Lymphodepletion followed by the transfer of T_Ns induces T_Es in TDLNs and inhibits tumor progression. (A) Mice were irradiated with 500 cGy and injected intravenously with 40 × 10⁶ spleen cells from naive mice. These mice were then inoculated subcutaneously with 1 × 10⁵ of MCA205 tumor cells along the midline of the abdomen. The resultant skin tumors were measured in 2 perpendicular directions 2 to 3 times per week, and the tumor areas (mm²) were recorded. (B) Sublethally irradiated lymphopenic mice were reconstituted with either Thy1.2, CD4, or CD8-depleted cell populations and then inoculated subcutaneously with MCA205 tumor cells. The depletion of Thy1.2⁺ or CD4⁺ cells, but not the depletion of CD8⁺ cells, abrogated the antitumor effects observed after lymphodepletion and reconstitution. (C) Sublethally irradiated mice (500cGy) were injected intravenously with either 40 × 10⁶ whole spleen cells or with 10 × 10⁶ magnetically isolated splenic T_Ns. Mice were then inoculated SD with MCA205 tumor cells to stimulate the TDLNs. Twelve days later, TDLN cells were harvested and activated in vitro with the method of CD3/IL-2 for 5 days. Ten million of activated TDLN cells were then adoptively transferred into a new group of mice with 3-day established pulmonary metastases. The TDLN cells from the mice reconstituted with whole-spleen cells and splenic T_Ns exhibited similar therapeutic effects. (D) Congenic Ly5.1⁺ spleen cells were labeled with CFSE and transferred into irradiated lymphopenic mice. These mice were then inoculated SD with 3 × 10⁶ tumor cells to stimulate the TDLNs. Twelve days later, TDLNs, NDLNs, and spleens were harvested and analyzed for CFSE staining intensity within the Ly5.1⁺ subset.