Tumor-reactive CD4⁺Trp1⁺ T cells develop class II–dependent killing activity. (a) 10 d after tumor challenge, tumor-bearing mice were treated or not with 5 Gy of RT, 50,000 CD4⁺Trp1⁺ cells, 5 Gy + CD4⁺Trp1⁺, or 5 Gy + CD4⁺Trp1⁺ + anti–CTLA-4 mAb. 7 d after therapy (day 17 after tumor challenge), all mice were injected with CFSE-labeled B cell targets. CFSE^high (5 µM) cells were also loaded with the class II–restricted peptide recognized by CD4⁺Trp1⁺ cells, whereas CFSE^low (0.5 µM) cells were used as a control population. 14–16 h after i.v. injection of CFSE targets, mice were sacrificed and in vivo killing activity was quantified in single-cell suspensions from the spleens of each mouse. Data are representative of three independent experiments (n = 3 mice per group). Horizontal bars represent means. (b–f) CD4⁺Trp1⁺ T cells were primed in vivo and expanded in vitro to allow analysis of their in vitro killing activity. Tumor-reactive in vivo–primed CD4⁺Trp1⁺ T cells were incubated at different ratios with CFSE-loaded spleen targets (b and d) or B16/BL6 and EL-4 tumor targets (c, e, and f). (b and d) CFSE^low B cell targets were also loaded with the class II–restricted peptide recognized by Trp1 cells, whereas CFSE^high cells were used as the control population. (c, e, and f) B16/BL6 cells were loaded with 0.5 µM CFSE, whereas EL-4 control tumor cells were loaded with 5 µM CFSE. Blocking anti–class II or anti-FASL antibodies were used at a final concentration of 50 µg/ml for all in vitro experiments (b–e). (f) In vitro killing of tumor targets was also tested in presence of 0.5 µM concanamycin A (PFN inhibitor) and/or 25 µM Z-AAD-CMK. In vitro killing activity was determined 12–14 h after initiation of the assay by quantifying the decrease of the target population in comparison to the control population, as described in Materials and methods. Data are representative of four independent experiments. Numbers in a–c indicate percentages. Error bars in d–f represent means ± SD.