mTEC Subsets Research Articles

Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here, we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny in mice. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium.

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here, we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny in mice. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

Acute irradiation causes a long-term disturbance in the heterogeneity and gene expression profile of medullary thymic epithelial cells.

The thymus has the ability to regenerate from acute injury caused by radiation, infection, and stressors. In addition to thymocytes, thymic epithelial cells in the medulla (mTECs), which are crucial for T cell self-tolerance by ectopically expressing and presenting thousands of tissue-specific antigens (TSAs), are damaged by these insults and recover thereafter. However, given recent discoveries on the high heterogeneity of mTECs, it remains to be determined whether the frequency and properties of mTEC subsets are restored during thymic recovery from radiation damage. Here we demonstrate that acute total body irradiation with a sublethal dose induces aftereffects on heterogeneity and gene expression of mTECs. Single-cell RNA-sequencing (scRNA-seq) analysis showed that irradiation reduces the frequency of mTECs expressing AIRE, which is a critical regulator of TSA expression, 15 days after irradiation. In contrast, transit-amplifying mTECs (TA-mTECs), which are progenitors of AIRE-expressing mTECs, and Ccl21a-expressing mTECs, were less affected. Interestingly, a detailed analysis of scRNA-seq data suggested that the proportion of a unique mTEC cluster expressing Ccl25 and a high level of TSAs was severely decreased by irradiation. In sum, we propose that the effects of acute irradiation disrupt the heterogeneity and properties of mTECs over an extended period, which potentially leads to an impairment of thymic T cell selection.

Embryonic keratin19+ progenitors generate multiple functionally distinct progeny to maintain epithelial diversity in the adult thymus medulla

The thymus medulla is a key site for immunoregulation and tolerance, and its functional specialisation is achieved through the complexity of medullary thymic epithelial cells (mTEC). While the importance of the medulla for thymus function is clear, the production and maintenance of mTEC diversity remains poorly understood. Here, using ontogenetic and inducible fate-mapping approaches, we identify mTEC-restricted progenitors as a cytokeratin19+ (K19+) TEC subset that emerges in the embryonic thymus. Importantly, labelling of a single cohort of K19+ TEC during embryogenesis sustains the production of multiple mTEC subsets into adulthood, including CCL21+ mTEClo, Aire+ mTEChi and thymic tuft cells. We show K19+ progenitors arise prior to the acquisition of multiple mTEC-defining features including RANK and CCL21 and are generated independently of the key mTEC regulator, Relb. In conclusion, we identify and define a multipotent mTEC progenitor that emerges during embryogenesis to support mTEC diversity into adult life.

Revelations in Thymic Epithelial Cell Biology and Heterogeneity from Single-Cell RNA Sequencing and Lineage Tracing Methodologies.

Thymic epithelial cells (TECs) make up the thymic microenvironments that support the generation of a functionally competent and self-tolerant T-cell repertoire. Cortical (c)TECs, present in the cortex, are essential for early thymocyte development including selection of thymocytes expressing functional TCRs (positive selection). Medullary (m)TECs, located in the medulla, play a key role in late thymocyte development, including depletion of self-reactive T cells (negative selection) and selection of regulatory T cells. In recent years, transcriptomic analysis by single-cell (sc)RNA sequencing (Seq) has revealed TEC heterogeneity previously masked by population-level RNA-Seq or phenotypic studies. We summarize the discoveries made possible by scRNA-Seq, including the identification of novel mTEC subsets, advances in understanding mTEC promiscuous gene expression, and TEC alterations from embryonic to adult stages. Whereas pseudotime analyses of scRNA-Seq data can suggest relationships between TEC subsets, experimental methods such as lineage tracing and reaggregate thymic organ culture (RTOC) are required to test these hypotheses. Lineage tracing - namely, of β5t or Aire expressing cells - has exposed progenitor and parent-daughter cellular relationships within TEC.

Mechanisms of Direct and Indirect Presentation of Self-Antigens in the Thymus.

The inevitability of evolution of the adaptive immune system with its mechanism of randomly rearranging segments of the T cell receptor (TCR) gene is the generation of self-reactive clones. For the sake of prevention of autoimmunity, these clones must be eliminated from the pool of circulating T cells. This process occurs largely in the thymic medulla where the strength of affinity between TCR and self-peptide MHC complexes is the factor determining thymocyte fate. Thus, the display of self-antigens in the thymus by thymic antigen presenting cells, which are comprised of medullary thymic epithelial (mTECs) and dendritic cells (DCs), is fundamental for the establishment of T cell central tolerance. Whereas mTECs produce and present antigens in a direct, self-autonomous manner, thymic DCs can acquire these mTEC-derived antigens by cooperative antigen transfer (CAT), and thus present them indirectly. While the basic characteristics for both direct and indirect presentation of self-antigens are currently known, recent reports that describe the heterogeneity of mTEC and DC subsets, their presentation capacity, and the potentially non-redundant roles in T cell selection processes represents another level of complexity which we are attempting to unravel. In this review, we underscore the seminal studies relevant to these topics with an emphasis on new observations pertinent to the mechanism of CAT and its cellular trajectories underpinning the preferential distribution of thymic epithelial cell-derived self-antigens to specific subsets of DC. Identification of molecular determinants which control CAT would significantly advance our understanding of how the cellularly targeted presentation of thymic self-antigens is functionally coupled to the T cell selection process.

The transcription factor Sox4 is required for thymic tuft cell development.

Medullary thymic epithelial cells (mTECs) help shape the thymic microenvironment for T-cell development by expressing a variety of peripheral tissue-restricted antigens (TRAs). The self-tolerance of T cells is established by negative selection of autoreactive T cells that bind to TRAs. To increase the diversity of TRAs, a fraction of mTECs terminally differentiates into distinct subsets resembling atypical types of epithelial cells in specific peripheral tissues. As such, thymic tuft cells that express peripheral tuft cell genes have recently emerged. Here, we show that the transcription factor SRY-box transcription factor 4 (Sox4) is highly expressed in mTECs and is essential for the development of thymic tuft cells. Mice lacking Sox4 specifically in TECs had a significantly reduced number of thymic tuft cells with no effect on the differentiation of other mTEC subsets, including autoimmune regulator (Aire)+ and Ccl21a+ mTECs. Furthermore, Sox4 expression was diminished in mice deficient in TEC-specific lymphotoxin β receptor (LTβR), indicating a role for the LTβR-Sox4 axis in the differentiation of thymic tuft cells. Given that Sox4 promotes differentiation of peripheral tuft cells, our findings suggest that mTECs employ the same transcriptional program as peripheral epithelial cells. This mechanism may explain how mTECs diversify peripheral antigen expression to project an immunological self within the thymic medulla.

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.

Differential expression of tissue-restricted antigens among mTEC is associated with distinct autoreactive T cell fates

Medullary thymic epithelial cells (mTEC) contribute to the development of T cell tolerance by expressing and presenting tissue-restricted antigens (TRA), so that developing T cells can assess the self-reactivity of their antigen receptors prior to leaving the thymus. mTEC are a heterogeneous population of cells that differentially express TRA. Whether mTEC subsets induce distinct autoreactive T cell fates remains unclear. Here, we establish bacterial artificial chromosome (BAC)-transgenic mouse lines with biased mTEClo or mTEChi expression of model antigens. The transgenic lines support negative selection of antigen-specific thymocytes depending on antigen dose. However, model antigen expression predominantly by mTEClo supports TCRαβ+ CD8αα intraepithelial lymphocyte development; meanwhile, mTEChi-restricted expression preferentially induces Treg differentiation of antigen-specific cells in these models to impact control of infectious agents and tumor growth. In summary, our data suggest that mTEC subsets may have a function in directing distinct mechanisms of T cell tolerance.

Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells

To induce central T‐cell tolerance, medullary thymic epithelial cells (mTEC) collectively express most protein‐coding genes, thereby presenting an extensive library of tissue‐restricted antigens (TRAs). To resolve mTEC diversity and whether promiscuous gene expression (PGE) is stochastic or coordinated, we sequenced transcriptomes of 6,894 single mTEC, enriching for 1,795 rare cells expressing either of two TRAs, TSPAN8 or GP2. Transcriptional heterogeneity allowed partitioning of mTEC into 15 reproducible subpopulations representing distinct maturational trajectories, stages and subtypes, including novel mTEC subsets, such as chemokine‐expressing and ciliated TEC, which warrant further characterisation. Unexpectedly, 50 modules of genes were robustly defined each showing patterns of co‐expression within individual cells, which were mainly not explicable by chromosomal location, biological pathway or tissue specificity. Further, TSPAN8+ and GP2+ mTEC were randomly dispersed within thymic medullary islands. Consequently, these data support observations that PGE exhibits ordered co‐expression, although mechanisms underlying this instruction remain biologically indeterminate. Ordered co‐expression and random spatial distribution of a diverse range of TRAs likely enhance their presentation and encounter with passing thymocytes, while maintaining mTEC identity.

Thymic expression of tissue-restricted self-antigens is a highly coordinated and evolutionary conserved process

Abstract Promiscuous gene expression (pGE) of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for tolerance imposition in the thymus. PGE is characterized on the one hand by inclusion of a broad range of TRAs and on the other hand by its mosaic patterns, whereby each antigen is only expressed in 1–3% of mTECs at a given point in time. Yet, this mosaic pattern at the single cell level faithfully adds up to the full repertoire of self-antigens at the population level. In order to analyze the regulatory mechanisms underlying this transcriptional heterogeneity among mTECs, we applied two complementing approaches, the isolation of minor mTEC subsets as defined by TRA-selected gene co-expression groups in conjunction with single cell mRNA sequencing. Different TRA-selected mTEC subfractions, each expressing distinct sets of genes in a mutually overlapping fashion, mapped to distinct stages of mTEC development. These co-expression patterns were evolutionary conserved between mouse and human (Rattay et al., J. Autoimmunity 2015). Applying an unbiased single cell mRNA sequencing approach, we extended these findings to the single cell level and showed that the mouse mTEC population essentially represents a composite of multiple co-expression groups (Brennecke et al., Nat. Immunology 2015). These co-expression groups may represent only snapshots of a continuum of changing co-expression groups along the lifetime of an individual mTEC, as captured in the model of “sliding co-expression groups” (Pinto et al., PNAS 2013). Continuous genome scanning would potentially enlarge the overall diversity of self-antigens displayed by a single mTEC.

Thymic Crosstalk Coordinates Medulla Organization and T-Cell Tolerance Induction.

The thymus ensures the generation of a functional and highly diverse T-cell repertoire. The thymic medulla, which is mainly composed of medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), provides a specialized microenvironment dedicated to the establishment of T-cell tolerance. mTECs play a privileged role in this pivotal process by their unique capacity to express a broad range of peripheral self-antigens that are presented to developing T cells. Reciprocally, developing T cells control mTEC differentiation and organization. These bidirectional interactions are commonly referred to as thymic crosstalk. This review focuses on the relative contributions of mTEC and DC subsets to the deletion of autoreactive T cells and the generation of natural regulatory T cells. We also summarize current knowledge regarding how hematopoietic cells conversely control the composition and complex three-dimensional organization of the thymic medulla.

Homeodomain-interacting protein kinase 2, a novel autoimmune regulator interaction partner, modulates promiscuous gene expression in medullary thymic epithelial cells.

Promiscuous expression of a plethora of tissue-restricted Ags (TRAs) by medullary thymic epithelial cells (mTECs) plays an essential role in T cell tolerance. Although the cellular mechanisms by which promiscuous gene expression (pGE) imposes T cell tolerance have been well characterized, the underlying molecular mechanisms remain poorly understood. The autoimmune regulator (AIRE) is to date the only validated molecule known to regulate pGE. AIRE is part of higher-order multiprotein complexes, which promote transcription, elongation, and splicing of a wide range of target genes. How AIRE and its partners mediate these various effects at the molecular level is still largely unclear. Using a yeast two-hybrid screen, we searched for novel AIRE-interacting proteins and identified the homeodomain-interacting protein kinase 2 (HIPK2) as a novel partner. HIPK2 partially colocalized with AIRE in nuclear bodies upon cotransfection and in human mTECs in situ. Moreover, HIPK2 phosphorylated AIRE in vitro and suppressed the coactivator activity of AIRE in a kinase-dependent manner. To evaluate the role of Hipk2 in modulating the function of AIRE in vivo, we compared whole-genome gene signatures of purified mTEC subsets from TEC-specific Hipk2 knockout mice with control mice and identified a small set of differentially expressed genes. Unexpectedly, most differentially expressed genes were confined to the CD80(lo) mTEC subset and preferentially included AIRE-independent TRAs. Thus, although it modulates gene expression in mTECs and in addition affects the size of the medullary compartment, TEC-specific HIPK2 deletion only mildly affects AIRE-directed pGE in vivo.

Thymus Medulla Formation and Central Tolerance Are Restored in IKKα−/− Mice That Express an IKKα Transgene in Keratin 5+ Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique capacity to present a diverse array of tissue restricted Ags that induce clonal deletion of self-reactive thymocytes. One mTEC subset expresses keratin 5 (K5) and K14, but fails to bind Ulex europaeus agglutinin-1 (UEA-1) lectin. A distinct mTEC subset binds UEA-1 and expresses K8, but not K5 or K14. Development of both mTEC subsets requires activation of the noncanonical NF-kappaB pathway. In this study, we show that mTEC development is severely impaired and autoimmune manifestations occur in mice that are deficient in IkappaB kinase (IKK)alpha, a required intermediate in the noncanonical NF-kappaB signaling pathway. Introduction of an IKKalpha transgene driven by a K5 promoter restores the K5(+)K14(+) mTEC subset in IKKalpha(-/-) mice. Unexpectedly, the K5-IKKalpha transgene also rescues the UEA-1 binding mTEC subset even though K5 expression is not detectable in these cells. In addition, expression of the K5-IKKalpha transgene ameliorates autoimmune symptoms in IKKalpha(-/-) mice. These data suggest that 1) medulla formation and central tolerance depend on activating the alternative NF-kappaB signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.