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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Josefowicz, S. Z., Lu, L. F. & Rudensky, A. Y. Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012).
Wing, J. B., Tanaka, A. & Sakaguchi, S. Human FOXP3+ regulatory T cell heterogeneity and function in autoimmunity and cancer. Immunity 50, 302–316 (2019).
Panduro, M., Benoist, C. & Mathis, D. Tissue Tregs. Annu. Rev. Immunol. 34, 609–633 (2016).
Hill, J. A. et al. Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature. Immunity 27, 786–800 (2007).
Ferraro, A. et al. Interindividual variation in human T regulatory cells. Proc. Natl Acad. Sci. USA 111, E1111–E1120 (2014).
Zemmour, D. et al. Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR. Nat. Immunol. 19, 291–301 (2018).
Ono, M. Control of regulatory T-cell differentiation and function by T-cell receptor signalling and Foxp3 transcription factor complexes. Immunology 160, 24–37 (2020).
Kwon, H. K., Chen, H. M., Mathis, D. & Benoist, C. Different molecular complexes that mediate transcriptional induction and repression by FoxP3. Nat. Immunol. 18, 1238–1248 (2017).
Campbell, D. J. & Koch, M. A. Phenotypical and functional specialization of FOXP3+ regulatory T cells. Nat. Rev. Immunol. 11, 119–130 (2011).
Li, C. et al. TCR transgenic mice reveal stepwise, multi-site acquisition of the distinctive fat-Treg phenotype. Cell 174, 285–299 (2018).
Dispirito, J. R. et al. Molecular diversification of regulatory T cells in nonlymphoid tissues. Sci. Immunol. 3, eaat5861 (2018).
Miragaia, R. J. et al. Single-cell transcriptomics of regulatory T cells reveals trajectories of tissue adaptation. Immunity 50, 493–504 (2019).
Powell, B. R., Buist, N. R. & Stenzel, P. An X-linked syndrome of diarrhea, polyendocrinopathy, and fatal infection in infancy. J. Pediatr. 100, 731–737 (1982).
Ramsdell, F. & Ziegler, S. F. FOXP3 and scurfy: how it all began. Nat. Rev. Immunol. 14, 343–349 (2014).
Barzaghi, F., Passerini, L. & Bacchetta, R. Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: a paradigm of immunodeficiency with autoimmunity. Front. Immunol. 3, 211 (2012).
d’Hennezel, E., Bin, D. K., Torgerson, T. & Piccirillo, C. A. The immunogenetics of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. J. Med. Genet. 49, 291–302 (2012).
Duclaux-Loras, R. et al. Clinical heterogeneity of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: a French multicenter retrospective study. Clin. Transl. Gastroenterol. 9, 201 (2018).
Gambineri, E. et al. Clinical, immunological, and molecular heterogeneity of 173 patients with the phenotype of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Front. Immunol. 9, 2411 (2018).
Barzaghi, F. et al. Long-term follow-up of IPEX syndrome patients after different therapeutic strategies: an international multicenter retrospective study. J. Allergy Clin. Immunol. 141, 1036–1049 (2018).
Godfrey, V. L., Wilkinson, J. E. & Russell, L. B. X-linked lymphoreticular disease in the scurfy (sf) mutant mouse. Am. J. Pathol. 138, 1379–1387 (1991).
Wan, Y. Y. & Flavell, R. A. Regulatory T-cell functions are subverted and converted owing to attenuated Foxp3 expression. Nature 445, 766–770 (2007).
Van Gool, F. et al. A mutation in the transcription factor Foxp3 drives T helper 2 effector function in regulatory T cells. Immunity 50, 362–377 (2019).
Lin, W. et al. Regulatory T cell development in the absence of functional Foxp3. Nat. Immunol. 8, 359–368 (2007).
Gavin, M. A. et al. Foxp3-dependent programme of regulatory T-cell differentiation. Nature 445, 771–775 (2007).
Charbonnier, L. M. et al. Functional reprogramming of regulatory T cells in the absence of Foxp3. Nat. Immunol. 20, 1208–1219 (2019).
Bacchetta, R. et al. Defective regulatory and effector T cell functions in patients with FOXP3 mutations. J. Clin. Invest. 116, 1713–1722 (2006).
Otsubo, K. et al. Identification of FOXP3-negative regulatory T-like (CD4+CD25+CD127low) cells in patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Clin. Immunol. 141, 111–120 (2011).
Boldt, A. et al. Differences in FOXP3 and CD127 expression in Treg-like cells in patients with IPEX syndrome. Clin. Immunol. 153, 109–111 (2014).
Walker, M. R. et al. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+. J. Clin. Invest. 112, 1437–1443 (2003).
Gavin, M. A. et al. Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development. Proc. Natl Acad. Sci. USA 103, 6659–6664 (2006).
Allan, S. E. et al. Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production. Int. Immunol. https://doi.org/10.1093/intimm/dxm014 (2007).
McMurchy, A. N. et al. A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells. Blood 121, 1265–1275 (2013).
Zemmour, D. et al. Flicr, a long noncoding RNA, modulates Foxp3 expression and autoimmunity. Proc. Natl Acad. Sci. USA 114, E3472–E3480 (2017).
Seddiki, N. et al. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J. Exp. Med. 203, 1693–1700 (2006).
Pesenacker, A. M. et al. A regulatory T-cell gene signature is a specific and sensitive biomarker to identify children with new-onset type 1 diabetes. Diabetes 65, 1031–1039 (2016).
Stoeckius, M. et al. Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol. 19, 224 (2018).
Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902 (2019).
Bakke, A. C., Purtzer, M. Z. & Wildin, R. S. Prospective immunological profiling in a case of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). Clin. Exp. Immunol. 137, 373–378 (2004).
Ziegler, S. F. FOXP3: of mice and men. Annu. Rev. Immunol. 24, 209–226 (2006).
Fontenot, J. D., Gavin, M. A. & Rudensky, A. Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4, 330–336 (2003).
Hori, S., Nomura, T. & Sakaguchi, S. Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057–1061 (2003).
Malek, T. R. & Ashwell, J. D. Interleukin 2 upregulates expression of its receptor on a T cell clone. J. Exp. Med. 161, 1575–1580 (1985).
Boyman, O. et al. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 311, 1924–1927 (2006).
Samstein, R. M. et al. Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification. Cell 151, 153–166 (2012).
Remedios, K. A. et al. The TNFRSF members CD27 and OX40 coordinately limit TH17 differentiation in regulatory T cells. Sci. Immunol. 3, eaau2042 (2018).
Kim, J. M., Rasmussen, J. P. & Rudensky, A. Y. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat. Immunol. 8, 191–197 (2007).
Sitrin, J. et al. Regulatory T cells control NK cells in an insulitic lesion by depriving them of IL-2. J. Exp. Med. 210, 1153–1165 (2013).
Cobbold, S. & Waldmann, H. Infectious tolerance. Curr. Opin. Immunol. 10, 518–524 (1998).
Plitas, G. et al. Regulatory T cells exhibit distinct features in human breast cancer. Immunity 45, 1122–1134 (2016).
Gambineri, E. et al. Clinical and molecular profile of a new series of patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: inconsistent correlation between forkhead box protein 3 expression and disease severity. J. Allergy Clin. Immunol. 122, 1105–1112 (2008).
Bettelli, E. et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238 (2006).
Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
Godec, J. et al. Compendium of immune signatures identifies conserved and species-specific biology in response to inflammation. Immunity 44, 194–206 (2016).
Wolock, S. L., Lopez, R. & Klein, A. M. Scrublet: computational identification of cell doublets in single-cell transcriptomic data. Cell Syst. 8, 281–291 (2019).
Aran, D. et al. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat. Immunol. 20, 163–172 (2019).
Becht, E. et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat. Biotechnol. https://doi.org/10.1038/nbt.4314 (2018).
Klein, A. M. et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161, 1187–1201 (2015).
Johansen, N. & Quon, G. scAlign: a tool for alignment, integration, and rare cell identification from scRNA-seq data. Genome Biol. 20, 166 (2019).
Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. Voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014).
Soneson, C. & Robinson, M. D. Bias, robustness and scalability in single-cell differential expression analysis. Nat. Methods 15, 255–261 (2018).
Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag, 2009).
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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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.
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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 Figs. 1–10.
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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DOI: https://doi.org/10.1038/s41590-021-00910-8
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