Search

The thymus is a unique primary lymphoid organ that supports the production of self‐tolerant T‐cells essential for adaptive immunity. Intrathymic microenvironments are microanatomically compartmentalised, forming defined cortical, and medullary regions each differentially supporting critical aspects of thymus‐dependent T‐cell maturation. Importantly, the specific functional properties of thymic cortical and medullary compartments are defined by highly specialised thymic epithelial cells (TEC). For example, in the medulla heterogenous medullary TEC (mTEC) contribute to the enforcement of central tolerance by supporting deletion of autoreactive T‐cell clones, thereby counterbalancing the potential for random T‐cell receptor generation to contribute to autoimmune disease. Recent advances have further shed light on the pathways and mechanisms that control heterogeneous mTEC development and how differential mTEC functionality contributes to control self‐tolerant T‐cell development. Here we discuss recent findings in relation to mTEC development and highlight examples of how mTEC diversity contribute to thymus medulla function. [ABSTRACT FROM AUTHOR]

As the primary site of T-cell development, the thymus dictates immune competency of the host. The rates of thymus function are not constant, and thymus regeneration is essential to restore new T-cell production following tissue damage from environmental factors and therapeutic interventions. Here, we show the alarmin interleukin (IL) 33 is a product of Sca1+ thymic mesenchyme both necessary and sufficient for thymus regeneration via a type 2 innate immune network. IL33 stimulates expansion of IL5-producing type 2 innate lymphoid cells (ILC2), which triggers a cellular switch in the intrathymic availability of IL4. This enables eosinophil production of IL4 to re-establish thymic mesenchyme prior to recovery of thymopoiesis-inducing epithelial compartments. Collectively, we identify a positive feedback mechanism of type 2 innate immunity that regulates the recovery of thymus function following tissue injury. Although thymic function declines with age, the thymus also has the ability to regenerate following injury. Here, the authors demonstrate that IL-33 and type-2 innate lymphoid cells trigger the expansion of eosinophils following radiation injury, which in turn produce IL-4 to stimulate the recovery of the thymus mesenchyme during thymus regeneration. [ABSTRACT FROM AUTHOR]

γδT cells are produced in the thymus throughout life and provide immunity at epithelial‐rich sites. Unlike conventional αβT cells, γδT‐cell development involves intrathymic acquisition of effector function, with priming for either IL17 or IFN‐γ production occurring during embryonic or adult life, respectively. How the thymus controls effector‐primed γδT‐cell generation in adulthood is poorly understood. Here, we distinguished de novo γδT cells from those undergoing thymus recirculation and/or retention using Rag2GFP mice alongside markers of maturation/effector priming including CD24, CD25, CD73, and IFN‐γ, the latter by crossing with IFN‐γYFP GREAT mice. We categorize newly developing γδT‐cells into an ordered sequence where CD25+CD73−IFN‐γYFP− precursors are followed sequentially by CD25−CD73+IFN‐γYFP− intermediates and CD25−CD73+IFN‐γYFP+ effectors. To determine intrathymic requirements controlling this sequence, we examined γδT‐cell development in Relb−/− thymus grafts that lack medullary microenvironments. Interestingly, medulla deficiency did not alter CD25+ γδT‐cell precursor generation, but significantly impaired development of effector primed stages. This impact on γδT‐cell priming was mirrored in plt/plt mice lacking the medullary chemoattractants CCL19 and CCL21, and also Ccl21a−/− but not Ccl19−/− mice. Collectively, we identify the medulla as an important site for effector priming during adult γδT‐cell development and demonstrate a specific role for the medullary epithelial product CCL21 in this process. [ABSTRACT FROM AUTHOR]

Autoimmune regulator+ (Aire) medullary thymic epithelial cells (mTECs) play a critical role in tolerance induction. Several studies demonstrated that Aire+mTECs differentiate further into Post‐Aire cells. Yet, the identification of terminal stages of mTEC maturation depends on unique fate‐mapping mouse models. Herein, we resolve this limitation by segmenting the mTEChi(MHCIIhiCD80hi) compartment into mTECA/hi (CD24−Sca1−), mTECB/hi (CD24+Sca1−), and mTECC/hi (CD24+Sca1+). While mTECA/hi included mostly Aire‐expressing cells, mTECB/hi contained Aire+ and Aire− cells and mTECC/hi were mainly composed of cells lacking Aire. The differential expression pattern of Aire led us to investigate the precursor‐product relationship between these subsets. Strikingly, transcriptomic analysis of mTECA/hi, mTECB/hi, and mTECC/hi sequentially mirrored the specific genetic program of Early‐, Late‐ and Post‐Aire mTECs. Corroborating their Post‐Aire nature, mTECC/hi downregulated the expression of tissue‐restricted antigens, acquired traits of differentiated keratinocytes, and were absent in Aire‐deficient mice. Collectively, our findings reveal a new and simple blueprint to survey late stages of mTEC differentiation. [ABSTRACT FROM AUTHOR]

Abstract: Members of the innate lymphoid cell (ILC) family have been implicated in the development of thymic microenvironments and the recovery of this architecture after damage. However, a detailed characterization of this family in the thymus is lacking. To better understand the thymic ILC compartment, we have utilized multiple in vivo models including the fate mapping of inhibitor of DNA binding‐2 (Id2) expression and the use of Id2 reporter mice. Our data demonstrate that ILCs are more prominent immediately after birth, but were rapidly diluted as the T‐cell development program increased. As observed in the embryonic thymus, CCR6+NKp46− lymphoid tissue inducer (LTi) cells were the main ILC3 population present, but numbers of these cells swiftly declined in the neonate and ILC3 were barely detectable in adult thymus. This loss of ILC3 means ILC2 are the dominant ILC population in the thymus. Thymic ILC2 were able to produce IL‐5 and IL‐13, were located within the medulla, and did not result from ILC3 plasticity. Furthermore, in WT mice, thymic ILC2 express little RANKL (receptor activator of nuclear factor kappa‐B ligand) arguing that functionally, these cells provide different signals to LTi cells in the thymus. Collectively, these data reveal a dynamic switch in the ILC populations of the thymus during neonatal development. [ABSTRACT FROM AUTHOR]

Most αβ T cells that form in the thymus are generated during mainstream conventional thymocyte development and involve the generation and selection of a diverse αβ TCR repertoire that recognizes self-peptide/MHC complexes. Additionally, the thymus also supports the production of T cell subsets that express αβ TCRs but display unique developmental and functional features distinct from conventional αβ T cells. These include multiple lineages of CD1d-restricted invariant NKT (iNKT) cells that express an invariant αβ TCR, branch off from mainstream thymocytes at the CD4+ CD8+ stage, and are potent producers of polarizing cytokines. Importantly, and despite their differences, iNKT cells and conventional αβ T cells share common requirements for thymic epithelial microenvironments during their development. Moreover, emerging evidence suggests that constitutive cytokine production by iNKT cells influences both conventional thymocyte development and the intrathymic formation of additional innate CD8+ αβ T cells with memory-like properties. In this article, we review evidence for an intrathymic innate lymphocyte network in which iNKT cells play key roles in multiple aspects of thymus function. [ABSTRACT FROM AUTHOR]

Mutations in the filaggrin gene (Flg) are associated with increased systemic levels of Th17 cells and increased IL-17A production following antigen exposure in both humans and mice. In addition to Th17 cells, γδ T cells can produce IL-17A. The differentiation of γδ T cells to either IFNγ or IL-17A-producing (γδT17) cells is mainly determined in the thymus. Interestingly, it has been reported that filaggrin is expressed in the Hassall bodies in the human thymic medulla. However, whether filaggrin affects γδ T cell development is not known. Here, we show that filaggrin-deficient flaky tail (ft/ft) mice have an increased number of γδT17 cells in the spleen, epidermis, and thymus compared to wild-type (WT) mice. We demonstrate that filaggrin is expressed in the mouse thymic medulla and that blocking the egress of cells from the thymus results in accumulation of Vγ2+ γδT17 cells in the thymus of adult ft/ft mice. Finally, we find increased T cell receptor expression levels on γδ T cells and increased levels of IL-6 and IL-23 in the thymus of ft/ft mice. These findings demonstrate that filaggrin is expressed in the mouse thymic medulla and that production of Vγ2+ γδT17 cells is dysregulated in filaggrin-deficient ft/ft mice. [ABSTRACT FROM AUTHOR]

Abstract: In the thymus, medullary thymic epithelial cells (mTEC) determine the fate of newly selected CD4+ and CD8+ single positive (SP) thymocytes. For example, mTEC expression of Aire controls intrathymic self‐antigen availability for negative selection. Interestingly, alterations in both Foxp3+ Regulatory T‐cells (T‐Reg) and conventional SP thymocytes in Aire−/− mice suggest additional, yet poorly understood, roles for Aire during intrathymic T‐cell development. To examine this, we analysed thymocytes from Aire−/− mice using Rag2GFP and Foxp3 expression, and a recently described CD69/MHCI subset definition of post‐selection CD4+ conventional thymocytes. We show that while Aire is dispensable for de novo generation of conventional αβT‐cells, it plays a key role in controlling the intrathymic T‐Reg pool. Surprisingly, a decline in intrathymic T‐Reg in Aire−/− mice maps to a reduction in mature recirculating Rag2GFP− T‐Reg that express CCR6 and re‐enter the thymus from the periphery. Furthermore, we show mTEC expression of the CCR6 ligand CCL20 is reduced in Aire−/− mice, and that CCR6 is required for T‐Reg recirculation back to the thymus. Collectively, our study re‐defines requirements for late stage intrathymic αβT‐cell development, and demonstrates that Aire controls a CCR6‐CCL20 axis that determines the developmental makeup of the intrathymic T‐Reg pool. [ABSTRACT FROM AUTHOR]

The thymus is a primary lymphoid tissue that supports the generation of αβT cells. In this review, we describe the processes that give rise to the thymus medulla, a site that nurtures self-tolerant T-cell generation following positive selection events that take place in the cortex. To summarize the developmental pathways that generate medullary thymic epithelial cells ( mTEC) from their immature progenitors, we describe work on both the initial emergence of the medulla during embryogenesis, and the maintenance of the medulla during postnatal stages. We also investigate the varying roles that receptors belonging to the tumor necrosis factor receptor superfamily have on thymus medulla development and formation, and highlight the impact that T-cell development has on thymus medulla formation. Finally, we examine the evidence that the thymic medulla plays an important role during the intrathymic generation of distinct αβT-cell subtypes. Collectively, these studies provide new insight into the development and functional importance of medullary microenvironments during self-tolerant T-cell production in the thymus. [ABSTRACT FROM AUTHOR]

In the thymus, interactions with both cortical and medullary microenvironments regulate the development of self-tolerant conventional CD4+ and CD8+ αβT cells expressing a wide range of αβTCR specificities. Additionally, the cortex is also required for the development of invariant NKT (iNKT) cells, a specialized subset of T cells that expresses a restricted αβTCR repertoire and is linked to the regulation of innate and adaptive immune responses. Although the role of the cortex in this process is to enable recognition of CD1d molecules expressed by CD4+CD8+ thymocyte precursors, the requirements for additional thymus microenvironments during iNKT cell development are unknown. In this study, we reveal a role for medullary thymic epithelial cells (mTECs) during iNKT cell development in the mouse thymus. This requirement for mTECs correlates with their expression of genes required for IL-15 trans-presentation, and we show that soluble IL-15/IL-15Rα complexes restore iNKT cell development in the absence of mTECs. Furthermore, mTEC development is abnormal in iNKT cell-deficient mice, and early stages in iNKT cell development trigger receptor activator for NF-κB ligand-mediated mTEC development. Collectively, our findings demonstrate that intrathymic iNKT cell development requires stepwise interactions with both the cortex and the medulla, emphasizing the importance of thymus compartmentalization in the generation of both diverse and invariant αβT cells. Moreover, the identification of a novel requirement for iNKT cells in thymus medulla development further highlights the role of both innate and adaptive immune cells in thymus medulla formation. [ABSTRACT FROM AUTHOR]

Thymocytes expressing the invariant Vγ5 γδT-cell receptor represent progenitors of dendritic epidermal T-cells (DETC) that play an important immune surveillance role in the skin. In contrast to the bulk of αβT-cell development, Vγ5+ DETC progenitor development occurs exclusively in fetal thymus. Whilst αβT-cell development is known to require chemokine receptor mediated migration through distinct thymus regions, culminating in medullary entry and thymic egress, the importance and control of intrathymic migration for DETC progenitors is unclear. We recently revealed a link between Vγ5+ DETC progenitor development and medullary thymic epithelial cells expressing Aire, a known regulator of thymic chemokine expression, demonstrating that normal Vγ5+ DETC progenitor development requires regulated intramedullary positioning. Here we investigate the role of chemokines and their receptors during intrathymic Vγ5+ DETC progenitor development and establishment of the DETC pool in the skin. We report that thymic medullary accumulation of Vγ5+ DETC progenitors is a G-protein coupled receptor dependent process. However, this process occurs independently of Aire’s influences on intrathymic chemokines, and in the absence of CCR4 and CCR7 expression by DETC progenitors. In contrast, analysis of epidermal γδT-cells at neonatal and adult stages in CCR4−/− mice reveals that reduced numbers of DETC in adult epidermis are not a consequence of diminished intrathymic embryonic development, nor deficiencies in initial epidermal seeding in the neonate. Collectively, our data reveal differences in the chemokine receptor requirements for intrathymic migration of αβ and invariant γδT-cells, and highlight a differential role for CCR4 in the maintenance, but not initial seeding, of DETC in the epidermis. [ABSTRACT FROM AUTHOR]

Abstract: The role of mesenchymal stromal cells (MSCs) in regulating immune responses in the thymus is currently unclear. Here we report the existence and role of a MSC population in the thymus that expresses the pericyte and MSC marker CD248 (endosialin). We show using a CD248-deficient mouse model, that CD248 expression on these cells is required for full post-natal thymus development and regeneration post-Salmonella infection. In CD248−/− mice the thymus is hypocellular and regeneration is poorer, with significant loss of all thymocyte populations. This identifies the requirement of CD248 to maintain optimal thymic cellularity post-partum and infection. [Copyright &y& Elsevier]

The thymus supports the production of self-tolerant T cells from immature precursors. Studying the mechanisms regulating the establishment and maintenance of stromal microenvironments within the thymus therefore is essential to our understanding of T-cell production and ultimately immune system functioning. Despite our ability to phenotypically define stromal cell compartments of the thymus, the mechanisms regulating their development and the ways by which they influence T-cell precursors are still unclear. Here, we review recent findings and highlight unresolved issues relating to the development and functioning of thymic stromal cells. [ABSTRACT FROM AUTHOR]

The vitamin A metabolite and transcriptional modulator retinoic acid (RA) is recognized as an important regulator of epithelial cell homeostasis in several tissues. Despite the known importance of the epithelial compartment of the thymus in T cell development and selection, the potential role of RA in the regulation of thymic cortical and medullary epithelial cell homeostasis has yet to be addressed. In this study, using fetal thymus organ cultures, we demonstrate that endogenous RA signaling promotes thymic epithelial cell (TEC) cell-cycle exit and restricts TEC cellularity preferentially in the cortical TEC compartment. Combined gene expression, biochemical, and functional analyses identified mesenchymal cells as the major source of RA in the embryonic thymus. In reaggregate culture experiments, thymic mesenchyme was required for RA-dependent regulation of TEC expansion, highlighting the importance of mesenchyme-derived RA in modulating TEC turnover. The RA-generating potential of mesenchymal cells was selectively maintained within a discrete Ly51intgp38+ subset of Ly51+ mesenchyme in the adult thymus, suggesting a continual role for mesenchymal cell-derived RA in postnatal TEC homeostasis. These findings identify RA signaling as a novel mechanism by which thymic mesenchyme influences TEC development. [ABSTRACT FROM AUTHOR]

The thymic medulla is critical for the enforcement of central tolerance. In addition to deletion of auto-reactive T-cells, the thymic medulla supports the maturation of heterogeneous natural αβT-cells linked to tolerance mechanisms. Natural IL-17-secreting CD4 + αβT-cells (nTh17) represent recently described natural αβT-cells that mature and undergo functional priming intrathymically. Despite a proposed potential to impact upon either protective or pathological inflammatory responses, the intrathymic mechanisms regulating the balance of nTh17 development are unclear. Here we compare the development of distinct natural αβT-cells in the thymus. We reveal that thymic stromal MHC class II expression and RelB-dependent medullary thymic epithelial cells (mTEC), including Aire + mTEC, are an essential requirement for nTh17 development. nTh17 demonstrate a partial, non-redundant requirement for both ICOS-ligand and CD80/86 costimulation, with a dispensable role for CD80/86 expression by thymic epithelial cells. Although mTEC constitutively expressed inducible nitric oxide synthase (iNOS), a critical negative regulator of conventional Th17 differentiation, iNOS was not essential to constrain thymic nTh17. These findings highlight the critical role of the thymic medulla in the differential regulation of novel natural αβT-cell subsets, and reveal additional layers of thymic medullary regulation of T-cell driven autoimmunity and inflammation. [ABSTRACT FROM AUTHOR]

αβT cell development depends upon serial migration of thymocyte precursors through cortical and medullary microenvironments, enabling specialized stromal cells to provide important signals at specific stages of their development. Although conventional αβT cells are subject to clonal deletion in the medulla, entry into the thymus medulla also fosters αβT cell differentiation. For example, during postnatal periods, the medulla is involved in the intrathymic generation of multiple αβT cell lineages, notably the induction of Foxp3+ regulatory T cell development and the completion of invariant NKT cell development. Although migration of conventional αβT cells to the medulla is mediated by the chemokine receptor CCR7, how other T cell subsets gain access to medullary areas during their normal development is not clear. In this study, we show that combining a panel of thymocyte maturation markers with cell surface analysis of CCR7 and CCR4 identifies distinct stages in the development of multiple αβT cell lineages in the thymus. Although Aire regulates expression of the CCR4 ligands CCL17 and CCL22, we show that CCR4 is dispensable for thymocyte migration and development in the adult thymus, demonstrating defective T cell development in Aire-/- mice is not because of a loss of CCR4-mediated migration. Moreover, we reveal that CCR7 controls the development of invariant NKT cells by enabling their access to IL-15 trans-presentation in the thymic medulla and influences the balance of early and late intrathymic stages of Foxp3+ regulatory T cell development. Collectively, our data identify novel roles for CCR7 during intrathymic T cell development, highlighting its importance in enabling multiple αβT cell lineages to access the thymic medulla. [ABSTRACT FROM AUTHOR]