Fig. 4

Notch1 in tumor tissues inhibited CTL activation in tumor-DLNs but not in spleen cells. Splenocytes isolated from tumor-bearing mice were analyzed for CD3⁺ CD8⁺ T cells and CD49b⁺ NK cells by flow cytometry. a Flow cytometry analysis of CD3⁺ CD8⁺ T lymphocytes in CD45⁺ gate in spleens. b Flow cytometry analysis of CD49b⁺ NK cells in CD45⁺ gate in spleens. c In vitro cytotoxicity of CD8⁺ tumor-DLNs cells from B16 tumor-bearing mice against B16 cells in co-culture system. CD8⁺ T effector cells purified by positive selection from tumor-DLNs of in C57BL/6 mice subcutaneously inoculated with Notch1 knockdown or overexpressing cells were co-cultured with wild-type B16 cells at the different ratio of 1:1, 5:1, and 25:1. Tumor-DLNs isolated from non-tumor bearing mice were used as a baseline control. d In vitro cytotoxicity of CD8⁺ splenocytes from B16 tumor-bearing mice against B16 cells in co-culture system. Spleens isolated from non-tumor bearing mice were used as a baseline control. e IFN-γ ELISPOT assay was performed in splenocytes and tumor-DLN cells isolated from B16 tumor-bearing mice and restimulated with irradiated B16. Data was pooled from 3 independent experiments. *, P < 0.05. "ns", no statistical significance