Lymphodepletion increases the percentage of CD4⁺CD25⁺Foxp3⁺ Tregs in LNs and tumor tissues. Irradiated mice were transferred intravenously with whole spleen cells and then inoculated with tumor cells in the right flank. Twelve days later, NDLNs (left inguinal), TDLNs (A), and tumor tissues (B) were harvested, and single-cell suspensions were prepared for FACS analysis. The percentage of CD4⁺CD25⁺Foxp3⁺ cells in the LNs was significantly increased in irradiated mice compared with nonirradiated mice. An elevated percentage of CD4⁺CD25⁺Foxp3⁺ cells was also observed in the tumor tissues of the irradiated mice. (C) Kinetics of the percentage of CD4⁺CD25⁺Foxp3⁺ cells (i), the absolute number of total LN cells (ii), and that of CD4⁺CD25⁺Foxp3⁺ cells (iii). Inguinal LNs were harvested at different time points after irradiation and analyzed by flow cytometry. (D) Normal mice were inoculated SD with 1.5 × 10⁶ MCA205 tumor cells. Twelve-day TDLN cells were harvested and activated in vitro using the anti-CD3/IL-2 method in the absence or presence of CD4⁺CD25⁺ cells isolated from the spleens of irradiated mice or nonirradiated mice at different ratios. Activated TDLN cells were tested for antigen-specific IFN-γ production after specific stimulation as indicated.