Thymic Epithelium Research Articles

MicroRNA-449a modulates medullary thymic epithelial cell differentiation

Medullary thymic epithelial cells (mTECs) ectopically express a diversity of peripheral tissue-restricted antigens (PTAs) and provide unique cues for the expansion, maturation and selection of a repertoire of functionally diverse T lymphocytes. Genetic deletion of all mature microRNAs in thymic epithelial cells (TECs) results in premature thymic involution, progressive disorganisation of the thymic epithelium, and alteration in thymic T cell lineage commitment, consequently eliciting autoimmune disorders. In the present study, we identified that microRNA-449a (miR-449a), a member of miR-449 cluster, regulated mTEC differentiation. Expression of miR-449a was induced by RANK ligand in mouse fetal thymus. In in vitro studies, overexpression of miR-449a induced thymic epithelial progenitor cells (TEPCs) differentiation into mature mTECs. Despite abundant expression of miR-449a in developing thymus, miR-449a-mutant mice exhibited normal thymic development. This might be partially due to in miR-449a-mutant thymus the up-regulation of miR-34a which shared similar seed sequence with miR-449a. However, thymic expression of miR-449/34 sponge which was able to neutralize the function of miR-449/34 family members significantly reduced the number of mature Ly51-MHCIIhi mTECs. Taken together, our data suggested that miR-449a modulated mTEC differentiation, and members of miR-34 cluster functioned redundantly to rescue miR-449a deficiency in thymus development.

EphB receptors, mainly EphB3, contribute to the proper development of cortical thymic epithelial cells

ABSTRACTEphB and their ligands ephrin-B are an important family of protein tyrosine kinase receptors involved in thymocyte-thymic epithelial cell interactions known to be key for the maturation of both thymic cell components. In the present study, we have analyzed the maturation of cortical thymic epithelium in EphB-deficient thymuses evaluating the relative relevance of EphB2 and EphB3 in the process. Results support a relationship between the epithelial hypocellularity of mutant thymuses and altered development of thymocytes, lower proportions of cycling thymic epithelial cells and increased epithelial cell apoptosis. Together, these factors induce delayed development of mutant cortical TECs, defined by the expression of different cell markers, i.e. Ly51, CD205, MHCII, CD40 and β5t. Furthermore, although both EphB2 and EphB3 are necessary for cortical thymic epithelial maturation, the relevance of EphB3 is greater since EphB3−/− thymic cortex exhibits a more severe phenotype than that of EphB2-deficient thymuses.

Ghrelin Protects the Thymic Epithelium From Conditioning-Regimen-Induced Damage and Promotes the Restoration of CD4+ T Cells in Mice After Bone Marrow Transplantation.

The delay in immune reconstitution after hematopoietic stem cell transplantation (HSCT), especially a delay in central immune reconstitution, leads to opportunistic infections and disease relapse after transplantation and affects the long-term outcome of HSCT. This delay is mainly attributable to thymic damage after myeloablative chemotherapy and radiotherapy. We established a model of allogeneic bone marrow transplantation (BMT) in mice and administered ghrelin (GRL) 7 days before the conditioning regimen or the day after BMT to explore the effect of GRL on thymus. All the GRL-treated mice, especially those administered GRL before the conditioning regimen, exhibited more intact thymic architecture and a more rapid restoration of CD4 T lymphocytes after BMT than those of the corresponding control mice. Moreover, the levels of T cell receptor excision circles were significantly higher in the mice treated with GRL before the conditioning regimen than in the control mice at 28 days after BMT. Our findings suggest that GRL may be a novel potential therapeutic approach to protecting the thymic epithelium from conditioning regimen-induced damage and promoting rapid and durable thymic and peripheral CD4 T cell recovery after HSCT.

Cooperative interaction of BMP signalling and Foxn1 gene dosage determines the size of the functionally active thymic epithelial compartment

Thymopoiesis strictly depends on the function of the Foxn1 transcription factor that is expressed in the thymic epithelium. During embryonic development, initial expression of the Foxn1 gene is induced in the pharyngeal endoderm by mesenchyme-derived BMP4 signals. Here, by engineering a time-delayed feedback system of BMP inhibition in mouse embryos, we demonstrate that thymopoiesis irreversibly fails if Foxn1 gene expression does not occur during a defining time span in mid-gestation. We also reveal an epistatic interaction between the extent of BMP signalling and the gene dosage of Foxn1. Our findings illustrate the complexities of the early steps of thymopoiesis and indicate that sporadic forms of thymic hypoplasia in humans may result from the interaction of genes affecting the magnitude of BMP signalling and Foxn1 expression.

Loss of thymic innate lymphoid cells leads to impaired thymopoiesis in experimental graft-versus-host disease

AbstractGraft-versus-host disease (GVHD) and posttransplant immunodeficiency are frequently related complications of allogeneic hematopoietic transplantation. Alloreactive donor T cells can damage thymic epithelium, thus limiting new T-cell development. Although the thymus has a remarkable capacity to regenerate after injury, endogenous thymic regeneration is impaired in GVHD. The mechanisms leading to this regenerative failure are largely unknown. Here we demonstrate in experimental mouse models that GVHD results in depletion of intrathymic group 3 innate lymphoid cells (ILC3s) necessary for thymic regeneration. Loss of thymic ILC3s resulted in deficiency of intrathymic interleukin-22 (IL-22) compared with transplant recipients without GVHD, thereby inhibiting IL-22–mediated protection of thymic epithelial cells (TECs) and impairing recovery of thymopoiesis. Conversely, abrogating IL-21 receptor signaling in donor T cells and inhibiting the elimination of thymic ILCs improved thymopoiesis in an IL-22–dependent fashion. We found that the thymopoietic impairment in GVHD associated with loss of ILCs could be improved by restoration of IL-22 signaling. Despite uninhibited alloreactivity, exogenous IL-22 administration posttransplant resulted in increased recovery of thymopoiesis and development of new thymus-derived peripheral T cells. Our study highlights the role of innate immune function in thymic regeneration and restoration of adaptive immunity posttransplant. Manipulation of the ILC–IL-22–TEC axis may be useful for augmenting immune reconstitution after clinical hematopoietic transplantation and other settings of T-cell deficiency.

Thymic epithelial cells require p53 to support their long-term function in thymopoiesis in mice

AbstractThymic epithelial cells (TECs) provide crucial microenvironments for T-cell development and tolerance induction. As the regular function of the thymus declines with age, it is of fundamental and clinical relevance to decipher new determinants that control TEC homeostasis in vivo. Beyond its recognized tumor suppressive function, p53 controls several immunoregulatory pathways. To study the cell-autonomous role of p53 in thymic epithelium functioning, we developed and analyzed mice with conditional inactivation of Trp53 in TECs (p53cKO). We report that loss of p53 primarily disrupts the integrity of medullary TEC (mTEC) niche, a defect that spreads to the adult cortical TEC compartment. Mechanistically, we found that p53 controls specific and broad programs of mTEC differentiation. Apart from restraining the expression and responsiveness of the receptor activator of NF-κB (RANK), which is central for mTEC differentiation, deficiency of p53 in TECs altered multiple functional modules of the mTEC transcriptome, including tissue-restricted antigen expression. As a result, p53cKO mice presented premature defects in mTEC-dependent regulatory T-cell differentiation and thymocyte maturation, which progressed to a failure in regular and regenerative thymopoiesis and peripheral T-cell homeostasis in the adulthood. Lastly, peripheral signs of altered immunological tolerance unfold in mutant mice and in immunodeficient mice that received p53cKO-derived thymocytes. Our findings position p53 as a novel molecular determinant of thymic epithelium function throughout life.

History of the thymus: from an "accident of evolution" to the programming of immunological self-tolerance

This synthesis presents the most important disruptions of conceptions about the thymus since its discovery in Antique Greece. For centuries, the thymus was considered as a vestigial organ, and its role in T-lymphocyte differentiation has been proposed only in the 1960's. Most recent studies attribute to the thymus an essential and unique role in the programming of central immunological self-tolerance. The basal mechanism implicated in this function is the transcription in thymic epithelium of genes encoding precursors of self-antigens. Processing of these latter leads to presentation of self-antigens by the major histocompatibility complex (MHC) machinery expressed by thymic epithelial cells and dendritic cells. During fetal life, this presentation drives negative selection of T-cell clones harboring receptors with high affinity for these MHC/self-antigen complexes. After birth, this presentation also promotes the generation of regulatory T cells specific for these complexes. A number of studies, as well as the identification of Aire and Fezf2 genes, have shown that a thymus dysfunction plays a crucial role in the development of organ-specific autoimmunity.

Thymic homing of activated CD4+ T cells induces degeneration of the thymic epithelium through excessive RANK signaling

Activated T cells have been shown to be able to recirculate into the thymus from the periphery. The present study was aimed to elucidate the functional consequences of thymic homing of activated T cells upon developing thymocytes and thymic epithelial cells (TEC). In the presence of activated T cells, especially CD4+ T cells, T cell development was found to be inhibited in thymic organ cultures with markedly reduced cellularity. Thymic transplantation demonstrated that the inhibitory effect was most likely due to a defective microenvironment. As the major component of the thymic stroma, the TEC compartment was severely disturbed after prolonged exposure to the activated T cells. In addition to reduced cell proliferation, TEC differentiation was heavily skewed to the mTEC lineage. Furthermore, we demonstrated that RANKL highly expressed by activated CD4+ T cells was primarily responsible for the detrimental effects. Presumably, excessive RANK signaling drove overproduction of mTECs and possibly exhaustion of epithelial progenitors, thereby facilitating the deterioration of the epithelial structures. These findings not only reveal a novel activity of activated T cells re-entering the thymus, but also provide a new perspective for understanding the mechanism underlying thymic involution.

Increased epithelial-free areas in thymuses with altered EphB-mediated thymocyte-thymic epithelial cell interactions.

Epithelial-free areas, present in both thymic cortex and medulla, have been studied in WT and EphB-deficient mice that have important alterations in the development of thymic epithelium due to the lack of proper thymocyte-thymic epithelial cell interactions. In both WT and mutant thymuses, the number and size of epithelial-free areas are significantly larger in the medulla than in the cortex. The two parameters show a reverse correlation: low numbers of these areas course with large epithelial-free areas and vice versa. However, their structure and cell content are similar in mutant and WT thymuses. Cortical epithelial-free areas just contain DP thymocytes, while the medullary ones consist of SP cells, blood vessels, mesenchyme-derived ER-TR7+ cells and components of the extracellular matrix (i.e., collagen IV, fibronectin, laminin). Other components, such as desmin, αSMA, PDGFRβ and Ng2, frequently associated with blood vessel walls, also appear. Vimentin, although present in medullary epithelial-free areas, does not co-express with epithelial cells. Other markers related to epithelial-mesenchymal transitions, such as Snail, Slug or FSP1, are not expressed. These results suggest that alterations in the cell interactions between distinct thymic cell components that induce both increased proportions of apoptotic thymic epithelial cells and altered behavior of the mesenchyme associated with the medullary vasculature could explain the appearance of these areas and their differences in the cortex and medulla.

Dissecting and modeling the emergent murine TEC compartment during ontogeny.

The origin of the thymic epithelium, i.e. the cortical (cTEC) and medullary (mTEC) epithelial cells, from bipotent stem cells through TEC progenitors and lineage-specific progeny still remains poorly understood. We sought to obtain an unbiased view of the incipient emergence of TEC subsets by following embryonic TEC development based on co-expression of EpCAM, CD80 and MHC class II (MHCII) on non-hematopoietic (CD45- ) thymic stromal cells in wild-type BL6 mice. Using a combination of ex vivo analysis, Re-aggregate Thymic Organ Culture (RTOC) reconstitution assays and mathematical modeling, we traced emergent lineage commitment in murine embryonic TECs. Both experimental and mathematical datasets supported the following developmental sequence: MHCII- CD80- → MHCIIlo CD80- → MHCIIhi CD80- → MHCIIhi CD80hi TECs, whereby MHCIIhi CD80- and MHCIIhi CD80hi TECs bear features of cTECs and mTECs respectively. These emergent MHCIIhi CD80- cTECs directly generate mature MHCIIhi CD80hi mTECs in vivo and in vitro, thus supporting the asynchronous model of TEC lineage commitment.

CT-guided percutaneous radiofrequency ablation in the treatment of stage I thymoma.

e18287 Background: Thymoma is an uncommon tumor originating from the epithelial cells of the thymic epithelium. It is commonly associated with myasthenia gravis. Though surgical resection might provide the hope of curing thymoma, potential risks of invasive trauma and post operation complications still exists. Video-assisted thoracoscopic surgery (VATS) has many advantages over traditional surgery including minor trauma. However, surgeons operating the equipment to perform VATS need to be highly specifically trained. Although CT-guided percutaneous radiofrequency ablation (RFA) has emerged as a minimally invasive and curative treatment in other tumors, it has never been used in the treatment of stage I thymoma. Methods: The current study enrolled 13 patients with stage I thymomas whom refused open surgery or VATS. As a result, all patients received first-line CT-guided percutaneous RFA as treatment. The procedure was clearly described and displayed for clinical promoting. Results: The feasibility and treatment effectiveness were also recorded. The median time of ablation was 15 mins (range, 10 - 20 mins). Minimum blood loss was associated with the puncture. All lesions were completely ablated (13/13, 100%). Only one patient out of thirteen had thymoma recurrence at 35.5 months after initial ablation treatment (1/13, 7.7%) at follow-up period (median 80.5 months, range, 64.6-116.9 months). There was no procedure-related death after RFA treatment. Myasthenia gravis was cured in most patients (10/13,76.9%), alleviated in 3 patients (23.1%). The most common complications recorded were fever (13/13, 100%), incisional pain (13/13, 100%), nausea (38.5%), pleural effusion (69.2%), pneumothorax (69.2%) and hemoptysis (84.6%). Conclusions: It is clinically applicable for CT-guided percutaneous RFA to be considered as a safe and effective treatment in stage I thymoma.

Small interference ITGA6 gene targeting in the human thymic epithelium differentially regulates the expression of immunological synapse-related genes

ABSTRACTThe thymus supports differentiation of T cell precursors. This process requires relocation of developing thymocytes throughout multiple microenvironments of the organ, mainly with thymic epithelial cells (TEC), which control intrathymic T cell differentiation influencing the formation and maintenance of the immunological synapse. In addition to the proteins of the major histocompatibility complex (MHC), this structure is supported by several adhesion molecules. During the process of thymopoiesis, we previously showed that laminin-mediated interactions are involved in the entrance of T-cell precursors into the thymus, as well as migration of differentiating thymocytes within the organ. Using small interference RNA strategy, we knocked-down the ITGA6 gene (which encodes the CD49f integrin α-chain) in cultured human TEC, generating a decrease in the expression of the corresponding CD49f subunit, in addition to modulation in several other genes related to cell adhesion and migration. Thymocyte adhesion to TEC was significantly impaired, comprising both immature and mature thymocyte subsets. Moreover, we found a modulation of the MHC, with a decrease in membrane expression of HLA-ABC, in contrast with increase in the expression of HLA-DR. Interestingly, the knockdown of the B2M gene (encoding the β-2 microglobulin of the HLA-ABC complex) increased CD49f expression levels, thus unraveling the existence of a cross-talk event in the reciprocal control of CD49f and HLA-ABC. Our data suggest that the expression levels of CD49f may be relevant in the general control of MHC expression by TEC and consequently the corresponding synapse with developing thymocytes mediated by the T-cell receptor.

Memory B cells are poised for robust antigen presentation by HLA-DO

Abstract The B cell is a uniquely efficient APC due to expression of BCR for antigen uptake and delivery to MHCII-enriched compartments (MIIC), and key regulatory molecules, invariant chain (Ii), HLA-DM and HLA-DO. All professional APCs express Ii and DM, whereas expression of DO is limited to B cells, certain DCs and thymic medullary epithelia. DO levels change dramatically in B cells entering or exiting the germinal center (GC). To determine effects of DO on other components of the MHCII pathway, we delineated the intracellular distribution of DO, DM, MHCII, and class II-associated CLIP in T2 cells with or without DO or DM expression using structured illumination microscopy. The distribution at steady state shows a very restricted lysosomal destination (LAMP1+) for DO and DM, and dual destinations (surface and LAMP1+) for CLIP and MHCII. DO expression increased overall CLIP compared to DM+DO-cells, but almost doubled the percent of CLIP co-localized with LAMP1, establishing peptide exchange potential (PEP) within MIICs, but not any other compartments or the cell surface. PEP enhancement associated with DO expression is likely due to its regulation of DM activity, as neither the overall CLIP level nor the percent of DR in MIICs increased. We then evaluated the extent of PEP in distinct primary B cell populations isolated from human tonsils, including memory (CD38−/IgD−), naïve (CD38−/IgD+), and GC (CD38+/IgD−) B cells. The measurement shows PEPMemory (significantly) > PEPNaïve > PEPGC−, although the percent of CLIP among all class II peptides is naïve > memory >> GC. These results argue that DO expression in memory B cells equips them for more efficient APC function upon re-encounter with antigen.

DNA breaks and chromatin structural changes enhance the transcription of autoimmune regulator target genes

The autoimmune regulator (AIRE) protein is the key factor in thymic negative selection of autoreactive T cells by promoting the ectopic expression of tissue-specific genes in the thymic medullary epithelium. Mutations in AIRE cause a monogenic autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. AIRE has been shown to promote DNA breaks via its interaction with topoisomerase 2 (TOP2). In this study, we investigated topoisomerase-induced DNA breaks and chromatin structural alterations in conjunction with AIRE-dependent gene expression. Using RNA sequencing, we found that inhibition of TOP2 religation activity by etoposide in AIRE-expressing cells had a synergistic effect on genes with low expression levels. AIRE-mediated transcription was not only enhanced by TOP2 inhibition but also by the TOP1 inhibitor camptothecin. The transcriptional activation was associated with structural rearrangements in chromatin, notably the accumulation of γH2AX and the exchange of histone H1 with HMGB1 at AIRE target gene promoters. In addition, we found the transcriptional up-regulation to co-occur with the chromatin structural changes within the genomic cluster of carcinoembryonic antigen-like cellular adhesion molecule genes. Overall, our results suggest that the presence of AIRE can trigger molecular events leading to an altered chromatin landscape and the enhanced transcription of low-expressed genes.

Irf4 Expression in Thymic Epithelium Is Critical for Thymic Regulatory T Cell Homeostasis.

The thymus is a primary lymphoid organ required for the induction and maintenance of central tolerance. The main function of the thymus is to generate an immunocompetent set of T cells not reactive to self. During negative selection in the thymus, thymocytes with autoreactive potential are either deleted or differentiated into regulatory T cells (Tregs). The molecular basis by which the thymus allows high-efficiency Treg induction remains largely unknown. In this study, we report that IFN regulatory factor 4 (Irf4) is highly expressed in murine thymic epithelium and is required to prime thymic epithelial cells (TEC) for effective Treg induction. TEC-specific Irf4 deficiency resulted in a significantly reduced thymic Treg compartment and increased susceptibility to mononuclear infiltrations in the salivary gland. We propose that Irf4 is imperative for thymic Treg homeostasis because it regulates TEC-specific expression of several chemokines and costimulatory molecules indicated in thymocyte development and Treg induction.

Thymic Epithelial Cells.

Intrathymic T cell development is a complex process that depends upon continuous guidance from thymus stromal cell microenvironments. The thymic epithelium within the thymic stroma comprises highly specialized cells with a high degree of anatomic, phenotypic, and functional heterogeneity. These properties are collectively required to bias thymocyte development toward production of self-tolerant and functionally competent T cells. The importance of thymic epithelial cells (TECs) is evidenced by clear links between their dysfunction and multiple diseases where autoimmunity and immunodeficiency are major components. Consequently, TECs are an attractive target for cell therapies to restore effective immune system function. The pathways and molecular regulators that control TEC development are becoming clearer, as are their influences on particular stages of T cell development. Here, we review both historical and the most recent advances in our understanding of the cellular and molecular mechanisms controlling TEC development, function, dysfunction, and regeneration.

Expression of PTEN and its pseudogene PTENP1, and promoter methylation of PTEN in non-tumourous thymus and thymic tumours

AimsMutation or promoter methylation of the phosphatase tensin homologue deleted on chromosome 10 tumour suppressor gene (PTEN) promotes some cancers. Moreover, PTENP1 (PTEN pseudogene) transcript regulates PTEN expression and is...

AB035. PS01.17: Primary thymic adenocarcinoma with signet ring cell features to a 39-year-old male: a case report

: We describe a case of primary thymic adenocarcinoma, an extremely uncommon neoplasm with only 43 cases reported in literature. Patient was a 39-year-old male who presented with an anterior mediastinal mass extending to the left supraclavicular area. He later developed bilateral pleural and pericardial effusions which led to his untimely demise. A small biopsy from the left supraclavicular area was done post-mortem which showed back to back arrangement of neoplastic glands and occasional signet-ring cells. The initial diagnosis was metastatic adenocarcinoma with signet-ring cell features, but extensive clinicoradiologic work-up failed to reveal a primary tumor elsewhere. Immunohistochemically, the tumor cells were reactive for CK-7, CEA, and CD5 and non-reactive for CK-20, TTF-1, NAPSIN A, AFP, PAX-8, CD-117, CA19-9, CA-125, CDX2, P63, CD45 and CD99. The morphologic and immunophenotypic similarities of thymic adenocarcinomas with signet ring cell features from the lungs, stomach, and pancreas can pose diagnostic challenges for surgical pathologists especially for small biopsy specimens where origin from a dysplastic thymic epithelium or a thymic cyst cannot be ascertained. Therefore, a thorough clinical and radiological work-up with immunohistochemical investigation is critical. The findings in this case further broaden the morphologic spectrum of thymic adenocarcinomas.

Evolution of thymic malignancy management in Japan

Although surgery for the thymus began as early as 1940 in Japan, resection of a thymic epithelial tumor continued to be challenging for clinical practitioners until the 1970’s. Thereafter, introduction of the Masaoka clinicopathological staging system, development of extended thymectomy procedure, advancements in understanding of the pathological and biological characteristics of these tumors, and establishment of the Japanese Association for Study of the Thymus (JART) led to significant progress in clinical practice for treating affected patients. Presently, surgical resection procedures for thymic epithelia tumors are safely performed for more than 2,000 patients each year in Japan, approximately 40% of which are achieved using video-assisted thoracoscopic surgery (VATS). JART and Japanese clinicians are currently involved in global collaboration research activities with the International Thymic Malignancy Interest Group (ITMIG) and International Association for the Study of Lung Cancer (IASLC).

MicroRNAs Regulate Thymic Epithelium in Age-Related Thymic Involution via Down- or Upregulation of Transcription Factors.

Age-related thymic involution is primarily induced by defects in nonhematopoietic thymic epithelial cells (TECs). It is characterized by dysfunction of multiple transcription factors (TFs), such as p63 and FoxN1, and also involves other TEC-associated regulators, such as Aire. These TFs and regulators are controlled by complicated regulatory networks, in which microRNAs (miRNAs) act as a key player. miRNAs can either directly target the 3′-UTRs (untranslated regions) of the TFs to suppress TF expression or target TF inhibitors to reduce or increase TF inhibitor expression and thereby indirectly enhance or inhibit TF expression. Here, we review the current understanding and recent studies about how miRNAs are involved in age-related thymic involution via regulation of TEC-autonomous TFs. We also discuss potential strategies for targeting miRNAs to rejuvenate age-related declined thymic function.