Treg lineage-restricted BACH2 expression is required for tumor immunosuppression. (A) Volume (left) and representative photographs (right) of tumors at the indicated time points following subcutaneous implantation of 3 × 10⁵ MC38 colorectal adenocarcinoma cells into the flanks of tamoxifen-treated animals of the indicated genotypes. (B) Mass of tumors dissected from euthanized Foxp3^{EGFP-Cre-ERT2} animals of the indicated Bach2 genotype on day 33 after implantation. (C) CD4⁺ and CD8⁺ Tconv density within tumors on day 33 after implantation. (D) Representative flow cytometry (left) and replicate measurements (right) of the frequency of IFN-γ– and TNF-α–expressing CD8⁺ and CD4⁺ Tconv cells following brief ex vivo restimulation of tumor-infiltrating lymphocytes with PMA/ionomycin in the presence of brefeldin A. (E) Density of IFN-γ–expressing CD8⁺ and CD4⁺ Tconv cells within tumors on day 33 after implantation. (F) Density of CD4⁺ Foxp3⁺ Treg cells within tumors on day 33 after implantation. (G) Ratio of Foxp3⁺ Treg cells to IFN-γ–expressing CD8⁺ T cells within tumors on day 33 after implantation. Data are pooled from three (A) or two (B–G) independently repeated experiments with 37–40 (A) and 14–18 (B–G) mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.005; Wilcoxon–Mann–Whitney test (A, B, E, and G) and unpaired two-tailed Student’s t test (C, D, and F). Numbers in gates show percentages. Bars and error show mean and SEM.