Zbtb32 displays similar binding propensity but opposing regulation with Bcl6 in CD8 ⁺ T cells. (A) Distribution of Zbtb32-binding peaks in CD8⁺ T cells across the genome. (B) Enrichment of known TF-binding motifs in binding peaks between WT and Zbtb32^−/− OT-I TILs. (C) GSEA results for comparing the enrichment DEGs in Bcl6⁺ and Tim-3⁺ CD8⁺ T cell subsets in WT and Zbtb32^−/− OT-I TILs (GEO accession: GSE114631). (D) Venn diagrams showing Zbtb32-regulated genes and DEGs in T_pex and T_tex CD8⁺ TILs (GEO accession: GSE114631) and Zbtb32- and Bcl6-regulated genes in CD8⁺ TILs. (E) Quantifications of cell ratio and specific molecules of transduced and RV control OT-I TILs, co-transferred into recipients 8 days after 1 × 10⁶ B16-OVA cell inoculation (n = 6–8 for each group). (F) Schematic diagram of the transfer of naïve OT-I cells. (G) Tumor growth in mice transplanted with 1 × 10⁶ B16-OVA cells after the transfer of 0.3 × 10⁶ naïve WT, Zbtb32^−/−, Bcl6^−/−, and Zbtb32^−/−Bcl6^−/− OT-I cells (n = 6 for each group). (H) ChIP-seq data showing Zbtb32 and Bcl6 occupancy at several classic genes in CD8⁺ T cells following 4-day activation in vitro. (I) Quantifications of specific genes expressions in Bcl6-, Zbtb32-, double OE, and RV control activated CD8⁺ T cells measured by RT-qPCR (n = 3 for each group). (J) Expression level of Prdm1 in Bcl6-, Bcl6-Z-, Zbtb32-, and Zbtb32-B OE activated CD8⁺ T cells measured by RT-qPCR (n = 3 for each group). GEO accessions: GSE182034, GSE182035. Data are shown as means ± SEM; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by paired (E) and unpaired (I and J) two-tailed Student’s t test and Bonferroni-corrected two-way ANOVA (G). Data shown are a representative (G, I, and J) or a pool (E) of two independent experiments.