Combination of CD4⁺Trp1⁺ ACT and CTLA-4 blockade enhances activation and differentiation of CD4⁺Trp1⁺ T^eff cells and reduces the number of T reg cells. (a–e) Tumor-bearing mice were treated at day 10 with or without 5 Gy of RT followed by transfer of 50,000 CD4⁺Trp1⁺ cells in the presence or absence of anti–CTLA-4 mAb. 8 d after therapy (day 18 after tumor challenge), mice were euthanized and numbers of CD4⁺Trp1⁺Foxp3⁻ cells (T^eff cells; a and d) and CD4⁺Trp1⁺Foxp3⁺ cells (T reg cells; b and e) were analyzed in LNs (a and b) and tumors (d and e). Numbers of CD4⁺Trp1⁺ T^eff and T reg cells in tumors (d and e) were calculated as described in Materials and methods. (g and h) Proportions of CD4⁺Trp1⁺ T^eff cells to CD4⁺Trp1⁺ T reg cells (g) and CD4⁺Trp1⁺ T^eff cells to total T reg cells (h) were also calculated from tumor samples. CD4⁺Trp1⁺ cells from LNs (c) and tumor samples (f) were restimulated ex vivo, and IFN-γ, TNF, and IL-2 secretion was determined on a per cell basis. (i) In a parallel experiment, tumors were dissected and fresh frozen in optimum cutting temperature solution, cut, and stained for DAPI (blue), CD31 (red), and CD4 (green). Samples were analyzed by confocal microscopy with a 20X water immersion objective. Bars, 50 µm. Images showing whole-tumor immunofluorescence are shown in Fig. S3. Data are representative of three independent experiments (n = 3 mice per group). Horizontal bars represent means.