1. Volume 24, Issue 6, Pages 1627-1638.e6 (August 2018)
Nr4a Receptors Regulate Development and Death of Labile Treg Precursors to Prevent Generation of Pathogenic Self-Reactive Cells 
Takashi Sekiya, Sana Hibino, Keita Saeki, Mitsuhiro Kanamori, Satoshi Takaki, Akihiko Yoshimura 
Cell Reports 
Volume 24, Issue 6, Pages e6 (August 2018)
DOI: /j.celrep
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2. Cell Reports 2018 24, 1627-1638.e6DOI: (10.1016/j.celrep.2018.07.008)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
Identification of Nr4a-Dependent and -Independent Treg Cell Developmental Programs
(A) Expression profiles of genes (rows) in the indicated cell types assessed by microarray. Genes that showed different expression levels (|log2| > 1) compared to wild-type Tconv cells (6,180 probes) were subjected to hierarchical clustering. Two independent biological replicates were analyzed.
(B) Expression patterns of Nr4a and Foxp3 proteins in each cell subset. Asterisk denotes some contamination of Foxp3+ cells in the Nr4a-TKO Treg population.
(C and D) Classification of genes upregulated (C) or suppressed (D) in Tregs. Values in each panel represent the number of probes in each cluster.
(E) qRT-PCR analysis for the indicated genes in the indicated cells. Results shown are presented relative to Hprt expression (n = 5 per group).
(F) ELISA analysis of cytokine levels.
(G) CpG methylation profiles at TSDRs in the indicated cell populations.
(E and F) Results shown in (E) and (F) are representative of two independent biological replicates, performed in triplicate, means ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005; n.s., not significant (one-way ANOVA with Bonferroni test).
See also Figures S1–S3.
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4. Figure 2
Requirement for Nr4a Factors in Full and Sustained Induction of Foxp3
(A) Flow cytometry of the cells, cultured with or without agonistic ligands for 48 hr.
(B) Flow cytometry of phosphorylation of Stat5 in the indicated cells, cultured with or without IL-2 for 1 hr.
(C) qRT-PCR of Foxp3 mRNA in the indicated thymocytes, cultured with IL-2 (100 U/mL) and TNFRSF agonists, with or without IFN-γ and IL-4-blocking antibodies, for 24 or 48 hr.
(D) Left: qRT-PCR of Foxp3 mRNA from the indicated cells, after culture with IL-2 (100 U/mL) and TNFRSF agonists, with or without IFN-γ and IL-4-blocking antibodies, for 24 or 48 hr. Right: mean average scores of Foxp3 levels of four biological replicates. Scores of day 0 were set to one. Results shown are representative of three (C) and four (D) independent biological replicates, performed in triplicate, means ± SD. ∗p < 0.05; ∗∗p < 0.01 [one-way ANOVA with Bonferroni test (C), two-way ANOVA with Sidak test (D)].
See also Figure S4.
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5. Figure 3
Requirement for Nr4a Factors in IL-2/CD25 and TNFSF/TNFRSF Signal-Mediated Induction of Foxp3
(A) Gating strategy to separate wild-type preTregs into Nr4a3hi and Nr4a3lo subsets.
(B) Flow cytometry of wild-type unfractionated preTregs, wild-type Nr4a3lo preTregs, and wild-type Nr4a3hi preTregs, cultured with or without IL-2 and a cocktail of TNFRSF agonists, for 48 hr.
(C) Flow cytometry of the indicated molecules in Nr4a3loFoxp3−, Nr4a3midFoxp3−, Nr4a3midFoxp3+, and Nr4a3hiFoxp3+ populations (black, red, blue, and green lines, respectively), fractionated as shown in the left panel. Thymocytes from wild-type Nr4a3GFPFoxp3hCD2-hCD52-KI double reporter mice were analyzed.
(D) Quantification of the results in (C). Mean fluorescence intensities (MFI) of Nr4a3loFoxp3− cells are set at one. Results shown are representative of three independent biological replicates, performed in triplicate, means ± SD. ∗p < 0.05; ∗∗∗p < (one-way ANOVA with Bonferroni test).
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6. Figure 4 Positive Feedback between Nr4a Factors and Foxp3, Eos
(A) Flow cytometry of wild-type preTregs from the thymus of Foxp3hCD2-hCD25-KINr4a3GFP mice, cultured with IL-2 for 72 hr.
(B) Quantification of the results in (A). Percentages of Nr4a3-GFP and GITR-positive cells in the indicated populations after culture are shown.
(C) Genome browser view of chromatin immunoprecipitation sequencing (ChIP-seq) and DNaseI-HS-seq profile maps. Distribution of Foxp3-binding sites (Samstein et al., 2012), trimethylation of histone H3K4 (Wei et al., 2009), and DNaseI hypersensitive sites (Samstein et al., 2012) at Nr4a loci in Tregs are shown. Peak calls and p values are shown for anti-Foxp3 ChIP-seq results. Regions analyzed in the reporter assay in Figure S5B are indicated.
(D) qRT-PCR analysis of wild-type and Nr4a-TKO Tregs, which were transduced with control retrovirus or Nr4a2-, hEOS-, or both Nr4a2- and hEOS-expressing retroviruses. Results shown are presented relative to Hprt expression. Results shown are representative of three independent biological replicates, each performed in triplicate, means ± SD. ∗p < 0.05; ∗∗p < Student’s t test (B), one-way ANOVA with Bonferroni test (D).
See also Figure S5.
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7. Figure 5
Generation of Pathogenic Self-Reactive Cells from Nr4a-TKO Treg-Fated Thymocytes
(A) Survival of wild-type, Scurfy, and Nr4a-TKO mice (n = 12–16 per group).
(B) Flow cytometry of Annexin V binding in the indicated cells.
(C) Quantification of the results in (B).
(D) qRT-PCR of Bcl2l11 mRNA expression in the indicated cell types.
(E) Flow cytometry of autologous mixed lymphocyte reactions (MLR) of wild-type, Nr4a-TKO, and Foxp3-KO Tconv, preTreg, and Treg responder cells. Each responder cell population was labeled with CFSE and co-cultured with T cell-depleted, irradiated splenocytes derived from the same mice from which the responder cells were obtained. Cells were cultured with or without polyclonal stimulation by anti-CD3ε antibodies. Results shown are representative of three (C) or five (D) independent biological replicates, each performed in triplicate, means ± SD. ∗p < 0.05; ∗∗p < Kaplan-Meier analysis with Mantel-Cox log-rank tests (A), one-way ANOVA with Bonferroni test (C and D).
See also Figure S6.
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8. Figure 6 Pathogenic Characteristics of Nr4a-TKO Treg-Fated Thymocytes
(A) Body weight of control CD3ε−/− mice or recipient CD3ε−/− mice transferred with the indicated cells (shown as body weights relative to day 0, means ± SD).
(B) H&E staining of liver sections from CD3ε−/− mice transferred with the indicated cells. Scale bars, 100 μm.
(C) Titers of antibodies to purified recombinant proteins (Coomassie-stained gel, top) in sera collected from CD3ε−/− mice transferred with the indicated cells (n = 8 per group). Horizontal lines indicate the mean.
(D) Flow cytometry of Annexin V binding, Ki-67, Foxp3, IL-17, and IFN-γ expression of total CD3+CD4+ cells pooled from the spleens and lymph nodes of CD3ε−/− recipient mice of the indicated cells.
(E) Quantification of the results in (D). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Two-way ANOVA with Sidak test (A), one-way ANOVA with Bonferroni test (C and E).
See also Figure S6.
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9. Figure 7
Pathogenic Characteristics of Gene Expression in G113 TCR-Fixed Nr4a-TKO Treg-Fated Thymocytes
(A) A schematic of experiments shown in (B)–(D).
(B and C) Flow cytometry of TCRβ expression of total thymocytes (B) and CD4, CD8, GITR, CD25, and Foxp3 expression of TCRβ+ thymocytes (C) from mixed bone marrow chimeric Rag2−/− mice reconstituted with the indicated donor cells.
(D) qRT-PCR for the indicated genes in wild-type and Nr4a-TKO CD25hiGITRhiCD4SP thymocytes. Results shown are presented relative to Hprt expression. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < (Student’s t test).
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