Fig. 5

| Phenotypic character of transferred CD45.2 T cells in B16-OVA mice and the in vivo anti-tumor activity of different treatments. a and b) Activated CD8⁺ T cells from the spleens of OT-1 mice were cocultured with B16-OVA cells at a ratio of 2:1 for 5 h and 10 h and treated with CAI (10 μM), DMF (20 μM) or a combination of CAI and DMF. In another experiment, sorted CD8⁺ T cells were treated with CAI (10 μM), 1-MT (0.2 mM) or a combination of CAI and 1-MT. B16-OVA cell apoptosis was analyzed by flow cytometry at 5 h and 10 h after drug treatment. c and d CD45.1 C57BL/6 mice bearing 3 × 3 mm OVA-B16 melanomas were subject to the adoptive transfer of OVA-specific CD45.2⁺CD8⁺ T cells (1 × 10⁷ cells/mouse) three times (every 5 days). At the same time, these mice were treated with PBS or CAI (20 mg/kg), 1-MT (5 mg/ml in drinking water), DMF (10 mg/kg), or CAI + 1-MT, CAI + DMF or anti-PD-1 neutralizing antibody (250 μg per mouse) for 20 days. Five days later, several of the mice (n = 6) were sacrificed to obtain TILs for flow cytometry, and the remaining mice continued to receive drug treatment. Anti-CD45.2 antibody was used to distinguish donor CD45.2 T cells from host and competitor cells. c and d Flow cytometry plots showing the expression of PD-1⁺ in CD45.2 ⁺ TILs from B16-OVA CD45.1 mice after various treatments (left: representative histogram plot; right: statistical histogram plot). e and f Tumor growth was measured (left), and long-term survival was analyzed (right). RAG1 KO mice bearing 3 × 3 mm OVA-B16 melanomas were subject to the adoptive transfer of OVA-specific CD45.1⁺CD8⁺ T cells (1 × 10⁷ cells/mouse) every 5 days. The mice were grouped and administered treatments as indicated above. g and h Tumors were harvested after 15 days of inoculation, dissociated into single-cell suspensions, and stained for flow cytometry (n = 6/group). The expression of PD-1⁺CD45.1⁺ TILs in B16-OVA CD45.2 mice after various treatments was analyzed by flow cytometry (left: representative histogram plot; right: statistical histogram plot). i and j Tumor growth curves of RAG1 KO mice (n = 7/group). Data are from three independent experiments, and the error bars represent the mean ± s.e.m. **p < 0.01, ***p < 0.001 by one-way ANOVA and Kaplan-Meier survival analysis