Fig. 7

Ablation of activin-A signaling renders CD4⁺ T cells exhausted via a Tox/Tox2-dictated transcriptional program. A Changes in gene expression (x-axis) and statistical significance (y-axis) in lung tumor-infiltrating CD4⁺ T cells from CD4/ALK4-KO versus WT mice, presented as a volcano plot. Log2 fold change (x axis) cutoff was set at ±0.58 and P value (y axis) cutoff was set at < 0.05. Down: Down-regulated, Up: Up-regulated, Not Signif: Not significant. B Pathway analysis of Differentially Expressed Genes. Terms and scores were retrieved from Molecular Signatures Database (MSigDB). C GSEA results for Differentially Expressed Genes (DEGs) against a gene set related to CD8⁺ T cell exhaustion, retrieved from Molecular Signatures Database (MSigDB). FDR: False Discovery Rate, p: p-value, ES: Enrichment Score, NES: Normalized Enrichment Score. D Representative immunoblots showing Tox/Tox2 expression. Quantification of relative Tox/Tox2 protein levels is depicted; TATA binding protein (TBP). Data are mean ± SEM and are representative of 3 independent experiments. E Representative flow cytometry plots (left) and cumulative MFI values (right) of Tox/Tox2 expression by ex vivo activin-A- or PBS-treated CD4⁺ T cells from lung tumors of LLC-OVA-inoculated C57BL/6 mice cells. F ChIP analyses demonstrating the binding of Tox/Tox2 on the Pdcd1, Lag3, Nfatc1 and Jun loci. Data are mean ± SEM and are representative of 2–3 independent experiments. Statistical significance was obtained by unpaired Student’s t-test; *P < 0.05 and **P < 0.01