Fig. 6: Epigenetic imprint of thymic antigen encounter persists in recent thymic emigrants with “anergic” phenotype. | Nature Communications

Fig. 6: Epigenetic imprint of thymic antigen encounter persists in recent thymic emigrants with “anergic” phenotype.

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. 6

a, b Gating scheme to identify Pop A - D among naïve CD4 splenic T cells (a) from mice harboring both Foxp3-RFP reporter and Nur77-eGFP reporter or CD25- SP4 thymocytes from Nur77-eGFP reporter on basis of Ly6C and GFP (b). Sequential gating in (a) excludes Foxp3+ Treg and defines Pop A-D among naïve CD62Lhi CD44lo T cells. c Gating scheme to identify analogous Pop A’-D’ on basis of Ly6C and proxy marker CD5. Histograms depict GFP expression in PopA’-D’ as gated in lefthand plot in either Nur77-GFP reporter (middle histograms) or RAG-GFP reporter (righthand histograms). d quantification of RAG-GFP+ fraction +/- SEM of Pop A’-D’ (as gated in c) from N = 3 biological replicate RAG-GFP animals. e, f schematic (e) depicts experimental design with adoptive transfer of thymocytes from Nur77-eGFP reporter mice into congenically marked CD45.1+ hosts. At sequential time points after transfer, recipient splenocytes were harvested. Following enrichment for CD4+ cells by bench-top negative selection, splenocytes were stained to detect cells of donor origin among CD25- naive CD4 T cell in Pop A-D (gating as in Supp Fig. 8b). Graph (f) depicts quantification of distribution across pop A-D of donor cells at sequential time points for N = 3 biological replicates +/- SEM. g Venn diagram depicts overlap between genes differentially expressed in Nur77/Nr4a1-hi vs. Lo SP4 semimature thymocytes (see Fig. 5a, GSE279344) and genes near differentially accessible regions (DAR) in the genome across Pops A-D (PMID: 36100367; GSE206074). To do so, DAR were collapsed by nearest gene. Heatmap depicts rpm in Pops A-D for most differentially accessible OCR near these genes (see also Supplementary Data 4e). h Venn diagram as in (e) but further filtered to identify Nr4a-dependent gene set. Heatmap depicts expression of those genes in our thymic RNAseq data set aligned against accessibility in Pop A-D ATACseq (PMID: 36100367; GSE206074; see also Supplementary Data 4f). Statistical analysis: d One-way Anova with Tukey’s multiple comparison correction. data in a–d are representative of at least N = 4 biological replicates. f Two-way Anova with Holm-Sidak multiple comparison correction. Source data are provided as a Source Data file.

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