Fig 1.
A schematic diagram of the workflow.
GEO, Gene expression omnibus; RSEM, RNA-Seq by Expectation–Maximization; DEGs, Differentially expressed genes; GO, Gene ontology; WGCNA, Weighted correlation network analysis; GSEA, Gene set enrichment analysis.
Table 1.
Homo sapiens and Mus musculus sample category information.
Fig 2.
Human differentially expressed gene (DEG) analysis.
(A) Heatmap constructed using 199 upregulated and 441 downregulated genes in exhausted T-cells. Significant changes are determined as fold change > 1.5 and False discovery rate (FDR) < 0.05. (B–C) Gene ontology (GO) and (D–E) Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis for human DEGs. Selected key biological processes in (B) upregulated and (C) downregulated genes. GO analysis is performed based on biological process. (D) All six enriched KEGG pathways in upregulated genes. (E) The top six enriched KEGG pathways in downregulated genes. GO terms and KEGG pathway remain when the FDR is < 0.05. (F–G) Functional protein association networks of upregulated genes. (F) Cytoscape software and Cytohubba plugin have been used to identify hub genes with top 10 interaction degrees via analyzing interactions files obtained from the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Red to yellow represents interaction degrees from top to bottom. (G) Interaction degrees of the hub genes. X-axis represents the number of adjacent genes.
Fig 3.
Co-expression analysis in human exhausted T-cells.
(A) Analysis of a set of soft thresholding powers. The left panel is the scale free fit index as a function of soft thresholding power. The right panel is mean connectivity as a function of the soft thresholding power. (B) Heatmap of module and trait correlation. Module is marked by unique color labels. ME: module eigengene. (C) Eigengene dendrogram and heatmap between modules and the exhaustion trait. The top figure represents the hierarchical clustering dendrogram of MEs and exhaustion trait. The bottom figure shows the eigengene network heatmap plot of the adjacencies including exhaustion trait. Each row and column corresponds to one ME or exhaustion trait. Red color represents positive correlation, while blue represents negative correlation. (D) Scatterplot of gene significance for exhaustion trait (y-axis) vs. membership in a selected module (x-axis).
Fig 4.
GO and KEGG pathway enrichment analysis for human red module genes.
(A) Selected key biological processes. (B) All enriched KEGG pathways. GO terms and KEGG pathway remain when the FDR is < 0.05.
Fig 5.
Overlap genes between human and mouse sets.
(A) Overlap of upregulated genes in exhausted T-cells and most correlative modules with exhaustion between human and mouse, as well as protein associated network of nine proteins in both the overlap of upregulated genes and selected modules. Red colored nodes: regulation of immune system process (GO:0002682). (B) and (C) show the expression of selected genes at the mRNA level in human and mouse datasets from exhausted (red) and non-exhausted (blue) T cells, respectively. Median values are indicated by lines in the box and whisker plot. Hinge values and whisker 1.5* interquartile range (IQR) values have been calculated using Tukey. Statistical analysis has been performed using an unpaired two-tailed Student’s t-test (*P < 0.05, ** P < 0.01, *** P < 0.001).
Fig 6.
GSEA of exhausted versus non-exhausted CD8+ T-cells.
(A–D) The four shared enriched gene sets in human exhausted T-cells with a P-value < 0.05. ES, enrichment score; NES, normalized enrichment score.