The CD8⁺ T activity significantly contributes to pA20-36–induced tumor growth inhibition. (A) Intracellular cytokines expression of tumor-infiltrating CD8⁺ T cells. Purified CD8⁺ T cells from pCNT- or pA20-36–treated tumor masses were incubated with A20 tumor cells for 3 hours and then analyzed for intracellular expression of IL-2, interferon-γ (IFN-γ), and granzyme B by flow cytometry. A representative experiment of 2 independent experiments with similar results is shown. Numbers in the quadrants indicate the percentage of cells. (B) Antigen-specific proliferation of tumor-infiltrating CD8⁺ T cells. Purified CD8⁺ T cells from tumor mass of pCNT- or pA20-36–treated mice were stained with CFSE, incubated with A20 or 5T33MM tumor cells (5 × 10³), and 1 week later measured by flow cytometry. Percentage of proliferating CD8⁺ T cells was calculated from CFSE profiles with the use of FlowJo software, as described in “Methods.” Values are the mean ± SD (n = 3). A representative experiment of 2 independent experiments with similar results is shown. (C) Cytotoxic activity of tumor-infiltrating CD8⁺ T cells. Purified CD8⁺ T cells (effector cells, E) from pCNT- or pA20-36–treated tumor masses were incubated with BATDA-labeled A20 or 5T33MM cells (5 × 10³) (target cells, T), at ratio of 50:1 and 100:1. BATDA-specific release was measured by time-resolved fluorometry. Maximal release of BATDA was measured by incubating target cells in lysis buffer containing 1% Triton X-100; spontaneous release was measured by incubating cells in medium alone. Cytolytic activity was calculated as follows: (experimental release − spontaneous release)/(Triton X-100 release × spontaneous release) × 100. Values are the mean ± SD (n = 3); statistical analysis was performed by Student t test. A representative experiment of 2 independent experiments with similar results is shown. (D) CD8⁺ T cells contributed to anti-tumor activity of pA20-36 peptide. BALB/c mice (n = 6/group) were antibody-mediated depleted of CD8⁺ T cells or left undepleted and were subcutaneously injected with A20 tumor cells (5 × 10⁶); mice were then treated intravenously with pA20-36 or scrambled pCNT (200 mg ≃ kg⁻¹ ≃ d⁻¹), beginning the day after tumor injection. Tumor volumes were evaluated at the indicated time after tumor injection. A representative experiment of 2 independent experiments is shown. Symbols represent the tumor volume of each individual mouse; horizontal lines indicate the mean. Statistical analysis was performed by 2-way analysis of variance.