Combined transient ablation and single-cell RNA-sequencing reveals the development of medullary thymic epithelial cells

Medullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene-expression patterns within the mTEC compartment are heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single-cell RNA-sequencing in Mus musculus. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. We propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

[1]  Yong Zhao,et al.  Thymic Epithelial Cells Contribute to Thymopoiesis and T Cell Development , 2020, Frontiers in Immunology.

[2]  Fabian J Theis,et al.  Generalizing RNA velocity to transient cell states through dynamical modeling , 2019, Nature Biotechnology.

[3]  Andrew C. Adey Integration of Single-Cell Genomics Datasets , 2019, Cell.

[4]  Paul J. Hoffman,et al.  Comprehensive Integration of Single-Cell Data , 2018, Cell.

[5]  Z. Hu,et al.  Live-cell imaging reveals the relative contributions of antigen-presenting cell subsets to thymic central tolerance , 2019, Nature Communications.

[6]  Samantha Riesenfeld,et al.  EmptyDrops: distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data , 2019, Genome Biology.

[7]  F. Dhalla,et al.  Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells , 2019, bioRxiv.

[8]  E. Anton,et al.  Single-cell transcriptomic analysis of mouse neocortical development , 2019, Nature Communications.

[9]  Lai Guan Ng,et al.  Dimensionality reduction for visualizing single-cell data using UMAP , 2018, Nature Biotechnology.

[10]  I. Amit,et al.  Lung Single-Cell Signaling Interaction Map Reveals Basophil Role in Macrophage Imprinting , 2018, Cell.

[11]  Charles J. Vaske,et al.  Transcriptional Programming of Normal and Inflamed Human Epidermis at Single-Cell Resolution , 2018, Cell reports.

[12]  J. Lee,et al.  Single-cell RNA sequencing technologies and bioinformatics pipelines , 2018, Experimental & Molecular Medicine.

[13]  Erik Sundström,et al.  RNA velocity of single cells , 2018, Nature.

[14]  Eyal David,et al.  Single-cell mapping of the thymic stroma identifies IL-25-producing tuft epithelial cells , 2018, Nature.

[15]  Ping Xu,et al.  A Single‐Cell Transcriptomic Atlas of Thymus Organogenesis Resolves Cell Types and Developmental Maturation , 2018, Immunity.

[16]  Mark S. Anderson,et al.  Thymic tuft cells promote an IL4-enriched medulla and shape thymocyte development , 2018, Nature.

[17]  Paul Hoffman,et al.  Integrating single-cell transcriptomic data across different conditions, technologies, and species , 2018, Nature Biotechnology.

[18]  J. Junker,et al.  Simultaneous lineage tracing and cell-type identification using CRISPR/Cas9-induced genetic scars , 2018, Nature Biotechnology.

[19]  J. Ramalho‐Santos Data , 2018, Nature.

[20]  James A. Gagnon,et al.  Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain , 2018, Nature Biotechnology.

[21]  R. Perniola Twenty Years of AIRE , 2018, Front. Immunol..

[22]  Mauro J. Muraro,et al.  Troy+ brain stem cells cycle through quiescence and regulate their number by sensing niche occupancy , 2018, Proceedings of the National Academy of Sciences.

[23]  Howard Y. Chang,et al.  Rapid chromatin repression by Aire provides precise control of immune tolerance , 2018, Nature Immunology.

[24]  Ricardo J. Miragaia,et al.  Single-cell RNA-sequencing resolves self-antigen expression during mTEC development , 2018, Scientific Reports.

[25]  S. Parnell,et al.  Redefining thymus medulla specialization for central tolerance , 2017, The Journal of experimental medicine.

[26]  John C Marioni,et al.  Detection and removal of barcode swapping in single-cell RNA-seq data , 2017, Nature Communications.

[27]  E. Maldonado,et al.  Transit-Amplifying Cells in the Fast Lane from Stem Cells towards Differentiation , 2017, Stem cells international.

[28]  Y. Hsu,et al.  Emerging roles of transit‐amplifying cells in tissue regeneration and cancer , 2017, Wiley interdisciplinary reviews. Developmental biology.

[29]  H. Kiyonari,et al.  Essential role of CCL21 in establishment of central self-tolerance in T cells , 2017, The Journal of experimental medicine.

[30]  Russell B. Fletcher,et al.  Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics , 2017, BMC Genomics.

[31]  A. Metspalu,et al.  DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes , 2017, The Journal of Biological Chemistry.

[32]  Grace X. Y. Zheng,et al.  Massively parallel digital transcriptional profiling of single cells , 2016, Nature Communications.

[33]  T. Kodama,et al.  Fezf2 Orchestrates a Thymic Program of Self-Antigen Expression for Immune Tolerance , 2015, Cell.

[34]  Sarah A Teichmann,et al.  Computational assignment of cell-cycle stage from single-cell transcriptome data. , 2015, Methods.

[35]  G. Anderson,et al.  Co-ordination of intrathymic self-representation , 2015, Nature Immunology.

[36]  David Zemmour,et al.  Aire controls gene expression in the thymic epithelium with ordered stochasticity , 2015, Nature Immunology.

[37]  Wolfgang Huber,et al.  Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells , 2015, Nature Immunology.

[38]  B. Becher,et al.  Alternative NF‐κB signaling regulates mTEC differentiation from podoplanin‐expressing precursors in the cortico‐medullary junction , 2015, European journal of immunology.

[39]  L. Romani,et al.  Thymosin α1: burying secrets in the thymus , 2015, Expert opinion on biological therapy.

[40]  T. Kaisho,et al.  Limitation of immune tolerance–inducing thymic epithelial cell development by Spi-B–mediated negative feedback regulation , 2014, The Journal of experimental medicine.

[41]  C. Ponting,et al.  Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia , 2014, Genome research.

[42]  D. Gray,et al.  Multilineage potential and self-renewal define an epithelial progenitor cell population in the adult thymus. , 2014, Cell Reports.

[43]  B. Kyewski,et al.  Adult Thymus Contains FoxN1− Epithelial Stem Cells that Are Bipotent for Medullary and Cortical Thymic Epithelial Lineages , 2014, Immunity.

[44]  L. Klein,et al.  Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see) , 2014, Nature Reviews Immunology.

[45]  Mark S. Anderson,et al.  Enhancement of an anti-tumor immune response by transient blockade of central T cell tolerance , 2014, The Journal of Experimental Medicine.

[46]  Cesare Furlanello,et al.  A promoter-level mammalian expression atlas , 2015 .

[47]  Mark S. Anderson,et al.  Lineage tracing and cell ablation identify a post-Aire-expressing thymic epithelial cell population. , 2013, Cell reports.

[48]  V. Gallo,et al.  FOXN1: A Master Regulator Gene of Thymic Epithelial Development Program , 2013, Front. Immunol..

[49]  Y. Takahama,et al.  Lymphotoxin β Receptor Regulates the Development of CCL21-Expressing Subset of Postnatal Medullary Thymic Epithelial Cells , 2013, The Journal of Immunology.

[50]  M. David,et al.  IRF7-Dependent IFN-β Production in Response to RANKL Promotes Medullary Thymic Epithelial Cell Development , 2013, The Journal of Immunology.

[51]  Y. Takahama,et al.  Lymphotoxin b Receptor Regulates the Development of CCL21-Expressing Subset of Postnatal Medullary Thymic Epithelial Cells , 2013 .

[52]  P. Peterson,et al.  Post-Aire Maturation of Thymic Medullary Epithelial Cells Involves Selective Expression of Keratinocyte-Specific Autoantigens , 2012, Front. Immun..

[53]  F. Watt,et al.  Lineage Tracing , 2012, Cell.

[54]  K. Hochedlinger,et al.  Sox2(+) adult stem and progenitor cells are important for tissue regeneration and survival of mice. , 2011, Cell stem cell.

[55]  Mark S. Anderson,et al.  Control of central and peripheral tolerance by Aire , 2011, Immunological reviews.

[56]  S. Bamforth,et al.  A Novel Role for Transcription Factor Lmo4 in Thymus Development Through Genetic Interaction with Cited2 , 2010, Developmental dynamics : an official publication of the American Association of Anatomists.

[57]  J. Miyazaki,et al.  Biphasic Aire expression in early embryos and in medullary thymic epithelial cells before end-stage terminal differentiation , 2010, The Journal of experimental medicine.

[58]  Allan R. Jones,et al.  A robust and high-throughput Cre reporting and characterization system for the whole mouse brain , 2009, Nature Neuroscience.

[59]  B. Kyewski,et al.  Promiscuous gene expression patterns in single medullary thymic epithelial cells argue for a stochastic mechanism , 2008, Proceedings of the National Academy of Sciences.

[60]  B. Kyewski,et al.  Promiscuous gene expression and the developmental dynamics of medullary thymic epithelial cells , 2007, European journal of immunology.

[61]  C. Benoist,et al.  Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire , 2007, The Journal of experimental medicine.

[62]  J. Penninger,et al.  RANK signals from CD4+3− inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla , 2007, The Journal of Experimental Medicine.

[63]  D. Gray,et al.  Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells. , 2006, Blood.

[64]  Amy Li,et al.  Side population in adult murine epidermis exhibits phenotypic and functional characteristics of keratinocyte stem cells , 2006, Proceedings of the National Academy of Sciences.

[65]  M. Herlyn,et al.  Isolation of a novel population of multipotent adult stem cells from human hair follicles. , 2006, The American journal of pathology.

[66]  Mark S. Anderson,et al.  Projection of an Immunological Self Shadow Within the Thymus by the Aire Protein , 2002, Science.

[67]  L. Peltonen,et al.  Aire deficient mice develop multiple features of APECED phenotype and show altered immune response. , 2002, Human molecular genetics.

[68]  D. Elder,et al.  Human hair follicle bulge cells are biochemically distinct and possess an epithelial stem cell phenotype. , 1999, The journal of investigative dermatology. Symposium proceedings.

[69]  M. Claesson,et al.  Thymic Epithelial Cells , 1990, Scandinavian journal of immunology.