CD8 tumor reactivity in mice given CD25-depleted T cells and in mice depleted of CD4 cells in vivo peaked at different times after transplantation. Some mice from the experiments shown in Figure 6B were euthanized on (A) day 11 or (B) day 18 after transplantation, and splenic CD8⁺ T cells were isolated. The CD8⁺ T cells were examined in IFN-γ ELISPOT assays with AGN2a tumor cell stimulators to detect the presence of IFN-γ–secreting tumor-specific CD8 T cells. The data represent 1 of 2 replicate experiments. (C) Splenic CD8 T cells were analyzed by flow cytometry on day 18 after transplantation for expression of PD-1, Tim-3, and annexin V. Splenocytes from each group were pooled from 3 mice, and the data are representative of 3 replicate experiments. (D) Average percentages of PD-1⁺, Tim-3⁺, and PD-1⁺/annexin V⁺ CD8⁺ T cells were calculated. Each bar represents the average of 6 mice, and statistical comparisons were determined using the Student t test (*P < .05, **P < .01). P values compare in vivo CD4-depleted mice to mice given unmanipulated T cells or CD25-depleted T cells.