Fig. 6

Notch1 expression in melanoma cells increased tumor cell-mediated inhibition of T cell proliferation and activation. a In vitro cytotoxicity of CD8⁺ T lymphocytes against Notch1 knockdown or overexpressing cells. CD8⁺ T lymphocytes isolated from wild-type B16 (unmodified) tumor-bearing mice were activated by CD3 antibody, CD28 antibody, and irradiated B16 cells for 3 days. Then the CD8⁺ effector cell were co-cultured with Notch1 knockdown or overexpressing cells at the different ratio of 1:1, 5:1, and 25:1. In vitro cytotoxicity of CD8⁺ T lymphocytes against Notch1 knockdown or overexpressing cells was detected by chromium release assay. b Lymphocyte colony formation of CD8⁺ T cell after co-cultured with B16 cells (magnification, 200×). Black arrows indicated lymphocyte colony formation. Total CD8⁺ T cells purified by positive selection from spleens of wild-type B16 tumor-bearing mice were co-cultured with Notch1 knockdown or overexpressing cells at the ratio of 1:1 in transwell chamber (Con A 5 μg/ml). c Number of lymphocyte colony. d The inhibition rate of lymphocyte proliferation by B16 cells was detected by CCK8 kit. e CD8⁺ T lymphocytes labeled with CFSE were analyzed for proliferation by flow cytometry. f The apoptosis of CD8⁺ T lymphocytes were analyzed by flow cytometry after 7 days. Results shown were representative of 3 independent experiments. *, P < 0.05. **, P < 0.01