Transcriptional analysis of WT and TGF-β receptor – deficient antigen-specific CD8 T cells activated in chronically infected mice. (A) Graph showing the experimental design of RNA-seq. (B) Volcano plots show log₂(fold-change) vs. −log₁₀ (adjusted P value) of selected genes upregulated or downregulated in KO P14 cells compared with WT P14 cells on D12 after transfer into mice with established chronic LCMV infection. The dotted lines indicate log₂(fold-change) = 0.6 or −0.6, and adjusted P = 0.05. (C) GSEA shows biological pathways enriched in KO P14 cells activated in mice with established chronic LCMV infection. Plotted by NES. (D) Heatmap showing the relative expression of metabolism-related genes in KO vs. WT P14 cells. Heatmaps generated using z-scores derived from normalized counts. (E) Representative FACS plots showed phosphorylation of S6 ribosome protein ex vivo on D15 after transfer. (F) GSEA-compared gene signatures of IL-7R^lo effector cells on D8 after acute LCMV Armstrong infection for enrichment in the transcriptional profiles of KO vs. WT P14 cells. (G) Representative FACS plots showed KLRG-1 expression on WT and KO P14 cells on D15 after transfer. (H) IFN-γ and TNF production after in vitro stimulation by subsets of WT and KO P14 cells harvested from spleen on D15 after transfer. Graphs showing the frequencies of IFN-γ–producing and TNF- and IFN-γ–producing cells among different subsets of WT and KO P14 cells. Paired Student’s t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (I) A summary of GSEA enrichments of WT and KO P14 cells for gene signatures of indicated cell types. Visualized by NES and −log₁₀ (FDR q value). Flow cytometry data in E, G, and H are representative of two independent experiments (n = 4–5 per experiment). RNA-seq datasets were generated from four biological replicates.