Zbtb32 upregulates Id2 to enhance CD8 ⁺ T cell–mediated tumor rejection. (A) Fold changes in the expression levels of different TFs in DEGs in Zbtb32^−/− and WT CD8⁺ TILs. (B) The occupancies of Zbtb32 and Bcl6 at the Id2 gene locus using ChIP-seq datasets. (C) ChIP-qPCR data showing the deposition of Zbtb32 at Id2 locus in CD8⁺ T cells in vitro activated for 4 days with or without Bcl6 overexpression. The detected sites are labeled by blue arrows in Fig. 7 B (n = 3 for each group). (D) Quantifications of Id2 expression in Bcl6-, Zbtb32-, double OE, and RV control activated CD8⁺ T cells measured by RT-qPCR (n = 3 for each group). (E)Id2 expression in WT and Zbtb32 OE CD8⁺ T cells (left panel) and Zbtb32 expression in WT and Id2 OE CD8⁺ T cells (right panel) measured by RT-qPCR. (F) Schematic diagram of Id2 rescue assay in B6 mice, transferred with 7 × 10⁵ transduced CD8⁺ T cells on day 8 after inoculation of 1 × 10⁶ B16-OVA cells. (G) Tumor growth in WT and Zbtb32^−/− mice transferred with activated WT or Zbtb32^−/− CD8⁺ OT-I cells with or without Id2 overexpression (n = 6 for each group). (H) Schematic diagram of Id2 rescue assay in B6 mice, co-transferred with 5 × 10⁵ transduced CD8⁺ T cells on day 8 after inoculation of 1 × 10⁶ B16-OVA cells. (I) Representative plots and quantifications of TCF1 and Tim-3 expressions in co-transferred WT and Zbtb32^−/− CD8⁺ TILs with or without Id2 overexpression (n = 4 for each group). (J) MFI and quantification of GzmB, IFNγ, and Ki-67 in WT and Zbtb32^−/− CD8⁺ OT-I TILs with or without Id2 overexpression (n = 4 for each group). Data are shown as means ± SEM; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by paired (I) and unpaired (C, D, E, and J) two-tailed Student’s t test and Bonferroni-corrected two-way ANOVA (G). Data shown are a representative (C–J) of two independent experiments.