BL/6 mice were injected subcutaneously with 2 × 105 MC38 OVA cells and immunodepleted by irradiation (2.5 Gy, twice). Naive OT-I CD8+ T cells were stimulated with OVA257-264 peptide for 24 hours and transfected with lentivirus for overexpression of ETV7 or vector as control (a). OT-I CD8+ T cells were then adoptively transferred by tail vein injection (i.v.) and injected intraperitoneally (i.p.). Tumor loads were measured (b). Protein expression (the percentage of CD8+ T cells as well as MFI) in tumor-infiltrating CD8+ T cells, including ETV7 (a), memory proteins, and exhaustion proteins was measured by FACS analysis (c) (n = 6 mice per group). d-f, C57BL/6 mice were injected intraperitoneally (i.p.) with 200 μg of α-CD4 antibody (clone GK1.5) or PBS twice a week to deplete CD4+ T cells or not. 7 days later, the mice were subcutaneously injected with 2×106 B16-OVA cells. Naive OT-I CD8+ T cells were stimulated with OVA257-264 peptide for 24 hours and transfected with lentivirus for overexpression of ETV7 or vector as control. After 3 days, OT-I CD8+ T cells were adoptively transferred by tail vein injection (i.v.). CD4+ T cells in spleen were analyzed by FACS (d). Tumors were photographed and weighed (e). The percentage and MFI of memory proteins and exhaustion proteins (f) were measured by FACS analysis (n = 5 mice per group). g, Related to Fig. 6. Mice were treated as described in Fig. 6a. Peripheral blood (10 µl) was collected and analyzed for lineage distribution and of B cells (B220+), T cells (CD3+), and myeloid cells (B220− CD3−) were analyzed by FACS at the 1st and 2nd month (n = 5 mice per group). Data are the mean ± SD. P-values are indicated. Significance was calculated by two-sided unpaired Student’s t-test.
