Cortical Thymic Epithelial Cells Research Articles

Functional Differences between Proteasome Subtypes.

Four proteasome subtypes are commonly present in mammalian tissues: standard proteasomes, which contain the standard catalytic subunits β1, β2 and β5; immunoproteasomes containing the immuno-subunits β1i, β2i and β5i; and two intermediate proteasomes, containing a mix of standard and immuno-subunits. Recent studies revealed the expression of two tissue-specific proteasome subtypes in cortical thymic epithelial cells and in testes: thymoproteasomes and spermatoproteasomes. In this review, we describe the mechanisms that enable the ATP- and ubiquitin-dependent as well as the ATP- and ubiquitin-independent degradation of proteins by the proteasome. We focus on understanding the role of the different proteasome subtypes in maintaining protein homeostasis in normal physiological conditions through the ATP- and ubiquitin-dependent degradation of proteins. Additionally, we discuss the role of each proteasome subtype in the ATP- and ubiquitin-independent degradation of disordered proteins. We also discuss the role of the proteasome in the generation of peptides presented by MHC class I molecules and the implication of having different proteasome subtypes for the peptide repertoire presented at the cell surface. Finally, we discuss the role of the immunoproteasome in immune cells and its modulation as a potential therapy for autoimmune diseases.

Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development.

In the thymus, the thymic epithelium provides a microenvironment essential for the development of functionally competent and self-tolerant T cells. Previous findings showed that modulation of Wnt/β-catenin signaling in mouse thymic epithelial cells (TECs) disrupts embryonic thymus organogenesis. However, the role of β-catenin in TECs for postnatal T-cell development remains to be elucidated. Here, we analyzed gain-of-function (GOF) and loss-of-function (LOF) of β-catenin highly specific in mouse TECs. We found that GOF of β-catenin in TECs results in severe thymic dysplasia and T-cell deficiency beginning from the embryonic period. By contrast, LOF of β-catenin in TECs reduces the number of cortical TECs and thymocytes modestly and only postnatally. These results indicate that fine-tuning of β-catenin expression within a permissive range is required for TECs to generate an optimal microenvironment to support postnatal T-cell development.

Insufficiency of CD205-positive cortical thymic epithelial cells in immature Japanese Black cattle with severe thymic abnormalities and poor prognosis

To investigate the involvement of thymic function in the development of diseases with poor prognosis in calves, this study conducted a survey for the assessment of thymus cell composition in immature Japanese Black cattle with poor prognosis. Histopathological evaluation of 47 cattle showed signs of acute thymic involution in most cases. Less than half of the cases had a cortex predominant over the medulla in the thymic parenchyma, and a quarter of the cases indicated severe histological condition with an unclear boundary between the cortex and medulla. Correlation analysis revealed a close relationship between the corresponding stages of acute involution, cortical occupancy, and the expression of CD4, CD8B, and CD205. When cases were grouped by cortical occupancy, the expression of CD4 and CD8B expression was lower in the severe group with less than 25 % cortical occupancy, and the expression of CD205 was lower in the group with an unclear cortical-medullary boundary. Meanwhile, there was no difference in the expressions of IL7, CD80, FEZF2, and FOXN1 according to cortical occupancy. Immunohistochemistry has shown that cytokeratin-positive thymic epithelial cells are more densely populated in the severe thymus. UEA-I-binding medullary thymic epithelial cells were also present, but CD205-positive cortical thymic epithelial cells were rare in severe thymus. Moreover, there were significantly fewer Ki-67-positive cells in cattle with severe thymus. Therefore, these results indicate that thymic histological abnormalities frequently occur in immature cattle with a poor prognosis, and the presence of CD205-positive cortical thymic epithelial cells is associated with the severity of the abnormalities.

Self-mediated positive selection of T cells sets an obstacle to the recognition of nonself

Adaptive immune recognition is mediated by the binding of peptide-human leukocyte antigen complexes by T cells. Positive selection of T cells in the thymus is a fundamental step in the generation of a responding T cell repertoire: only those T cells survive that recognize human peptides presented on the surface of cortical thymic epithelial cells. We propose that while this step is essential for optimal immune function, the process results in a defective T cell repertoire because it is mediated by self-peptides. To test our hypothesis, we focused on amino acid motifs of peptides in contact with T cell receptors. We found that motifs rarely or not found in the human proteome are unlikely to be recognized by the immune system just like the ones that are not expressed in cortical thymic epithelial cells or not presented on their surface. Peptides carrying such motifs were especially dissimilar to human proteins. Importantly, we present our main findings on two independent T cell activation datasets and directly demonstrate the absence of naïve T cells in the repertoire of healthy individuals. We also show that T cell cross-reactivity is unable to compensate for the absence of positively selected T cells. Additionally, we show that the proposed mechanism could influence the risk for different infectious diseases. In sum, our results suggest a side effect of T cell positive selection, which could explain the nonresponsiveness to many nonself peptides and could improve the understanding of adaptive immune recognition.

PSMB11 regulates gene expression in cortical thymic epithelial cells.

The PSMB11 proteasomal subunit, expressed only in cortical thymic epithelial cells (cTECs), is essential for the development of functional CD8+ Tcells. An attractive yet unproven theory holds that PSMB11 generates unique major histocompatibility complex class I (MHC I)-associated peptides required for positive selection. We recently reported that PSMB11 regulates the expression of hundreds of genes in cTECs, mainly by differential proteolysis of transcription factors. Thereby, PSMB11 maintains the distinctness of cTECs relative to medullary TECs (mTECs) and promotes cortex-to-medulla migration of developing thymocytes. These conclusions have been challenged by Ohigashi and colleagues, who suggest that their data show that PSMB11 uniquely controls antigen presentation without affecting cTEC biology. Here, we perform a comprehensive reanalysis of transcriptomic and proteomic data from the Ohigashi lab and confirm our original conclusions. This Matters Arising paper is in response to Ohigashi etal. (2019), published in Cell Reports. See also the response by Ohigashi and Takahama (2021), published in this issue of Cell Reports.

Specific impact of β5t on proteasome subunit composition in cortical thymic epithelial cells.

SUMMARYβ5t is a cortical thymic epithelial cell (cTEC)-specific component of the thymoproteasome, which is essential for the optimal production of functionally competent CD8+ T cells. Our recent analysis showed a specific impact of β5t on proteasome subunit composition in cTECs, supporting the possibility that the thymoproteasome optimizes CD8+ T cell development through the production of MHC-I-associated unique self-peptides in cTECs. However, a recent article reports that β5t regulates the expression of hundreds of cTEC genes and affects both CD4+ and CD8+ thymocytes by causing oxidative stress in thymocytes. The authors further analyze our published data and describe that they confirm their conclusions. Here, we examine the issues that they raise and conclude that, rather than regulating hundreds of genes in cTECs, β5t has a highly specific impact in cTECs on proteasome subunit composition. This Matters Arising Response article addresses the Apavaloaei et al. (2021) Matters Arising paper, published concurrently in Cell Reports.

Recognition of Multiple Hybrid Insulin Peptides by a Single Highly Diabetogenic T-Cell Receptor.

The mechanisms underlying the major histocompatibility complex class II (MHCII) type 1 diabetes (T1D) association remain incompletely understood. We have previously shown that thymocytes expressing the highly diabetogenic, I-Ag7-restricted 4.1-T-cell receptor (TCR) are MHCII-promiscuous, and that, in MHCII-heterozygous mice, they sequentially undergo positive and negative selection/Treg deviation by recognizing pro- and anti-diabetogenic MHCII molecules on cortical thymic epithelial cells and medullary hematopoietic antigen-presenting cells (APCs), respectively. Here, we use a novel autoantigen discovery approach to define the antigenic specificity of this TCR in the context of I-Ag7. This was done by screening the ability of random epitope–GS linker–I-chain fusion pools to form agonistic peptide–MHCII complexes on the surface of I- chain-transgenic artificial APCs. Pool deconvolution, I-Ag7-binding register-fixing, TCR contact residue mapping, and alanine scanning mutagenesis resulted in the identification of a 4.1-TCR recognition motif XL(G/A)XEXE(D/E)X that was shared by seven agonistic hybrid insulin peptides (HIPs) resulting from the fusion of several different chromogranin A and/or insulin C fragments, including post-translationally modified variants. These data validate a novel, highly sensitive MHCII-restricted epitope discovery approach for orphan TCRs and suggest thymic selection of autoantigen-promiscuous TCRs as a mechanism for the murine T1D–I-Ag7-association.

Dual roles for LUBAC signaling in thymic epithelial cell development and survival.

Thymic epithelial cells (TECs) form a unique microenvironment that orchestrates T cell differentiation and immunological tolerance. Despite the importance of TECs for adaptive immunity, there is an incomplete understanding of the signalling networks that support their differentiation and survival. We report that the linear ubiquitin chain assembly complex (LUBAC) is essential for medullary TEC (mTEC) differentiation, cortical TEC survival and prevention of premature thymic atrophy. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, severely impaired expansion of the thymic medulla and AIRE-expressing cells. Furthermore, HOIL-1-deficiency caused early thymic atrophy due to Caspase-8/MLKL-dependent apoptosis/necroptosis of cortical TECs. By contrast, deficiency in the LUBAC component, SHARPIN, caused relatively mild defects only in mTECs. These distinct roles for LUBAC components in TECs correlate with their function in linear ubiquitination, NFκB activation and cell survival. Thus, our findings reveal dual roles for LUBAC signaling in TEC differentiation and survival.

Regulation of thymic T regulatory cell differentiation by TECs in health and disease

The thymus produces self-limiting and self-tolerant T cells through the interaction between thymocytes and thymus epithelial cells (TECs), thereby generating central immune tolerance. The TECs are composed of cortical and medullary thymic epithelial cells, which regulate the positive and negative selection of T cells, respectively. During the process of negative selection, thymocytes with self-reactive ability are deleted or differentiated into regulatory T cells (Tregs). Tregs are a subset of suppressor T cells that are important for maintaining immune homeostasis. The differentiation and development of Tregs depend on the development of TECs and other underlying molecular mechanisms. Tregs regulated by thymic epithelial cells are closely related to human health and are significant in autoimmune diseases, thymoma and pregnancy. In this review, we summarize the current molecular and transcriptional regulatory mechanisms by which TECs affect the development and function of thymic Tregs. We also review the pathophysiological models of thymic epithelial cells regulating thymic Tregs in human diseases and specific physiological conditions.

Delayed maturation of thymic epithelium in mice with specific deletion of \u03b2-catenin gene in FoxN1 positive cells

Wnt signalling pathways have been reported to be involved in thymus development but their precise role in the development of both thymic epithelium (TE) and thymocytes is controversial. Herein, we examined embryonic, postnatal and adult thymi of mice with a specific deletion of β-catenin gene in FoxN1+ thymic epithelial cells (TECs). Together with a high postnatal mouse mortality, the analysis showed severe thymic hypocellularity, largely due an important reduction in numbers of developing thymocytes, and delayed, partially blocked maturation of mutant TECs. Affected TECs included largely cortical (c) TEC subsets, such as immature MTS20+ TECs, Ly51+ cTECs and a remarkable, rare Ly51+MTS20+MHCIIhi cell subpopulation previously reported to contain thymic epithelial progenitor cells (TEPCs) (Ulyanchenko et al., Cell Rep 14:2819–2832, 2016). In addition, altered postnatal organization of mutant thymic medulla failed to organize a unique, central epithelial area. This delayed maturation of TE cell components correlated with low transcript production of some molecules reported to be masters for TEC maturation, such as EphB2, EphB3 and RANK. Changes in the thymic lymphoid component became particularly evident after birth, when molecules expressed by TECs and involved in early T-cell maturation, such as CCL25, CXCL12 and Dll4, exhibited minimal values. This represented a partial blockade of the progression of DN to DP cells and reduced proportions of this last thymocyte subset. At 1 month, in correlation with a significant increase in transcript production, the DP cell percentage increased in correlation with a significant fall in the number of mature TCRαβhi thymocytes and peripheral T lymphocytes.

IPSC-Derived Thymic Epithelial Progenitor Cells As Cell-Based Therapy to Restore Thymic Function in Hematopoietic Stem Cell Transplant Recipients

Hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for many malignant and non-malignant diseases to date. The thymus plays a key role in post-transplant immune reconstitution. The outcome of a HSCT and the recipient thymic function are intimately intertwined. In fact, the production of mature T cells occurs in the thymus and depends on the interaction of hematopoietic progenitor cells with non-hematopoietic stromal elements, notably thymic epithelial cells (TECs). Through positive and negative selection, a healthy thymus generates a broad and self-tolerant T cell receptor (TCR) repertoire that protects from infection and prevents autoimmunity. In addition, the thymus gives rise to regulatory T-cells which confine immune responses and further promote central tolerance. At odds with its importance for successful transplant outcomes, the thymus is exquisitely sensitive to a wide range of insults encountered during the peri-transplant period, including cytotoxic agents, steroids and irradiation. In the allogeneic transplant setting, acute graft-versus-host disease (GVHD) can cause further thymic injury. Thymic injury in turn, leads to susceptibility to infections and autoimmune complications. Moreover, it is now widely recognized that thymic injury leading to defective deletion of self-reactive T-cells along with misguided T-helper cell polarization and B-cell activation constitute major drivers of chronic GVHD. Together these represent the leading causes of non-relapse-related transplant mortality and morbidity. As such, HSCT can precipitate a vicious cycle, in which the success of the HSCT procedure hinges on intact thymic function, but the HSCT process itself often causes irrecoverable thymic injury (Figure1). Thymic function naturally declines with age with little to no endogenous potential to regenerate. Current treatment options for thymic injury during HSCT are limited to symptomatic therapy. Therefore, novel approaches to cure irreversible thymic injury are critically needed.To address this need, our goal is to generate fully functional thymic epithelial progenitor cells (TEPCs) from hPSCs as a stem cell-based therapy for thymic regeneration and restoration of immune competence in HSCT recipients. Building on the efficient induction of definitive endoderm from human pluripotent stem cells (hPSCs) by directing signals controlling lineage bifurcations, we have developed a novel platform that drives TEPC development through sequential induction of the intermediaries anterior foregut (AFG) and pharyngeal pouch endoderm (PPE). Here we show that TEPCs derived in vitro from hPSCs or hESCs express the key thymic transcription factor FOXN1 along with Keratin 5 and Keratin 8, the primary cytoskeletal building blocks of medullary and cortical TECs, respectively. In addition, in vitro derived TEPCs express MHC class II to varying degrees (Figure 2). To optimize our in vitro platform, modifications of key-signaling pathways in the differentiation protocol are validated by comparing the transcriptome of hPSC-derived TEPCs to that of primary human medullary and cortical thymic epithelial cells using next generation sequencing. Our ongoing work is testing the capacity of hPSC-derived TEPCs to promote T-cell maturation, positive and negative selection resulting in the development of a broad and self-tolerant TCR repertoire from adoptively transferred human HSPCs in a humanized mouse model.Through sequential validation of fidelity, functionality, transplantability and sustainability of hPSC-derived TEPCs, we strive to develop an experimental model that can be translated by a clinical Good Manufacturing Practice (cGMP) facility for use in bone marrow transplant recipients.Ultimately, the potential applications of in vitro derived TEPCs are not confined to restoring thymic function in the HSCT setting. TEPCs derived from solid organ donors hold the promise of inducing tolerance to donor tissues, thereby preventing rejection of the transplanted donor organ. Leveraging the body's own ability to educate T-cells through regenerating thymic tissues could represent a novel approach to addressing key challenges in transplantation immunology. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow-Derived Mesenchymal Stromal Cells Promote Thymopoiesis and Function Recovery in Acute Graft-Versus-Host Disease Murine Models

Background: The thymus is the primary lymphoid organ of T-cells development and plays a crucial role for immune tolerance following allogeneic hematopoietic stem cell transplantation (allo-HSCT). The toxicity of chemoradiotherapy conditioning regimen and acute graft-versus-host disease (aGVHD) damage the thymic structure and function, which result in the production of autoreactive T-cells and the reduction of natural regulatory T cells (nTregs) to aggravate the development of aGVHD. Nowadays, mesenchymal stromal cells (MSCs) as multipotent stem cells, have been widely recognized as a promising therapeutic strategy for aGVHD. However, the well-known mechanisms of MSCs ameliorating aGVHD mainly focus on their immunoregulatory effect on peripheral immune compartments. The contribution of MSCs to thymus, the central immunological organs, is rarely reported.Methods: In this study, we carried out studies in a murine aGVHD models of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c). We randomly divided aGVHD mice into two groups, including MSCs group and control group without MSCs treatment. In MSCs group, aGVHD mice were intravenously administrated GFP-labelled bone marrow (BM) derived MSCs at a dose of 5×105 cells/mice at 7 day after allo-HSCT. The mice were killed at 1d, 2d, 4d and 20d after MSCs injection in MSCs group and the corresponding time in control group, respectively. Thymic tissues were collected and manufactured into frozen sections or thymic single-cell suspension. And then we analyzed the distribution of GFP-MSCs in thymus and thymic size, the expression of thymic T cells subsets (CD4+CD8-, CD4-CD8+, CD4+CD8+ T, CD4+CD25+Foxp3+Tregs), thymic epithelial cells (TECs) substes (CD45-CD326+Ly51+ cortical TECs and CD45-CD326+UEA-1+ medullary TECs) and the level of T cell receptor rearrangement excision circles (TRECs) in thymus between the two groups to evaluate the repair effect of MSCs for thymus.Results: We found that MSCs treated mice have longer survival and alleviate the clinical signs of aGVHD compared to the control group (P<0.05). Next, we observed that GFP-MSCs can home to thymic tissue and mainly locate in the corticomedullary boundary at 2d, 4da after MSCs infusion. MSCs treated thymus exhibited a more organized corticomedullary architecture, compared with the conrol group. Significant increases in thymus size and weight, as well as the number of total thymocytes were seen in MSCs group. To further investigate whether MSCs promote thymopoiesis and ameliorate thymic functions after allo-HSCT, we found that in MSCs group the proportion of thymic T cells subets composition (CD4+CD8-, CD4-CD8+, CD4+CD8+), especially Foxp3+ Tregs, and the expression of TECs, especially cTECs, were significantly higher than control group at 20d after the MSCs infusion (P<0.001). Furthermore, MSCs administration resulted in a remarkable increase in the levels of TRECs in the thymocyte at 20d after the MSCs infusion compared with the control group (P=0.007).Conclusions: Collectively, our data proposed a new mechanism for MSCs ameliorating aGVHD by central immune organ, including promoting thymopoiesis and enhancing thymic output. DisclosuresNo relevant conflicts of interest to declare.

Indispensable epigenetic control of thymic epithelial cell development and function by polycomb repressive complex 2

Thymic T cell development and T cell receptor repertoire selection are dependent on essential molecular cues provided by thymic epithelial cells (TEC). TEC development and function are regulated by their epigenetic landscape, in which the repressive H3K27me3 epigenetic marks are catalyzed by polycomb repressive complex 2 (PRC2). Here we show that a TEC-targeted deficiency of PRC2 function results in a hypoplastic thymus with reduced ability to express antigens and select a normal repertoire of T cells. The absence of PRC2 activity reveals a transcriptomically distinct medullary TEC lineage that incompletely off-sets the shortage of canonically-derived medullary TEC whereas cortical TEC numbers remain unchanged. This alternative TEC development is associated with the generation of reduced TCR diversity. Hence, normal PRC2 activity and placement of H3K27me3 marks are required for TEC lineage differentiation and function and, in their absence, the thymus is unable to compensate for the loss of a normal TEC scaffold.

The Early Postnatal Life: A Dynamic Period in Thymic Epithelial Cell Differentiation.

The microenvironments formed by cortical (c) and medullary (m) thymic epithelial cells (TECs) play a non-redundant role in the generation of functionally diverse and self-tolerant T cells. The role of TECs during the first weeks of the murine postnatal life is particularly challenging due to the significant augment in T cell production. Here, we critically review recent studies centered on the timely coordination between the expansion and maturation of TECs during this period and their specialized role in T cell development and selection. We further discuss how aging impacts on the pool of TEC progenitors and maintenance of functionally thymic epithelial microenvironments, and the implications of these chances in the capacity of the thymus to sustain regular thymopoiesis throughout life.

Thymic epithelial cells require the autophagy-related protein Vps34 for CD4+ T cell selection

Abstract The generation of a functional, self-tolerant T cell repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). The presentation of self-antigens to developing thymocytes involves a variety of cellular processes, including proteolysis, endocytosis, vesicle trafficking, and autophagy. In our current study, we focus on the class III PI3K, Vps34, which has been implicated in autophagy and vesicle trafficking. To address the contribution of Vps34 to TEC function, we have generated mice with a TEC-specific Vps34 deficiency. Conditional mutants display progressively hypoplastic thymi with reduced TEC cellularity and altered TEC subset ratio. Additionally, when TCR transgenic lines were bred to these animals we found profound defects in the positive selection of the CD4 T lymphocyte lineage but not the CD8 T lymphocyte lineage. Interestingly, cortical TECs from mutant mice display an increased abundance of unprocessed antigen bound to surface MHCII molecules indicating altered antigen processing. Collectively, these studies identify a role for the PI3K Vps34 in maintaining TEC homeostasis and contributing to the repertoire of selecting ligands processed and presented by TECs.

The Role of Proteasomes in the Thymus.

The thymus provides a microenvironment that supports the generation and selection of T cells. Cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) are essential components of the thymic microenvironment and present MHC-associated self-antigens to developing thymocytes for the generation of immunocompetent and self-tolerant T cells. Proteasomes are multicomponent protease complexes that degrade ubiquitinated proteins and produce peptides that are destined to be associated with MHC class I molecules. cTECs specifically express thymoproteasomes that are essential for optimal positive selection of CD8+ T cells, whereas mTECs, which contribute to the establishment of self-tolerance in T cells, express immunoproteasomes. Immunoproteasomes are also detectable in dendritic cells and developing thymocytes, additionally contributing to T cell development in the thymus. In this review, we summarize the functions of proteasomes expressed in the thymus, focusing on recent findings pertaining to the functions of the thymoproteasomes and the immunoproteasomes.

Role of immunoproteasomes and thymoproteasomes in health and disease

The proteasome is a multisubunit protease that degrades intracellular proteins into small peptides. Besides playing a pivotal role in many cellular processes indispensable for survival, it is involved in the production of peptides presented by major histocompatibility complex class I molecules. In addition to the standard proteasome shared in all eukaryotes, jawed vertebrates have two specialized forms of proteasome known as immunoproteasomes and thymoproteasomes. The immunoproteasome, which contains cytokine-inducible catalytic subunits with distinct cleavage specificities, produces peptides presented by class I molecules more efficiently than the standard proteasome. The thymoproteasome, which contains a unique catalytic subunit β5t, is a tissue-specific proteasome expressed exclusively in cortical thymic epithelial cells. It plays a critical role in CD8+ cytotoxic T cell development via positive selection. This review provides a brief overview on the structure and function of these specialized forms of proteasome and their involvement in human disease.

In Pursuit of Adult Progenitors of Thymic Epithelial Cells.

Peripheral T cells capable of discriminating between self and non-self antigens are major components of a robust adaptive immune system. The development of self-tolerant T cells is orchestrated by thymic epithelial cells (TECs), which are localized in the thymic cortex (cortical TECs, cTECs) and medulla (medullary TECs, mTECs). cTECs and mTECs are essential for differentiation, proliferation, and positive and negative selection of thymocytes. Recent advances in single-cell RNA-sequencing technology have revealed a previously unknown degree of TEC heterogeneity, but we still lack a clear picture of the identity of TEC progenitors in the adult thymus. In this review, we describe both earlier and recent findings that shed light on features of these elusive adult progenitors in the context of tissue homeostasis, as well as recovery from stress-induced thymic atrophy.

Thymoproteasome optimizes positive selection of CD8+ T cells without contribution of negative selection.

Functionally competent and self-tolerant T cell repertoire is shaped through positive and negative selection in the cortical and medullary microenvironments of the thymus. The thymoproteasome specifically expressed in the cortical thymic epithelium is essential for the optimal generation of CD8+ T cells. Although how the thymoproteasome governs the generation of CD8+ T cells is not fully understood, accumulating evidence suggests that the thymoproteasome optimizes CD8+ T cell production through the processing of self-peptides associated with MHC class I molecules expressed by cortical thymic epithelial cells. In this review, we describe recent advances in the mechanism of thymoproteasome-dependent generation of CD8+ T cells, focusing on the process of cortical positive selection independent of apoptosis-mediated negative selection.

Descriptive and functional characterization of epidermal growth factor‑like domain 8 in mouse cortical thymic epithelial cells by integrated analysis of gene expression signatures and networks.

Epidermal growth factor-like domain 8 (EGFL8), a newly identified member of the EGFL family, and plays negative regulatory roles in mouse thymic epithelial cells (TECs) and thymocytes. However, the role of EGFL8 in these cells remains poorly understood. In the present study, in order to characterize the function of EGFL8, genome-wide expression profiles in EGFL8-overexpressing or -silenced mouse cortical TECs (cTECs) were analyzed. Microarray analysis revealed that 458 genes exhibited a >2-fold change in expression levels in the EGFL8-overexpressing vs. the EGFL8-silenced cTECs. Several genes involved in a number of cellular processes, such as the cell cycle, proliferation, growth, migration and differentiation, as well as in apoptosis, reactive oxygen species generation, chemotaxis and immune responses, were differentially expressed in the EGFL8-overexpressing or -silenced cTECs. WST-1 analysis revealed that that the overexpression of EGFL8 inhibited cTEC proliferation. To investigate the underlying mechanisms of EGFL8 in the regulation of cTEC function, genes related to essential cellular functions were selected. Reverse transcription-polymerase chain reaction analysis revealed that EGFL8 knockdown upregulated the expression of cluster differentiation 74 (CD74), Fas ligand (FasL), C-X-C motif chemokine ligand 5 (CXCL5), CXCL10, CXCL16, C-C motif chemokine ligand 20 (CCL20), vascular endothelial growth factor-A (VEGF-A), interferon regulatory factor 7 (Irf7), insulin-like growth factor binding protein-4 (IGFBP-4), thrombospondin 1 (Thbs1) and nuclear factor κB subunit 2 (NF-κB2) genes, and downregulated the expression of angiopoietin-like 1 (Angptl1), and neuropilin-1 (Nrp1) genes. Additionally, EGFL8 silencing enhanced the expression of anti-apoptotic molecules, such as B-cell lymphoma-2 (Bcl-2) and Bcl-extra large (Bcl-xL), and that of cell cycle-regulating molecules, such as cyclin-dependent kinase 1 (CDK1), CDK4, CDK6 and cyclin D1. Moreover, gene network analysis revealed that EGFL8 exerted negative effects on VEGF-A gene expression. Hence, the altered expression of several genes associated with EGFL8 expression in cTECs highlights the important physiological processes in which EGFL8 is involved, and provides insight into its biological functions.