PMC

(A) Heatmap indicating relative expression of indicated genes within global transcriptional profiles of tumors from Bach2^+/+ and Bach2^–/– (KO) animals. (B) Wet weight of tumors dissected from WT and Bach2^–/– animals at day 18 following implantation. (C) Density of CD4⁺ and CD8⁺ T cells normalized by tumor mass. (D) Immunohistochemical analysis of CD8⁺ T cells in sections of B16 tumors from mice of indicated genotypes at day 18 after implantation. Scale bar: 200 μm. (E) Frequency of Ki67⁺ cells among CD8⁺ T cells and CD4⁺ Foxp3^– conventional T (Tconv) cells at day 18 after implantation. (F) IFN-γ expression among intratumoral CD4⁺ and CD8⁺ T cells at day 18 after implantation. (G) Growth of subcutaneous B16 tumors in littermate mice of the indicated genotypes at selected time points following implantation. Significant differences from growth in Bach2^+/+ mice are indicated. Error bars represent mean ± SEM. Results are representative of ≥2 independent experiments. **P < 0.01; ***P < 0.005; ****P < 0.001, 2-tailed Student’s t tests.

(A) Representative flow cytometry and (B) replicate measurements of CD4 and Foxp3 expression among T cells infiltrating B16 tumors at day 18 following implantation. Numbers in gates represent percentages of gated cells. (C) Rag1^–/– mice were reconstituted with 1:1 mixtures of BM cells from Foxp3^DTR mice and either WT or Bach2-deficient (KO) mice and administered PBS or DTx at indicated time points relative to implantation. Tumor growth was measured after subcutaneous implantation of 1.25 × 10⁵ B16 cells at indicated time points following implantation. Significant differences in tumor area compared with the WT:Foxp3^DTR → Rag1^–/– (PBS) group are indicated. Representative photographs of tumors at day 18 after implantation are shown. Rulers show millimeters. (D) Rag1^–/– mice were reconstituted with 1:1 mixtures of BM cells from Bach2-deficient and Foxp3^DTR mice and administered PBS or DTx in conjunction with α-CD8– and/or α–IFN-γ–depleting monoclonal antibodies or isotype control antibodies (IgG) at indicated time points relative to implantation. Tumor growth was measured at indicated time points after subcutaneous implantation of 2.5 × 10⁵ B16 cells. All data are representative of ≥2 independent experiments. Error bars represent mean ± SEM. **P < 0.01; ***P < 0.005; ****P < 0.001, 2-tailed Student’s t tests.

(A and B) Growth of subcutaneous (A) B16 and (B) EL-4 tumors in littermate Bach2^+/+ (WT) and Bach2^–/– mice at indicated time points following implantation. (A) Representative photographs of tumors at day 18 after implantation (inset). Rulers show millimeters. Error bars represent mean ± SEM. *P < 0.05; ***P < 0.005; ****P < 0.001, 2-tailed Student’s t tests. (C) Principal component analysis of transcriptional profiles from tumors of Bach2^+/+ (blue) and Bach2^–/– (red) mice at day 18 following implantation of B16 tumor cells. Principal component 3 is indicated by the axis perpendicular to the x and y axes. (D) Global differences in transcriptional profiles of tumors from Bach2-deficient and WT mice. 3,623 differentially expressed genes identified in the analysis (adjusted P < 0.05; fold change > 2, 2-tailed Student’s t tests) are shown. (E and F) Gene set enrichment analysis of global transcriptional differences between tumors from Bach2-deficient and WT mice. Positions of genes from indicated gene sets within a list of gene expression differences rank ordered by fold change are indicated. Kolmogorov-Smirnov statistic was used to calculate statistical significance. All data are representative of ≥2 independent experiments. NES, normalized enrichment score.