Cy conditioning of the tumor-bearing mice prevents aberrant CD4⁺ T-cell differentiation and generates activated polyfunctional effector cells. The timeline of the procedure is outlined. On day 17 after tumor inoculation, some mice were treated with 150 mg/kg Cy before receiving T-cell transfer the next day. (A) At 7 days after T-cell transfer, spleen cells were examined for donor cell division status. Numbers indicate the percentage of the donor cells in spleen. (B) Absolute number of donor CD4⁺ T cells recovered from spleen (total splenocyte count × percent donor CD4⁺ T cells). Data are from 2 independent experiments and are shown as mean ± SD of at least 4 mice per group. (C) Expression profiles of Foxp3 and PD-1 on donor CD4⁺ T cells. Numbers indicate the percentage of cells in the corresponding quadrant. (D) Cytokine intracellular staining on divided donor CD4⁺ T cells. Purified CD4⁺ T cells were stimulated with HA peptide–pulsed fresh splenocytes for 5 hours before intracellular staining for IL-2, IFN-γ, and TNF-α. Plots shown are gated on the divided donor CD4⁺ T cells. Numbers represent the percentage of cells in each quadrant in divided donor CD4⁺ T cells. (E) Annexin V and CD127 (IL-7R) expression profiles on donor CD4⁺ T cells. Solid curve in each histogram represents divided donor cells, and shaded curve represents undivided donor cells. Numbers indicate percent of the gated population for divided donor cells. Results shown are representative of 3 independent experiments.