The duration of favorable immune-stimulatory environment created by chemotherapy is transient. (A) Kinetics of cellular restoration in mice spleens after Cy treatment. BALB/c mice were treated or not treated with 150 mg/kg Cy. Spleen cell numbers were enumerated at the indicated time points. Results are shown as mean ± SD of 3 mice each group. (B) Transient duration of favorable immune-stimulatory environment in postchemotherapy hosts. The schema delineates the timeline of the experimental procedure. All tumor-bearing mice were treated with Cy on the same day but received T-cell transfer at different time points. To normalize for the expansions of donor cells that were transferred at different time, a group of tumor-free mice (No tumor, −Cy) were given the same amount of donor cells as the Cy-treated tumor-bearing mice (A20HA, +Cy). Mice in each group were always analyzed 7 days after T-cell transfer. At the time of analysis, mouse spleen cells were prepared and analyzed by FACS. The profiles of cell division (Thy1.1 staining), PD-1, and Foxp3 expression of the donor CD4⁺ T cells are shown as dot plots. Numbers represent the percentage of each individual subset in the donor population. (C) Summary of the results shown in panel B. To determine the fold expansion of donor cells, the absolute numbers of donor cells (calculated as total splenocyte count × percent donor cells in spleen) in Cy-treated tumor-bearing mice were divided by the average number of donor cells in no Cy-treated tumor-free mice. Results are shown as mean ± SD of 3 mice each group.