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Single-cell analysis of FOXP3 deficiencies in humans and mice unmasks intrinsic and extrinsic CD4+ T cell perturbations

Nature Immunology volume 22, pages 607–619 (2021)Cite this article

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Abstract

FOXP3 deficiency in mice and in patients with immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome results in fatal autoimmunity by altering regulatory T (Treg) cells. CD4+ T cells in patients with IPEX syndrome and Foxp3-deficient mice were analyzed by single-cell cytometry and RNA-sequencing, revealing heterogeneous Treg-like cells, some very similar to normal Treg cells, others more distant. Conventional T cells showed no widespread activation or helper T cell bias, but a monomorphic disease signature affected all CD4+ T cells. This signature proved to be cell extrinsic since it was extinguished in mixed bone marrow chimeric mice and heterozygous mothers of patients with IPEX syndrome. Normal Treg cells exerted dominant suppression, quenching the disease signature and revealing in mutant Treg-like cells a small cluster of genes regulated cell-intrinsically by FOXP3, including key homeostatic regulators. We propose a two-step pathogenesis model: cell-intrinsic downregulation of core FOXP3-dependent genes destabilizes Treg cells, de-repressing systemic mediators that imprint the disease signature on all T cells, furthering Treg cell dysfunction. Accordingly, interleukin-2 treatment improved the Treg-like compartment and survival.

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Fig. 1: Identification of Treg-like cells in IPEX syndrome by flow cytometry.
Fig. 2: Transcriptional changes in IPEX Treg and Tconv cells by population RNA-seq.
Fig. 3: scRNA-seq reveals the heterogeneous effect of FOXP3 deficiency in IPEX Treg cells.
Fig. 4: Normal Tconv phenotypes in patients with IPEX syndrome.
Fig. 5: scRNA-seq reveals heterogeneous effects of Foxp3 ablation in ∆Foxp3 mice.
Fig. 6: Cell-intrinsic and extrinsic effects of FOXP3 deficiency in Treg and Tconv cells of IPEX female carriers (mothers).
Fig. 7: Cell-intrinsic and extrinsic effects of Foxp3 deficiency in Treg and Tconv cells in mice.
Fig. 8: IL-2 therapy mitigates Foxp3-deficiency disease.

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Data availability

The data reported in this paper have been deposited in the Gene Expression Omnibus database under SuperSeries accession no. GSE168492: GSE166866 (human population RNA-seq), GSE166860 (mouse population RNA-seq), GSE167976 (human scRNA-seq) and GSE167575 (mouse scRNA-seq).

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Acknowledgements

We thank M. Levings and A. Rudensky for insightful discussions and K. Hattori, C. Araneo, K. Seddu and the Klarman Cell Observatory team for help with mice, cell sorting and single-cell profiling. This work was funded by grants from the National Institutes of Health to C. Benoist and D.M. (AI116834 and AI150686), T.A.C. (AI085090) and L.M.C. (AI153174); the Institut National de la Santé et Recherche Médicale; the European Union Seventh Framework (269037 and 261387) and Horizon 2020 (693762); and the Agence Nationale pour la Recherche (Investissement d’Avenir ANR-10-IAHU-01) to I.A., E.S., M.D., J.L., M.C., B.N., F.R.L., F.R. and N.C.B. J.L. was supported by an INSERM Poste d’Accueil and an Arthur Sachs scholarship.

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Authors and Affiliations

  1. Department of Immunology, Harvard Medical School, Boston, MA, USA

    David Zemmour, Juliette Leon, Diane Mathis & Christophe Benoist

  2. Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    David Zemmour

  3. Division of Immunology, Boston Children’s Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Louis-Marie Charbonnier, Mehdi Benamar, Carlo Brugnara & Talal A. Chatila

  4. INSERM UMR 1163, University of Paris, Imagine Institute, Paris, France

    Juliette Leon, Emmanuelle Six, Marianne Delville, Julien Zuber, Benedicte Neven, Frank M. Ruemmele, Nadine Cerf-Bensussan, Frederic Rieux-Laucat & Isabelle André

  5. Division of Pediatric Allergy and Immunology, Meram Medical Faculty, Necmettin Erbakan University, Konya, Turkey

    Sevgi Keles

  6. Necker Children’s Hospital, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France

    Marianne Delville, Julien Zuber, Benedicte Neven, Frank M. Ruemmele & Nadine Cerf-Bensussan

  7. Division of Pediatric Allergy and Immunology, Marmara University School of Medicine, Istanbul, Turkey

    Safa Baris

  8. Istanbul Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, Istanbul, Turkey

    Safa Baris

  9. Division of Allergy and Immunology, Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, UT, USA

    Karin Chen

  10. Division of Immunology, Department of Pediatrics, University of Washington and Seattle Children’s Research Institute, Seattle, WA, USA

    Karin Chen

  11. Division of Allergy, Immunology, and Rheumatology, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Maria I. Garcia-Lloret

  12. Biotherapy Department and Clinical Investigation Center, Assistance Publique Hopitaux Paris, Inserm, Paris, France

    Marina Cavazzana

  13. Université de Paris, Paris, France

    Marina Cavazzana

  14. Institut Imagine, Paris, France

    Marina Cavazzana & Isabelle André

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Contributions

D.Z., L.M.C., J.L. and M.B. performed the experiments; E.S., S.K., M.D., S.B., J.Z., K.C., B.N., M.I.G.L., F.R., N.C.B., F.R.L., M.C., I.A., T.A.C., L.M.C. and C. Bruganara provided samples and discussed interpretations; D.Z., L.M.C., J.L., T.A.C., I.A., C. Benoist and D.M. designed the study and analyzed and interpreted the data; D.Z., J.L., C. Benoist and D.M. wrote the manuscript.

Corresponding authors

Correspondence to Diane Mathis or Christophe Benoist.

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The authors declare no competing interests.

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Peer review information Nature Immunology thanks Fred Ramsdell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Zoltan Fehervari 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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Supplementary information

Supplementary Information

Supplementary Figs. 1–10.

Reporting Summary

Supplementary Table 1

Clinical characteristics of healthy donors and patients with IPEX syndrome by cohort (summary table and singular values).

Supplementary Table 2

IPEX signature genes with their average IPEX/HD fold change in Treg and Tconv cells.

Supplementary Table 3

CD4+ signatures significantly enriched in the IPEX signature.

Supplementary Table 4

List of human and mouse scRNA-seq datasets (human and mice) with quality control metrics.

Supplementary Table 5

Treg cell signature genes with their average Treg/Tconv fold change in ΔFoxp3 mice, WT mice, BMC ΔFoxp3 cells in BMCs and WT cells in BMCs.

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Zemmour, D., Charbonnier, LM., Leon, J. et al. Single-cell analysis of FOXP3 deficiencies in humans and mice unmasks intrinsic and extrinsic CD4+ T cell perturbations. Nat Immunol 22, 607–619 (2021). https://doi.org/10.1038/s41590-021-00910-8

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