Thymic epithelial cells: working class heroes for T cell development and repertoire selection

The thymus represents an epithelial-mesenchymal tissue, anatomically structured into discrete cortical and medullary regions that contain phenotypically and functionally distinct stromal cells, as well as thymocytes at defined stages of maturation. The stepwise progression of thymocyte development seems to require serial migration through these distinct thymic regions, where interactions with cortical thymic epithelial cell (cTEC) and medullary thymic epithelial cell (mTEC) subsets take place. Recent work on TEC subsets provides insight into T cell development and selection, such as the importance of tumour necrosis factor (TNF) receptor superfamily members in thymus medulla development, and the specialised antigen processing/presentation capacity of the thymic cortex for positive selection. Here, we summarise current knowledge on the development and function of the thymic microenvironment, paying particular attention to the cortical and medullary epithelial compartments.     

Expression of housekeeping and immunoproteasome subunit genes is differentially regulated in positively and negatively selecting thymic stroma subsets

The expression of housekeeping and/or immunoproteasomes in isolated thymic stroma subsets has so far not been analyzed but may have important consequences for self peptide repertoires presented by MHC class I molecules during positive and negative thymic selection. Here we determined the expression of housekeeping and immunoproteasome beta subunits and of PA28 in positively and negatively selecting stroma subsets. Positively selecting cortical thymic epithelial cells (cTEC) expressed only housekeeping but no immunoproteasome beta subunit mRNA and proteins. However, immunoproteasome beta subunits could be induced in cTEC by infection with Listeria monocytogenes or injection of IFN-gamma. In negatively selecting stroma including medullary epithelial cells and dendritic cells, incomplete and low representation of housekeeping beta subunit proteins but high and complete expression of immunoproteasome beta subunit proteins suggests absence of proper housekeeping proteasomes and predominance of immunoproteasomes. Expression of immunoproteasome beta subunits in negatively selecting stroma was independent of IFN-gamma receptor as shown in knockout (KO) mice. Absence of LMP2 altered thymic selection of the MHC class I-restricted transgenic P14 TCR in KO mice. The data suggest that negative selection may primarily involve immunoproteasome peptide repertoires and that peripheral infection may influence peptide repertoires involved in positive selection.     

Thymus‐specific serine protease regulates positive selection of a subset of CD4+ thymocytes

Thymus‐specific serine protease (TSSP) was initially reported as a putative protease specifically expressed in the endosomal compartment of cortical thymic epithelial cells (cTEC). As such, TSSP is potentially involved in the presentation of the self‐peptides that are bound to MHC class II molecules expressed at the cTEC surface and are involved in the positive selection of CD4⁺ thymocytes. We tested this hypothesis by generating mutant mice deprived of Prss16, the gene encoding TSSP. TSSP‐deficient mice produced normal numbers of T cells, despite a decrease in the percentage of cTEC expressing high surface levels of MHC class II. By using sensitive transgenic models expressing MHC class II‐restricted TCR transgenes (Marilyn and OT‐II), we showed that the absence of TSSP markedly impaired the selection of Marilyn and OT‐II CD4⁺ T cells. In contrast, selection of CD8⁺ T cells expressing an MHC class I‐restricted TCR transgene (OT‐I) was unaffected. Therefore, TSSP is involved in the positive selection of some CD4⁺ T lymphocytes and likely constitutes the first serine protease to play a function in the intrathymic presentation of self‐peptides bound to MHC class II complexes.     

Identification and characterization of major histocompatibility complex class II compartments in cortical thymic epithelial cells

Previous studies have concentrated on elucidating the subcellular localization of major histocompatibility (MHC) class II molecules mainly in B cells, macrophages, and dendritic cells. Despite very rich cell-surface expression of MHC class II molecules by cortical thymic epithelial cells (cTECs), little is known regarding the expression of these molecules by cTECs at the subcellular level. In the present study we focused on the identification and characterization of MHC class II compartments (MIICs) in cTECs in situ by immunogold electron microscopy (IEM). We found that MHC class II molecules were located exclusively in the cytoplasmic vacuoles, and we identified these MHC class II molecule-containing cytoplasmic vacuoles as MIICs in cTECs. These MIICs were immunopositive for early endosomal, late endosomal, and lysosomal markers. Moreover, in these MIICs, MHC class II molecules were colocalized with cathepsin L, H2-DM, class II-associated invariant chain (Ii), and class II-associated invariant chain peptide (CLIP). Similarly, Ii molecules were colocalized with endosomal and lysosomal markers, cathepsin L, and H2-DM in the vacuoles. Taken together, these results suggest that MIICs in cTECs represent conventional endocytic compartments. The colocalization of MHC class II molecule or Ii with cathepsin L and H2-DM in the MIICs suggests that MIICs in cTECs may be sites of Ii degradation and peptide loading.     

Thymic stromal organization is regulated by the specificity of T cell receptor/major histocompatibility complex interactions

The thymic architecture is normally compartmentalized into a central medulla surrounded by a peripheral cortical region. We investigated how compartmentalization of the thymic stroma is regulated using T cell receptor (TCR)-transgenic mouse models. Our studies show that the signals generated by TCR/peptide/major histocompatibility complex interactions regulate thymic stromal cell compartmentalization. In TCR-transgenic mice, normal stromal cell compartmentalization occurs when the transgenic TCR is expressed on a background that does not result in skewing toward either positive or negative selection. In models representing strong positive selection, the thymic stromal elements do not fully organize into a central medulla. Instead, small medullary foci are dispersed throughout the thymus with some regions residing directly under the capsule. The highest degree of disorganization in medullary epithelial regions is observed in TCR-transgenic mice that exhibit negative selection. Although the medullary foci lack central organization, the expression in these regions of CD80, CD86 and CD40, as well as the clustering of dendritic cells, is similar to that observed in medullae of wild-type mice. Thus, the organization of the medulla appears to occur in two stages: (1) small medullary epithelial regions that are dispersed in fetal thymi expand and associate with antigen-presenting cells, and (2) the expanded medullary foci organize into a central medullary compartment. Our data suggest a model in which this second stage of stromal cell organization is increasingly inhibited as the normal balance of TCR-mediated signals is skewed by higher-avidity interactions between thymocytes and antigen-presenting cells.     

Immunosuppression with cyclosporin A alters the thymic microenvironment

The effect of cyclosporin A (CyA) immunosuppression on the murine thymic microenvironment and T lymphocyte development has been analysed using monoclonal antibodies to epithelial and lymphocyte subpopulations, macrophages and major histocompatibility complex (MHC) class II antigens in immunohistochemistry and flow cytometry. The major microenvironmental target for CyA-induced damage was the thymic medulla, where a reduction in all epithelial cell subsets, dendritic cells and macrophages was observed. In contrast, the thymic cortex appeared essentially normal. CyA had no detectable effect on the intensity of microenvironmental expression of MHC class II molecules in either cortex or medulla, although the number of MHC class II positive medullary cells was reduced after CyA treatment. CyA also had a differential effect on the thymic lymphocyte populations where there was little change in the Thy-1 bright, CD5 dull, CD4+, CD8+ cortical thymocytes but a depletion of the Thy-1 dull, CD5 bright, CD4 or CD8 single-positive medullary cells. This lymphocyte loss may be due partly to increased migration from thymus to spleen and other peripheral lymphoid organs, and partly to a block in the differentiation stage from cortical to medullary lymphocyte. The thymic microenvironment and lymphocyte subpopulations recover rapidly after cessation of CyA treatment, although there may be longer term functional defects resulting from the CyA-induced injury.     

Phenotypic Mapping of The Chicken Embryonic Thymic Microenvironment Developing Within an Organ Culture System

The chicken thymic microenvironment, as it developed in an embryonic thymus organ culture system, was phenotypically mapped using a panel of mAb defining both epithelial and nonepithelial stromal cell antigens. We have previously reported that thymocyte proliferation and differentiation will proceed for up to 6–8 days in thymus organ culture, hence demonstrating the functional integrity of the thymic microenvironment in vitro. During this time, the stromal component reflected that of the normal embryo with cortical and medullary epithelial areas readily identifiable by both morphology and surface-antigen expression. An abundance of subcapsular and cortical epithelial antigens was detected in the cultured thymus, particularly those normally expressed by the epithelium lining the capsule, trabeculae, and vascular regions (type epithelium) in the adult and embryonic thymus. Medullary epithelial antigens developed in organ culture, although were present in lower frequency than observed in the age-matched embryonic thymus. MHC class II expression by both epithelial and nonepithelial cells was maintained at high levels throughout the culture period. With increasing time in culture, the ratio of epithelial to nonepithelial cells decreased, concurrent with a decrease in thymocyte frequency and suggestive of a bidirectional interaction between these two cell types. Thus, a functionally intact thymic microenvironment appears to be maintained in embryonic thymus organ culture, a model that is currently being exploited to assess the role of stromal antigens, as defined by our mAb, in the process of thymopoiesis.     

NF-κB-inducing kinase contributes to normal development of cortical thymic epithelial cells: its possible role in shaping a proper T-cell repertoire

Nuclear factor (NF)-κB-inducing kinase (NIK) is known to be a critical regulator of multiple aspects of the immune response. Although the role of NIK in the development of medullary thymic epithelial cells (mTECs) has been well documented, the impact of NIK on the differentiation and function of cortical thymic epithelial cells (cTECs) remains ambiguous. To investigate the possible involvement of NIK in cTEC differentiation, we have compared the gene expression and function of cTECs from a NIK-mutant mouse, alymphoplasia (aly/aly) with those of cTECs from wild-type (WT) mice. Flow cytometric analyses revealed that expression levels of MHC class II, but not MHC class I or other TEC markers, were higher in aly/aly cells than in WT cells. Notably, the proportion of MHC class II^hi+ cTECs was elevated in aly/aly mice. We also demonstrated that expression of Ccl5 mRNA in the MHC class II^hi+ subset of aly/aly cTECs was decreased compared with that in WT cells, implying an abnormal pattern of gene expression in aly/aly cTECs. Analyses of bone marrow chimera using aly/aly or aly/+ mice as hosts suggested that Vβ usage and CD5 expression on WT T-cells were altered when they matured in aly/aly thymi. These results collectively indicate that NIK may be involved in controlling the function of cTEC in selecting a proper T-cell repertoire.     

The intrathymic microenvironment: expression of lectin receptors and lectin-like molecules of differentiation antigens and MHC gene products

The expression of MHC products, differentiation antigens and lectin receptors has been investigated in the various cell types populating different compartments of the thymus. The ultrastructural classification of suspended thymic epithelial cells was facilitated by using a technique that preserves cortical nursing cells or medullary epithelial cell clusters. A subset of peanut lectin positive lymphocytes could be distinguished by their ability to bind soybean lectin also. This subset corresponds to the large proliferating lymphocytes that populate the area between the thymic capsule and the cortex. Ia and H-2 D/K antigens could be detected on nearly all epithelial and lymphoid cells. Expression of H-2 antigens, however, is more pronounced on medullary epithelial cells. T-cell differentiation antigens such as Thy-1 and Lyt-1 could be demonstrated not only on lymphocytes, but, interestingly enough, on cortical epithelial cells as well. These latter cells, in addition, exhibit a cell membrane-bound lectin with a specificity for D-galactose which might well be the structure responsible for binding the galactosyl residues of the peanut lectin receptor of thymic lymphocytes. Binding sites for a large set of lectins could be demonstrated on both, thymic lymphocytes and epithelium. The intrathymic differentiation pathway of T-lineage cells is discussed with regard to those lymphocytic and epithelial cell surface structures considered to enable cellular interaction.     

Role of thymic cortex-specific self-peptides in positive selection of T cells

During T cell development in the thymus, a virgin repertoire of diverse TCRαβ recognition specificities in immature thymocytes is selected through positive and negative selection to form an immunocompetent and self-tolerant repertoire of mature T cells. Positive selection supports the survival of thymocytes that receive weak signals of low-avidity TCR engagement, whereas negative selection deletes potentially harmful self-reactive thymocytes upon high-avidity TCR engagement. Early studies have highlighted the role of TCR interaction with polymorphic MHC determinants in positive selection, while negative selection imposes TCR specificity to peptide antigens displayed by MHC molecules. However, recent advances in the biology of thymic stromal cells have indicated that the formation of an immunocompetent TCR repertoire requires positive selection by thymic cortical epithelial cells expressing a unique protein degradation machinery, suggesting the role of self-peptide repertoire specifically expressed by thymic cortical epithelial cells in the development of the acquired immune system.     

T-cell selection in the thymus: New routes toward the identification of the self-peptide ligandome presented by thymic epithelial cells

Within the thymus, thymic epithelial cells (TECs) provide a dedicated niche for the selection of functional T cells expressing a highly variable and self-tolerant T-cell receptor (TCR) repertoire. In this minireview, we start by summarizing recent studies that have improved our understanding on the composition of cortical TEC and medullary TEC microenvironments. Next, we focus on the molecular processes that control the function of TECs in T-cell selection. In particular, we discuss the role of cortical TECs in positive selection and the pathways employed by these cells to generate and present selecting self-peptides:MHC II complexes. Several studies have underscored the role of the β5t-containing thymoproteasome in the production of unique MHC I-bound peptides critical for CD8 T-cell selection. Contrarily, the identity of the molecular determinants that regulate the generation of MHC II-bound self-peptides capable of positive selecting CD4 T cells is far more uncertain. We highlight recent advances that interconnect the autophagy-lysosomal pathway, the presentation of specific sets of self-peptide:MHC II complexes, and the diversification of CD4 TCR repertoire. Lastly, we discuss how these findings may open up new avenues for deciphering the identity of the MHC I and MHC II ligandome in the thymus.     

Activation of cortical and inhibited differentiation of medullary epithelial cells in the thymus of lymphotoxin-beta receptor-deficient mice: an ultrastructural study

The reciprocal influences of thymic lymphocyte and nonlymphocyte populations, i.e. thymic cross-talk, are necessary for the proper maturation of thymocytes and the development/maintenance of thymic stromal microenvironments. Although the molecular influences exerted by thymic stromal cells on maturing thymocytes have been extensively studied, the identity of signalling molecules used by thymocytes to influence the thymic stromal cells is still largely unknown. Our study provides the first ultrastructural evidence that the functional lymphotoxin-beta receptor (LTbetaR) signalling pathway is engaged in the cross-talk between thymocytes and the thymic stromal cell population. We show that LTbetaR signalling is of the utmost significance for the preservation of the subcellular integrity of all thymic epithelial cells. In the absence of LTbetaR there is (1) hypertrophy and activation of cortical thymic epithelial cells, (2) the complete loss of fully differentiated medullary thymic epithelial cells, and (3) the inhibited differentiation of remaining medullary thymic epithelial cells with the appearance of prominent intercellular cysts in the thymic medulla.     

Spatial (Tbata) expression in mature medullary thymic epithelial cells

The Spatial gene is expressed in highly polarized cell types such as testis germ cells, brain neurons and thymic epithelial cells (TEC). Its expression was documented in testis and brain but poorly characterized in thymus. Here, we characterize for the first time Spatial‐expressing TEC throughout ontogeny and adult mouse thymus. Spatial is expressed in thymic‐fated domain by embryonic day E10.5 and persists in subcapsular, cortical, medullary epithelial cells and in MTS24⁺ progenitor TEC. Using mouse strains in which thymocyte development is blocked at various stages, we show that Spatial expression is independent of thymocyte‐derived signals during thymus organogenesis. Analyses on purified thymic cell subsets show that Spatial short isoforms are expressed in cortical TEC (cTEC) and mature medullary TEC (mTEC). Spatial long isoforms were detected in the same TEC population. Spatial presents a nuclear distribution specific to mature mTEC expressing UEA1 and Aire. Aire‐ and RANKL‐deficient mice revealed that Spatial expression is drastically reduced in the thymus of these mutants. These findings reveal a critical function of Aire in regulating Spatial expression, which is compatible with promiscuous Spatial gene expression.     

In vitro positive selection of alpha beta TCR transgenic thymocytes by a conditionally immortalized cortical epithelial clone

Development of mature CD4 and CD8 single-positive T cells requires a process known as positive selection, which depends on the specific recognition of self-peptide-MHC complexes on thymic stromal cells by immature CD4+CD8+ thymocytes. We have used an in vitro reaggregate system to study the positive selection of thymocytes by conditionally immortalized thymic epithelial clones. Thymocytes from mice transgenic for the F5 alpha beta TCR, specific for a peptide from the influenza nucleoprotein in the context of H-2Db, are positively selected in the H- 2b MHC background, but fail to mature in mice expressing the H-2q haplotype. Development of embryonic day 15 F5 H-2q transgenic thymocytes was followed in reaggregate cultures supplemented with H-2b- expressing epithelial clones. A conditionally immortalized cortical epithelial clone, derived from H-2Kb-tsA58 transgenic mice, was found to be as efficient as freshly isolated thymic stromal cells in positively selecting CD8 transgenic thymocytes. In contrast, an H-2b- expressing kidney epithelial clone did not augment positive selection above background levels, implying that the effect of the thymic epithelial clone was not merely the presentation of selecting MHC molecules. Mature transgenic thymocytes generated in reaggregate cultures were able to differentiate into functionally competent cytotoxic T cells. This model provides an important in vitro system for the detailed study of the specific molecular interactions leading to positive selection of developing thymocytes.      

Influence of a thymic hormone on cultured epithelial cells of the thymus

Addition of cortisone to primary cultures of mouse thymic stromal cells resulted in increased growth of medullary epithelial cells while cortical epithelial subpopulations were inhibited or destroyed. These adverse effects on cortical cells were ameliorated by the thymic hormone THF. Because thymocytes are also sensitive to both cortisone and THF we sought to eliminate them from the cultures by deoxyguanosine treatment. This treatment differentiated even more strongly between growth of cortical epithelial cells which were inhibited and medullary epithelium that was very strongly stimulated. These effects are discussed in relation to involution of the thymus with aging.     

Human intrathymic T cell differentiation

The human thymus develops early on in fetal gestation with morphologic maturity reached by the beginning of the second trimester. Endodermal epithelial tissue from the third pharyngeal pouch gives rise to TE3+ cortical thymic epithelium while ectodermal epithelial tissue from the third pharyngeal cleft invaginates and splits during development to give rise to A2B5/TE4+ medullary and subcapsular cortical thymic epithelium. Fetal liver CD7+ T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression on thymocytes of other T lineage surface molecules. Human thymic epithelial cells grown in vitro bind to mature and immature thymocytes via CD2 and CD11a/CD18 (LFA-1) molecules on thymocytes and by CD58 (LFA-3) and CD54 (ICAM-1) molecules on thymic epithelial cells. Thymic epithelial cells produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF--molecules that likely are important in various stages of thymocyte activation and differentiation. Thymocytes can be activated via several cell surface molecules including CD2, CD3/TCR, and CD28 molecules. Finally, CD7+ CD4-CD8- CD3- thymocytes give rise to T cells of both the TCRab+ and TCR gd+ lineages.     

Analysis of the thymic microenvironment by monoclonal antibodies with special reference to thymic nurse cells

Two hybridoma cell lines secreting monoclonal antibodies against stromal tissues of mouse thymus were produced using the spleen cells of BALB/C mice immunized with newborn thymic homogenate of C57BL/6 mice emulsified in Freund's complete adjuvant. The monoclonal antibody Th-3 reacted with stromal cells in the thymic cortex and the monoclonal antibody Th-4 reacted with stromal cells in the thymic medulla. The stromal cells revealed by Th-3 showed a meshwork structure in the cortex, and formed a monolayered border at the cortical surface and around the vasculature. Each mesh of this structure was connected to each other, forming a complex labyrinth and being open toward the medullary area. Neither lymphoid cells nor any cells in any other organs were reacted with this Th-3 antibody. However, the reactivity of Th-3 with the thymic cortical stromal cells was observed not only in C57BL/6 mice which had been used as source of antigen, but also in other strains of mice such as C3H and BALB/C. Immunoelectron microscopy revealed that Th-3 monoclonal antibody was reactive with some component, diffusely present in the cytoplasm of cortical epithelial cells. The pattern of Th-3 positive meshwork in the thymic cortex was quite similar to that stained by either anti-IA or anti-IE antibody, but the Th-3 positive reaction was not inhibited by these anti-IA and anti-IE antibodies. Thymic nurse cells prepared by the method of Wekerle were positive for Th-3 antibody. On the contrary, Th-4 reacted only with epithelial cells in the thymic medulla. It was suggested that Th-3 monoclonal antibody detected some antigen specific to so called thymic nurse cells.     

Differential processing of self-antigens by subsets of thymic stromal cells

The stromal network of the thymus provides a unique environment that supports the development of mature CD4(+) and CD8(+) T cells expressing a very diverse repertoire of T cell receptors (TCR) with limited reactivity to self-antigens. Thymic cortical epithelial cells (cTECs) are specialized antigen-presenting cells (APCs) that promote the positive selection of developing thymocytes while medullary thymic epithelial cells (mTECs) and thymic dendritic cells (tDCs) induce central tolerance to self-antigens. Recent studies showed that cTECs express a unique set of proteases involved in the generation of self-peptides presented by major-histocompatibility encoded molecules (pMHC) and consequently may express a unique set of pMHC complexes. Conversely, the stromal cells of the medulla developed several mechanisms to mirror as closely as possible the constellation of self-peptides derived from peripheral tissues. Here, we discuss how these different features allow for the development of a highly diverse but poorly self-reactive repertoire of functional T cells.