Promiscuous gene expression and the developmental dynamics of medullary thymic epithelial cells

Thymic epithelial cells (TEC) form the structural and functional microenvironment necessary for the establishment and quality control of the T cell repertoire. In addition, they provide an ectopic source of numerous tissue-restricted antigens (TRA), a feature called promiscuous gene expression (pGE). How the regulation of pGE is related to the cell biology of TEC subset(s), e.g. their turnover and developmental interrelationship is still poorly understood. The observation that pGE is foremost a property of phenotypically and functionally mature medullary TEC (mTEC) implies that the full implementation of pGE is contingent on mTEC differentiation. Here, we show that the emergence of TEC subsets and pGE is tightly correlated during ontogeny and we provide evidence that mature CD80pos mTEC develop from an immature CD80neg subset. This differentiation step proceeds continuously in the postnatal thymus. While mature mTEC turnover in 2 to 3 weeks, immature mTEC encompass a smaller cycling and a larger non-cycling pool. The latter might serve as a reservoir of committed precursors, which sustain this renewal process. Our data document that mTEC represent a highly dynamic cell population, and they imply that the availability and display of TRA in the thymus undergoes a perpetual temporal and spatial reorganization.     

Adult thymic epithelial cell (TEC) progenitors and TEC stem cells: Models and mechanisms for TEC development and maintenance

The thymus is the primary lymphoid organ for generating self‐restricted and self‐tolerant functional T cells. Its two distinct anatomical regions, the cortex and the medulla, are involved in positive and negative selection, respectively. Thymic epithelial cells (TECs) constitute the framework of this tissue and function as major stromal components. Extensive studies for more than a decade have revealed how TECs are generated during organogenesis; progenitors specific for medullary TECs (mTECs) and cortical TECs (cTECs) as well as bipotent progenitors for both lineages have been identified, and the signaling pathways required for the development and maturation of mTECs have been elucidated. However, little is known about the initial commitment of mTECs and cTECs during ontogeny, and how regeneration of both lineages is sustained in the postnatal/adult thymus. Recently, stem cell activities in TECs have been demonstrated, and TEC progenitors have been identified in the postnatal thymus. In this review, recent advances in studying the development and maintenance of TECs are summarized, and the possible mechanisms of thymic regeneration and involution are discussed.     

Intermediate expression of CCRL1 reveals novel subpopulations of medullary thymic epithelial cells that emerge in the postnatal thymus

Cortical and medullary thymic epithelial cells (cTECs and mTECs, respectively) provide inductive microenvironments for T‐cell development and selection. The differentiation pathway of cTEC/mTEC lineages downstream of common bipotent progenitors at discrete stages of development remains unresolved. Using IL‐7/CCRL1 dual reporter mice that identify specialized TEC subsets, we show that the stepwise acquisition of chemokine (C–C motif) receptor‐like 1 (CCRL1) is a late determinant of cTEC differentiation. Although cTECs expressing high CCRL1 levels (CCRL1^hi) develop normally in immunocompetent and Rag2^?/?thymi, their differentiation is partially blocked in Rag2^?/?Il2rg^?/? counterparts. These results unravel a novel checkpoint in cTEC maturation that is regulated by the cross‐talk between TECs and immature thymocytes. Additionally, we identify new Ulex europaeus agglutinin 1 (UEA)⁺ mTEC subtypes expressing intermediate CCRL1 levels (CCRL1^int) that conspicuously emerge in the postnatal thymus and differentially express Tnfrsf11a, Ccl21, and Aire. While rare in fetal and in Rag2^?/? thymi, CCRL1^int mTECs are restored in Rag2^?/?Marilyn TCR‐Tg mice, indicating that the appearance of postnatal‐restricted mTECs is closely linked with T‐cell selection. Our findings suggest that alternative temporally restricted routes of new mTEC differentiation contribute to the establishment of the medullary niche in the postnatal thymus.     

Serial progression of cortical and medullary thymic epithelial microenvironments

Thymic epithelial cells (TECs) provide key instructive signals for T‐cell differentiation. Thymic cortical (cTECs) and medullary (mTECs) epithelial cells constitute two functionally distinct microenvironments for T‐cell development, which derive from a common bipotent TEC progenitor. While seminal studies have partially elucidated events downstream of bipotent TECs in relation to the emergence of mTECs and their progenitors, the control and timing of the emergence of the cTEC lineage, particularly in relation to that of mTEC progenitors, has remained elusive. In this review, we describe distinct models that explain cTEC/mTEC lineage divergence from common bipotent progenitors. In particular, we summarize recent studies in mice providing evidence that mTECs, including the auto‐immune regulator⁺ subset, derive from progenitors initially endowed with phenotypic properties typically associated with the cTEC lineage. These observations support a novel “serial progression” model of TEC development, in which progenitors serially acquire cTEC lineage markers, prior to their commitment to the mTEC differentiation pathway. Gaining a better understanding of the phenotypic properties of early stages in TEC progenitor development should help in determining the mechanisms regulating cTEC/mTEC lineage development, and in strategies aimed at thymus reconstitution involving TEC therapy.     

Bipotency of thymic epithelial progenitors comes in sequence

In the thymus, in order to become MHC‐restricted self‐tolerant T cells, developing thymocytes need to interact with cortical and medullary thymic epithelial cells (TECs). Although the presence of a common bipotent progenitor for these functionally and structurally distinct epithelial subsets has been clearly established, the initial developmental stages of these bipotent cells have not been well characterized. In this issue of the European Journal of Immunology, Baik et al. [Eur. J. Immunol. 2013.43: 589–594] focus on the phenotypical changes of the early bipotent populations and show how the cortical and medullary markers are sequentially acquired during TEC development. These findings argue against a binary model in which both cortical and medullary lineages diverge simultaneously from lineage‐negative TEC progenitors and highlight an unexpected overlap in the phenotypic properties of these bipotent TECs with their lineage‐restricted counterparts.     

CCRL1 marks heterogeneity in cortical and medullary thymic epithelial cells

Cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs), which play essential roles in the establishment of a functionally competent and self‐tolerant repertoire of T cells, are derived from common thymic epithelial progenitor cells (pTECs). Recent findings indicate that mTECs are derived from cells that express molecules that are abundant in cTECs rather than mTECs, and provide fresh insight into the characteristics of pTECs and their diversification pathways into TEC subpopulations. In this issue of the European Journal of Immunology, Ribeiro et al. [Eur. J. Immunol. 2014. 44: 2918–2924] focus on CCRL1, an atypical chemokine receptor that is highly expressed by cTECs rather than mTECs, and show that CCRL1‐expressing embryonic TECs can give rise to mTECs. Interestingly, Ribeiro et al. further report that a fraction of postnatal mTECs express CCRL1 at a low level, suggesting novel complexity in mTECs.     

Thymocytes and RelB-dependent medullary epithelial cells provide growth-promoting and organization signals,respectively, to thymic medullary stromal cells

The thymic medulla is composed of distinct epithelial cell subsets, defined in this report by the reactivity of two novel antibodies, 95 and 29, raised against mouse thymic epithelial cell lines. These antibodies were used to probe the development of medulla in wild-type or mutant thymuses. In CD3σ-deficient mice where thymocyte maturation is arrested at the CD4^? CD8^? stage, few scattered 95⁺ and 29⁺ epithelial cells are found. When few mature thymocytes develop as in CD3-ζ/η mice, expansion and organization of 95⁺ but not 29⁺ cells, becomes detectable. In RelB-deficient mice, T cell maturation proceeds normally but negative selection is inefficient due to the lack of thymic medulla and dendritic cells. Strikingly, 29⁺ epithelial cells are absent and 95⁺ medullary epithelial cells are scattered throughout the thymus, intermingling with CDR1⁺ cortical epithelium. In chimeric mice lacking only dendritic cells, the corticomedullary junction persists and both 95⁺ and 29⁺ epithelial cells are localized in the medulla. These results suggest that two types of signals are required for development of thymic medulla. A growth signal depends upon the presence of maturing thymocytes, but organization of the thymic medulla requires the presence of activated 29⁺ medullary epithelial cells.     

Relb acts downstream of medullary thymic epithelial stem cells and is essential for the emergence of RANK+ medullary epithelial progenitors

Thymic epithelial cells (TECs) provide essential signals for αβT‐cell development, and medullary TECs (mTECs) control T‐cell tolerance through both negative selection and Foxp3⁺ regulatory T (Treg) cell development. Although heterogeneity within the mTEC compartment is well studied, the molecular regulators of specific stages of mTEC development are still poorly understood. Given the importance of the RANK‐RANKL axis in thymus medulla formation, we have used RANK Venus reporter mice to analyze the ontogeny of RANK⁺ TECs during development and correlated RANK expression with mTEC stem cells defined by SSEA‐1. In addition, we have investigated how requirements for the key regulators Foxn1 and Relb map to specific stages of mTEC development. Here, we show SSEA‐1⁺ mTEC stem cells emerge prior to RANK expression and are present in both nude and Relb^?/? mice, providing direct evidence that mTEC lineage specification occurs independently of Foxn1 and Relb. In contrast, we show that Relb is necessary for the effective production of downstream RANK⁺ mTEC progenitors. Collectively, our work defines stage‐specific requirements for critical TEC regulators during medulla development, including the timing of Relb dependency, and provides new information on mechanisms controlling mTEC specification.     

GPR109A expressed on medullary thymic epithelial cells affects thymic Treg development

Regulatory T cells (Treg) maintain immune homeostasis due to their anti-inflammatory functions. They can be generated either centrally in the thymus or in peripheral organs. Metabolites such as short-chain fatty acids produced by intestinal microbiota can induce peripheral Treg differentiation, by activating G-protein-coupled-receptors like GPR109A. In this study, we identified a novel role for GPR109A in thymic Treg development. We found that Gpr109a^−/− mice had increased Treg under basal conditions in multiple organs compared with WT mice. GPR109A was not expressed on T cells but on medullary thymic epithelial cells (mTECs), as revealed by single-cell RNA sequencing in both mice and humans and confirmed by flow cytometry in mice. mTECs isolated from Gpr109a^−/− mice had higher expression of autoimmune regulator (AIRE), the key regulator of Treg development, while the subset of mTECs that did not express Gpr109a in the WT displayed increased Aire expression and also enhanced signaling related to mTEC functionality. Increased thymic Treg in Gpr109a^−/− mice was associated with protection from experimental autoimmune encephalomyelitis, with ameliorated clinical signs and reduced inflammation. This work identifies a novel role for GPR109A and possibly the gut microbiota, on thymic Treg development via its regulation of mTECs.     

A new monoclonal antibody to human subcapsular thymic epithelial cells

Summary A monoclonal antibody, termed K-20, was generated against an anaplastic thymic carcinoma cell line, Ty-82. Subcapsular thymic epithelial cells of the thymus and blood vessels in various organs were shown to react with the K-20 monoclonal antibody by immunohistochemical staining. Immunofluorescent study revealed that various haematopoietic fresh cells and cell lines did not show any significant reactivity with K-20, except for one Epstein-Barr-virus-carrying lymphoma cell line (SP-50B). Western immunoblotting and affinity purification procedure revealed that K-20 was directed to a protein with a molecular weight of 28 kDa. K-20 is unique in its restrictive reactivity with human subcapsular thymic epithelial cells.     

识别胸腺髓质上皮细胞和胸腺树突状细胞的单链抗体的筛选

目的　利用噬菌体展示技术寻找胸腺基质细胞表面的新抗原。方法　用培养的胸腺基质细胞系MTDC作为靶抗原富集初级噬菌体抗体库 ,从经过富集后的次级抗体库中挑选克隆 ,制备单链抗体 ,并在冰冻切片及培养的细胞系上检测抗体的特异性。结果　从经 4轮富集的次级抗体库中挑选到一个新的克隆。用此克隆制备的单链抗体可同时辨认胸腺髓质上皮细胞亚群和胸腺树突状细胞亚群。结论　胸腺髓质上皮细胞和胸腺树突状细胞均具有异质性 ,同时噬菌体展示技术可以作为寻找细胞表面新分子的强有力工具。     

Generation of both cortical and Aire+ medullary thymic epithelial compartments from CD205+ progenitors

In the adult thymus, the development of self‐tolerant thymocytes requires interactions with thymic epithelial cells (TECs). Although both cortical and medullary TECs (cTECs/mTECs) are known to arise from common bipotent TEC progenitors, the phenotype of these progenitors and the timing of the emergence of these distinct lineages remain unclear. Here, we have investigated the phenotype and developmental properties of bipotent TEC progenitors during cTEC/mTEC lineage development. We show that TEC progenitors can undergo a stepwise acquisition of first cTEC and then mTEC hallmarks, resulting in the emergence of a progenitor population simultaneously expressing the cTEC marker CD205 and the mTEC regulator Receptor Activator of NF‐κB (RANK). In vivo analysis reveals the capacity of CD205⁺ TECs to generate functionally competent cortical and medullary microenvironments containing both cTECs and Aire⁺ mTECs. Thus, TEC development involves a stage in which bipotent progenitors can co‐express hallmarks of the cTEC and mTEC lineages through sequential acquisition, arguing against a simple binary model in which both lineages diverge simultaneously from bipotent lineage negative TEC progenitors. Rather, our data reveal an unexpected overlap in the phenotypic properties of these bipotent TECs with their lineage‐restricted counterparts.     

Abnormal development of thymic dendritic and epithelial cells in human X-linked severe combined immunodeficiency

The X-linked form of severe combined immunodeficiency (X-SCID) is caused by mutations in the common cytokine receptor gamma chain and results in lack of T and NK cells and defective B cells. Without immune reconstitution, X-SCID patients typically die from infection during infancy. This report describes thymic epithelial (TE), lymphocyte, and dendritic cell (DC) differentiation in the thymic microenvironment of seven X-SCID patients who died before or after treatment for their immunodeficiency. X-SCID thymus consisted predominately of TE cells without grossly evident corticomedullary distinction. CD3+ and CD1a+ developing T cells and CD83+ thymic DC were reduced >50-fold when compared to age- and gender-matched control thymus (P < 0.001). TE expression of epithelial differentiation markers CK14, involucrin, and high molecular weight cytokeratins also differed in X-SCID versus normal thymus. These histopathologic findings indicate that in addition to T cells, thymic DC development and differentiation of TE cells are also abnormal in X-SCID.     

Regulatory T cells in thymic epithelium-induced tolerance. I. Suppression of mature peripheral non-tolerant T cells

Athymic mice grafted at birth with allogeneic thymic epithelium (TE) display life-long tolerance to tissue grafts of the TE donor strain, in spite of harboring peripheral T cells capable of rejecting those grafts. Tolerance is maintained in these chimeras by TE-specific regulatory CD4 T cells. We presently address the quantification and the mechanisms of this dominant tolerance process. C57BL/6 mice containing variable but defined numbers of peripheral, resident T cells received cell transfers of graded numbers of peripheral T cells from B6(BALB E10) chimeras (C57BL/6 nude mice grafted with TE from 10-day-old BALB/c embryos), resulting in a series of animals containing a wide range of donor (tolerant) versus host (non-tolerant) T cell chimerism. Increasing the relative representation of donor T cells results in a progressive delay in the rejection of BALB/c skin grafts, life-long tolerance being achieved at a ratio of tolerant and non-tolerant T cell populations of 1. In recipients displaying full tolerance, graftreactive non-tolerant T cells were not deleted, anergized or committed to noninflammatory functions. Thus, sorted host T cells from tolerant recipients readily rejected BALB/c skin grafts upon transfer to immunodeficient animals. Finally, measurements of “helper” and inflammatory activities, as well as interleukin-4 and interferon-γ production, failed to discriminate between T cell populations from tolerant and non-tolerant animals after specific in vitro stimulation. We conclude that: (a) TE-selected regulatory T cells can suppress, in a quantitative manner, in vivo T cell responses against major and minor histocompatibility antigens expressed by the TE and, (b) this suppressive activity neither inactivates mature non-tolerant T cells, nor does it seem to drive their differentiation along noninflammatory pathways.