Figure 4

TGFβ insensitive Pmel-1 remain activated in immunosuppressive tumor microenvironment. On day 14, 21, and 28 post co-adoptive transfer of DN and mock transduced Pmel-1 to B16 tumor-bearing and non tumor-bearing mice, splenocytes and tumor-infiltrating lymphocytes (TILs) of treated mice were analyzed for IFN-γ production by intracellular cytokine staining. Between 2x10⁴-3x10⁵ TILs and 1x10⁷-6x10⁷ splenocytes were collected on each day of experimentation. Figure4A shows representative dot plots of TILs harvested from DN/mock mixture pmel-1 injected mice stimulated with either IL2 + control OVA peptide or IL2 + melanoma specific gp100_25-33 peptide. About 25-35% of DN TGFβ RII tumor-infiltrating lymphocytes were actively secreting IFN-γ upon re-stimulation compared to about 10% of mock-transduced TILs (4B). In both tumor-bearing and non tumor-bearing mice (all mice received DC vaccinations), a higher % of DN Pmel-1 splenocytes produce IFN-γ on day 14 and 21 post adoptive transfer compared to mock Pmel-1 splenocytes (4B). Experiment was performed twice with similar results.