Translational repression of 5’ TOP mRNAs in chronically activated CD8+ T cells in vitro
In vitro chronically activated CD8+ T cells show hallmarks of T cell exhaustion. (a) Schematic for in vitro generation of Tacute, Tre-act, and Tchron cells by anti-CD3 and anti-CD28 antibody activation. Each 2-day activation is indicated in Tchron (Act. 1, Act. 2, etc). (b) IFN-γ and TNFα secretion of Tchron and Tre-act measured by ELISA (n = 3 donors, error bars represent mean ± standard deviation). Of note, Tre-act (in blue) shows the final activation (ending on day 14), not the initial activation. p values were calculated using unpaired two-tailed t tests. (c) Representative flow cytometry histograms of the inhibitory receptors LAG3, TIM3, PD1, and CD39 (n = 4 donors). (d) Principal component analysis of RNA-seq data from Tacute, Tre-act, and Tchron cells in four donors. (e) Differential expression analysis of RNA-seq data comparing Tchron to Tre-act cells. Genes with significant changes in expression are in red (|log2(fold change)| >1 and adjusted p-value <0.05). (f) GSEA of MYC target genes, mTORC1 signalling pathway genes, and glycolysis genes in Tchron versus Tre-act cells.
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