(2021), International Journal of Cancer eCollection 2020. Harris, J. E. et al. This study thus identifies NR4A1 as a key general regulator in the induction of T cell dysfunction, and a potential target for tumour immunotherapy. For each sample, the total number of identified islands is listed, followed by the number of islands for each genomic region (with the percentage of the total). Transcriptional regulatory network for the establishment of CD8. 17, 304–314 (2016). c, Haematoxylin and eosin staining of colon tissues. Super-shift experiments (lane 3) were conducted in the presence of anti-c-Jun antibody. Fig. See this image and copyright information in PMC. Xindong Liu or Xiu-Wu Bian or Chen Dong. 199, 15–24 (2004). Using the AP-1 consensus motif as a probe, DNA binding was performed on nuclear extracts from empty vector or RV-Nr4a1-GFP-transduced CD4+ T cells stimulated with PMA/ionomycin (PMA/Iono) for the indicated time periods. have filed a patent application (PCT/CN2018/072044) on the role of NR4A1 in T cells. 2 Distribution of H3K4me3 and H3K27me3 peaks in mouse CD4, Extended Data Fig. Nat. The library was screened (using hypergeometric enrichment calculations in the HOMER program) for reliable NR4A1 motifs; each enriched motif with a specific P value. Eight days after infection, mice were euthanized and donor cells were assessed. 2015;16(1):38-46. doi: 10.2174/1389450115666141120112818. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. NR4A1 is stably overexpressed in T tol cells. Exhausted T cells were initially characterized in mice infected with chronic lymphocytic choriomeningitis virus (LCMV), but a similar T cell phenotype was also later reported in humans with hepatitis C virus (HCV) or human immunodeficiency virus (HIV) infections, and in human cancer. helped with plasmid construction and EMSA experiments. 2020 Nov;50(11):1643-1652. doi: 10.1002/eji.202048869. Epub 2015 Sep 22. Bethesda, MD 20894, Copyright b, Hierarchical clustering and principal component analysis of six types of CD4+ T cells (Ttol, naive, TH1, TH2, TH17 and nTreg). National Library of Medicine PLoS One. Annu. An OCT-1-binding consensus probe was used as a control. Bottom, the classical NR4A1-binding consensus sequence. Fig. The nuclear orphan receptor NR4A1 and NR4A3 as tumor suppressors in hematologic neoplasms. and C.D. SICER v.1.1 was used to identify significant peaks (FDR of 5%), with input DNA (ChIP–seq) as the control. Extended Data Fig. Molecular and transcriptional basis of CD4+ T cell dysfunction during chronic infection. A.W., B.Z., X.W. Immunity 21, 167–177 (2004). NR4A1 binding also promotes acetylation of histone 3 at lysine 27 (H3K27ac), leading to activation of tolerance-related genes. (2021), Current Opinion in Virology n = 4 per group. 7 NR4A1 deficiency promotes CD8, Extended Data Fig. 7. n = 5 mice (8 weeks old) per group. Hierarchical clusters of gene modules of transcription factors, nucleosomes, ribosomes, spliceosome/RNA processing, mitochondria, ubiquitination and proteasome are shown. Nat. Co-inhibitory molecule B7 superfamily member 1 expressed by tumor-infiltrating myeloid cells induces dysfunction of anti-tumor CD8+ T cells. The role of metabolic processes during T cell activation has been increasingly acknowledged, and recent data suggest an impact of T cell immunometabolism on T cell function and T cell-mediated autoimmunity. Fig. 4, 882–890 (2003). Chen J, López-Moyado IF, Seo H, Lio CJ, Hempleman LJ, Sekiya T, Yoshimura A, Scott-Browne JP, Rao A. h, H3K27ac and NR4A1 peaks at Pdcd1, Havcr2 and Lag3 loci. c, Flow cytometry analysis and quantification of the T cell exhaustion surface markers PD-1 and TIM-3 in tumour-infiltrating donor T cells. Fig. Anderson, A. C., Joller, N. & Kuchroo, V. K. Lag-3, Tim-3, and TIGIT: co-inhibitory receptors with specialized functions in immune regulation. Four weeks after infection, wild-type and Nr4a1−/− CD8+ T cells in the spleen were analysed by flow cytometry. 2015 Dec;129(12):1151-61. doi: 10.1042/CS20150346. Accessibility Immunity 48, 773–786.e5 (2018). Xie X, Song X, Yuan S, Cai H, Chen Y, Chang X, Liang B, Huang D. Clin Sci (Lond). and X.-W.B. Macián, F. et al. The E3 ubiquitin ligase GRAIL regulates T cell tolerance and regulatory T cell function by mediating T cell receptor-CD3 degradation. 2. h, Flow cytometry analysis and quantification of IFNγ and TNF expression in tumour-infiltrating donor cells after OT-I peptide restimulation. Chan, C. J. et al. h, H3K4me3 and H3K27me3 modifications as well as gene expression profiles were normalized separately with GeneSpring software. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Immunol. n = 4 per group. and JavaScript. C.D. March 13, 2019. Immunol. ); an Institute Project grant (SWH2015QN07 and SWH2016HWHZ-01 to X.L. 7, 1130–1132 (2006). A distinct gene module for dysfunction uncoupled from activation in tumor-infiltrating T cells. This file contains Supplementary Tables 1-7 – see Supplementary Information file for full legends. 8. Fig. n = 4 per group. 2021 Feb 7;24(3):102166. doi: 10.1016/j.isci.2021.102166. 'Nur'turing tumor T cell tolerance and exhaustion: novel function for Nuclear Receptor Nur77 in immunity. Nat. The transcription factor NFAT promotes exhaustion of activated CD8+ T cells. eCollection 2020. Lith SC, van Os BW, Seijkens TTP, de Vries CJM. Deletion of NR4A1 in T cells results in overexpression of IL-2…. 2019 May;19(5):251. doi: 10.1038/s41568-019-0138-4. Extended Data Fig. Science 354, 1165–1169 (2016). TxStart, start of transcription; TxEnd, end of transcription. g, H3K4me3 and H3K27me3 histone modifications across the T cell-anergy-related genes Egr3, Dgkz and Cdkn2a, and the T cell-exhaustion-related genes Lag3, Pdcd1 and Tigit, for indicated T cell subsets. EMBO J. EMBO Rep. 9, 50–55 (2008). 8600 Rockville Pike Jeon, M. S. et al. Mice were euthanized 6 days after T cell transfer. Histone acetylation regulates orphan nuclear receptor NR4A1 expression in hypercholesterolaemia. T cells expressing chimeric antigen receptors (CAR T cells) targeting human CD19 (hCD19) have shown clinical efficacy against B cell malignancies 1,2.CAR T cells have been less effective against solid … and C.D. ); the National Natural Science Foundation of China (31630022 and 91642201 to C.D., and 31770973 to X.L. known to lead to T cell dysfunction, and further analyses indicated that NR4A1 mediates T cell exhaustion by antagonizing the AP-1-mediated transcriptional programme. 3: Disruption of NR4A1 prevents T cell exhaustion caused by tumour and viral infection. 5. There were also increased expression of the TCA components Dlst and Idh3a. Intriguingly, although c-JUN overexpression increased expansion of CAR T cells… 3. 1. Exp Mol Med. We identify positive feedback loops connecting TOX and NR4A and suggest that diminished expression or activity of these transcription factors … Scott-Browne, J. P. et al. However, the molecular mechanisms that underlie this dysfunction remain unclear. 5. We identify the nuclear receptor Nur77 as central regulator of T cell … 23, 515–548 (2005). The recent discovery that Nur77 plays a crucial role in T cell tolerance and exhaustion … We show that an initiating NFAT induces secondary transcription factors of the TOX and NR4A families, which are critical for CD8 + T cell exhaustion downstream of NFAT. Immunol. NR4A1-mediated transcriptional regulation of T…. Data are representative of two individual experiments. Dynamic changes in chromatin accessibility occur in CD8+ T cells responding to viral infection. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells. show that Nr4a3, but not Nr4a1, is NFAT pathway dependent, resulting in restricted Nr4a3-Timer expression during T cell development. 4: NR4A1-mediated transcriptional regulation of T cell tolerance. Eight days later, donor-derived T cells were sorted from spleens and assessed. Notably, the transcription factor NR4A1 is stably expressed at high levels in tolerant T cells. S.X. Whyte, W. A. et al. Cell 155, 934–947 (2013). Immunol. Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction. 3. These authors contributed equally: Xindong Liu, Yun Wang, Huiping Lu. If the development of terminal exhaustion is a linear process (Beltra et al., 2020), and because the transcriptional profile of T E cells appears the most functional, T M cells might give rise to T E cells, which in turn might develop into T … Nat. BATF and NR4A1 could regulate PD-1 to inhibit T cell function [ 30, 31 ]; EGR2 targets … All data are representative of two independent experiments and graphs show mean ± s.d. 2021 Feb 2;11:622509. doi: 10.3389/fimmu.2020.622509. However, the molecular mechanisms that underlie this dysfunction remain unclear. d, Equal amounts of CD45.1+ wild-type and CD45.2+Nr4a1−/− bone marrow cells (4 × 106 per mouse) were transferred into Rag1−/− mice. 25, 2623–2633 (2006). Trends Immunol. Nr4a reporter mice are useful tools for studying TCR signaling. Science 354, 1160–1165 (2016). Another possible interpretation is that c-JUN competes for chromatin binding with NR4A family members, which regulate T cell exhaustion, as c-JUN and NR4A1 share a substantial number of common chromatin-binding sites . e, Flow cytometry analysis of IFNγ, IL-4, IL-17A and FOXP3 in RV-vector-GFP- or RV-Nr4a1-GFP-infected CD4+ T cells under TH1, TH2, TH17 and iTreg cell polarization conditions. c, Flow cytometry analysis and quantification of KLRG1 and CD127 expression in Nr4a1−/− and Nr4a1+/− P14 cells in the spleen. Epub 2019 Feb 27. Deletion of NR4A1 in T cells exacerbates colitis. Nan Fang Yi Ke Da Xue Xue Bao. We thank the C.D., X.L. a, Summaries of H3K4me3 and H3K27me3 peaks in mouse CD4+ T cells including Ttol, TH1, TH2, TH17, nTreg, iTreg and naive T cells. 1. d, qRT–PCR measurement of gene expression in RV-Nr4a1-GFP- and empty vector-transduced CD4+ T cells under neutral polarization conditions. J.L., J.H., W.J. The colour coding indicates the expression level of 10,462 genes, with 0 as the median. Data are representative of three individual experiments and graphs show mean ± s.d. Correspondence to Disruption of NR4A1 prevents T cell exhaustion caused by tumour and viral infection. P values were calculated using a two-sided unpaired Student’s t-test. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Liu, X., Wang, Y., Lu, H. et al. 6. P values were calculated using a two-sided unpaired Student’s t-test. BACH2 regulates CD8+ T cell differentiation by controlling access of AP-1 factors to enhancers. 1 Comparison of gene expression profiles among CD4, Extended Data Fig. Role of the Orphan Nuclear Receptor NR4A Family in T-Cell Biology. a, Flow cytometry analysis and quantification of EOMES and T-bet expression in Nr4a1−/− and Nr4a1+/− P14 cells in the spleen. Source data. Extended Data Fig. b, Distribution of H3K4me3 and H3K27me3 modifications within gene bodies in mouse CD4+ T cells. Whereas anti–CTLA-4 seems to act by depleting intratumoral regulatory T cells (1, 2), antibodies to PD-1 or PD-L1 act by overcoming a hyporesponsive state, termed “exhaustion” or “dysfunction,” that … Data are representative of two individual experiments and graphs show mean ± s.d. Nat Rev Cancer. A growing number of transcription factors have been implicated in T cell exhaustion including T-BET, EOMES, Spry2, BLIMP-1, VHL, FOXO1, IRF, BATF, and NFATC1 (96,101) and more recently, the NR4A family members, NR4A1… Nat. Recently, a pattern of chromatin accessibility enriched for consensus motifs for Nr4a and NFAT transcription factors was specifically associated with T-cell exhaustion (32). Epub 2020 Oct 25. The function of T cells is tightly regulated by a combinational co-stimulatory signal, and dominance of negative co-stimulation results in T cell dysfunction2. Nat. ChIP–seq and data analysis of NR4A1 and H3K27ac in CD4 +…, Extended Data Fig. Immunol. Rev. Careers. 4 Deletion of NR4A1 in T cells results in overexpression of IL-2 and IFNγ. X.L. Nat. X.L. Wenzl K, Troppan K, Neumeister P, Deutsch AJ. a–c, Naive OT-II cells were transferred into naive CD45.1+ (B6SJL) recipient mice, followed by either intraperitoneal injection of OVA emulsified in CFA (activated group), or intravenous injection of soluble OT-II peptide (500 μg per mouse) twice at day 0 and day 3 (tolerant group). NR4A1-mediated transcriptional regulation of T cell tolerance. Safford, M. et al. d, Experimental strategy for assay of adoptively transferred T cells in tumour-bearing mice. The receptors CD96 and CD226 oppose each other in the regulation of natural killer cell functions. Comparison of gene…, Extended Data Fig. i, Flow cytometry analysis and quantification of CD107A expression in tumour-infiltrating donor cells. Histogram indicates the number of peaks at various distances from NR4A1 to c-Jun peak summits. Fig. 1. wrote the manuscript and supervised the study. d. Flow cytometry analysis of donor-derived CD25+FOXP3+ Treg cells in lamina propria (LP), mesenteric lymph nodes (mLN) and spleens (SP) 28 days after T cell transfer. The expression of NR4A1, a TF that induces T cell exhaustion [27, 28], was upregulated at later stages of the exhaustion state. Jennings et al. & Gajewski, T. F. Molecular regulation of T-cell anergy. from MD Anderson Cancer Center; and NIH grants (R01HL143520, R56AI125269, R21AI120012 and R03CA219760 to R.N.). Eight weeks after reconstitution, mice were infected with LCMV-clone 13. volume 567, pages525–529(2019)Cite this article. Graphs show ChIP–qPCR measurement of c-Jun enrichment at the Jund locus and Pgpep1 and Naf1 promoter regions. Detailed information for ChIP–seq samples is provided in Supplementary Table 5. n = 1 for ChIP–seq samples. n = 3 mice per group. conducted ATAC-seq and data analysis. Immunity 44, 989–1004 (2016). Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Mice were euthanized four weeks after T cell transfer. Google Scholar. e, NR4A1-binding consensus in CD4+ T cells. Immunity 40, 289–302 (2014). Enhanced T cell responses due to diacylglycerol kinase ζ deficiency. 4. Nat. 18, 173–183 (2017). helped with sample preparation for ChIP–seq. Extended Data Fig. For d, f–h, SICER v.1.1 was used to identify significant peaks (FDR of 5%) with input DNA (ChIP–seq) as the control. Transcriptional mechanisms underlying lymphocyte tolerance. 14, 230–237 (2013). T-cell activation in the absence of Nr4a1 results in increased expression of the ETC components Atp5c1, Atp5f1, Atp5h, Cox6a1, Cox6b2, Cox7b, Ndufa6, Ndufa5, Ndufa8 and Ndufa11 . P values were calculated using a two-sided unpaired Student’s t-test. Please enable it to take advantage of the complete set of features! & Greenberg, P. D. Tolerance and exhaustion: defining mechanisms of T cell dysfunction. Exhausted CD8+T Cells in the Tumor Immune Microenvironment: New Pathways to Therapy. The Transcription factors such as HIF-1α, NR4A1, TOX, Eomes, T-bet, Blimp-1, NFAT and BATF regulate PD-1 expression and have been implicated in T cell exhaustion and dysfunction. OD, optical density. P values were calculated using a two-sided unpaired Student’s t-test. Article  All of the samples, except nTreg cells, were duplicated. eCollection 2016. Essential role of the E3 ubiquitin ligase Cbl-b in T cell anergy induction. d, Percentage of genes associated with H3K4me3 alone (K4), H3K27me3 alone (K27), both H3K4me3 and H3K27me3 (K4&K27), or neither (None), for each T cell subset as indicated. Martinez, G. J. et al. f, Genome-wide H3K4me3 and H3K27me3 histone modifications across the gene loci for three master transcription factors (Tbx21, Gata3 and Rorc) for indicated T cell subsets. Here, using an in vitro T cell tolerance induction system in mice, we characterize genome-wide epigenetic and gene expression features in tolerant T cells, and show that they are distinct from effector and regulatory T cells. Nature Thank you for visiting nature.com. f, Genome-wide co-localization of c-Jun with NR4A1-binding sites in CD4+ T cells. analysed the microarray and ChIP–seq data. Immunity 45, 1327–1340 (2016). The mouse genome (mm9) is divided into four regions: promoter (1 kb upstream and downstream of the TSS), exon, intron and intergenic regions. d, e, Chimeric mice were generated by transferring equal amounts of CD45.1+ wild-type and CD45.2+ Nr4a1−/− bone marrow cells into Rag1−/− mice. d, Venn diagram of NR4A1-regulated genes, Ttol cell-related genes and NR4A1-targeted genes. Greenwald, R. J., Freeman, G. J. d, Ingenuity pathway analysis (IPA) of differentially expressed genes in Ttol cells, with twofold as the cut-off. Sekiya T, Kagawa S, Masaki K, Fukunaga K, Yoshimura A, Takaki S. iScience. a–c, CD45.1+CD45.2+ (B6SJL × C57BL6) recipient mice were injected subcutaneously with E.G7 tumour cells (5 × 105 cells per mouse) in one flank. Egr-2 and Egr-3 are negative regulators of T cell activation. Donor-derived T cells were collected from the lamina propria and analysed for IFNγ and IL-17A expression by flow cytometry. Kalekar, L. A. et al. c, ELISA measurement of IFNγ and IL-2 expression in splenocytes from wild-type and Nr4a1−/− mice in the context of food oral tolerance. Kitagawa, Y. et al. You are using a browser version with limited support for CSS. N.S., not significant. Source data, a, qRT–PCR measurement of expression of Nr4a1, Il2 and Ifng in wild-type and Nr4a1−/− CD4+ and CD8+ T cells stimulated with plate-coated anti-CD3 and anti-CD28 at indicated time points. Odagiu L, May J, Boulet S, Baldwin TA, Labrecque N. Front Endocrinol (Lausanne). Article  & Sharpe, A. H. The B7 family revisited. Nat. Pie chart represents the genome-wide distribution of NR4A1 occupancy in promoter, exon, intron and intergenic regions in RV-Nr4a1-transduced CD4+ T cells, after peak calling and subtraction of IgG control peaks. Notably, the transcription factor NR4A1 is stably expressed at high levels in tolerant T cells. Subsequent studies showed that the severity of T cell exhaustion typically correlated with the number of inhibitory receptors that were co-expressed. 9 NR4A1 inhibits recruitment of AP-1 components in CD4, https://doi.org/10.1038/s41586-019-0979-8, Remodeling the chromatin landscape in T lymphocytes by a division of labor among transcription factors, Regulation of peripheral Th/Treg differentiation and suppression of airway inflammation by Nr4a transcription factors, Origin and fine-tuning of effector CD8 T cell subpopulations in chronic infection, Long‐term in vitro expansion ensures increased yield of central memory T cells as perspective for manufacturing challenges, Tolerance induction in memory CD4 T cells is partial and reversible. Source data, a–c, Congenic CD45.1+B6SJL mice received equal amounts of CD45.2+ Nr4a1−/− cells or Nr4a1+/− P14 cells (1 × 104 per mouse) and were infected with LCMV-Armstrong at the dosage of 2 × 105 PFU. Schietinger, A. Donor-derived T cells were collected from tumour, draining lymph nodes and spleens, and subjected to flow cytometry analysis. NR4A1 binding also promotes acetylation of histone 3 at lysine 27 (H3K27ac), leading to activation of tolerance-related genes. CD4+ T cell anergy prevents autoimmunity and generates regulatory T cell precursors. b, Caeca and colons were collected from Rag1−/− mice with wild-type or Nr4a1−/− CD4+ T cells. and X.-W.B. NR4A1 is required for T cell tolerance formation. 3 NR4A1 is stably overexpressed in T. Extended Data Fig. Immunity 32, 670–680 (2010). The function of T cells is tightly regulated by a combinational co-stimulatory signal, and dominance of negative co-stimulation results in T cell dysfunction2. c, Comparison of global distribution of H3K27me3 modifications at promoter, exon, intron and intergenic regions among different T cell subsets. T cell dysfunction – in the form of exhaustion, anergy, or tolerance – is known to limit the effector functions of T cells, but the molecular mechanisms that induce … Genome-wide distributions and heatmaps are shown for H3K27ac around TSS and super-enhancer regions. Here, using an in vitro T cell tolerance induction system in mice, we characterize genome-wide epigenetic and gene expression features in tolerant T cells, and show that they are distinct from effector and regulatory T cells. Immunity 30, 155–167 (2009). Data availability. NR4A transcription factors limit CAR T cell function in solid tumours. ISSN 1476-4687 (online). Immunol. e, Sizes of E.G7 tumour. a, b, Flow cytometry analysis and quantification of IFNγ expression (a) and TNF expression (b) in donor-derived T cells from tumours. FOIA b, Flow cytometry analysis of IFNγ and IL-17A expression in wild-type and Nr4a1−/− CD4+ and CD8+ T cells from lymph nodes (LN) and spleens (SP). g, Histogram showing PD-1, TIM-3 and BCL-2 expression in tumour-infiltrating donor T cells. Crawford, A. et al. ); the Natural Science Foundation Project of Chongqing (CSTC2014JCYJYS10001 to X.L. X.L. PBS-treated mice were used as a control group. 17, 851–860 (2016). This study thus identifies NR4A1 as a key general regulator in the induction of T cell dysfunction, and a potential target for tumour immunotherapy. Would you like email updates of new search results? Nature. Extended Data Fig. T cell exhaustion has emerged as an important topic of investigation, relevant not only in T cell biology but also in the clinic because the presence of exhausted cells correlates with poor … The colour coding depicts the normalized value for each gene based on the following scales: expression (−2 to +2), H3K4me3 (−2 to +3) and H3K27me3 (−1 to +3), with 0 as the median. PubMed Google Scholar. Chr, chromosome. Mechanistically, NR4A1 is preferentially recruited to binding sites of the transcription factor AP-1, where it represses effector-gene expression by inhibiting AP-1 function. & Dong, C. Molecular mechanisms of T-cell tolerance. b, qRT–PCR measurement of T cell activation-related and tolerance-related genes. Six-week-old mice; n = 3 per group (a–c); n = 4 per group (d). T-cell tolerance or function is determined by combinatorial costimulatory signals. Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China, Xindong Liu, Yun Wang, Rui Huang, Jin Wu, Qiwen Zhao, Senlin Xu, Shicang Yu, Yan Wang & Xiu-Wu Bian, Huiping Lu, Jing Li, Jing Hao, Qi Wu, Xiaohu Wang, Wei Jin, Ling Ni & Chen Dong, Institute for Systems Biology, Seattle, WA, USA, Institute of Immunology, Third Military Medical University (Army Medical University), Chongqing, China, MD Anderson Cancer Center, Houston, TX, USA, Andrei Alekseev, Hiep Khong, Tenghui Chen, Aibo Wang & Roza Nurieva, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China, Medical Research Institute, School of Medicine, Wuhan University, Wuhan, China, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Shanghai, China, You can also search for this author in Nurieva, R. et al. In their study, the authors hypothesized a role for NR4A1 in CD8 + T cell exhaustion based on the observation that Nr4a1 transcription is abolished and that the NBRE motif is lost in open chromatin regions in CD8 + TILs following anti-PD1 treatment, a treatment that reinvigorate exhausted T … 241, 133–144 (2011). Guidance of regulatory T cell development by Satb1-dependent super-enhancer establishment. This file contains the source data for tumour growth. ChIP–seq and microarray data have been deposited in the GEO, with accession code GSE96969. helped with colon tissue histology staining. NR4A1 inhibits recruitment of AP-1 components in CD4 + T cells. ADS  laboratory members for their assistance and O. M. Conneely for the Nr4a1−/− mouse strain. CAS  https://doi.org/10.1038/s41586-019-0979-8, DOI: https://doi.org/10.1038/s41586-019-0979-8, Immunological Reviews g, H3K27ac and NR4A1 peaks at the Hivep3 locus in naive and 6-h-activated wild-type and NR4A1 knockout CD4+ T cells, as well as RV-vector- and RV-Nr4a1-transduced CD4+ T cells. Nr4a3-Timer requires stronger and/or longer TCR signals than Nr4a1 … c, Naive CD4+ T cells were sorted from wild-type and Nr4a1−/− mice and activated with plated anti-CD3 and anti-CD28 for 36 or 72 h. Activated T cells were collected and subjected to ChIP assay using anti-c-Jun antibody, followed by qPCR analysis. Roychoudhuri, R. et al. Pauken, K. E. et al. Article  c, Correlation analysis of CD4+ T cell subsets using a k shared nearest neighbours classification algorithm. Singer, M. et al. 173, 7331–7338 (2004). e, ChIP–qPCR measurement of c-Jun enrichment at the Il2 locus in wild-type and Nr4a1−/− CD4+ T cells activated with plated anti-CD3 and anti-CD28 for 36 or 72 h. f, H3K27ac and NR4A1 peaks at the Nr4a1, Bach2, Nt5e and Samhd1 loci in RV-vector- and RV-Nr4a1-transduced CD4+ T cells.