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. 2022 Mar 15;132(6):e147908. doi: 10.1172/JCI147908

Figure 4. NANOG blocks CD8+ T cell infiltration through HDAC1-mediated epigenetic repression of CXCL10.

Figure 4

(A) Correlation plot of NANOG and T cell infiltration signatures in pan-tumor types. Correlation and 2-tailed P values were assessed using the Pearson’s correlation coefficient and unpaired Student’s t test. (B) Pearson’s correlation of NANOG signature expression with indicated transcripts of T cell–recruiting chemokines. RSEM, relative SEM. (C) qPCR analysis of Cxcl9 and Cxcl10 mRNA expression. (D) Western blot analysis of the expression of CXCL10. (E–G) CT26-no insert or CT26-Nanog cells were transfected with siGFP or siHdac1. WCL, whole-cell lysate; SUP, supernatant. (E) Western blot analysis of CXCL10 expression. (F) qPCR analysis of Cxcl10 mRNA expression. (G) Relative occupancy of AcH3K14, AcH3K27, and HDAC1 in the Cxcl10 promoters was assessed by qChIP analysis. The ChIP data values represent ratios relative to the input. (H) Transwell T cell migration assay using CT26-Nanog-siGFP or CT26-Nanog-siHdac1 cells were treated with IgG or anti-CXCL10. (I) Western blot analysis of the expression of GFP (CXCL10) in CT26-no insert or CT26-Nanog WT cells transduced with Cxcl10 WT or Cxcl10 MT. (J–L) CT26-no insert, CT26-Nanog-Cxcl10 WT, or CT26-Nanog-Cxcl10 MT tumor–bearing mice were treated with anti–PD-1 antibody. (J) Tumor growth curves. (K) Flow cytometric profiles of tumor-infiltrating CD3+CD8+ T cells. (L) Ratio of granzyme B+ to tumor-infiltrating CD3+CD8+ T cells. Five mice from each group were used for in vivo experiments. (D, E, and I) β-Actin was used as an internal loading control. Results in the graphs represent 3 independent experiments performed in triplicate. Data represent the mean ± SD. *P < 0.05, **P <0.01, and ***P < 0.001, by 1-way ANOVA (C, F, G, H, K, and L) and 2-way ANOVA (J).