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

Nature Immunology volume 21, pages 1267–1279 (2020)Cite this article

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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. Nr4a1–3 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.

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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.

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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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  1. These authors contributed equally: Corey Tan, Ryosuke Hiwa.

Authors and Affiliations

  1. Division of Rheumatology, Rosalind Russell and Ephraim P. Engleman Arthritis Research Center, Department of Medicine, University of California, San Francisco, CA, USA

    Corey Tan, Ryosuke Hiwa, James L. Mueller, Vivasvan Vykunta, Kenta Hibiya, Mark Noviski, Jeremy F. Brooks, Jose Garcia, Cheryl Heyn & Julie Zikherman

  2. Biomedical Sciences (BMS) Graduate Program, University of California, San Francisco, CA, USA

    Corey Tan & Mark Noviski

  3. HHMI Medical Fellows Program, School of Medicine, University of California, San Francisco, CA, USA

    John Huizar

  4. J. David Gladstone Institutes, San Francisco, CA, USA

    Zhongmei Li & Alexander Marson

  5. Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA, USA

    Zhongmei Li & Alexander Marson

  6. Department of Microbiology and Immunology, University of California, San Francisco, CA, USA

    Zhongmei Li & Alexander Marson

  7. Innovative Genomics Institute, University of California, Berkeley, CA, USA

    Zhongmei Li & Alexander Marson

  8. Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA

    Zhongmei Li & Alexander Marson

  9. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Zhongmei Li & Alexander Marson

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Contributions

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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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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