B16 melanoma up-regulates class II expression in vivo in an IFN-γ–dependent manner and are direct targets of cytotoxic CD4⁺Trp1⁺ T cells. (a) In vivo class II expression by tumor cells from mice receiving either CD4⁺Trp1⁺ cells alone or in combination with 5 Gy of RT and in the presence or absence of blocking anti–CTLA-4 and neutralizing IFN-γ. Mice were treated at day 10 after challenge with B16/BL6 melanoma expressing Thy1.1 and sacrificed 7 d after initiation of therapy (day 17 after tumor). Single-cell suspensions of tumors were analyzed for MHC class II levels by gating in Thy1.1-positive cells. Numbers indicate percentages. (b) Quantification of class II expression by tumors in vivo shown as cumulative data from three independent experiments (n = 3 mice per group). Horizontal bars represent means. (c) CD4⁺Trp1⁺ T cells were primed in vivo and expanded in vitro to allow analysis of their antitumor activity upon retransfer into tumor-bearing CII^−/− recipient mice. In brief, 10 d after tumor challenge, MHC CII^−/− recipient mice were treated with 5 Gy of RT, primed CD4⁺Tpr1⁺ cells, and anti–CTLA-4. Half of the mice were also treated with 200 µg of blocking anti–class II antibody every 3 d, and tumor growth was monitored over time. Data are representative of two independent experiments (n = 5 mice per group). Error bars represent means ± SEM.