NR4A nuclear receptors restrain B cell responses to antigen when second signals are absent or limiting

Abstract

Antigen stimulation (signal 1) triggers B cell proliferation and primes B cells to recruit, engage and respond to T cell help (signal 2). Failure to receive signal 2 within a defined time window results in B cell apoptosis, yet the mechanisms that enforce dependence on co-stimulation are incompletely understood. Nr4a13 encode a small family of orphan nuclear receptors that are rapidly induced by B cell antigen receptor stimulation. Here, we show that Nr4a1 and Nr4a3 play partially redundant roles to restrain B cell responses to antigen in the absence of co-stimulation and do so, in part, by repressing the expression of BATF and, consequently, MYC. The NR4A family also restrains B cell access to T cell help by repressing expression of the T cell chemokines CCL3 and CCL4, as well as CD86 and ICAM1. Such NR4A-mediated regulation plays a role specifically under conditions of competition for limiting T cell help.

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Fig. 1: NUR77/Nr4a1 expression scales with BCR stimulation in vitro and in vivo.
Fig. 2: NUR77/Nr4a1 promotes BCR-induced cell death and restrains BCR-induced proliferation of B cells in vitro in a cell-intrinsic manner.
Fig. 3: NUR77/Nr4a1 restrains survival and proliferation of B cells that receive signal 1 in the absence of signal 2.
Fig. 4: NUR77/Nr4a1 selectively restrains humoral immune responses to T cell–independent type II immunogens in vivo.
Fig. 5: Conditional elimination of NR4A function in B cells reveals cooperative repression of BCR-induced B cell expansion.
Fig. 6: NUR77/Nr4a1 restrains a subset of BCR-induced PRGs.
Fig. 7: BATF mediates negative regulation of MYC expression and proliferation by NUR77/Nr4a1.
Fig. 8: NUR77/Nr4a1 restrains B cell competition for T cell help under limiting conditions.

Data availability

Raw and processed data files for the RNA-seq analyses (corresponding to Fig. 6a,b) have been deposited in the NCBI Gene Expression Omnibus under accession code GSE146747 and are provided in Supplementary Data 1. All other data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

References

  1. 1.

    Bretscher, P. & Cohn, M. A theory of self–nonself discrimination. Science 169, 1042–1049 (1970).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2.

    Cyster, J. G. & Allen, C. D. C. B cell responses: cell interaction dynamics and decisions. Cell 177, 524–540 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Damdinsuren, B. et al. Single round of antigen receptor signaling programs naive B cells to receive T cell help. Immunity 32, 355–366 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Turner, J. S., Marthi, M., Benet, Z. L. & Grigorova, I. Transiently antigen-primed B cells return to naive-like state in absence of T-cell help. Nat. Commun. 8, 15072 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    Akkaya, M. et al. Second signals rescue B cells from activation-induced mitochondrial dysfunction and death. Nat. Immunol. 19, 871–884 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Winoto, A. & Littman, D. R. Nuclear hormone receptors in T lymphocytes. Cell 109, S57–S66 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. 7.

    Lin, B. et al. Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell 116, 527–540 (2004).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8.

    Thompson, J. & Winoto, A. During negative selection, Nur77 family proteins translocate to mitochondria where they associate with Bcl-2 and expose its proapoptotic BH3 domain. J. Exp. Med. 205, 1029–1036 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Calnan, B. J., Szychowski, S., Chan, F. K., Cado, D. & Winoto, A. A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection. Immunity 3, 273–282 (1995).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. 10.

    Sekiya, T. et al. Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis. Nat. Immunol. 14, 230–237 (2013).

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Chen, J. et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature 567, 530–534 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Liu, X. et al. Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction. Nature 567, 525–529 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Cheng, L. E. C., Chan, F. K., Cado, D. & Winoto, A. Functional redundancy of the Nur77 and Nor-1 orphan steroid receptors in T-cell apoptosis. EMBO J. 16, 1865–1875 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Moran, A. E. et al. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J. Exp. Med. 208, 1279–1289 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Mullican, S. E. et al. Abrogation of nuclear receptors Nr4a3 and Nr4a1 leads to development of acute myeloid leukemia. Nat. Med. 13, 730–735 (2007).

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Mueller, J., Matloubian, M. & Zikherman, J. Cutting edge: an in vivo reporter reveals active B cell receptor signaling in the germinal center. J. Immunol. 194, 2993–2997 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Zikherman, J., Parameswaran, R. & Weiss, A. Endogenous antigen tunes the responsiveness of naive B cells but not T cells. Nature 489, 160–164 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Noviski, M. et al. Optimal development of mature B cells requires recognition of endogenous antigens. J. Immunol. 203, 418–428 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Tan, C. et al. Nur77 links chronic antigen stimulation to B cell tolerance by restricting the survival of self-reactive B cells in the periphery. J. Immunol. 202, 2907–2923 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Huang, B., Pei, H. Z., Chang, H. W. & Baek, S. H. The E3 ubiquitin ligase Trim13 regulates Nur77 stability via casein kinase 2α. Sci. Rep. 8, 13895 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  21. 21.

    Paus, D. et al. Antigen recognition strength regulates the choice between extrafollicular plasma cell and germinal center B cell differentiation. J. Exp. Med. 203, 1081–1091 (2006).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Sonoda, E. et al. B cell development under the condition of allelic inclusion. Immunity 6, 225–233 (1997).

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Lee, S. L. et al. Unimpaired thymic and peripheral T cell death in mice lacking the nuclear receptor NGFI-B (Nur77). Science 269, 532–535 (1995).

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Hobeika, E. et al. Testing gene function early in the B cell lineage in mb1-cre mice. Proc. Natl Acad. Sci. USA 103, 13789–13794 (2006).

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Kraus, M., Alimzhanov, M. B., Rajewsky, N. & Rajewsky, K. Survival of resting mature B lymphocytes depends on BCR signaling via the Igα/β heterodimer. Cell 117, 787–800 (2004).

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Huizar, J., Tan, C., Noviski, M., Mueller, J. L. & Zikherman, J. Nur77 is upregulated in B-1a cells by chronic self-antigen stimulation and limits generation of natural IgM plasma cells. Immunohorizons 1, 188–197 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Fowler, T. et al. Divergence of transcriptional landscape occurs early in B cell activation. Epigenet. Chromatin 8, 20 (2015).

    Article  CAS  Google Scholar 

  28. 28.

    Zaretsky, I. et al. ICAMs support B cell interactions with T follicular helper cells and promote clonal selection. J. Exp. Med. 214, 3435–3448 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29.

    Shiow, L. R. et al. CD69 acts downstream of interferon-α/β to inhibit S1P1 and lymphocyte egress from lymphoid organs. Nature 440, 540–544 (2006).

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Li, S. et al. The transcription factors Egr2 and Egr3 are essential for the control of inflammation and antigen-induced proliferation of B and T cells. Immunity 37, 685–696 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Betz, B. C. et al. Batf coordinates multiple aspects of B and T cell function required for normal antibody responses. J. Exp. Med. 207, 933–942 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Taub, D. D., Conlon, K., Lloyd, A. R., Oppenheim, J. J. & Kelvin, D. J. Preferential migration of activated CD4+ and CD8+ T cells in response to MIP-1α and MIP-1β. Science 260, 355–358 (1993).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. 33.

    Shrestha, B. et al. B cell-derived vascular endothelial growth factor A promotes lymphangiogenesis and high endothelial venule expansion in lymph nodes. J. Immunol. 184, 4819–4826 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Krzysiek, R. et al. Antigen receptor engagement selectively induces macrophage inflammatory protein-1 α (MIP-1α) and MIP-1β chemokine production in human B cells. J. Immunol. 162, 4455–4463 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Nowyhed, H. N., Huynh, T. R., Thomas, G. D., Blatchley, A. & Hedrick, C. C. Cutting edge: the orphan nuclear receptor Nr4a1 regulates CD8+ T cell expansion and effector function through direct repression of Irf4. J. Immunol. 195, 3515–3519 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36.

    Finkin, S., Hartweger, H., Oliveira, T. Y., Kara, E. E. & Nussenzweig, M. C. Protein amounts of the MYC transcription factor determine germinal center B cell division capacity. Immunity 51, 324–336.e5 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Caro-Maldonado, A. et al. Metabolic reprogramming is required for antibody production that is suppressed in anergic but exaggerated in chronically BAFF-exposed B cells. J. Immunol. 192, 3626–3636 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Heinzel, S. et al. A Myc-dependent division timer complements a cell-death timer to regulate T cell and B cell responses. Nat. Immunol. 18, 96–103 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Ma, Y. et al. CRISPR/Cas9 screens reveal Epstein–Barr virus-transformed B cell host dependency factors. Cell Host Microbe 21, 580–591.e7 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Inoue, T. et al. The transcription factor Foxo1 controls germinal center B cell proliferation in response to T cell help. J. Exp. Med. 214, 1181–1198 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Wingate, A. D. & Arthur, J. S. Post-translational control of Nur77. Biochem. Soc. Trans. 34, 1107–1109 (2006).

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Masuyama, N. et al. Akt inhibits the orphan nuclear receptor Nur77 and T-cell apoptosis. J. Biol. Chem. 276, 32799–32805 (2001).

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Pekarsky, Y. et al. Akt phosphorylates and regulates the orphan nuclear receptor Nur77. Proc. Natl Acad. Sci. USA 98, 3690–3694 (2001).

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Glasmacher, E. et al. A genomic regulatory element that directs assembly and function of immune-specific AP-1–IRF complexes. Science 338, 975–980 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Li, P. et al. BATF–JUN is critical for IRF4-mediated transcription in T cells. Nature 490, 543–546 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Schraml, B. U. et al. The AP-1 transcription factor Batf controls TH17 differentiation. Nature 460, 405–409 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Thomas, M. D., Kremer, C. S., Ravichandran, K. S., Rajewsky, K. & Bender, T. P. c-Myb is critical for B cell development and maintenance of follicular B cells. Immunity 23, 275–286 (2005).

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Schwickert, T. A. et al. A dynamic T cell–limited checkpoint regulates affinity-dependent B cell entry into the germinal center. J. Exp. Med. 208, 1243–1252 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49.

    Kuraoka, M. et al. Complex antigens drive permissive clonal selection in germinal centers. Immunity 44, 542–552 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Zhan, Y. et al. Cytosporone B is an agonist for nuclear orphan receptor Nur77. Nat. Chem. Biol. 4, 548–556 (2008).

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Zhan, Y. et al. The orphan nuclear receptor Nur77 regulates LKB1 localization and activates AMPK. Nat. Chem. Biol. 8, 897–904 (2012).

    CAS  PubMed  Article  Google Scholar 

  52. 52.

    Goodnow, C. C. et al. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334, 676–682 (1988).

    CAS  PubMed  Article  Google Scholar 

  53. 53.

    Barnden, M. J., Allison, J., Heath, W. R. & Carbone, F. R. Defective TCR expression in transgenic mice constructed using cDNA-based α- and β-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76, 34–40 (1998).

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Renshaw, B. R. et al. Humoral immune responses in CD40 ligand–deficient mice. J. Exp. Med. 180, 1889–1900 (1994).

    CAS  PubMed  Article  Google Scholar 

  55. 55.

    Mombaerts, P. et al. Mutations in T-cell antigen receptor genes α and β block thymocyte development at different stages. Nature 360, 225–231 (1992).

    CAS  PubMed  Article  Google Scholar 

  56. 56.

    Wholey, W. Y. et al. Synthetic liposomal mimics of biological viruses for the study of immune responses to infection and vaccination. Bioconjug. Chem. 31, 685–697 (2020).

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

We thank our funders: NIAID 5T32AI007334-28 (C.T.), the HHMI Medical Research Fellows program (J.H.), NIAMS R01AR069520 (J.Z.) the Rheumatology Research Foundation Innovative Research Award (J.Z.), and the Uehara Memorial Foundation Research Fellowship (R.H.). A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund, is an investigator at the Chan Zuckerberg Biohub and is a recipient of The Cancer Research Institute Lloyd J. Old STAR grant. A.M. has received funds from the Innovative Genomics Institute and Parker Institute for Cancer Immunotherapy.

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C.T., R.H., J.L.M., V.V., K.H., M.N., J.H., J.F.B. and J.Z. conceived of and designed the experiments. C.T., R.H., J.L.M., V.V., K.H., M.N., J.H., J.F.B., J.G. and C.H. performed the experiments. C.T., R.H., J.L.M., V.V., K.H., M.N., J.H. and J.Z. analyzed the data. J.L.M., Z.L. and A.M. generated the Nr4a3−/− mice. C.T. and J.Z. wrote the manuscript. C.T., R.H., J.L.M., M.N., J.F.B. and J.Z. edited the manuscript.

Corresponding author

Correspondence to Julie Zikherman.

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Competing interests

A.M. is a co-founder of Arsenal BioSciences and Spotlight Therapeutics and serves on their boards of directors and scientific advisory boards. A.M. has served as an advisor to Juno Therapeutics, was a member of the scientific advisory board at PACT Pharma and was an advisor to Trizell. A.M. owns stock in Arsenal BioSciences, Spotlight Therapeutics and PACT Pharma. The Marson Laboratory has received sponsored research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead and Anthem Blue Cross Blue Shield. J.Z. serves as a scientific consultant for Walking Fish Therapeutics.

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Peer review information Peer reviewer reports are available. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 NUR77 does not alter proximal BCR signal transduction or BAFFR expression.

a, b, NUR77-EGFP lymphocytes were treated as in Fig. 1h except for only 2 h. Endogenous NUR77 (A) and EGFP (B) in B220 + B cells was assessed via flow. c, Splenocytes from Nr4a1+/+ and Nr4a1−/− mice were loaded with Indo-1 dye and stained to identify CD23 + B cells. Samples were collected on a flow cytometer for 20 s, and then for 3 m following stimulation with either anti-IgM or anti-IgD. Intracellular calcium is depicted for CD23 + B cells. d, e, Splenocytes from Nr4a1+/+ and Nr4a1−/− mice were stimulated with anti-IgM for 5 m, fixed, permeabilized, and then stained for either pErk (B) or pS6 (C) followed by staining to identify CD23 + B220 + B cells. f, Splenocytes from CD45.1 + Nr4a1+/+ and CD45.2 + Nr4a1−/− mice were mixed 1:1, and then either stimulated with 10 μg/mL anti-IgM or media alone for 4 h. After stimulus wash-out and 15 m rest, cells were re-stimulated with anti-IgD for 5 m and processed as in B, C to detect intra-cellular pErk. g, h, i, Lymphocytes from CD45.1 + Nr4a1+/+ and CD45.2 + Nr4a1−/− mice were mixed 1:1 and co-cultured for 72 h with anti-IgM + /- BAFF 20 ng/ml as described. Cells were then stained to detect CD45.1, CD45.2, B220, and BAFFR. G. Plots depict surface expression of BAFFR in B and T cells either ex vivo or after culture + /- anti-IgM. H. Graph depicts BAFFR MFI in B220 + B cells. I. Graph depicts ratio of CD45.2 + Nr4a1−/− relative to CD45.1 + Nr4a1+/+ B220 + B cells after 72 h culture normalized to input ratio. Data in C-F reflect N = 2 biological replicates and in G-I reflect N = 3 biological replicates. Mean + /- SEM displayed for all graphs. Statistical significance was assessed with two-way ANOVA with Tukey’s (H); two-tailed unpaired student’s t-test with Holm-Sidak (I). **p < 0.01, ****p < 0.0001.

Extended Data Fig. 2 NUR77 restrains survival and proliferation of BCR-stimulated B cells in a cell-intrinsic manner.

a, Graph depicts live CD45.2 + B cell number corresponding to Fig. 2a,b samples. b, Lymphocytes from NUR77-EGFP mice were stimulated with anti-IgM + /- 20 ng/mL BAFF for 24 h. Graph depicts GFP MFI of live B cells. c, d, Graphs depict the ratio of CD45.2 + Nr4a1−/− or Nr4a1+/+ B cells relative to co-cultured WT CD45.1 + B cells, normalized to the input ratio, and correspond to Fig. 2. C, D samples. e, Splenocytes from Nr4a1+/+, Nr4a1+/– and Nr4a1−/− mice were stimulated with 10 μg/mL anti-IgM for 2 h. Graph depicts MFI of endogenous NUR77 expression in CD23 + B cells. f-h, Either MACS-purified B cells or total lymphocytes from CD45.2 + Nr4a1−/− and CD45.1 + Nr4a1+/+ mice were co-cultured in a 1:1 ratio with anti-IgM for 72 h. f, Histograms depict CTV dilution after culture with 6.4μg/ml anti-IgM. g, Graph depicts ratio of Nr4a1−/− CD45.2+ relative to co-cultured WT CD45.1+ purified B cells, normalized to the input ratio. h, Graph depicts division index of co-cultured purified B cells. i-j, Lymphocytes from either Nr4a1+/+ or Nr4a1−/− CD45.2+ IgHEL Tg mice were treated as in Fig. 2a. i, Graph depicts ratio of CD45.2 + B cells of each genotype relative to co-cultured CD45.1+ WT B cells, normalized to the input ratio. j, Graph depicts division index of CD45.2 + B cells for each genotype. k, l, Lymphocytes from either mb1-cre, mb1-cre Nr4a1fl/fl, or Nr4a1−/− mice were mixed 1:1 with CD45.1+ lymphocytes and treated as in Fig. 2a. k, l, Graphs depicts total B cell number (K) and division index (L) of each genotype. Data in this figure depict N = 3 biological replicates. Mean + /- SEM displayed for all graphs. Statistical significance was assessed with two-tailed unpaired student’s t-test with Holm-Sidak (A-D, G-J); two-way ANOVA with Tukey’s (K, L). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Extended Data Fig. 3 Regulation of NUR77 expression by co-stimulation.

a-c, Lymphocytes from WT mice were stimulated with the given doses of the indicated stimulus over a time course. Graphs depict endogenous NUR77 expression in B cells as determined via intracellular flow staining. Graphs in this figure depict N = 3 biological replicates for all panels. Mean + /- SEM displayed for all graphs.

Extended Data Fig. 4 NUR77 restrains B cell expansion in the absence of signal two.

a-c, Lymphocytes from CD45.1 + Nr4a1+/+ and CD45.2 + Nr4a1−/− mice were mixed 1:1, loaded with CTV, and co-cultured for 72 h with the given stimulus. Cells were then stained to detect CD45.1, CD45.2, B220, CTV via flow cytometry. a, Representative histograms depict CTV dilution of Nr4a1+/+ and Nr4a1−/− B cells stimulated with: 10 μg/mL LPS, 2 μM CPG, or 10 μg/mL anti-IgM + /- either 1 μg/mL anti-CD40 or 10 ng/mL IL-4. b, Graph depicts ratio of CD45.2 + Nr4a1−/− B cells relative to co-cultured CD45.1 + Nr4a1+/+ B cells, normalized to the input ratio. c, Graph depict % of B cells that divided at least once as determined by CTV dilution. d, Mixed co-cultures were generated as described above in (D-F) in the presence of low dose anti-IgM 1 μg/mL. Co-stimulation with 1 μg/mL anti-CD40 was added at the indicated timepoint, and cells were harvested for analysis after a total of 72 h in culture each condition. Shown are representative histograms depicting CTV dilution of live B cells of each co-cultured genotype. Histograms are representative of 4 biological replicates. e, mb1-cre and mb1-cre Nr4a1f/f (Nr4a1cKO) mice were immunized IP with 100 μg NP-Ficoll (as in Fig. 4b); anti-NP IgG3 titers were determined via ELISA at serial time points. f, Model: Ag induces NUR77 expression which restrains B cell proliferation and promotes apoptosis. Receipt of co-stimulatory signals bypasses restraint imposed by NUR77 by supplying robust proliferation and survival signals. Graphs in this figure depict N = 3 biological replicates for all panels except E which depicts N = 5 biological replicates. Mean + /- SEM displayed for all graphs. Statistical significance was assessed with one-way ANOVA with Dunnett’s (B); two-tailed unpaired student’s t-test with Holm-Sidak (C, E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Extended Data Fig. 5 Compensatory expression of Nr4a genes in Nr4a1−/− B cells and generation of Nr4a3−/− mice.

a-d, Lymphocytes from Nr4a1+/+ and Nr4a1−/− mice harboring NUR77-EGFP BAC Tg were stimulated with 10 μg/mL anti-IgM for the indicated times. qPCR was performed to determine relative expression of Nr4a1, Nr4a2, Nr4a3, and GFP transcripts. Samples were also subjected to surface staining in parallel to detect % B cells via flow cytometry. Mean % B cells for Nr4a1+/+ samples was 37.6 ± 0.15 (SEM), and for Nr4a1−/− samples was 33.5 ± 1.02 (SEM). N = 3 biological replicates for all conditions. Nr4a1+/+ samples correspond to data in Fig. 1a–c. e, Lymphocytes from reporter Nr4a1+/+ and Nr4a1−/− mice were stimulated with the given doses of anti-IgM for 24 h. Graph depicts GFP MFI in B cells as determined by flow cytometry. f, Left: Schematic showing the extent of nucleotide deletion in exon 3 of the Nr4a3 gene harboring ATG translation initiation site, resulting in the introduction of a premature stop codon. Right: Representative PCR showing absence of the WT Nr4a3 gene and the presence of a truncated product in Nr4a3−/− mice. g, h, MACS-purified B cells from Nr4a1+/+, Nr4a1−/−, and Nr4a3−/− splenocytes were stimulated with 10 μg/mL anti-IgM for the indicated times. qPCR was performed to determine relative expression of Nr4a3 (G) and Nr4a1 (H) transcripts. i, Thymocytes from WT, Nr4a1−/− and Nr4a3−/− mice were incubated + /- PMA and ionomycin for 2 h. Subsequently whole cell lysates were blotted with Ab to detect NUR77, NOR-1, MYC, and GAPDH. Red arrow indicates presence of low abundance truncated NOR-1 protein in Nr4a3−/− mice. Graphs in this figure depict N = 3 biological replicates for all panels. Mean + /- SEM displayed for all graphs. Statistical significance was assessed with two-tailed unpaired student’s t-test with Holm-Sidak (A-E); two-way ANOVA with Tukey’s (G, H). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data

Extended Data Fig. 6 B and T cell development and homeostasis in NR4A-deficient mice.

a-c, Splenocytes from WT, Nr4a1−/− and Nr4a3−/− mice were stained to detect T cell subsets. a, Plots depict gating. CM = central memory. EM = effector memory. b, c, Graphs depict T cell subsets. d, Thymocytes and splenocytes from WT, Nr4a1−/− and Nr4a3−/− mice were permeabilized and stained to detect Foxp3. Graph depicts % Tregs in CD4 + gate. e-g, Splenocytes from WT, Nr4a1−/− and Nr4a3−/− mice were stained to detect B cell subsets. e, Plots of B220 + cells (top row) to identify marginal zone (MZ). Bottom row depicts B220 + subsets after exclusion of MZ. f, g, Graphs depict B cell subsets (F) and IgM/IgD MFI on B220 + CD23 + splenocytes (G). h, i, Non-competitive chimeras were generated by reconstituting lethally irradiated CD45.1+ WT mice with BM from either CD45.2 + mb1-cre mice (cre chimera), or CD45.2+ cDKO chimera for 8-10 weeks. Graphs depict splenic T cell subsets as gated in A. N = 5 chimeras / genotype. j, k, Splenocytes from from mb1-cre, Nr4a1cKO, and cDKO mice were stained to detect B cells. Graphs depict % B220 + cells (J) and IgM/IgD MFI on B220 + splenic B cells (K). l, Graph depicts splenic subsets from chimeras above (H, I) as gated in E. m, n, Irradiated CD45.1+ recipients were reconstituted with 1:1 BM mix from donor A (CD45.1/2 WT mice) and donor B (either CD45.2 + mb1-cre control or CD45.2+ cDKO mice) for 10-12 weeks. Graphs depict relative reconstitution of B cell developmental subsets in the BM and spleen. N = 5 for chimeras each. Panels depict N = 3 biological replicates except N = 5 got chimeras (H, I, L, M, N). Mean + /- SEM for all graphs. Statistical significance was assessed with two-way ANOVA with Tukey’s (B-D, F, G, K); two-tailed unpaired student’s t-test with Holm-Sidak (H, I, L); one-way ANOVA with Tukey’s (J). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Extended Data Fig. 7 NUR77/Nr4a1 and NOR-1/Nr4a3 redundantly restrain BCR-induced B cell expansion.

a-c, Samples correspond to those described in Fig. 5e,f. a, Histograms depict CTV dilution in CD45.2 + B cells (black histogram) and co-cultured CD45.1 WT B cells (shaded gray histogram), and are representative of at least 3 mice/genotype. b, Shown is the ratio of CD45.2+ WT, Nr4a1−/− or Nr4a3−/− B cells relative to co-cultured CD45.1+ WT B cells (+ 20 ng/ml BAFF), normalized to the unstimulated condition. c, Graph depicts division index for each genotype. d, e, Samples correspond to those described in Fig. 5i,j, cultured in the presence of 20 ng/ml BAFF. d, Shown is the ratio of each CD45.2 + B cell genotype relative to co-cultured CD45.1+ WT B cells, normalized to the unstimulated condition. e, Graph depicts division index for each genotype. f-k, Competitive bone marrow chimeras were generated as described in Extended Data Fig. 5h, i. 10-12 weeks after reconstitution, lymphocytes were harvested from chimeras, CTV loaded and cultured with given stimuli (anti-IgM doses + /- 20 ng/ml BAFF + /- 10 ng/ml IL-4). N = 5 chimeras of each genotype were analyzed. f, g, h, Graphs depict ratio of CD45.2+ cre+ or cDKO B cells relative to CD45.1/2 WT B cells from each chimera, normalized to unstimulated condition. i, j, k, Graphs depict division index for CD45.2+ cre+ or cDKO B cells from each chimera. Graphs in this figure depict N = 3 biological replicates for panels (D-E) except (F-K) as noted above which represent N = 5 chimeras each. Mean + /- SEM displayed for all graphs. Statistical significance was assessed with two-way ANOVA with Tukey’s (B-E); two-tailed unpaired student’s t-test with Holm-Sidak (F-K). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Extended Data Fig. 8 Validation of NUR77 targets in BCR-stimulated B cells.

a, Relative expression of Batf transcript assessed by qPCR in purified B cells corresponding to Extended Data Fig. 5a–c samples. b, Lymphocytes from Nr4a1+/+ and Nr4a1−/− mice were stimulated with anti-IgM for 24 hours. Graph depicts MFI of BATF in B cells. c, Lymphocytes from competitive bone marrow chimeras (as in Extended Data Fig. 6m,n) were stimulated with anti-IgM for 24 h. Graph depicts MFI of BATF in CD45.2+ Cre-only or cDKO B cells. d, Graph depicts FPKM of Cd69 corresponding to Fig. 6a. e, Lymphocytes from CD45.1 + Nr4a1+/+ and CD45.2+Nr4a1−/− mice were co-cultured with anti-IgM for 24 h. Graph depicts MFI of CD69 on B cells. f, Relative expression of Cd69 transcript assessed by qPCR in purified B cells corresponding to Fig. 6j, K samples. g, Experiment performed as in Fig. 6o. Graph depicts MFI of CD69 on B cells. h, Relative expression of Cd69 transcript assessed by qPCR in purified B cells corresponding to Fig. 6l,m. i, Experiment performed as in Fig. 6q. Graph depicts MFI of CD69 on B cells. j, Experiment performed as in C above. Graph depicts MFI of CD69 on B cells. k-o, Experiments performed as in H-J, but Egr2 transcript and protein assayed instead. p, Experiment performed as in C above. Graph depicts MFI of IRF4 in B cells. q, Model: Nr4a1 and Nr4a3 feedback to restrain expression of other primary response genes (PRGs) induced by BCR stimulation. Data in this figure depict N = 3 biological replicates for all panels except in C, D, J, K, O, P which have N = 4. Mean +/− SEM displayed for all graphs. Statistical significance was assessed with two-tailed unpaired student’s t-test with Holm-Sidak (A-E, J-L, O, P); two-way ANOVA with Tukey’s (F-I, M, N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Extended Data Fig. 9 Regulation of MYC and BATF by NUR77.

a, Experiment performed as in Fig. 6o. Graph depicts MFI of intracellular MYC expression on B cells as determined via flow cytometry. b, Experiment performed as in Fig. 6q. Graph depicts MFI of intracellular MYC expression on B cells as determined via flow cytometry. c, Experiment performed as in Extended Data Fig. 8c with competitive chimeras described in Extended Data Fig. 6m,n. Graph depicts MFI of intracellular MYC expression on B cells as determined via flow cytometry. d, Publicly available ATAC-seq data (Immgen.org) was accessed to identify all consensus NR4A DNA binding motifs located within OCR < 100 kb from Batf gene in follicular mature B cells. The closest, most prominent peak was identified -20kB downstream of 3’ Batf exons, and tracks were displayed using UCSC genome browser. e, Lymphocytes from Batf+/+, Batf+/–, and Batf−/− mice were stimulated with the given doses of anti-IgM for 24 h. Graph depicts intracellular BATF expression in B cells as determined via flow cytometry. Data in this figure depict N = 3 biological replicates. Mean +/− SEM displayed for all graphs. Statistical significance was assessed with two-way ANOVA with Tukey’s (A, B); two-tailed unpaired student’s t-test with Holm-Sidak (C). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Extended Data Fig. 10 NUR77 modulates access to T cell help under competitive conditions.

a, Experiment performed as in Fig. 6o. Graph depicts MFI of surface CD86 expression on B cells as determined via flow cytometry. b, Experiment performed as in Extended Data Fig. 8c with competitive chimeras described in Extended Data Fig. 6m,n. Graph depicts MFI of surface CD86 expression on B cells as determined via flow cytometry. c, d, Lymphocytes harvested from CD45.1+Nr4a1+/+ and CD45.2+ Nr4a1−/− mice were mixed 1:1 and co-cultured with the given doses of anti-IgM for 24 h (C) or 48 h (D). Graphs depict MFI of ICAM-1 surface expression on each genotype of B cells as determined via flow cytometry. e, f, Schematic of adoptive transfer experimental design for Fig. 8. B cells were purified from splenocytes harvested from Nr4a1+/+ B1-8 Tg CD45.1/2+ and Nr4a1−/− B1-8 Tg CD45.2+ mice via bench-top negative selection, mixed 1:1, and loaded with CTV. 2x106-3x106 B cells were then adoptively transferred +/− OTII splenocytes into either WT or CD40L−/− hosts. Host mice were then immunized IP one day later with 100 μg NP-OVA/alum, followed by spleen harvest on d4. g, Graph depicts adoptive transfer data from experiments described in E, F above and presented in Fig. 8h with addition of unimmunized controls and CD40L−/− hosts that did not receive adoptive transferred OTII cells (shaded gray points). Data in this figure depict N = 3 biological replicates except B (N = 4) and G where individual biological samples are plotted. Mean +/− SEM displayed for all other graphs. Statistical significance was assessed with two-way ANOVA with Tukey’s (A); two-tailed unpaired student’s t-test with Holm-Sidak (B-D). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Supplementary Data 1

RNA-seq, RSEM FPKM and edgeR DEG lists.

Supplementary Data 2

Exact P values for all figures and extended data figures.

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Source Data Fig. 5

Unprocessed western blots corresponding to Fig. 5b.

Source Data Extended Data Fig. 5

Unprocessed western blots corresponding to Extended Data Fig. 5i.

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Tan, C., Hiwa, R., Mueller, J.L. et al. NR4A nuclear receptors restrain B cell responses to antigen when second signals are absent or limiting. Nat Immunol 21, 1267–1279 (2020). https://doi.org/10.1038/s41590-020-0765-7

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