Responses of CD8⁺ T cells from tumor-bearing mice treated with AdNK4 and DCs in an antigen-specific manner.

(A) Antigen-specific proliferation. C57Bl/6 mice with 8-day established subcutaneous E.G7-OVA tumors were inoculated with AdNK4 (AdNK4 + DCs) or AdNull (AdNull + DCs) followed 3 days later by intratumoral injection of DCs or inoculated with AdNK4 followed by the injection of PBS (AdNK4 + PBS). Controls included untreated tumor-bearing mice (no treatment). Nine days after the last treatment, splenic CD8⁺ T cells were isolated from treated or nontreated mice using the magnetic beads. In 96-well culture plates, 5 × 10⁵ CD8⁺ T cells were cocultured for 4 days with 5 × 10⁴ irradiated DCs with or without 50 μg/mL ovalbumin. The number of viable cells was determined using the MTS assay. The data are presented as the percentage increase over baseline on the initiation of the coculture. (B) IFN-γ production. In the termination of the coculture described above, the culture medium was collected, and the levels of murine IFN-γ were assayed by ELISA. For both panels, results are shown as the means ± the standard error (n = 3 per data point).