Rejection of established tumors depends on IFN-γ secretion by CD4⁺Trp1⁺ T cells and is independent of TNF and endogenous T, B, and NK cells. Tumor-bearing mice were irradiated at day 10 with 5 Gy followed or not by treatment with 50,000 CD4⁺Trp1⁺ cells and anti–CTLA-4 mAb. Tumor growth was measured and data are presented for each independent mouse. (a) Mice received only 5 Gy (left) or 5 Gy + CD4⁺Trp1⁺ + anti–CTLA-4 with control antibody or with neutralizing anti-TNF or anti–IFN-γ antibody. Two further groups included IFN-γ^−/− and IFN-γR^−/− recipients treated at day 10 with 5 Gy, 50,000 CD4⁺Trp1⁺ cells, and anti–CTLA-4. (b) In a separate experiment, recipient PFN^−/− and Rag^−/− mice were compared with wild-type mice for their capacity to reject established melanoma after treatment at day 10 with 5 Gy, 50,000 CD4⁺Trp1⁺ cells, and anti–CTLA-4. (c and d) Mean tumor growth in mice treated with 5 Gy of RT + CD4⁺Trp1⁺ cells in the presence (blue line) or absence (red line) of anti–CTLA-4. Mice used for this set of experiments were either wild-type C57BL/6 (c) or CTLA-4 human Tg (d) mice in which anti–CTLA-4 antibodies will only block CTLA-4 on the transferred CD4⁺Trp1⁺ cells. Data are representative of at least two independent experiments (n = 5 mice per group).