CTLA4-CD28 chimera gene modification of both CD4 and CD8 T cells potentiates the efficacy of adoptive T-cell therapy in a syngenic melanoma model. Seven days after B6 mice were subcutaneously injected with B16 melanoma cells, the mice were lymphodepleted by total body irradiation (4 Gy). Then, the mice were either left untreated or injected intravenously with the retrovirus-transduced CD4 and CD8 T cells. Regulatory T cell–depleted polyclonal CD4 T cells (CD4⁺CD25⁻) purified from normal B6 mice and Pmel-1 CD8 T cells were transduced with the retroviruses and rested for 48 hours before the transfer. Equal numbers of the transduced Pmel-1 T cells and CD4 T cells (2 × 10⁶) were used for the adoptive transfer. (A) Mean tumor size of 5 mice per group was recorded. Results are representative of at least 2 separate experiments (*P = .0105; Wilcoxon matched pairs test). (B) Survival data of the mice from all the experiments are summarized (n = 10 for Pmel-1 EV only; n = 15 for all other groups; **P = .002; log-rank test). (C-F) Four weeks after the adoptive transfer, peripheral blood leukocytes were counted and analyzed by flow cytometry. The retrovirus-transduced CD4 or Pmel-1 T cells were identified as CD4⁺GFP⁺ or CD8⁺GFP⁺ cells. The representative flow cytometry profiles for CD4 T cells (C) and Pmel-1 T cells (D) are displayed. The absolute cell numbers of the transduced CD4 (E) and Pmel-1 T cells (F) were calculated from the 2 independent experiments. P values, Student t test. (G) B16 tumor-bearing mice generated as described in panel A were lymphodepleted and injected with various combinations of the retrovirus-transduced polyclonal CD4 and Pmel-1 T cells at 1:1 ratio (2 × 10⁶). Mean tumor size of 5 mice per group was recorded. (H) B16 tumor-bearing mice generated as described in panel A were lymphodepleted and injected with EV-transduced polyclonal CD4 and EV-transduced Pmel-1 T cells or CTC28-transduced polyclonal CD4 and CTC28-transduced Pmel-1 T cells at 1:1 ratio (2 × 10⁶). Anti–IL-2 antibody or control rat IgG (100 μg/head) was injected intravenously every 3 days beginning from the day of the cell transfer. Mean tumor size of 5 mice per group was recorded. Results are representative of 2 independent experiments (G-H; ***P = .0001; ****P = .0006; *****P = .0021; Wilcoxon matched pairs test).