Combination treatment leads to a shift from T reg to T eff cells within the tumor. (A–D) TILs of tumor-bearing brains were analyzed by flow cytometry at days 21 and 35, 2 wk after initiation of treatment; two independent experiments pooled, one-way ANOVA with Bonferroni post-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (A) Total number of NK and T cells, pregated on CD45^hi/CD11b⁻ cells. (B) Ratio of CD8⁺ and CD4⁺IFN-γ⁺ T cells per CD4⁺FoxP3⁺ T cells; n ≥ 5/group; (C) BrdU and Ki67 labeling of T reg and T eff cells (pregated on CD45⁺CD11b⁻NK1.1⁻) in percent positive cells and (D) ratio of total numbers of T reg versus T eff cells; n ≥ 6/group; (E) Perforin-1 gene expression in TILs sorted from treated animals (n = 3/group). Shown is the normalized fold expression (ΔCT) in relation to HPRT expression; error bars are the SEM of replicate wells. Individual samples were pooled before sorting, which precluded statistical assessment. VM/Dk or B6 WT mice were injected i.c. with SMA-560 cells (n ≥ 7/group; F) and B16-F10 melanoma cells (n ≥ 10/group; G), respectively. Arrows show initiation of treatment. Osmotic minipumps delivered IL-12Fc or PBS i.t. as indicated by a horizontal line above graph. In addition, animals received i.p. injections with αCTLA-4 mAbs or PBS, indicated by vertical tick marks; two independent experiments pooled. Survival statistics according to Log-rank test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F) p(PBS/PBS vs. IL-12Fc/αCTLA-4) = 0.0182, p(IL-12Fc/PBS vs. IL-12Fc/αCTLA-4) = 0.0155, p(PBS/αCTLA-4 vs. IL-12Fc/αCTLA-4) = 0.0177. (G) p(PBS/PBS vs. IL-12Fc/αCTLA-4) = 0.0174. Histology below survival curves shows tumor burden at initiation of treatment, overview pictures. and higher magnification pictures of the indicated regions (arrowheads). Bars: 100 µm (F); 200 µm (G).