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. 2021 Apr 22;10:e65435. doi: 10.7554/eLife.65435

Figure 6. Thymocytes with low self-reactivity retain a preselection gene expression program marked by elevated expression of ion channel genes.

RNA-seq of OT-1, F5, and TG6 thymocytes at the early positive selection, late positive selection, and mature CD8SP stages. All samples have three biological replicates except for TG6 late positive selection, which has two biological replicates. (a, b) Gene set enrichment analysis (GSEA) was performed on all pairwise combinations of TCRtg samples, and the gene sets were filtered for enrichment in the order of self-reactivity (OT-1>F5>TG6) (a) or in the inverse order of self-reactivity (TG6>F5>OT-1) (b). Normalized enrichment scores (NES) are from the OT-1 vs TG6 (a) or TG6 vs OT-1 (b) comparison. Abbreviations: self-reactive (SR), ribonucleoprotein (RNP), nucleic acid (NA), transmembrane (TM). (c) Heatmap of differentially expressed (padj<0.05 for OT-1 vs TG6 comparison) ion channel genes (see Materials and methods) across all three stages and all three transgenic models. Gene expression values normalized by the DESeq2 variance stabilizing transformation were averaged over biological replicates and scaled per row. Heatmap is hierarchically clustered into four groups, numbered in the order of expression during development. Leading-edge genes from GSEA are in bold. (d) (Top) Plot of expression level in DP CD69− thymocytes versus fold difference for DN4 versus DP CD69− (left side of X-axis) or DP CD69− versus DP CD69+ (right side of X-axis) for ImmGen microarray data. The 11 genes chosen to represent the ‘preselection DP’ gene signature are indicated with red dots. (Bottom) Heatmap of normalized expression (as in (c)) of the 11 representative preselection DP genes in TG6, F5, and OT-1 thymocytes at the indicated stage of development.

Figure 6.

Figure 6—figure supplement 1. Gene expression differences between TG6 and OT-1 thymocytes at different stages of development.

Figure 6—figure supplement 1.

Plots of significance versus fold change for the OT-1 vs TG6 comparison at the early positive selection, late positive selection, and mature SP stages of development. The numbers in boxes denote the number of genes significantly upregulated for TG6 (upper left) and for OT-1 (upper right). Genes discussed in this study are indicated in color: green (ion channel genes), red (translation and ribosome genes), blue (T cell activation/effector function genes), purple (cell division genes), and orange (other). Other genes with very significant p-values and/or high fold change are labeled in gray.
Figure 6—figure supplement 2. Thymocytes with low self-reactivity retain a preselection gene expression program marked by elevated expression of ion channel genes.

Figure 6—figure supplement 2.

(a) Ion channel genes from the manually curated list in Figure 6b that exhibit significantly different (padj<0.05, fold change>0.5) expression between OT1 versus TG6 thymocytes at the indicated stages of development. Positive values (green) indicate genes enriched in TG6; negative values (red) indicate genes enriched in OT-1. (b) Normalized read counts (see Materials and methods) for the indicated genes at each stage of development. Red circles: OT-1; blue squares: F5; green triangles: TG6. (c) (Left) Heatmap of normalized expression of the ion transport genes (Group 2 genes from Figure 6d) in wild thymocytes at the indicated stages of development (scaled per row). Data are from the ImmGen microarray database. (Bottom) Graphic representation of the average gene expression pattern. (d) Normalized enrichment score (NES) for the top 15 gene sets enriched in DP CD69− cells compared to DN4 and DP CD69+thymocytes, using gene set enrichment analysis (GSEA) and the Gene Ontology database. Black bars indicate gene sets relating to ion channels.