Figure 7. PD-1–laIL-2 specifically reactivates PD-1⁺TIM3⁺ tumor-specific CD8⁺ T cells.

(A–D) CD4⁺, PD-1^–CD8⁺, PD-1⁺TIM3^–CD8⁺, and PD-1⁺TIM3⁺CD8⁺ T cells from A20 tumor-bearing mice were sorted out and cocultured with irradiated A20 cells in the presence of Erb–laIL-2 or PD-1–laIL-2 for the IFN-γ ELISPOT assay. Experimental scheme (A) and spots from PD-1^–CD8⁺ (B), PD-1⁺TIM3^–CD8⁺ (C) and PD-1⁺TIM3⁺CD8⁺ (D) T cells are shown. (E) PD-1 and TIM3 expressions on tetramer⁺CD8⁺ T cells in tumors from MC38 tumor-bearing mice (n = 5). (F–I) Splenocytes were stimulated with anti-CD3 and anti-CD28 antibodies. Five days later, PD-1⁺TIM3⁺CD8⁺ T cells were sorted out and labeled with CFSE. Then, the cells were cultured in 96-well plates in the presence of anti-CD3, Erb–laIL-2 plus anti–PD-1 (combo), or PD-1–laIL-2 for 2 days. The T cell clusters and CFSE expression were assayed with an Incucyte instrument. Total areas of the cluster are shown in F. Mean CFSE intensity is shown in G. (H) Percentage of CFSE low cells. (I) MFI of CFSE low cells. (J) A20 tumor-bearing mice (n = 5/group) were treated with equal molar amounts of Erb–laIL-2 (20 μg), anti–PD-1 (10 μg), or PD-1–laIL-2 (20 μg) on day 19. Six days later, T cells from the tumor were analyzed. Data represent mean ± SEM. The P value was determined by 2-way ANOVA with Tukey’s multiple comparisons test (D) or 1-way ANOVA with Tukey’s multiple comparisons test (E–J). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.