Depletion of Tregs after reconstitution increases the number of T_Es in the TDLNs. (A) Irradiated mice were reconstituted with 40 × 10⁶ naive spleen cells and then inoculated SD with 3 × 10⁶ MCA205 tumor cells in the right flank. Next, these mice were treated with PC61. The numbers of cells in the TDLNs, NDLNs, and spleens were counted on the indicated day after tumor inoculation. Treatment with PC61 significantly increased the number of TDLN cells; however, this treatment did not affect the number of NDLN cells or spleen cells. (B) Twelve-day TDLNs were harvested from mice reconstituted with normal spleen cells (40 × 10⁶) that were with PC61 treatment or left untreated. Each dot indicates the mean number of TDLN cells from separate experiments (n = 3 to 6 mice per group). (C) Reconstituted mice were inoculated SD with MCA205 tumor cells and then treated with PC61 or left untreated. Twelve days later, TDLN cells were harvested and activated in vitro as described in “Activation of tumor-draining lymph-node cells.” Mice bearing 3-day established pulmonary metastases were treated with 3 or 10 × 10⁶ activated TDLN cells. The TDLN cells from PC61-treated mice showed greater antitumor efficacy. (D) Twelve-day TDLN cells were harvested from reconstituted mice that were treated with PC61 or left untreated. These TDLN cells were activated in vitro with the method of CD3/IL-2 and tested for IFN-γ production after specific or nonspecific stimulation. A representative result from 3 independent experiments is shown. The further depletion of Tregs after sublethal irradiation and reconstitution increased tumor-antigen specific T cells in the TDLNs.