(A) LLC1-Ova tumor–bearing mice (n = 5) received sublethal irradiation followed by i.v. transfer of OT-I WT and A2BR^–/– CD8⁺ T cells treated ex vivo with αCD3/αCD28 with or without α4-1BB. The group of mice with transfer of α4-1BB–treated T cells received additional doses of α4-1BB treatment (100 μg/mouse) by i.p. daily once for 2 days. Tumors were harvested and weighed 22 days after T cell transfer. Similar experiments were conducted in E0771-SIY–bearing CD90.1 mice with transfer of 2C WT and A2BR^–/– CD8⁺ T cells and B16F10-bearing CD90.1 mice with transfer of Pmel WT and A2BR^–/– CD8⁺ T cells as well as in MC38-bearing CD90.1 mice with transfer of polyclonal tumor-reactive WT and A2BR^–/– CD8⁺ T cells. (B) Tumor growth and survival curves of E0771-SIY–bearing mice (n = 5–8) were plotted. Survival curves of B16F10-bearing mice (n = 5) (C) and MC38-bearing mice (n = 5) (D) were plotted. In parallel, E0771-SIY tumors (n = 5) treated as in A were harvested for immune characterization of Ki67⁺IFN-γ ⁺ CD8⁺ T cell counts (E), and frequency of TCF1⁺PD-1⁺ (F) or TOX⁺PD-1⁺ (G) cells in transferred tumor-infiltrating CD90.2⁺CD8⁺ T cells. Similarly, relative counts (H) and frequency (I) of transferred CD8⁺ T cells, frequency of exhausted-like subset TOX⁺PD-1⁺ (J) or CD39⁺PD-1⁺ cells (K) among transferred CD8⁺ T cell infiltrates were determined in B16F10-bearing mice treated as in C. The gating strategy is shown in Supplemental Figure 10. Data (means ± SEM) are representative of 2 (A, B, and E–K) or 3 (C) independent experiments. (A, B, and E–K) One-way ANOVA in combination with Dunnet’s test to correct for multiple comparisons and the log-rank test for survival curves (B–D) were used. *P < 0.05; **P < 0.01; ***P < 0.001.