Fig. 5: Transcriptional program induced in thymocytes fated for deletion is conserved in normal thymic repertoire and enriched for anergy-associated gene. | Nature Communications

Fig. 5: Transcriptional program induced in thymocytes fated for deletion is conserved in normal thymic repertoire and enriched for anergy-associated gene.

From: Nr4a1 and Nr4a3 redundantly control clonal deletion and contribute to an anergy-like transcriptome in auto-reactive thymocytes to impose tolerance in mice

Fig. 5

a Post-sort semimature CD25- SP4 thymocytes from Nur77-eGFP reporter gated as in Supp Fig. 8a and sorted according to lo, med, hi GFP expression, overlaid on pre-sort population. Biological triplicate samples subjected to bulk RNAseq analysis (GSE279344 and Supplementary Data 3a). b PCA plot of RNAseq analysis for samples in (a). c TPM values from GFPlo, med, hi SP4 samples sequenced as in (a) are plotted for GFP, Nr4a1-3, and Bcl2l11. These data represent biological triplicate samples subjected to bulk RNAseq analysis. Statistical analysis in Supplementary Data 3a. d Heatmap depicts normalized expression (z-score) of all genes with significant differential expression ( | log2FC | ≥ 1, adj p value/FDR ≤ 0.05) in any pairwise comparison. Genes are ordered by log2FC. e Volcano plot depicts significantly differentially expressed genes ( | log2FC | ≥ 1, FDR ≤ 0.05) between Nur77-GFPhi and -GFPlo CD4 T cells in black, while genes not passing significance thresholds are shown in light gray. Genes of interest are highlighted in green and annotated with their gene symbol. (GSE279344 and Supplementary Data 3a show Deseq2 analysis). f Gene sets from two differential expression analyses — OT-II from RIPmOVA hosts vs. WT hosts, and CD4 T cell Nur77-GFPhi vs. GFPlo — were each filtered for FDR ≤ 0.05 and for genes appearing in both sets. FC FC plot depicts genes with |log2FC | ≥ 1 in at least one gene set in black, while genes with |log2FC | ≤ 1 in both gene sets are shown in light gray. Genes of interest are highlighted in green and annotated with their gene symbol. See also Supplementary Data 3b. g Gene set enrichment analysis (GSEA) was undertaken to compare the list of genes upregulated in OT-II thymocytes from RIPmOVA hosts relative to WT hosts (log2Fc ≥ 1, fdr <0.05) to the genome-wide data set of TPM in Nur77-eGFP-hi and lo semimature SP4 as in (a) above (GSE279344). h Heatmap depicts dataset GSE178782 encompassing transcriptome of naĂ¯ve (CD62Lhi CD44lo FR4lo CD73lo CD25-) CD4 T cells of WT polyclonal genotype +/- 3 hr anti-CD3 stimulation as well as ex vivo naturally occurring anergic CD4 T cells (CD62Llo CD44hi FR4hi CD73hi CD25-). Genes included in the heatmap are those identified in our data set (Fig. 5a) that are DEG in OT-II thymocytes in RIPmOVA hosts relative to WT hosts (log2FC ≥ 1, fdr<0.05, N = 286). Modules depict selected genes that are either primary response genes, anergy-associated genes, or naĂ¯ve markers. See also Supplementary Data 4a. i, j GSEA was undertaken to compare either (i) genes upregulated in OT-II from RIPmOVA hosts (as in g above), or (j) genes upregulated in Nur77-eGFP-hi vs. -lo SP4 (log2FC ≥ 1, padj> 0.05 against genome-wide data set of TPM in naive and anergic WT CD4 T cells as in (h) above, (GSE178782).

Back to article page