Fig. 5: HDAC2-AS2 targeted CDK9 to impair the function of CD8+ T cells. | Nature Communications

Fig. 5: HDAC2-AS2 targeted CDK9 to impair the function of CD8+ T cells.

From: HBV-associated hepatocellular carcinomas inhibit antitumor CD8+ T cell via the long noncoding RNA HDAC2-AS2

Fig. 5

a Silver staining of the SDS-PAGE gel containing aliquots of samples derived from proteins pulled down by HDAC2-AS2 (right lane) or its antisense RNA (left lane) in mouse CD8+ T cells. The arrows indicate the gels submitted for mass-spectrometry, identifying CDK9 as the band unique to HDAC2-AS2. Representative images are shown from three independent replicates. b Western blot analysis of the interaction of CDK9 with HDAC2-AS2 (treated the same as panel a). Representative images are shown from three independent replicates. c RIP analysis of the interaction between CDK9 and HDAC2-AS2 (three independent replicates). d The expression of CDK9 in naïve and activated mouse CD8+ T cells (24, 48, 72 h) was detected by confocal microscopy. Scale bar: 5 μm. Representative images are shown from three independent replicates. e Western blot analysis of CDK9 expression in nuclear and cytoplasmic fractions of activated mouse CD8+ T cells and MHCC97H cells. Representative images are shown from three independent replicates. f Activated mouse CD8+ T cells were treated with EVs from Hepa1-6-Ctrl or HDAC2-AS2 cells for 48 h, mock is PBS treatment. CDK9 expression was detected by western blotting. EVs were purified by ultracentrifugation. Representative images are shown from three independent replicates. g The expression of CDK9 in activated mouse CD8+ T cells of retrovirus ctrl and retrovirus-HDAC2-AS2 infection was observed by confocal microscopy. Scale bar: 5 μm. Representative images are shown from three independent replicates. h FISH assay showing the localization of HDAC2-AS2 and CDK9 in activated human CD8+ T cells of lentivirus ctrl and lentivirus-HDAC2-AS2 infection. Scale bar: 5 μm. Representative images are shown from three independent replicates. i Experimental layout for single-cell Ti-ATAC-seq 2 to analyze chromatin accessibility and T cell receptor (TCR) clonality. j The UMAP projection displays T cells from HCC patients, illustrating the landscape of TCR clonality (left; indicated by the sizes of TCR clonotypes) and chromatin accessibility (right) from Ti-ATAC-seq 2 datasets. Each color represents a T-cell cluster. k The chromatin accessibility, indicated by gene scores of the indicated genes, is overlaid on the UMAP embedding. l Violin plot showing the chromatin accessibility as indicated by gene scores of CDK9 of the T-cell clusters. Each color represents a T-cell cluster, the cells of each cluster were obtained from patients (n = 9). m–o Violin plots showing the gene scores of indicated genes (m) and ChromVAR TF-motif bias-corrected deviation scores of indicated TF regulators (n) across the CD8 T-cell clusters. The cytotoxicity of the cluster is indicated by the gene scores of GZMK and PRF1, whereas the exhaustion of the cluster is indicated by the gene scores of TOX and PDCD1. (o). Boxplot showing the T-cell clonal expansion as indicated by the log10 (clonotype size +1) across the CD8 T-cell clusters. The boxplots denote the median with a quartile range (25–75%), and the length of whiskers represents 1.5x the IQR. The cells of each cluster were obtained from patients (n = 9). Statistical analyses were determined by a two-tailed Student’s t-test (c, o). Source data are provided as a Source Data file. Cyto cytoplasmic, Nuc nuclear, RV retrovirus.

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