Radio-resistant recipient Tregs inhibit the development of antitumor immunity in reconstituted lymphopenic mice. (A) Forty million GFP⁺ spleen cells were transferred into irradiated mice that were then inoculated SD with MCA205 cells. TDLNs and tumors were harvested on day 12, and single-cell suspensions were analyzed for GFP expression within the gated CD4⁺Foxp3⁺ Tregs subset. The percentage of GFP⁻ recipient cells among CD4⁺Foxp3⁺ cells is indicated. (B) Irradiated mice were reconstituted with GFP⁺ spleen cells (40 × 10⁶) and injected SD with MCA205 tumor cells. Single-cell suspensions were prepared from the TDLNs and were analyzed using FACS. The percentage of the transferred donor cells in fresh TDLNs is indicated by the percentage of GFP⁺ cells (i). TDLN cells from mice reconstituted with GFP⁺ spleen cells were activated with the method of CD3/IL-2. Activated TDLN cells were further stimulated with MCA205 tumor digests and stained for the detection IFN-γ as described in “Intracellular IFN-γ staining.” The majority of tumor-specific cells were primed from the transferred donor GFP⁺ cells (ii). (C) GFP⁺ spleen cells were magnetically depleted of CD25⁺ cells. These CD25⁻GFP⁺ cells were transferred intravenously into irradiated mice. Twelve days later, LNs were harvested and stained for FACS analysis. More than 90% of the CD4⁺Foxp3⁺ cells were GFP⁻ recipient cells. (D) Irradiated mice reconstituted with CD25⁻ depleted spleen cells were injected with MCA205 tumor cells. These mice were then treated with anti–CD25 Abs to further inhibit the recipient Tregs. The depletion of the residual recipient-derived Tregs significantly augmented antitumor immunity. (E) TDLNs were harvested from mice reconstituted with CD25− spleen cells that were treated with anti–CD25 mAb or left untreated. Three million of activated TDLN cells were transferred into mice bearing 3-day established pulmonary metastases. The depletion of recipient Tregs enhanced the generation of T_Es in the TDLNs.