Isolation,Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.     

DKK1 Mediated Inhibition of Wnt Signaling in Postnatal Mice Leads to Loss of TEC Progenitors and Thymic Degeneration

Background

Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus.

Methodology/Principal Findings

Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of ΔNP63⁺ Foxn1⁺ and Aire⁺ TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments.

Conclusions/Significance

Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.     

mTORC1 in Thymic Epithelial Cells Is Critical for Thymopoiesis,T-Cell Generation,and Temporal Control of γδT17 Development and TCRγ/δ Recombination

Thymus is crucial for generation of a diverse repertoire of T cells essential for adaptive immunity. Although thymic epithelial cells (TECs) are crucial for thymopoiesis and T cell generation, how TEC development and function are controlled is poorly understood. We report here that mTOR complex 1 (mTORC1) in TECs plays critical roles in thymopoiesis and thymus function. Acute deletion of mTORC1 in adult mice caused severe thymic involution. TEC-specific deficiency of mTORC1 (mTORC1KO) impaired TEC maturation and function such as decreased expression of thymotropic chemokines, decreased medullary TEC to cortical TEC ratios, and altered thymic architecture, leading to severe thymic atrophy, reduced recruitment of early thymic progenitors, and impaired development of virtually all T-cell lineages. Strikingly, temporal control of IL-17-producing γδT (γδT17) cell differentiation and TCRVγ/δ recombination in fetal thymus is lost in mTORC1KO thymus, leading to elevated γδT17 differentiation and rearranging of fetal specific TCRVγ/δ in adulthood. Thus, mTORC1 is central for TEC development/function and establishment of thymic environment for proper T cell development, and modulating mTORC1 activity can be a strategy for preventing thymic involution/atrophy.     

Can thymic epithelial cells be infected by human T-lymphotropic virus type 1?

The human T-lymphotropic virus type-1 (HTLV-1) is the cause of adult T cell leukaemias/lymphoma. Because thymic epithelial cells (TEC) express recently defined receptors for the virus, it seemed conceivable that these cells might be a target for HTLV-1 infection. We developed an in vitro co-culture system comprising HTLV-1+-infected T cells and human TECs. Infected T cells did adhere to TECs and, after 24 h, the viral proteins gp46 and p19 were observed in TECs. After incubating TECs with culture supernatants from HTLV-1+-infected T cells, we detected gp46 on TEC membranes and the HTLV-1 tax gene integrated in the TEC genome. In conclusion, the human thymic epithelium can be infected in vitro by HTLV-1, not only via cell-cell contact, but also via exposure to virus-containing medium.     

Loss of Pten Disrupts the Thymic Epithelium and Alters Thymic Function

The thymus is the site of T cell development and selection. In addition to lymphocytes, the thymus is composed of several types of stromal cells that are exquisitely organized to create the appropriate environment and microenvironment to support the development and selection of maturing T cells. Thymic epithelial cells (TECs) are one of the more important cell types in the thymic stroma, and they play a critical role in selecting functional T cell clones and supporting their development. In this study, we used a mouse genetics approach to investigate the consequences of deleting the Pten tumor suppressor gene in the TEC compartment of the developing thymus. We found that PTEN deficiency in TECs results in a smaller thymus with significantly disordered architecture and histology. Accordingly, loss of PTEN function also results in decreased T cells with a shift in the distribution of T cell subtypes towards CD8⁺ T cells. These experiments demonstrate that PTEN is critically required for the development of a functional thymic epithelium in mice. This work may help better understand the effects that certain medical conditions or clinical interventions have upon the thymus and immune function.     

Transgenic expression of cyclin D1 in thymic epithelial precursors promotes epithelial and T cell development

We previously reported that precursors within the keratin (K) 8+5+ thymic epithelial cell (TEC) subset generate the major cortical K8+5- TEC population in a process dependent on T lineage commitment. This report demonstrates that expression of a cyclin D1 transgene in K8+5+ TECs expands this subset and promotes TEC and thymocyte development. Cyclin D1 transgene expression is not sufficient to induce TEC differentiation in the absence of T lineage-committed thymocytes because TECs from both hCD3epsilon transgenic and hCD3epsilon/cyclin D1 double transgenic mice remain blocked at the K8+5+ maturation stage. However, enforced cyclin D1 expression does expand the developmental window during which K8+5+ cells can differentiate in response to normal hemopoietic precursors. Thus, enhancement of thymic function may be achieved by manipulating the growth and/or survival of TEC precursors within the K8+5+ subset.     

Identification of MiR-205 As a MicroRNA That Is Highly Expressed in Medullary Thymic Epithelial Cells

Thymic epithelial cells (TECs) support T cell development in the thymus. Cortical thymic epithelial cells (cTECs) facilitate positive selection of developing thymocytes whereas medullary thymic epithelial cells (mTECs) facilitate the deletion of self-reactive thymocytes in order to prevent autoimmunity. The mTEC compartment is highly dynamic with continuous maturation and turnover, but the genetic regulation of these processes remains poorly understood. MicroRNAs (miRNAs) are important regulators of TEC genetic programs since miRNA-deficient TECs are severely defective. However, the individual miRNAs important for TEC maintenance and function and their mechanisms of action remain unknown. Here, we demonstrate that miR-205 is highly and preferentially expressed in mTECs during both thymic ontogeny and in the postnatal thymus. This distinct expression is suggestive of functional importance for TEC biology. Genetic ablation of miR-205 in TECs, however, neither revealed a role for miR-205 in TEC function during homeostatic conditions nor during recovery from thymic stress conditions. Thus, despite its distinct expression, miR-205 on its own is largely dispensable for mTEC biology.     

p73 is expressed in human thymic epithelial cells.

The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis.     

Identification of Novel Thymic Epithelial Cell Subsets Whose Differentiation Is Regulated by RANKL and Traf6

Thymic epithelial cells (TECs) are critical for the normal development and function of the thymus. Here, we examined the developmental stages of TECs using quantitative assessment of the cortical and medullary markers Keratin 5 and Keratin 8 (K5 and K8) respectively, in normal and gain/loss of function mutant animals. Gain of function mice overexpressed RANKL in T cells, whereas loss of function animals lacked expression of Traf6 in TECs (Traf6ΔTEC). Assessment of K5 and K8 expression in conjunction with other TEC markers in wild type mice identified novel cortical and medullary TEC populations, expressing different combinations of these markers. RANKL overexpression led to expansion of all medullary TECs (mTECs) and enlargement of the thymic medulla. This in turn associated with a block in thymocyte development and loss of CD4⁺CD8⁺, CD4⁺ and CD8⁺ thymocytes. In contrast, Traf6 deletion inhibited the production of most TEC populations including cortical TECs (cTECs), defined by absence of UEA-1 binding and LY51 expression, but had no apparent effect on thymocyte development. These results reveal a large degree of heterogeneity within the TEC compartment and the existence of several populations exhibiting concomitant expression of cortical, medullary and epithelial markers and whose production is regulated by RANKL and Traf6.     

MTOR signaling is essential for the development of thymic epithelial cells and the induction of central immune tolerance

Thymic epithelial cells (TECs) are critical for the establishment and maintenance of appropriate microenvironment for the positive and negative selection of thymocytes and the induction of central immune tolerance. Yet, little about the molecular regulatory network on TEC development and function is understood. Here, we demonstrate that MTOR (mechanistic target of rapamycin [serine/threonine kinase]) is essential for proper development and functional maturation of TECs. Pharmacological inhibition of MTOR activity by rapamycin (RPM) causes severe thymic atrophy and reduction of TECs. TEC-specific deletion of Mtor causes the severe reduction of mTECs, the blockage of thymocyte differentiation and output, the reduced generation of thymic regulatory T (Treg) cells and the impaired expression of tissue-restricted antigens (TRAs) including Fabp2, Ins1, Tff3 and Chrna1 molecules. Importantly, specific deletion of Mtor in TECs causes autoimmune diseases characterized by enhanced tissue immune cell infiltration and the presence of autoreactive antibodies. Mechanistically, Mtor deletion causes overdegradation of CTNNB1/Beta-Catenin due to excessive autophagy and the attenuation of WNT (wingless-type MMTV integration site family) signaling in TECs. Selective inhibition of autophagy significantly rescued the poor mTEC development caused by Mtor deficiency. Altogether, MTOR is essential for TEC development and maturation by regulating proliferation and WNT signaling activity through autophagy. The present study also implies that long-term usage of RPM might increase the risk of autoimmunity by impairing TEC maturation and function.     

Autophagy and T-cell education in the thymus: Eat yourself to know yourself

During intrathymic generation of the T cell repertoire, a series of selection processes ensure that only self-MHC (Major Histocompatibility Complex) restricted and self-tolerant T cells are allowed to survive. Interactions with MHC ligands on the surface of thymic epithelial cells (TECs) play a pivotal role in the decision-making of developing thymocytes. A number of distinct cell-biological features of TECs have emerged that may predispose them to serve non-redundant functions in thymocyte “education”. Thus, cortical TECs express a rather unique set of proteolytic enzymes for antigen processing in the context of positive selection, whereas medullary TECs "ectopically" express a plethora of otherwise strictly tissue-restricted antigens (TRAs), a property that obviously has evolved to make these self-antigens "visible" to developing thymocytes for negative selection. One of the latest additions to this growing list of functional adaptations of TECs is their constitutively high rate of autophagy. Recently, we have provided evidence that autophagy in TECs shuttles cytoplasmic self-antigens into the MHC class II loading pathway for positive selection of T cells and tolerance induction.     

Role of the p63-FoxN1 regulatory axis in thymic epithelial cell homeostasis during aging

The p63 gene regulates thymic epithelial cell (TEC) proliferation, whereas FoxN1 regulates their differentiation. However, their collaborative role in the regulation of TEC homeostasis during thymic aging is largely unknown. In murine models, the proportion of TAp63⁺, but not ΔNp63⁺, TECs was increased with age, which was associated with an age-related increase in senescent cell clusters, characterized by SA-β-Gal⁺ and p21⁺ cells. Intrathymic infusion of exogenous TAp63 cDNA into young wild-type (WT) mice led to an increase in senescent cell clusters. Blockade of TEC differentiation via conditional FoxN1 gene knockout accelerated the appearance of this phenotype to early middle age, whereas intrathymic infusion of exogenous FoxN1 cDNA into aged WT mice brought only a modest reduction in the proportion of TAp63⁺ TECs, but an increase in ΔNp63⁺ TECs in the partially rejuvenated thymus. Meanwhile, we found that the increased TAp63⁺ population contained a high proportion of phosphorylated-p53 TECs, which may be involved in the induction of cellular senescence. Thus, TAp63 levels are positively correlated with TEC senescence but inversely correlated with expression of FoxN1 and FoxN1-regulated TEC differentiation. Thereby, the p63-FoxN1 regulatory axis in regulation of postnatal TEC homeostasis has been revealed.     

Cell‐type‐specific role of lamin‐B1 in thymus development and its inflammation‐driven reduction in thymus aging

Cellular architectural proteins often participate in organ development and maintenance. Although functional decay of some of these proteins during aging is known, the cell‐type‐specific developmental role and the cause and consequence of their subsequent decay remain to be established especially in mammals. By studying lamins, the nuclear structural proteins, we demonstrate that lamin‐B1 functions specifically in the thymic epithelial cells (TECs) for proper thymus organogenesis. An up‐regulation of proinflammatory cytokines in the intra‐thymic myeloid immune cells during aging accompanies a gradual reduction of lamin‐B1 in adult TECs. We show that these cytokines can cause senescence and lamin‐B1 reduction of the young adult TECs. Lamin‐B1 supports the expression of TEC genes that can help maintain the adult TEC subtypes we identified by single‐cell RNA‐sequencing, thymic architecture, and function. Thus, structural proteins involved in organ building and maintenance can undergo inflammation‐driven decay which can in turn contribute to age‐associated organ degeneration.     

Tendon tissue engineering: progress, challenges, and translation to the clinic

The tissue engineering field has made great strides in understanding how different aspects of tissue engineered constructs (TECs) and the culture process affect final tendon repair. However, there remain significant challenges in developing strategies that will lead to a clinically effective and commercially successful product. In an effort to increase repair quality, a better understanding of normal development, and how it differs from adult tendon healing, may provide strategies to improve tissue engineering. As tendon tissue engineering continues to improve, the field needs to employ more clinically relevant models of tendon injury such as degenerative tendons. We need to translate successes to larger animal models to begin exploring the clinical implications of our treatments. By advancing the models used to validate our TECs, we can help convince our toughest customer, the surgeon, that our products will be clinically efficacious. As we address these challenges in musculoskeletal tissue engineering, the field still needs to address the commercialization of products developed in the laboratory. TEC commercialization faces numerous challenges because each injury and patient is unique. This review aims to provide tissue engineers with a summary of important issues related to engineering tendon repairs and potential strategies for producing clinically successful products.     

Declining expression of a single epithelial cell‐autonomous gene accelerates age‐related thymic involution

Age‐related thymic involution may be triggered by gene expression changes in lymphohematopoietic and/or nonhematopoietic thymic epithelial cells (TECs). The role of epithelial cell‐autonomous gene FoxN1 may be involved in the process, but it is still a puzzle because of the shortage of evidence from gradual loss‐of‐function and exogenous gain‐of‐function studies. Using our recently generated loxP‐floxed‐FoxN1(fx) mouse carrying the ubiquitous CreER^T (uCreER^T) transgene with a low dose of spontaneous activation, which causes gradual FoxN1 deletion with age, we found that the uCreER^T‐fx/fx mice showed an accelerated age‐related thymic involution owing to progressive loss of FoxN1⁺ TECs. The thymic aging phenotypes were clearly observable as early as at 3–6 months of age, resembling the naturally aged (18–22‐month‐old) murine thymus. By intrathymically supplying aged wild‐type mice with exogenous FoxN1‐cDNA, thymic involution and defective peripheral CD4⁺ T‐cell function could be partially rescued. The results support the notion that decline of a single epithelial cell‐autonomous gene FoxN1 levels with age causes primary deterioration in TECs followed by impairment of the total postnatal thymic microenvironment, and potentially triggers age‐related thymic involution in mice.