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Disrupting Roquin-1 interaction with Regnase-1 induces autoimmunity and enhances antitumor responses

Nature Immunology volume 22, pages 1563–1576 (2021)Cite this article

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Abstract

Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies.

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Fig. 1: Roquin-1/2 and Regnase-1 maintain quiescence of T cells.
Fig. 2: Roquin-1/2 and Regnase-1 control TFH differentiation and humoral autoimmunity.
Fig. 3: Inactivation of Roquin-1/2 or Regnase-1 in CD8+ T cells enhances cytotoxicity.
Fig. 4: Functional definition of Roquin-1 interaction with Regnase-1.
Fig. 5: Molecular determinants of Roquin-1 interactions with Regnase-1.
Fig. 6: Inhibition of Roquin-1 interaction with Regnase-1 breaks T cell quiescence.
Fig. 7: Combined heterozygosity of Roquin-1E212K/M199R shows a synthetic phenotype.
Fig. 8: Disrupting Roquin-1 interaction with Regnase-1 improves effector function of tumor-specific T cells.

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Data availability

All data are provided in the article and its supplementary files or from the corresponding author upon reasonable request. For analysis of the Roquin structure the publicly available structural datasets of Roquin-1 ROQ domain bound to RNA (PDB 4QI2) and Roquin-1 ROQ-HEPN domain (PDB 4TXA) were used. Source data are provided with this paper.

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Acknowledgements

We thank D.H. Busch (TU Munich) for critical reading of the manuscript. We thank C. Keplinger (Helmholtz Zentrum München) and L. Esser and J. Klein (Ludwig-Maximilians-Universität, Munich) for excellent technical support. We thank A. Blutke, J. Kranich and R. Schieweck (Ludwig-Maximilians-Universität, Munich) for support with histology and polysome profiles and the BMC Core Facility (Ludwig-Maximilians-Universität, Munich) for support in flow cytometry and the immunoanalytics platform for their support in chromium release assays (E. Noessner, Helmholtz Zentrum München). For the provision of NanoBret constructs we thank K.-P. Knobeloch (University Clinic Freiburg) and D. Zehn (TU Munich) for providing the MigR1-OVA-GFP retroviral plasmid. The work was supported by the German Research Foundation grants SPP-1935 (to D.N. and V.H.), SFB-TRR338 (projects C02 to V.H. and C05 to S.T.), SFB-1054 (project A03) as well as HE3359/7-1 and HE3359/8-1 to V.H. and under Germany’s Excellence Strategy within the framework of the Munich Cluster for Systems Neurology (EXC 2145 SyNergy, ID 390857198 to W.W.) and SFB-870 (project A13 to W.W.) as well as grants from the Wilhelm Sander, Fritz Thyssen, Else Kröner-Fresenius and Krebshilfe foundations to V.H.

Author information

Author notes

  1. These authors contributed equally: Gesine Behrens, Stephanie L. Edelmann, Timsse Raj.

Authors and Affiliations

  1. Institute for Immunology, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität in Munich, Planegg-Martinsried, Germany

    Gesine Behrens, Timsse Raj, Nina Kronbeck, Elaine H. Wong, Christine Hohn & Vigo Heissmeyer

  2. Research Unit Molecular Immune Regulation, Helmholtz Zentrum München, Munich, Germany

    Stephanie L. Edelmann, Lisa Kifinger, Laura S. de Jonge & Vigo Heissmeyer

  3. Institute of Pharmaceutical Biotechnology, Ulm University, Ulm, Germany

    Thomas Monecke & Dierk Niessing

  4. Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany

    Elena Davydova & Dierk Niessing

  5. Institute of Developmental Genetics, Helmholtz Zentrum München, Neuherberg, Germany

    Florian Giesert & Wolfgang Wurst

  6. Cancer and Immunometabolism Research Group at the Gene Center, Ludwig-Maximilians-Universität in Munich, Munich, Germany

    Martin E. Kirmaier & Sebastian Theurich

  7. Department of Medicine III, LMU University Hospital, Ludwig-Maximilians-Universität in Munich, Munich, Germany

    Martin E. Kirmaier & Sebastian Theurich

  8. Core Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine at the Biomedical Center, Ludwig-Maximilians-Universität in Munich, Planegg-Martinsried, Germany

    Mariano Gonzalez Pisfil

  9. Department of Basic Medical Science, School of Medicine, University of Missouri-Kansas City, Kansas City, MO, USA

    Mingui Fu

  10. Department of Pathology, New York University School of Medicine, New York, NY, USA

    Stefan Feske

  11. Institute of Clinical Neuroimmunology, University Hospital and Biomedical Center, Ludwig-Maximilians-Universität in Munich, Planegg-Martinsried, Germany

    Naoto Kawakami

  12. German Center for Neurodegenerative Diseases (DZNE) Site Munich, Munich, Germany

    Wolfgang Wurst

  13. Technische Universität München, Lehrstuhl für Entwicklungsgenetik c/o Helmholtz Zentrum München, Munich, Germany

    Wolfgang Wurst

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  1. Gesine Behrens
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Contributions

G.B., S.L.E, T.R., N. Kronbeck and V.H. conceived the project and designed experiments with input from S.F., D.N. and N. Kawakami. G.B., S.L.E., T.R. and N. Kronbeck performed and analyzed most of the experiments. T.M. performed in vitro pulldown experiments, E.D. performed EMSA experiments and SPR assays and E.W. performed ELISA measurements. L.K. performed NanoBret assays and, together with M.G.P., FLIM/FRET experiments and C.H. contributed to kinetic expression analyses. M.K. and L.D.J. performed seahorse measurements with supervision from S.T. F.G. and W.W. generated mice expressing Roquin-1L209Y and M.F. provided the Zc3h12afl mouse line. The manuscript was written by G.B. and V.H. with critical input from S.F.

Corresponding author

Correspondence to Vigo Heissmeyer.

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The authors declare no competing interests.

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Peer review information Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. 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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Roquin-1/2 and Regnase-1 maintain quiescence of T cells.

(a, b) Analysis of mixed bone marrow chimeric mice using either WT (CD45.2) and WT (CD45.1) or TKOT (CD45.2) and WT (CD45.1) bone marrow cells injected into lethally irradiated CD45.1/2 recipient mice. Flow cytometry analysis of CD45.1 and CD45.2 cell populations (a) or Treg cells (b) in splenocytes from recipient mice 9 weeks after reconstitution (WT CD45.1/WT CD45.2 recipients: n = 2, WT CD45.1/TKO CD45.2 recipients: n = 3, analyzed in one experiment). (c) H&E sections of lungs showing alveoli from WT, DKOT, KOT and TKOT mice at the age of 6-8 weeks (Representative data of n ≥3 individual mice). (d, e) Analysis of CD45.2+ CD3+ T cells from Cre-ERt2 (WT), Rc3h1/2fl/fl; Cre-ERt2 (iDKO), Zc3h12afl/fl; Cre-ERt2 (iKO) and Rc3h1/2fl/fl; Zc3h12afl/fl; Cre-ERt2 (iTKO) mice that were adoptively transferred into WT CD45.1+ mice. Recipient mice were treated with tamoxifen by oral gavage to induce deletion of floxed alleles. On day 8 post transfer, T cells were analyzed for their ability to acquire an effector/memory phenotype (d) or to proliferate (e) within the host (n = 6 biological replicates).

Source data

Extended Data Fig. 2 Roquin-1/2 and Regnase-1 control metabolism and humoral autoimmunity.

CD4+ (a–e) or CD8+ (f, g) T cells from Cre-ERt2 (WT), Rc3h1/2fl/fl; Cre-ERt2 (iDKO), Zc3h12afl/fl; Cre-ERt2 (iKO) and Rc3h1/2fl/fl; Zc3h12afl/fl; Cd4-Cre-ERt2 (iTKO) were treated with 4´-OH tamoxifen in vitro to induce deletion of floxed alleles. T cells were activated in vitro and expanded with IL-2 medium for 2d. IL-2 was withdrawn overnight before T cells were restimulated with anti-CD3/28 prior to glycolytic (a, b, f) and mitochondrial stress tests (c–e, g). Shown are calculated ratios for ECAR (mpH/min/10000 cells) (a–c) and OCR (pmol/min/10000 cells) (d–e) relative to WT, respectively. Naïve CD45.2+ CD4+ T cells from Cd4-Cre-ERt2 (WT), Rc3h1/2fl/fl; Cd4-Cre-ERt2 (iDKO), Zc3h12afl/fl; Cd4-Cre-ERt2 (iKO) and Rc3h1/2fl/fl; Zc3h12afl/fl; Cd4-Cre-ERt2 (iTKO) mice were adoptively transferred into CD45.1+ recipient mice. Mice were treated with tamoxifen by oral gavage to induce deletion of floxed alleles. Adoptively transferred cells were identified by congenic markers on day 8 (h) or accumulation of germinal center B (i) or plasma cells (j) was determined on day 49 after induced deletion. (a–e) Data are presented as mean + /- SD of n = 3 biological replicates analyzed over 3 independent experiments, (f, g) representative of 3 independent experiments, (h) n = 6 analyzed mice, or (i, j) WT: n = 6, iDKO: n = 5, iKO: n = 4, iTKO: n = 6 in 2 independent experiments. Statistical significance was calculated by one-way ANOVA with Dunnett’s post-hoc test.

Source data

Extended Data Fig. 3 Describing phenotypes of DKOT, KOT and TKOT CD8+ T cells.

Flow cytometry analysis of markers of activation and exhaustion (a) and BATF transcription factor expression (b) in splenic WT, DKOT, KOT and TKOT CD8+ T cells. Data are representative of at least 5 individual 6-12 week old mice per genotype in at least two independent experiments. Flow cytometry analysis to Fig. 3e, f: intracellular cytokine staining of IFN-γ, TNF (c) and IL-2 (d) after PMA/ionomycin stimulation in CD8+ T cells of splenocytes from WT, DKOT, KOT and TKOT mice for 4 h. Histogram analysis to Fig. 3g: Granzyme B expression in splenic WT, DKOT, KOT and TKOT CD8+ T cells (e).

Extended Data Fig. 4 Regnase paralogs cannot complement for Roquin loss of function.

(a) Workflow of in vitro reconstitution experiments. (b) Mutations introduced in Regnase-1 coding sequence (marked in red) for the generation of an antibody-invisible GFP-tagged Regnase-1 construct (GFP-Regnase-1invis). (c) Flow cytometry analysis of Regnase-1 expression after retroviral transduction of WT CD4+ T cells with GFP-Regnase-1 or GFP-Regnase-1invis constructs. (d) WT or iDKO CD4+ T cells were retrovirally transduced with GFP-Regnase-1 or GFP-Regnase-1invis. Contour plots of flow cytometry analysis of CTLA-4 expression in dependence of the GFP expression level. (e, f) Flow cytometry analysis of ICOS or Regnase-1 expression after retroviral transduction of iKO or iDKO CD4+ T cells with the indicated GFP-fusion protein. Contour plots of histograms shown in Fig. 4c. (d–f) Data are representative of n = 3 independent experiments.

Extended Data Fig. 5 Dissecting protein domains of Roquin-1 sufficient for cooperative target regulation with Regnase-1.

(a) Schematic representation of Roquin-1 domain organization with indication of M199R mutation. iDKO (b, c) or WT (d) CD4+ T cells were retrovirally transduced with GFP, GFP-Roquin-1 or GFP-Roquin-1aa1-510 constructs. Histograms of flow cytometry analysis of ICOS, Regnase-1 or Ox40 expression, as indicated, in GFP+ cells with indication of respective geometric MFI value. (c) Contour plots of histograms depicted in (b). (e) iDKO CD4+ T cells were retrovirally transduced with the constructs encoding GFP, GFP-Roquin-1 or GFP-Roquin-1 mutant proteins. Flow cytometry analysis of Regnase-1 expression and quantification of fold suppression of Regnase-1 expression level in GFP+ cells relative to cells expressing GFP control construct. (b–e) Data are representative of n = 3 independent experiments. (e) Data are presented as mean + /- SEM of n = 3 independent experiments.

Source data

Extended Data Fig. 6 Functional validation of point mutations in Roquin-1 that reduce cooperation with Regnase-1.

(a) Structure of the Roquin-1 HEPNN/ROQ/HEPNC domain with a bound RNA stem-loop marked in green. All amino acids tested are colored, essential amino acids for Roquin-1 and Regnase-1 interaction in the ROQ domain are marked in orange, non-essential ones in yellow and amino acid M199 in blue. WT (b) or iDKO (c) CD4+ T cells were retrovirally transduced with GFP, GFP-Roquin-1 or the indicated GFP-Roquin-1 mutants. Contour plots of flow cytometry measurement of indicated targets in GFP+ cells after 16 h of doxycycline induction of GFP-tagged constructs. (b) Contour plots of histograms shown in Fig. 4f or (c) shown in Fig. 4g. (d) iDKO CD4+ T cells were retrovirally transduced with GFP, GFP-Roquin-1 or the indicated GFP-Roquin-1 mutants and sorted for GFP+ cells 6 h after doxycycline induced expression of the respective constructs. The levels of Zc3h12a, Icos or Tnfrsf4 mRNAs in GFP+ cells were determined via RT-qPCR, normalized to YWHAZ and calculated as fold suppression of the respective construct relative to cells expressing the GFP control construct. Data show mean + /-SEM of n = 3 independent experiments. (e) Representative images of iDKO CD4+ T cells transduced with the indicated GFP-Roquin-1 WT or mutant proteins, stained with anti-Rck (P-body marker) antibody and analyzed via Image Stream. (b, c) Data are representative of n = 3 independent experiments or (e) n = 2 independent experiments.

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Extended Data Fig. 7 Molecular determinants of Roquin-1 interaction with Regnase-1.

(a) Lysates of WT or iKO CD4+ T cells were subjected to immunoprecipitation (IP) with antibodies against Regnase-1. Input lysates before IP and eluates from beads after IP were analyzed in immunoblots with antibodies against Roquin-1 or Regnase-1. (b, d) Fluorescence microscopy images of HeLa cells transfected with BFP-Rck and GFP-Regnase-1 (GFP-Reg-1) and stained with ER staining dye (b), or of cells transfected with GFP-Regnase-1aa112-297 and mCherry-Roquin-1aa1-510 (d). Quantification of GFP fluorescence lifetime in the cytoplasm (cyto) and nucleus (nuc) (e) of transfected cells shown in (d). (c) Cytoplasmic lysates of CD4+ T cells were fractionated after sucrose gradient centrifugation and distribution of Roquin, Regnase-1 and Rpl7a proteins in the individual fractions analyzed via immunoblots, as indicated. Above, representative absorbance profile obtained during fractionation of gradients with indication of localization of 40 S and 60 S ribosomal subunits, 80 S monosomes and polysomes. (f, g) SDS-PAGE of competitive in vitro GST-pulldown experiments using GST-regnase-1D141N and SUMO-roquin-1aa2-440 in combination with the indicated untagged Roquin-1aa2-440 double or single mutants. Purified proteins before pulldown (IN), supernatants of wash steps (W) as well as eluted proteins (E) were loaded. Asterisks mark the migration of degradation products of GST-regnase-1. (h) Quantification of eluted mutant Roquin-1aa2-440 relative to SUMO-roquin-1aa2-440 wild-type protein of in vitro GST-pulldown experiments depicted in (g). (b, d, f, g) Depicted are representative data of n = 3 independent experiments or (a, c) of n = 2 independent experiments. (e) Data are presented as mean + /- SEM or (h) mean + /-SD and (h) statistical significance was calculated using t tests.

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Extended Data Fig. 8 Phenotypes of mice with mutations impairing Roquin-1 interaction with Regnase-1.

Contour plots of flow cytometry analysis of CD4+ T cells (a), TFH cells (b) and GC B cells (c) from spleens of 9-14 weeks old WT, Rc3h1M199R/fl;Cd4-Cre, Rc3h1M199R/fl;Vav-Cre and Rc3h1M199R/M199R mice. Contour plots are representative of WT, Rc3h1M199R/M199R: n = 6, Rc3h1M199R/fl; Cd4-Cre, Rc3h1M199R/fl; Vav-Cre: n = 5 (a), or WT, Rc3h1M199R/M199R: n = 8, Rc3h1M199R/fl; Cd4-Cre: n = 6, Rc3h1M199R/fl; Vav-Cre: n = 7 (b, c) analyzed mice in at least 3 independent experiments.

Extended Data Fig. 9 Exemplary gating strategy.

Cells were pregated on lymphocytes (FSC-A/SSC-A), single cells (SSC-H/SSC-W and FSC-H/FSC-W) and live cells (Fixable blue -) prior to gating on cell populations of interest.

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Behrens, G., Edelmann, S.L., Raj, T. et al. Disrupting Roquin-1 interaction with Regnase-1 induces autoimmunity and enhances antitumor responses. Nat Immunol 22, 1563–1576 (2021). https://doi.org/10.1038/s41590-021-01064-3

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