Transposable elements regulate thymus development and function.

Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN ɑ/ß. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.

Thymic-Epithelial-Cell-Dependent Microenvironment Influences Proliferation and Apoptosis of Leukemic Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological cancer characterized by the infiltration of immature T-cells in the bone marrow. Aberrant NOTCH signaling in T-ALL is mainly triggered by activating mutations of NOTCH1 and overexpression of NOTCH3, and rarely is it linked to NOTCH3-activating mutations. Besides the known critical role of NOTCH, the nature of intrathymic microenvironment-dependent mechanisms able to render immature thymocytes, presumably pre-leukemic cells, capable of escaping thymus retention and infiltrating the bone marrow is still unclear. An important challenge is understanding how leukemic cells shape their tumor microenvironment to increase their ability to infiltrate and survive within. Our previous data indicated that hyperactive NOTCH3 affects the CXCL12/CXCR4 system and may interfere with T-cell/stroma interactions within the thymus. This study aims to identify the biological effects of the reciprocal interactions between human leukemic cell lines and thymic epithelial cell (TEC)-derived soluble factors in modulating NOTCH signaling and survival programs of T-ALL cells and TECs. The overarching hypothesis is that this crosstalk can influence the progressive stages of T-cell development driving T-cell leukemia. Thus, we investigated the effect of extracellular space conditioned by T-ALL cell lines (Jurkat, TALL1, and Loucy) and TECs and studied their reciprocal regulation of cell cycle and survival. In support, we also detected metabolic changes as potential drivers of leukemic cell survival. Our studies could shed light on T-cell/stroma crosstalk to human leukemic cells and propose our culture system to test pharmacological treatment for T-ALL.

Thymus as Incontrovertible Target of Future Immune Modulatory Therapeutics.

Thymus plays a crucial role in cellular immunity by acting as a warehouse for proliferating and differentiating lymphocytes. Thymic stromal cells educate T-cells to differentiate self from non-self antigens while nurse cells and thymoproteasome play a major role in the maturation and differentiation of T-cells. The thymic conditions dictate T-cells to cope with the risk of cancer development. A study was designed to demonstrate potential mechanisms behind the failure to eliminate tumors and impaired immune surveillance as well as the impact of delay in thymus regression on cancer and autoimmune disorders. Scientific literature from Pubmed; Scopus; WOS; JSTOR; National Library of Medicine Bethesda, Maryland; The New York Academy of Medicine; Library of Speech Rehabilitation, NY; St. Thomas' Hospital Library; The Wills Library of Guys Hospital; Repository of Kings College London; and Oxford Academic repository was explored for pathological, physiological, immunological and toxicological studies of thymus. Studies have shown that systemic chemotherapy may lead to micro inflammatory environment within thymus where conventionally and dynamically metastasized dormant cells seek refuge. The malfunctioning of the thymus and defective T and Treg cells, bypassing negative selection, contributes to autoimmune disorders, while AIRE and Fezf2 play significant roles in thymic epithelial cell solidity. Different vitamins, TCM, and live cell therapy are effective therapeutics. Vitamin A, C, D, and E, selenium and zinc, cinobufagin and dietary polysaccharides, and glandular extracts and live cell injections have strong potential to restore immune system function and thymus health. Moreover, the relationship between different ages/stages of thymus and their corresponding T-cell mediated anti-tumor immune response needs further exploration.

Primary and secondary defects of the thymus.

The thymus is the primary site of T-cell development, enabling generation, and selection of a diverse repertoire of T cells that recognize non-self, whilst remaining tolerant to self- antigens. Severe congenital disorders of thymic development (athymia) can be fatal if left untreated due to infections, and thymic tissue implantation is the only cure. While newborn screening for severe combined immune deficiency has allowed improved detection at birth of congenital athymia, thymic disorders acquired later in life are still underrecognized and assessing the quality of thymic function in such conditions remains a challenge. The thymus is sensitive to injury elicited from a variety of endogenous and exogenous factors, and its self-renewal capacity decreases with age. Secondary and age-related forms of thymic dysfunction may lead to an increased risk of infections, malignancy, and autoimmunity. Promising results have been obtained in preclinical models and clinical trials upon administration of soluble factors promoting thymic regeneration, but to date no therapy is approved for clinical use. In this review we provide a background on thymus development, function, and age-related involution. We discuss disease mechanisms, diagnostic, and therapeutic approaches for primary and secondary thymic defects.

Ligustilide prevents thymic immune senescence by regulating Thymosin ß15-dependent spatial distribution of thymic epithelial cells.

BACKGROUND: Thymus is the most crucial organ connecting immunity and aging. The progressive senescence of thymic epithelial cells (TECs) leads to the involution of thymus under aging, chronic stress and other factors. Ligustilide (LIG) is a major active component of the anti-aging Chinese herbal medicine Angelica sinensis (Oliv.) Diels, but its role in preventing TEC-based thymic aging remains elusive. PURPOSE: This study explored the protective role of Ligustilide in alleviating ADM (adriamycin) -induced thymic immune senescence and its underlying molecular mechanisms. METHOD: The protective effect of Ligustilide on ADM-induced thymic atrophy was examined by mouse and organotypic models, and conformed by SA-ß-gal staining in TECs. The abnormal spatial distribution of TECs in the senescent thymus was analyzed using H&E, immunofluorescence and flow cytometry. The possible mechanisms of Ligustilide in ADM-induced thymic aging were elucidated by qPCR, fluorescence labeling and Western blot. The mechanism of Ligustilide was subsequently validated through actin polymerization inhibitor, genetic engineering to regulate Thymosin ß15 (Tß15) and Tß4 expression, molecular docking and ß Thymosin-G-actin cross-linking assay. RESULTS: At a 5 mg/kg dose, Ligustilide markedly ameliorated ADM-induced weight loss and limb grip weakness in mice. It also reversed thymic damage and restored positive selection impaired by ADM. In vitro, ADM disrupted thymic structure, reduced TECs number and hindered double negative (DN) T cell differentiation. Ligustilide counteracted these effects, promoted TEC proliferation and reticular differentiation, leading to an increase in CD4+ single positive (CD4SP) T cell proportion. Mechanistically, ADM diminished the microfilament quantity in immortalized TECs (iTECs), and lowered the expression of cytoskeletal marker proteins. Molecular docking and cross-linking assay revealed that Ligustilide inhibited the protein binding between G-actin and Tß15 by inhibiting the formation of the Tß15-G-actin complex, thus enhancing the microfilament assembly capacity in TECs. CONCLUSION: This study, for the first time, reveals that Ligustilide can attenuate actin depolymerization, protects TECs from ADM-induced acute aging by inhibiting the binding of Tß15 to G-actin, thereby improving thymic immune function. Moreover, it underscores the interesting role of Ligustilide in maintaining cytoskeletal assembly and network structure of TECs, offering a novel perspective for deeper understanding of anti thymic aging.

Culture of Mouse Thymic Epithelial Cells in Serum-Free Medium.

Primary cell culture systems are widely used as a valuable method for analyzing the biological functions of specific cells in vitro. Recently, various serum-free primary cell culture methods have been developed that do not involve the use of animal serums. Since the thymus is comprised of many cell types, such as thymocytes, thymic epithelial cells, macrophages, and fibroblasts, thymic epithelial cells must be isolated for their functional analysis in vitro. This chapter describes the detailed protocol for the selective primary culture of thymic epithelial cells using defined serum-free medium.

Medullary Thymic Epithelial Cell Antigen-presentation Assays.

Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance. By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3+ regulatory T cells. Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4+ T cells. We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4+ T cells, peptide-loading of mTEC, and the co-culture between these two cell types. Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4+ T cells. This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4+ T cells in an antigen-specific context. Key features • This protocol builds upon the method used by Lopes et al. (2018 and 2022) and Charaix et al. (2022). • This protocol requires transgenic mice, such as OTIIxRag2-/- mice and the cognate peptide OVA323-339, to assess mTEC antigen presentation to CD4+ T cells. • This requires specific equipment such as a Miltenyi Biotec AutoMACS® Pro Separator, a BD FACSAriaTM III cell sorter, and a BD® LSR II flow cytometer.

Insm1 regulates mTEC development and immune tolerance.

The expression of self-antigens in medullary thymic epithelial cells (mTECs) is essential for the establishment of immune tolerance, but the regulatory network that controls the generation and maintenance of the multitude of cell populations expressing self-antigens is poorly understood. Here, we show that Insm1, a zinc finger protein with known functions in neuroendocrine and neuronal cells, is broadly coexpressed with an autoimmune regulator (Aire) in mTECs. Insm1 expression is undetectable in most mimetic cell populations derived from mTECs but persists in neuroendocrine mimetic cells. Mutation of Insm1 in mice downregulated Aire expression, dysregulated the gene expression program of mTECs, and altered mTEC subpopulations and the expression of tissue-restricted antigens. Consistent with these findings, loss of Insm1 resulted in autoimmune responses in multiple peripheral tissues. We found that Insm1 regulates gene expression in mTECs by binding to chromatin. Interestingly, the majority of the Insm1 binding sites are co-occupied by Aire and enriched in superenhancer regions. Together, our data demonstrate the important role of Insm1 in the regulation of the repertoire of self-antigens needed to establish immune tolerance.

Epithelial-mesenchymal transition in age-associated thymic involution: Mechanisms and therapeutic implications.

The thymus is a critical immune organ with endocrine and immune functions that plays important roles in the physiological and pathological processes of the body. However, with aging, the thymus undergoes degenerative changes leading to decreased production and output of naive T cells and the secretion of thymic hormones and related cytokines, thereby promoting the occurrence and development of various age-associated diseases. Therefore, identifying essential processes that regulate age-associated thymic involution is crucial for long-term control of thymic involution and age-associated disease progression. Epithelial-mesenchymal transition (EMT) is a well-established process involved in organ aging and functional impairment through tissue fibrosis in several organs, such as the heart and kidney. In the thymus, EMT promotes fibrosis and potentially adipogenesis, leading to thymic involution. This review focuses on the factors involved in thymic involution, including oxidative stress, inflammation, and hormones, from the perspective of EMT. Furthermore, current interventions for reversing age-associated thymic involution by targeting EMT-associated processes are summarized. Understanding the key mechanisms of thymic involution through EMT as an entry point may promote the development of new therapies and clinical agents to reverse thymic involution and age-associated disease.

Lin28 regulates thymic growth and involution and correlates with MHCII expression in thymic epithelial cells.

Thymic epithelial cells (TECs) are essential for T cell development in the thymus, yet the mechanisms governing their differentiation are not well understood. Lin28, known for its roles in embryonic development, stem cell pluripotency, and regulating cell proliferation and differentiation, is expressed in endodermal epithelial cells during embryogenesis and persists in adult epithelia, implying postnatal functions. However, the detailed expression and function of Lin28 in TECs remain unknown. In this study, we examined the expression patterns of Lin28 and its target Let-7g in fetal and postnatal TECs and discovered opposing expression patterns during postnatal thymic growth, which correlated with FOXN1 and MHCII expression. Specifically, Lin28b showed high expression in MHCIIhi TECs, whereas Let-7g was expressed in MHCIIlo TECs. Deletion of Lin28a and Lin28b specifically in TECs resulted in reduced MHCII expression and overall TEC numbers. Conversely, overexpression of Lin28a increased total TEC and thymocyte numbers by promoting the proliferation of MHCIIlo TECs. Additionally, our data strongly suggest that Lin28 and Let-7g expression is reliant on FOXN1 to some extent. These findings suggest a critical role for Lin28 in regulating the development and differentiation of TECs by modulating MHCII expression and TEC proliferation throughout thymic ontogeny and involution. Our study provides insights into the mechanisms underlying TEC differentiation and highlights the significance of Lin28 in orchestrating these processes.

Primary explants of the postnatal thymus allow the expansion of clonogenic thymic epithelial cells that constitute thymospheres.

BACKGROUND: Thymic epithelial cells (TECs) are responsible for shaping the repertoires of T cells, where their postnatal regeneration depends on a subset of clonogenic TECs. Despite the implications for regenerative medicine, their cultivation and expansion remain challenging. Primary explant cell culture is a technique that allows the seeding and expansion of difficult-to-culture cells. Here, we report a reliable and simple culture system to obtain functional TECs and thymic interstitial cells (TICs). METHODS: To establish primary thymic explants, we harvested 1 mm cleaned fragments of thymus from 5-week-old C57/BL6 mice. Tissue fragments of a complete thymic lobe were placed in the center of a Petri dish with 1 mL of DMEM/F-12 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin‒streptomycin. To compare, thymic explants were also cultivated by using serum-free DMEM/F-12 medium supplemented with 10% KnockOut™. RESULTS: We obtained high numbers of functional clonogenic TECs and TICs from primary thymic explants cultivated with DMEM/F-12 with 20% FBS. These cells exhibited a highly proliferative and migration profile and were able to constitute thymospheres. Furthermore, all the subtypes of medullary TECs were identified in this system. They express functional markers to shape T-cell and type 2 innate lymphoid cells repertoires, such as Aire, IL25, CCL21 and CD80. Finally, we also found that ≥ 70% of lineage negative TICs expressed high amounts of Aire and IL25. CONCLUSION: Thymic explants are an efficient method to obtain functional clonogenic TECs, all mTEC subsets and different TICs Aire+IL25+ with high regenerative capacity.

Fish Collagen Peptides Enhance Thymopoietic Gene Expression, Cell Proliferation, Thymocyte Adherence, and Cytoprotection in Thymic Epithelial Cells via Activation of the Nuclear Factor-κB Pathway, Leading to Thymus Regeneration after Cyclophosphamide-Induced Injury.

Prolonged thymic involution results in decreased thymopoiesis and thymic output, leading to peripheral T-cell deficiency. Since the thymic-dependent pathway is the only means of generating fully mature T cells, the identification of strategies to enhance thymic regeneration is crucial in developing therapeutic interventions to revert immune suppression in immunocompromised patients. The present study clearly shows that fish collagen peptides (FCPs) stimulate activities of thymic epithelial cells (TECs), including cell proliferation, thymocyte adhesion, and the gene expression of thymopoietic factors such as FGF-7, IGF-1, BMP-4, VEGF-A, IL-7, IL-21, RANKL, LTß, IL-22R, RANK, LTßR, SDF-1, CCL21, CCL25, CXCL5, Dll1, Dll4, Wnt4, CD40, CD80, CD86, ICAM-1, VCAM-1, FoxN1, leptin, cathepsin L, CK5, and CK8 through the NF-κB signal transduction pathway. Furthermore, our study also revealed the cytoprotective effects of FCPs on TECs against cyclophosphamide-induced cellular injury through the NF-κB signaling pathway. Importantly, FCPs exhibited a significant capability to facilitate thymic regeneration in mice after cyclophosphamide-induced damage via the NF-κB pathway. Taken together, this study sheds light on the role of FCPs in TEC function, thymopoiesis, and thymic regeneration, providing greater insight into the development of novel therapeutic strategies for effective thymus repopulation for numerous clinical conditions in which immune reconstitution is required.

Administration of recombinant FOXN1 protein attenuates Alzheimer's pathology in mice.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia in older adults and characterized by progressive loss of memory and cognitive functions that are associated with amyloid-beta (Aß) plaques and neurofibrillary tangles. Immune cells play an important role in the clearance of Aß deposits and neurofibrillary tangles. T cells are the major component of the immune system. The thymus is the primary organ for T cell generation. T cell development in the thymus depends on thymic epithelial cells (TECs). However, TECs undergo both qualitative and quantitative loss over time. We have previously reported that a recombinant (r) protein containing FOXN1 and a protein transduction domain can increase the number of TECs and subsequently increases the number of T cells in mice. In this study we determined the ability of rFOXN1 to affect cognitive performance and AD pathology in mice. METHODS: Aged 3xTg-AD and APP/PS1 AD mice were injected with rFOXN1 or control protein. Cognitive performance, AD pathology, the thymic microenvironment and immune cells were then analyzed. RESULTS: Administration of rFOXN1 into AD mice improves cognitive performance and reduces Aß plaque load and phosphorylated tau in the brain. This is related to rejuvenating the aged thymic microenvironment, which results in enhanced T cell generation in the thymus, leading to increased number of T cells, especially IFNγ-producing T cells, in the spleen and the choroid plexus (CP), enhanced expression of immune cell trafficking molecules in the CP, and increased migration of monocyte-derived macrophages into the brain. Furthermore, the production of anti-Aß antibodies in the serum and the brain, and the macrophage phagocytosis of Aß are enhanced in rFOXN1-treated AD mice. CONCLUSIONS: Our results suggest that rFOXN1 protein has the potential to provide a novel approach to treat AD patients.

Intrathymic somatotropic circuitry: consequences upon thymus involution.

Growth hormone (GH) is a classic pituitary-derived hormone crucial to body growth and metabolism. In the pituitary gland, GH production is stimulated by GH-releasing hormone and inhibited by somatostatin. GH secretion can also be induced by other peptides, such as ghrelin, which interacts with receptors present in somatotropic cells. It is well established that GH acts directly on target cells or indirectly by stimulating the production of insulin-like growth factors (IGFs), particularly IGF-1. Notably, such somatotropic circuitry is also involved in the development and function of immune cells and organs, including the thymus. Interestingly, GH, IGF-1, ghrelin, and somatostatin are expressed in the thymus in the lymphoid and microenvironmental compartments, where they stimulate the secretion of soluble factors and extracellular matrix molecules involved in the general process of intrathymic T-cell development. Clinical trials in which GH was used to treat immunocompromised patients successfully recovered thymic function. Additionally, there is evidence that the reduction in the function of the somatotropic axis is associated with age-related thymus atrophy. Treatment with GH, IGF-1 or ghrelin can restore thymopoiesis of old animals, thus in keeping with a clinical study showing that treatment with GH, associated with metformin and dehydroepiandrosterone, could induce thymus regeneration in healthy aged individuals. In conclusion, the molecules of the somatotrophic axis can be envisioned as potential therapeutic targets for thymus regeneration in age-related or pathological thymus involution.

Histogenetic and disease-relevant phenotypes in thymic epithelial tumors (TETs): The potential significance for future TET classification.

Thymic epithelial tumors (TETs) encompass morphologically various subtypes. Thus, it would be meaningful to explore the expression phenotypes that delineate each TET subtype or overarching multiple subtypes. If these profiles are related to thymic physiology, they will improve our biological understanding of TETs and may contribute to the establishment of a more rational TET classification. Against this background, pathologists have attempted to identify histogenetic features in TETs for a long time. As part of this work, our group has reported several TET expression profiles that are histotype-dependent and related to the nature of thymic epithelial cells (TECs). For example, we found that beta5t, a constituent of thymoproteasome unique to cortical TECs, is expressed mainly in type B thymomas, for which the nomenclature of cortical thymoma was once considered. Another example is the discovery that most thymic carcinomas, especially thymic squamous cell carcinomas, exhibit expression profiles similar to tuft cells, a recently discovered special type of medullary TEC. This review outlines the currently reported histogenetic phenotypes of TETs, including those related to thymoma-associated myasthenia gravis, summarizes their genetic signatures, and provides a perspective for the future direction of TET classification.

Butyrophilin 2a2 (Btn2a2) expression on thymic epithelial cells promotes central T cell tolerance and prevents autoimmune disease.

Butyrophilins are surface receptors belonging to the immunoglobulin superfamily. While several members of the butyrophilin family have been implicated in the development of unconventional T cells, butyrophilin 2a2 (Btn2a2) has been shown to inhibit conventional T cell activation. Here, we demonstrate that in steady state, the primary source of Btn2a2 are thymic epithelial cells (TEC). Absence of Btn2a2 alters thymic T cell maturation and bypasses central tolerance mechanisms. Furthermore, Btn2a2-/- mice develop spontaneous autoimmunity resembling human primary Sjögren's Syndrome (pSS), including formation of tertiary lymphoid structures (TLS) in target organs. Ligation of Btn2a2 on developing thymocytes is associated with reduced TCR signaling and CD5 levels, while absence of Btn2a2 results in increased TCR signaling and CD5 levels. These results define a novel role for Btn2a2 in promoting central tolerance by modulating TCR signaling strength and indicate a potential mechanism of pSS development.

The Influence of Feeder Cell-Derived Extracellular Matrix Density on Thymic Epithelial Cell Culture.

The thymus is responsible for the selection and development of T cells, having an essential role in the establishment of adaptive immunity. Thymic epithelial cells (TECs) are key players in T cell development interacting with thymocytes in the thymic 3D environment. Feeder-layer cells have been frequently used as platforms for the successful establishment of TEC cultures. Nevertheless, the role of the feeder cell-derived extracellular matrix (ECM) on TEC cultures was not previously reported. Therefore, this work aimed at assessing the effect of the ECM produced by feeder cells cultured at two different densities on the establishment of TEC culture. Due to the high surface area and porosity, electrospun fibrous meshes were used to support ECM deposition. The feeder cell-derived ECM was efficiently recovered after decellularization, maintaining the composition of major proteins. All the decellularized matrices were permeable and showed an increase in surface mechanical properties after decellularization. TEC cultures confirmed that the ECM density impacts cellular performance, with higher densities showing a decreased cellular activity. Our findings provide evidence that feeder cell-derived ECM is a suitable substrate for TEC culture and can potentially be applied in thymus bioengineering.

Generation of functional thymic organoids from human pluripotent stem cells.

The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system but involutes with age, resulting in reduced naive T cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro, but functional thymic epithelial cells (TECs) only form months after transplantation of TEPs in vivo. We show the generation of TECs in vitro in isogenic stem cell-derived thymic organoids (sTOs) consisting of TEPs, hematopoietic progenitor cells, and mesenchymal cells, differentiated from the same hPSC line. sTOs support T cell development, express key markers of negative selection, including the autoimmune regulator (AIRE) protein, and facilitate regulatory T cell development. sTOs provide the basis for functional patient-specific thymic organoid models, allowing for the study of human thymus function, T cell development, and transplant immunity.

Foxn1 overexpression promotes thymic epithelial progenitor cell proliferation and mTEC maintenance, but does not prevent thymic involution.

The transcription factor FOXN1 is essential for fetal thymic epithelial cell (TEC) differentiation and proliferation. Postnatally, Foxn1 levels vary widely between TEC subsets, from low/undetectable in putative TEC progenitors to highest in differentiated TEC subsets. Correct Foxn1 expression is required to maintain the postnatal microenvironment; premature downregulation of Foxn1 causes a rapid involution-like phenotype, and transgenic overexpression can cause thymic hyperplasia and/or delayed involution. We investigated a K5.Foxn1 transgene that drives overexpression in mouse TECs, but causes neither hyperplasia nor delay or prevention of aging-related involution. Similarly, this transgene cannot rescue thymus size in Foxn1lacZ/lacZ mice, which undergo premature involution as a result of reduced Foxn1 levels. However, TEC differentiation and cortico-medullary organization are maintained with aging in both K5.Foxn1 and Foxn1lacZ/lacZ mice. Analysis of candidate TEC markers showed co-expression of progenitor and differentiation markers as well as increased proliferation in Plet1+ TECs associated with Foxn1 expression. These results demonstrate that the functions of FOXN1 in promoting TEC proliferation and differentiation are separable and context dependent, and suggest that modulating Foxn1 levels can regulate the balance of proliferation and differentiation in TEC progenitors.

SARS-CoV-2 infection of thymus induces loss of function that correlates with disease severity.

BACKGROUND: Lymphopenia, particularly when restricted to the T-cell compartment, has been described as one of the major clinical hallmarks in patients with coronavirus disease 2019 (COVID-19) and proposed as an indicator of disease severity. Although several mechanisms fostering COVID-19-related lymphopenia have been described, including cell apoptosis and tissue homing, the underlying causes of the decline in T-cell count and function are still not completely understood. OBJECTIVE: Given that viral infections can directly target thymic microenvironment and impair the process of T-cell generation, we sought to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on thymic function. METHODS: We performed molecular quantification of T-cell receptor excision circles and κ-deleting recombination excision circles to assess, respectively, T- and B-cell neogenesis in SARS-CoV-2-infected patients. We developed a system for in vitro culture of primary human thymic epithelial cells (TECs) to mechanistically investigate the impact of SARS-CoV-2 on TEC function. RESULTS: We showed that patients with COVID-19 had reduced thymic function that was inversely associated with the severity of the disease. We found that angiotensin-converting enzyme 2, through which SARS-CoV-2 enters the host cells, was expressed by thymic epithelium, and in particular by medullary TECs. We also demonstrated that SARS-CoV-2 can target TECs and downregulate critical genes and pathways associated with epithelial cell adhesion and survival. CONCLUSIONS: Our data demonstrate that the human thymus is a target of SARS-CoV-2 and thymic function is altered following infection. These findings expand our current knowledge of the effects of SARS-CoV-2 infection on T-cell homeostasis and suggest that monitoring thymic activity may be a useful marker to predict disease severity and progression.