Figure 9.
Zbtb32 and Bcl6 exert antagonistic functions in tumor-specific CD8+T cells. (A) Consensus binding sequences of Zbtb32 and Bcl6. (B) Quantification of the ratios of ChIP-seq overlapped peaks bound by Zbtb32 and specific TFs in total Zbtb32-binding peaks in CD8+ T cells. (C) Heatmaps of read density profiles of Zbtb32 and other TFs ChIP-seq in CD8+ T cells surrounding Zbtb32-binding peaks, respectively. (D) Zbtb32, with or without Bcl6 overexpression, and Bcl6 occupancy at several classic gene loci were aligned with assay for ATAC-seq tracks of WT and Zbtb32−/− from B16-OVA tumor (GEO accession: GSE123236). The binding peaks on Prdm1, Batf, Id2, Havcr2, Il2rb, Gzmb, and Gzmk loci are shown. (E) ChIP-qPCR results showing the deposition of Zbtb32 at specific gene loci in CD8+ T cells following 4-day activation in vitro with or without Bcl6 overexpression. The detected sites are labeled by blue arrow in Fig. S5 H (n = 3 for each group). (F) Schematic diagram of the transfer with 7 × 105 transduced OT-I cells on day 8. (G) The tumor growth in mice transferred with 0.7 × 106 activated CD8+ T cells with Zbtb32, Zbtb32-B, Bcl6, or Bcl6-Z overexpression on day 8 after inoculation with 1 × 106 B16-OVA cells (n = 6 for each group). (H) Schematic diagram of the co-transfer with 1.5×105 WT and 1.5 × 105Zbtb32−/− naïve OT-I cells. (I) Quantifications of relative cell ratios in Zbtb32, Zbtb32-B, Bcl6, and Bcl6-Z OE activated CD8+ T cells in comparison with RV control CD8+ T cells, co-transferred into recipients on day 8 after 1 × 106 B16-OVA cells inoculation (n = 4 for each group). (J) Quantifications of specific molecules of transduced and RV control OT-I TILs in a co-transfer assay (n = 4 for each group). (K) ChIP-qPCR results demonstrating the deposition of NCoR1 at Prdm1 gene locus in CD8+ T cells following 4-day activation in vitro with or without Bcl6 overexpression (n = 3 for each group). GEO accession: GSE182034. Data are shown as means ± SEM; ns, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001 by paired (J) and unpaired (E, I, and K) two-tailed Student’s t test and Bonferroni-corrected two-way ANOVA (G). Data shown are a representative (E, G, I, and J) or a pool (K) of two independent experiments. ATAC, transposase-accessible chromatin; Zbtb32-B, Zbtb32 with the Bcl6 BTB domain; Bcl6-Z, Bcl6 with the Zbtb32 BTB domain. Refer to the image caption for details.

Zbtb32 and Bcl6 exert antagonistic functions in tumor-specific CD8 + T cells. (A) Consensus binding sequences of Zbtb32 and Bcl6. (B) Quantification of the ratios of ChIP-seq overlapped peaks bound by Zbtb32 and specific TFs in total Zbtb32-binding peaks in CD8+ T cells. (C) Heatmaps of read density profiles of Zbtb32 and other TFs ChIP-seq in CD8+ T cells surrounding Zbtb32-binding peaks, respectively. (D) Zbtb32, with or without Bcl6 overexpression, and Bcl6 occupancy at several classic gene loci were aligned with assay for ATAC-seq tracks of WT and Zbtb32−/− from B16-OVA tumor (GEO accession: GSE123236). The binding peaks on Prdm1, Batf, Id2, Havcr2, Il2rb, Gzmb, and Gzmk loci are shown. (E) ChIP-qPCR results showing the deposition of Zbtb32 at specific gene loci in CD8+ T cells following 4-day activation in vitro with or without Bcl6 overexpression. The detected sites are labeled by blue arrow in Fig. S5 H (n = 3 for each group). (F) Schematic diagram of the transfer with 7 × 105 transduced OT-I cells on day 8. (G) The tumor growth in mice transferred with 0.7 × 106 activated CD8+ T cells with Zbtb32, Zbtb32-B, Bcl6, or Bcl6-Z overexpression on day 8 after inoculation with 1 × 106 B16-OVA cells (n = 6 for each group). (H) Schematic diagram of the co-transfer with 1.5×105 WT and 1.5 × 105Zbtb32−/− naïve OT-I cells. (I) Quantifications of relative cell ratios in Zbtb32, Zbtb32-B, Bcl6, and Bcl6-Z OE activated CD8+ T cells in comparison with RV control CD8+ T cells, co-transferred into recipients on day 8 after 1 × 106 B16-OVA cells inoculation (n = 4 for each group). (J) Quantifications of specific molecules of transduced and RV control OT-I TILs in a co-transfer assay (n = 4 for each group). (K) ChIP-qPCR results demonstrating the deposition of NCoR1 at Prdm1 gene locus in CD8+ T cells following 4-day activation in vitro with or without Bcl6 overexpression (n = 3 for each group). GEO accession: GSE182034. Data are shown as means ± SEM; ns, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001 by paired (J) and unpaired (E, I, and K) two-tailed Student’s t test and Bonferroni-corrected two-way ANOVA (G). Data shown are a representative (E, G, I, and J) or a pool (K) of two independent experiments. ATAC, transposase-accessible chromatin; Zbtb32-B, Zbtb32 with the Bcl6 BTB domain; Bcl6-Z, Bcl6 with the Zbtb32 BTB domain.

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