Development of immunocompetent T cells in the thymus is required for effective defence against all types of pathogens, including viruses, bacteria and fungi. To this end, T cells undergo a very strict educational program in the thymus, during which both non-functional and self-reactive T cell clones are eliminated by means of positive and negative selection1.Thymic epithelial cells (TECs) have an indispensable role in these processes, and previous studies have shown the notable heterogeneity of these cells2-7. Here, using multiomic analysis, we provide further insights into the functional and developmental diversity of TECs in mice, and reveal a detailed atlas of the TEC compartment according to cell transcriptional states and chromatin landscapes. Our analysis highlights unconventional TEC subsets that are similar to functionally well-defined parenchymal populations, including endocrine cells, microfold cells and myocytes. By focusing on the endocrine and microfold TEC populations, we show that endocrine TECs require Insm1 for their development and are crucial to maintaining thymus cellularity in a ghrelin-dependent manner; by contrast, microfold TECs require Spib for their development and are essential for the generation of thymic IgA+ plasma cells. Collectively, our study reveals that medullary TECs have the potential to differentiate into various types of molecularly distinct and functionally defined cells, which not only contribute to the induction of central tolerance, but also regulate the homeostasis of other thymus-resident populations.

Bioinformatics Unit Department of Life Sciences Core Facilities Weizmann Institute of Science Rehovot Israel

Department of Cell Biology Faculty of Science Charles University Prague Czech Republic

Department of Immunology and Regenerative Biology Weizmann Institute of Science Rehovot Israel

Department of Veterinary Resources Weizmann Institute of Science Rehovot Israel

Division of Biomedical Sciences School of Medicine University of California Riverside Riverside CA USA

Flow Cytometry Unit Department of Life Sciences Core Facilities Weizmann Institute of Science Rehovot Israel

MICC Cell Observatory Department of Life Sciences Core Facilities Weizmann Institute of Science Rehovot Israel

The Nancy and Stephen Grand Israel National Center for Personalized Medicine Weizmann Institute of Science Rehovot Israel

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Nature. 2023 Dec;624(7991):E4. doi: 10.1038/s41586-023-06881-0. PubMed

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Klein, L., Kyewski, B., Allen, P. M. & Hogquist, K. A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don’t see). Nat. Rev. Immunol. 14, 377–391 (2014). PubMed DOI PMC

Bornstein, C. et al. Single-cell mapping of the thymic stroma identifies IL-25-producing tuft epithelial cells. Nature 559, 622–626 (2018). PubMed DOI

Baran-Gale, J. et al. Ageing compromises mouse thymus function and remodels epithelial cell differentiation. eLife 9, e56221 (2020). PubMed DOI PMC

Bautista, J. L. et al. Single-cell transcriptional profiling of human thymic stroma uncovers novel cellular heterogeneity in the thymic medulla. Nat. Commun. 12, 1096 (2021). PubMed DOI PMC

Dhalla, F. et al. Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells. EMBO J. 39, e101828 (2020). PubMed DOI

Park, J. E. et al. A cell atlas of human thymic development defines T cell repertoire formation. Science 367, eaay3224 (2020). PubMed DOI PMC

Michelson, D. A., Hase, K., Kaisho, T., Benoist, C. & Mathis, D. Thymic epithelial cells co-opt lineage-defining transcription factors to eliminate autoreactive T cells. Cell 185, 2542–2558 (2022). PubMed DOI PMC

Abramson, J. & Anderson, G. Thymic epithelial cells. Annu. Rev. Immunol. 35, 85–118 (2017). PubMed DOI

Sansom, S. N. et al. Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia. Genome Res. 24, 1918–1931 (2014). PubMed DOI PMC

Anderson, M. S. et al. Projection of an immunological self shadow within the thymus by the Aire protein. Science 298, 1395–1401 (2002). PubMed DOI

Metzger, T. C. et al. Lineage tracing and cell ablation identifiy a post-Aire expressing thymic epithelial cell population. Cell Rep. 5, 166–179 (2013). PubMed DOI

Miller, C. N. et al. Thymic tuft cells promote an IL-4-enriched medulla and shape thymocyte development. Nature 559, 627–631 (2018). PubMed DOI PMC

Miragaia, R. J. et al. Single-cell RNA-sequencing resolves self-antigen expression during mTEC development. Sci. Rep. 8, 685 (2018). PubMed DOI PMC

Miyao, T. et al. Integrative analysis of scRNA-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator. eLife 11, e73998 (2022). PubMed DOI PMC

Wang, X. et al. Post-Aire maturation of thymic medullary epithelial cells involves selective expression of keratinocyte-specific autoantigens. Front. Immunol. 3, 19 (2012). PubMed DOI PMC

Goldstein, J. D. et al. IL-36 signaling in keratinocytes controls early IL-23 production in psoriasis-like dermatitis. Life Sci. Alliance 3, e202000688 (2020). PubMed DOI PMC

Wang, W., Yu, X., Wu, C. & Jin, H. IL-36γ inhibits differentiation and induces inflammation of keratinocyte via Wnt signaling pathway in psoriasis. Int. J. Med. Sci. 14, 1002–1007 (2017). PubMed DOI PMC

Mabbott, N. A., Donaldson, D. S., Ohno, H., Williams, I. R. & Mahajan, A. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium. Mucosal Immunol. 6, 666–677 (2013). PubMed DOI PMC

Onder, L. et al. Alternative NF-κB signaling regulates mTEC differentiation from podoplanin-expressing precursors in the cortico-medullary junction. Eur. J. Immunol. 45, 2218–2231 (2015). PubMed DOI

Wells, K. L. et al. Combined transient ablation and single-cell RNA sequencing reveals the development of medullary thymic epithelial cells. eLife 9, e60188 (2020). PubMed DOI PMC

Goldfarb, Y. et al. Mechanistic dissection of dominant AIRE mutations in mouse models reveals AIRE autoregulation. J. Exp. Med. 218, e20201076 (2021). PubMed DOI PMC

Borromeo, M. D. et al. ASCL1 and NEUROD1 reveal heterogeneity in pulmonary neuroendocrine tumors and regulate distinct genetic programs. Cell Rep. 16, 1259–1272 (2016). PubMed DOI PMC

Osipovich, A. B. et al. Insm1 promotes endocrine cell differentiation by modulating the expression of a network of genes that includes Neurog3 and Ripply3. Development 141, 2939–2949 (2014). PubMed DOI PMC

Jia, S. et al. Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic β-cell function. EMBO J. 34, 1417–1433 (2015). PubMed DOI PMC

Henry, C., Close, A.-F. & Buteau, J. A critical role for the neural zinc factor ST18 in pancreatic β-cell apoptosis. J. Biol. Chem. 289, 8413–8419 (2014). PubMed DOI PMC

Guo, T. et al. ISL1 promotes pancreatic islet cell proliferation. PLoS One 6, e22387 (2011). PubMed DOI PMC

Gehart, H. et al. Identification of enteroendocrine regulators by real-time single-cell differentiation mapping. Cell 176, 1158–1173 (2019). PubMed DOI

Fothergill, L. J. et al. Distribution and co-expression patterns of specific cell markers of enteroendocrine cells in pig gastric epithelium. Cell Tissue Res. 378, 457–469 (2019). PubMed DOI PMC

Jiang, W., Anderson, M. S., Bronson, R., Mathis, D. & Benoist, C. Modifier loci condition autoimmunity provoked by Aire deficiency. J. Exp. Med. 202, 805–815 (2005). PubMed DOI PMC

Tuncel, J., Benoist, C. & Mathis, D. T cell anergy in perinatal mice is promoted by T reg cells and prevented by IL-33. J. Exp. Med. 216, 1328–1344 (2019). PubMed DOI PMC

Dixit, V. D. et al. Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J. Clin. Invest. 114, 57–66 (2004). PubMed DOI PMC

Dixit, V. D. et al. Ghrelin promotes thymopoiesis during aging. J. Clin. Invest. 117, 2778–2790 (2007). PubMed DOI PMC

Kobayashi, N., Takahashi, D., Takano, S., Kimura, S. & Hase, K. The roles of Peyer’s patches and microfold cells in the gut immune system: relevance to autoimmune diseases. Front. Immunol. 10, 2345 (2019). PubMed DOI PMC

Kanaya, T. et al. The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells. Nat. Immunol. 13, 729–736 (2012). PubMed DOI PMC

Akiyama, N. et al. Limitation of immune tolerance-inducing thymic epithelial cell development by Spi-B-mediated negative feedback regulation. J. Exp. Med. 211, 2425 (2014). PubMed DOI PMC

Kimura, S. et al. Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity. Nat. Commun. 11, 234 (2020). PubMed DOI PMC

McCarthy, N. I. et al. Osteoprotegerin-mediated homeostasis of Rank PubMed DOI PMC

Dillon, A. & Lo, D. D. M cells: intelligent engineering of mucosal immune surveillance. Front. Immunol. 10, 1499 (2019). PubMed DOI PMC

Wang, J., Gusti, V., Saraswati, A. & Lo, D. D. Convergent and divergent development among M cell lineages in mouse mucosal epithelium. J. Immunol. 187, 5277–5285 (2011). PubMed DOI

Komban, R. J. et al. Activated Peyer’s patch B cells sample antigen directly from M cells in the subepithelial dome. Nat. Commun. 10, 2423 (2019). PubMed DOI PMC

Sakhon, O. S. et al. M cell-derived vesicles suggest a unique pathway for trans-epithelial antigen delivery. Tissue Barriers 3, e1004975 (2015). PubMed DOI PMC

Cerutti, A. The regulation of IgA class switching. Nat. Rev. Immunol. 8, 421–434 (2008). PubMed DOI PMC

López-Fraga, M., Fernández, R., Albar, J. P. & Hahne, M. Biologically active APRIL is secreted following intracellular processing in the Golgi apparatus by furin convertase. EMBO Rep. 2, 945–951 (2001). PubMed DOI PMC

de Lau, W. et al. Peyer’s patch M cells derived from Lgr5 PubMed DOI PMC

Meredith, M., Zemmour, D., Mathis, D. & Benoist, C. Aire controls gene expression in the thymic epithelium with ordered stochasticity. Nat. Immunol. 16, 942–949 (2015). PubMed DOI PMC

Lucas, B. et al. Diversity in medullary thymic epithelial cells controls the activity and availability of iNKT cells. Nat. Commun. 11, 2198 (2020). PubMed DOI PMC

Rios, D. et al. Antigen sampling by intestinal M cells is the principal pathway initiating mucosal IgA production to commensal enteric bacteria. Mucosal Immunol. 9, 907–916 (2016). PubMed DOI

Kim, Y.-I. et al. CX PubMed DOI

Reboldi, A. et al. Mucosal immunology: IgA production requires B cell interaction with subepithelial dendritic cells in Peyer’s patches. Science 352, aaf4822 (2016). PubMed DOI PMC

Vobořil, M. et al. A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer. eLife 11, e71578 (2022). PubMed DOI PMC

Vollmann, E. H. et al. Specialized transendothelial dendritic cells mediate thymic T-cell selection against blood-borne macromolecules. Nat. Commun. 12, 6230 (2021). PubMed DOI PMC

Gardner, J. M. et al. Deletional tolerance mediated by extrathymic Aire-expressing cells. Science 321, 843 (2008). PubMed DOI PMC

Su, G. H. et al. Defective B cell receptor-mediated responses in mice lacking the Ets protein, Spi-B. EMBO J. 16, 7118–7129 (1997). PubMed DOI PMC

Pacary, E. et al. Proneural transcription factors regulate different steps of cortical neuron migration through rnd-mediated inhibition of RhoA signaling. Neuron 69, 1069–1084 (2011). PubMed DOI

Jung, S. et al. Analysis of fractalkine receptor CX PubMed DOI PMC

Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013). PubMed DOI PMC

Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016). PubMed DOI PMC

Xu, H. et al. Sequence determinants of improved CRISPR sgRNA design. Genome Res 25, 1147–1157 (2015). PubMed DOI PMC

Concordet, J. P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242–W245 (2018). PubMed DOI PMC

Jaitin, D. A. et al. Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types. Science 343, 776–779 (2014). PubMed DOI PMC

Kohen, R. et al. UTAP: user-friendly transcriptome analysis pipeline. BMC Bioinformatics 20, 154 (2019). PubMed DOI PMC

Stuart, T. et al. Comprehensive integration of Single-cell data. Cell 177, 1888–1902 (2019). PubMed DOI PMC

Stuart, T., Srivastava, A., Madad, S., Lareau, C. A. & Satija, R. Single-cell chromatin state analysis with Signac. Nat. Methods 18, 1333–1341 (2021). PubMed DOI PMC

Hafemeister, C. & Satija, R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol. 20, 296 (2019). PubMed DOI PMC

Zhang, Y. et al. Model-based Analysis of ChIP–Seq (MACS). Genome Biol. 9, R137 (2008). PubMed DOI PMC

Schep, A. N., Wu, B., Buenrostro, J. D. & Greenleaf, W. J. chromVAR: inferring transcription-factor-associated accessibility from single-cell epigenomic data. Nat. Methods 14, 975–978 (2017). PubMed DOI PMC

Khan, A. et al. JASPAR 2018: update of the open-access database of transcription factor binding profiles and its web framework. Nucleic Acids Res. 46, D260–D266 (2018). PubMed DOI

Boyle, E. I. et al. GO::TermFinder—open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes. Bioinformatics 20, 3710–3715 (2004). PubMed DOI

Kachitvichyanukul, V. & Schmeiser, B. Computer generation of hypergeometric random variates. J. Stat. Comput. Sim. 22, 127–145 (2007). DOI

Benjaminit, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995).

Stoeckius, M. et al. Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol. 19, 224 (2018). PubMed DOI PMC

Jin, S. et al. Inference and analysis of cell–cell communication using CellChat. Nat. Commun. 12, 1088 (2021). PubMed DOI PMC

Autoantibodies against type I IFNs in humans with alternative NF-κB pathway deficiency