T cell development depends critically on the thymic stroma-in particular, the diverse array of functionally distinct thymic epithelial cell (TEC) types. However, a robust in vitro thymus model mimicking the native thymus and compatible with medium-/high-throughput analyses is currently lacking. Here, we demonstrate a high-density microwell-array-based miniaturized thymus organoid (mTO) model that supports T cell commitment and development, possesses key organizational characteristics of the native thymus, and is compatible with live imaging and medium-/high-throughput applications. We establish the minimum cellular input required for a functional mTO and show that mTO TEC phenotype and complexity closely mirror those of the native thymus. Finally, we use an mTO to probe the role of fetal thymic mesenchyme, revealing a requirement beyond maintenance of Foxn1 in differentiation/maintenance of mature TEC sub-populations. Collectively, mTOs present an invitro model of the native thymus adaptable to medium-/high-throughput applications and validated for exploration of thymus and thymus organoid biology.

Inducible gene expression circuits enable precise control over target gene activation and are widely used in direct reprogramming. However, their usability is often compromised by DNA methylation-induced silencing, especially in iPSCs. This deactivates genetic circuits in engineered iPSCs preventing them from being used for long-term scalable expansion of desired cell types. A2-ubiquitous chromatin opening elements (A2UCOE) have been recognized for their anti-silencing properties, but they have not been used in human iPSCs with inducible systems for direct reprogramming. This study investigates the role of A2UCOE in inducible systems and identifies strategies to eliminate associated gene leakage enabling long-term use of engineered human iPSCs.

We developed a compact all-in-one gene circuit - containing a doxycycline-inducible Tet-On system, 863 bp of A2UCOE, and FOXN1, a transcription factor critical for thymic epithelial cell (TEC) differentiation - easily deployed to new genomic sites. However, we observed significant FOXN1 gene leakage even without doxycycline, which is a novel limitation of A2UCOE. This leakage resulted in premature differentiation of iPSCs into TECs, limiting its continued use. To further investigate the relationship between A2UCOE and gene leakage, we generated A2UCOE fragments of varying lengths (1337 bp, 749 bp, and 547 bp) and found that all fragments, regardless of length, caused significant gene leakage. To solve this issue, we tested different spacer sequences between A2UCOE and the inducible promoter and found that the SV40 poly-A terminator fully eliminated FOXN1 leakage, and we show this effect is not due to AT- or GC-content. Unexpectedly, this architecture further enhanced anti-silencing effects > 60% providing prolonged stability for at least 30 days.

This study reveals a novel limitation of A2UCOE in inducible systems, specifically its contribution to gene leakage, which compromise sensitive systems like direct reprogramming of iPSCs. The inclusion of an SV40 poly-A sequence provides a practical solution and genomic architecture to improve the functionality of A2UCOE-based circuits. It also suggests investigating how termination of transcription modulates gene silencing as a novel design parameter. These findings have significant implications for the design of robust gene circuits, particularly in applications involving iPSCs, regenerative medicine, and cell therapy.

The thymus is a central immune organ for T cell development and plays an extremely important role in immune and aging. The unique physiological processes that occur in women, such as the menstrual cycle, pregnancy, and menopause, contribute to sexual dimorphism in thymic immunity. Thymic epithelial cells (TECs) are key stromal cells that affect thymus development and degeneration. Interestingly, TECs in women have stronger proliferation potentiality and ability for output of T cells than those in men. In comparison to men, women exhibit higher susceptibility to autoimmune disease, which can be attributed to lower AIRE expression in the female thymus, which is influenced by fluctuating hormone levels. In this review, we summarize the principles of female thymus regulation by hormones, particularly the influence of female sex hormones in the development and function of TECs, as well as the underlying mechanisms, with the aim of providing new ideas and strategies to inhibit or slow down female thymus degeneration.

The thymus is crucial for optimal T-cell development by facilitating the generation and selection of a diverse repertoire of T cells that can recognize foreign antigens while promoting tolerance to self-antigens. A number of inborn errors of immunity causing complete or partial defects in thymic development (athymia) and/or impaired thymic function have been increasingly recognized that manifest clinically with a combination of life-threatening infections, severe multiorgan autoimmunity, and/or cardiac, craniofacial, ectodermal, and endocrine abnormalities. The introduction of newborn screening programs and the advent of thymic transplantation show promise for early detection and improving the outcomes of patients with certain thymic inborn errors of immunity. We discuss our current understanding of the genetics, immunopathogenesis, diagnosis, and treatment of inborn errors of immunity that impair thymic development and/or function.

Immunohistochemistry for keratins 5, 8, 14, and 18 was performed on Japanese Black calf thymuses at various stages of acute thymic involution. Keratins 5 and 14 were predominantly localized in the thymic medulla, while keratins 8 and 18 were broadly distributed throughout the parenchyma. Despite thymic involution, the distribution patterns of these keratins remained consistent. The cortical area, assessed by keratin 5 staining, progressively decreased with involution but retained approximately 40% of the total parenchyma even at the most severe stage. These results suggest that the thymic cortex shrinks but does not completely disappear during acute thymic involution in calves.

Spaceflight imposes a constellation of physiological challenges-cosmic radiation, microgravity, disrupted circadian rhythms, and psychosocial stress-that critically compromise astronaut health. Among the most vulnerable organs is the thymus, a cornerstone of immune system functionality, tasked with generating naive T cells essential for adaptive immunity. The thymus is particularly sensitive to spaceflight conditions, as its role in maintaining immune homeostasis is tightly regulated by a balance of systemic and local factors easily disrupted in space. Cosmic radiation, an omnipresent hazard beyond Earth's magnetosphere, accelerates DNA damage and cellular senescence in thymic epithelial cells, impairing thymopoiesis and increasing the risk of immune dysregulation. Microgravity and circadian rhythm disruption exacerbate this by altering immune cell migration patterns and stromal support, critical for T-cell development. Psychosocial stressors, including prolonged isolation and mission-induced anxiety, further compound thymic atrophy by elevating systemic glucocorticoid levels. Ground-based analogs simulating cosmic radiation and microgravity have been instrumental in elucidating mechanisms of thymic involution and its downstream effects on immunity. These models reveal that long-duration missions result in diminished naive T-cell output, leaving astronauts vulnerable to infections and possibly at high risk for developing neoplasia. Advances in countermeasures, such as pharmacological interventions targeting thymic regeneration and bioengineering approaches to protect thymic architecture, are emerging as vital strategies to preserve immune resilience during prolonged space exploration. Focusing on the thymus as a central hub of immune vulnerability underscores its pivotal role in spaceflight-induced health risks. Understanding these dynamics will not only enhance the safety of human space missions but also provide critical insights into thymus biology under extreme conditions.

Reconstitution of the thymus is essential for assessing thymic function following injury. However, the currently employed cytoreductive regimes unvaryingly affect the thymic microenvironment, thereby impeding the recovery of T lymphopoiesis. The thymic stroma is composed of epithelial and mesenchymal cells. Thymic mesenchymal cells originate from the Neural crest (NC) and mesoderm and contribute to thymus organogenesis, yet their role in thymic regeneration is unclear. In this study, using transgenic mice expressing NC-specific Cre and Cre-driven DT receptors, we investigated the role of NC-derived mesenchymal cells in thymic regeneration following total body irradiation. We revealed that NC-derived mesenchymal cells have reduced susceptibility to irradiation and induce the upregulation of hematopoietic factors that promote thymus regeneration after irradiation. Additionally, using adult thymic organ culture and renal capsule transplantation, depletion of NC-derived mesenchymal cells resulted in a reduction of DN1-like early T-cell progenitors (ETP) and impaired thymic regeneration. Furthermore, among the numerous factors upregulated by NC-derived mesenchymal cells, Periostin and Flt3L were markedly increased after irradiation and promoted abundance of DN1-like ETPs during thymic reconstitution. Collectively, these findings highlight the importance of NC-derived mesenchymal cells in thymic regeneration.

The thymus is an essential component of the immune system responsible for producing T cells. It is anatomically divided into two main regions: the outer cortex and the inner medulla. This chapter summarizes our current understanding of thymic stromal cell functions, with a particular focus on the interactions between these cells and T cells. This exploration aims to shed light on the pathogenesis of immune disorders, including autoimmunity.

Congenital athymia is a life-limiting disorder due to rare inborn errors of immunity causing impaired thymus organogenesis or abnormal thymic stromal cell development and function. Athymic infants have a T-lymphocyte-negative, B-lymphocyte-positive, natural killer cell-positive immunophenotype with profound T-lymphocyte deficiency and are susceptible to severe infections and autoimmunity. Patients variably display syndromic features. Expanding access to newborn screening for severe combined immunodeficiency and T lymphocytopenia and broad genetic testing, including next-generation sequencing technologies, increasingly facilitate their timely identification. The recommended first-line treatment is allogeneic thymus transplantation, which is a specialized procedure available in Europe and the United States. Outcomes for athymic patients are best with early diagnosis and thymus transplantation before the development of infectious and inflammatory complications. These guidelines on behalf of the European Society for Immunodeficiencies provide a comprehensive review for clinicians who manage patients with inborn thymic stromal cell defects; they offer clinical practice recommendations focused on the diagnosis, investigation, risk stratification, and management of congenital athymia with the aim of improving patient outcomes.

The thymus is a central organ that controls T cell development. Thymic epithelial cells (TECs) create a unique microenvironment essential for the differentiation of major histocompatibility complex (MHC)-restricted and self-tolerant T cells. TECs present a complex of self-peptides and MHC molecules (self-pMHCs) to immature T cells and regulate their survival and differentiation based on their affinity for self-pMHCs. The processing of self-peptides in TECs depends on bulk protein degradation systems, specifically autophagy and proteasomes. Studies using autophagy- and proteasome-deficient mouse models have demonstrated that these degradation systems in TECs are indispensable for maintaining immune homeostasis. Although autophagy and proteasomes are ubiquitous in nearly all eukaryotic cells, TECs exhibit unique characteristics in their autophagy and proteasome functions. Autophagy in TECs is constitutively active and independent of stress responses, while TEC proteasomes contain specialized catalytic subunits. This review summarizes the distinctive characteristics of autophagy and proteasomes in TECs and their roles in immune system regulation.

Age-related thymic involution is characterized by the loss of T cell development and the supporting epithelial network, which are replaced by adipose tissue. We previously showed that aging functionally impairs lymphohematopoietic progenitor cells, including thymic early T cell progenitors (ETPs), contributing to thymic involution. Considering that the thymic microenvironment is essential for thymocyte incubation, we aimed to investigate its role in age-related thymic involution and the mechanisms underlying these changes. The challenge in studying these processes led us to transplant T cell-depleted fetal thymus tissue into the kidney capsule of aged mice. This model allowed us to identify the mechanisms driving age-related changes in the thymic microenvironment and to assess whether these changes could be reversed. Flow cytometry was used to detect naïve T cells (CD62L+CD44-), including CD4 CD8 double-negative, double-positive, and single-positive T cells. Real-time PCR was used to detect and quantify signal-joint T cell receptor excision circles. We rearranged δRec-ΨJα in murine peripheral blood leukocytes to evaluate the thymic output of newly developed naïve T cells in the mice and gene expression in the thymus. Age-related thymic involution decreased naïve T cells and increased memory T cells, while fetal thymus transplantation improved thymic output and T cell production and reversed the impairment of thymopoiesis due to thymic involution in aged mice. Furthermore, the expression of key cytokines was restored and ETPs in the aged mice showed normal thymic T cell development. Our study suggests that degenerative changes in the thymic microenvironment are the primary cause of thymic dysfunction, leading to immunosenescence associated with age-related thymic involution.

T cell development in the thymus is dependent on the thymic microenvironment, in which thymic epithelial cells (TECs) are the major component. However, TECs undergo both a qualitative and quantitative loss during aging, which is believed to be the major factor responsible for age-dependent thymic atrophy. FOXN1 plays a critical role in TEC development and adult TECs maintenance. We have previously reported that intrathymic injection of a recombinant (r) protein containing murine FOXN1 and a protein transduction domain increases the number of TECs in mice, leading to enhanced thymopoiesis. However, intrathymic injection may not be an ideal choice for clinical applications. In this study, we produced a rFOXN1 fusion protein containing the N-terminal of CCR9, human FOXN1 and a protein transduction domain. When injected intravenously into 14-month-old mice, the rFOXN1 fusion protein enters the thymus and TECs, and enhances thymopoiesis, resulting in increased T cell generation in the thymus and increased number of T cells in peripheral lymphoid organ. Our results suggest that the rFOXN1 fusion protein has the potential to be used in preventing and treating T cell immunodeficiency in older adults.

The thymus is an important site for the establishment of an appropriate immune response through positive and negative selection of developing T cells. During selection, developing T cells interact with cortical and medullary thymic epithelial cells (TECs), termed cTECs and mTECs, respectively. Using a Foxn1Cre+/-SKIfl/fl mouse model, we found that TEC-specific deletion of SKI reduced the mTEC compartment in the thymus and that tissue-restricted Ag expression in mTECs was altered. This decrease in the medullary area led to a decrease in CD4 thymocyte cellularity; however, mature CD4 cellularity in the spleen remained normal. Interestingly, naive CD4 T cells purified from SKI-deleted mice showed a defect in proliferation in vitro after global TCR stimulation, and these mice were significantly protected from developing experimental autoimmune encephalomyelitis compared with the control mice. Overall, our findings suggest that SKI signaling in the thymus regulates mTEC differentiation and function as well as downstream peripheral T cell responses and provide evidence for targeting SKI in T cell-driven autoimmune diseases such as multiple sclerosis.

T cell development is fundamental to immune system establishment, yet how this development changes with age remains poorly understood. Here, we construct a transcriptional and epigenetic atlas of T cell developmental programs in neonatal and adult mice, revealing the ontogeny of divergent gene regulatory programs and their link to age-related differences in phenotype and function. Specifically, we identify a gene module that diverges with age from the earliest stages of genesis and includes programs that govern effector response and cell cycle regulation. Moreover, we reveal that neonates possess more accessible chromatin during early thymocyte development, likely establishing poised gene expression programs that manifest later in thymocyte development. Finally, we leverage this atlas, employing a CRISPR-based perturbation approach coupled with single-cell RNA sequencing as a readout to uncover a conserved transcriptional regulator, Zbtb20, that contributes to age-dependent differences in T cell development. Altogether, our study defines transcriptional and epigenetic programs that regulate age-specific differences in T cell development.

The thymus is the key immune organ for the development of T cells. Different populations of thymic stromal cells interact with T cells, thereby controlling the dynamic development of T cells through their differentiation and function. Proteostasis represents a balance between protein expression, folding, and modification and protein clearance, and its fluctuation usually depends at least partially on related protein regulatory systems for further survival and effects. However, in terms of the substantial requirement for self-antigens and their processing burden, increasing evidence highlights that protein regulation contributes to the physiological effects of thymic stromal cells. Impaired proteostasis may expedite the progression of thymic involution and dysfunction, accompanied by the development of autoimmune diseases or thymoma. Hence, in this review, we summarize the regulation of proteostasis within different types of thymic stromal cells under physiological and pathological conditions to identify potential targets for thymic regeneration and immunotherapy.

Thymic epithelial cells (TECs) orchestrate T cell development by imposing positive and negative selection on thymocytes. Current studies on TEC biology are hampered by the absence of long-term ex vivo culture platforms, while the cells driving TEC self-renewal remain to be identified. Here, we generate long-term (>2 years) expandable 3D TEC organoids from the adult mouse thymus. For further analysis, we generated single and double FoxN1-P2A-Clover, Aire-P2A-tdTomato, and Cldn4-P2A-tdTomato reporter lines by CRISPR knockin. Single-cell analyses of expanding clonal organoids reveal cells with bipotent stem/progenitor phenotypes. These clonal organoids can be induced to express Foxn1 and to generate functional cortical- and Aire-expressing medullary-like TECs upon RANK ligand + retinoic acid treatment. TEC organoids support T cell development from immature thymocytes in vitro as well as in vivo upon transplantation into athymic nude mice. This organoid-based platform allows in vitro study of TEC biology and offers a potential strategy for ex vivo T cell development.

Thymic epithelial tumors (TETs) are rare mediastinal cancers originating from the thymus, classified in two main histotypes: thymoma and thymic carcinoma (TC). TETs affect a primary lymphoid organ playing a critical role in keeping T-cell homeostasis and ensuring an adequate immunological tolerance against "self". In particular, thymomas and not TC are frequently associated with autoimmune diseases (ADs), with Myasthenia Gravis being the most common AD present in 30% of patients with thymoma. This comorbidity, in addition to negatively affecting the quality and duration of patients' life, reduces the spectrum of the available therapeutic options. Indeed, the presence of autoimmunity represents an exclusion criteria for the administration of the newest immunotherapeutic treatments with checkpoint inhibitors. The pathophysiological correlation between TETs and autoimmunity remains a mystery. Several studies have demonstrated the presence of a residual and active thymopoiesis in adult patients affected by thymomas, especially in mixed and lymphocytic-rich thymomas, currently known as type AB and B thymomas. The aim of this review is to provide the state of art in regard to the histological features of the different TET histotype, to the role of the different immune cells infiltrating tumor microenvironments and their impact in the break of central immunologic thymic tolerance in thymomas. We discuss here both cellular and molecular immunologic mechanisms inducing the onset of autoimmunity in TETs, limiting the portfolio of therapeutic strategies against TETs and greatly impacting the prognosis of associated autoimmune diseases.

T cells play critical role in multiple immune processes including antigen response, tumor immunity, inflammation, self-tolerance maintenance and autoimmune diseases et. Fetal liver or bone marrow-derived thymus-seeding progenitors (TSPs) settle in thymus and undergo T cell-lineage commitment, proliferation, T cell receptor (TCR) rearrangement, and thymic selections driven by microenvironment composed of thymic epithelial cells (TEC), dendritic cells (DC), macrophage and B cells, thus generating T cells with diverse TCR repertoire immunocompetent but not self-reactive. Additionally, some self-reactive thymocytes give rise to Treg with the help of TEC and DC, serving for immune tolerance. The sequential proliferation, cell fate decision, and selection during T cell development and self-tolerance establishment are tightly regulated to ensure the proper immune response without autoimmune reaction. There are remarkable progresses in understanding of the regulatory mechanisms regarding ubiquitination in T cell development and the establishment of self-tolerance in the past few years, which holds great potential for further therapeutic interventions in immune-related diseases.

The thymus, a central primary lymphoid organ of the immune system, plays a key role in T cell development. Surprisingly, the thymus is quite neglected with regards to standardized pathology approaches and practices for assessing structure and function. Most studies use multispectral flow cytometry to define the dynamic composition of the thymus at the cell population level, but they are limited by lack of contextual insight. This knowledge gap hinders our understanding of various thymic conditions and pathologies, particularly how they affect thymic architecture, and subsequently, immune competence. Here, we introduce a digital pathology pipeline to address these challenges. Our approach can be coupled to analytical algorithms and utilizes rationalized morphometric assessments of thymic tissue, ranging from tissue-wide down to microanatomical and ultrastructural levels. This pipeline enables the quantitative assessment of putative changes and adaptations of thymic structure to stimuli, offering valuable insights into the pathophysiology of thymic disorders. This versatile pipeline can be applied to a wide range of conditions that may directly or indirectly affect thymic structure, ranging from various cytotoxic stimuli inducing acute thymic involution to autoimmune diseases, such as myasthenia gravis. Here, we demonstrate applicability of the method in a mouse model of age-dependent thymic involution, both by confirming established knowledge, and by providing novel insights on intrathymic remodeling in the aged thymus. Our orthogonal pipeline, with its high versatility and depth of analysis, promises to be a valuable and practical toolset for both basic and translational immunology laboratories investigating thymic function and disease.

The role of natural killer group 2D (NKG2D) in peripheral T cells as a costimulatory receptor is well established. However, its contribution to T cell thymic education and functional imprint is unknown. Here, we report significant changes in development, receptor signaling, transcriptional program, and function in T cells from mice lacking NKG2D signaling. In C57BL/6 (B6) and OT-I mice, we found that NKG2D deficiency results in Vβ chain usage changes and stagnation of the double-positive stage in thymic T cell development. We found that the expression of CD5 and CD45 in thymocytes from NKG2D deficient mice were reduced, indicating a direct influence of NKG2D on the strength of T cell receptor (TCR) signaling during the developmental stage of T cells. Depicting the functional consequences of NKG2D, peripheral OT-I NKG2D-deficient cells were unresponsive to ovalbumin peptide stimulation. Paradoxically, while αCD3/CD28 agonist antibodies led to phenotypic T cell activation, their ability to produce cytokines remained severely compromised. We found that OT-I NKG2D-deficient cells activate STAT5 in response to interleukin-15 but were unable to phosphorylate ERK or S6 upon TCR engagement, underpinning a defect in TCR signaling. Finally, we showed that NKG2D is expressed in mouse and human thymic T cells at the double-negative stage, suggesting an evolutionarily conserved function during T cell development. The data presented in this study indicate that NKG2D impacts thymic T cell development at a fundamental level by reducing the TCR threshold and affecting the functional imprint of the thymic progeny. In summary, understanding the impact of NKG2D on thymic T cell development and TCR signaling contributes to our knowledge of immune system regulation, immune dysregulation, and the design of immunotherapies.

The thymus is one of the most crucial immunological organs, undergoing visible age-related shrinkage. Thymic epithelial cells (TECs) play a vital role in maintaining the normal function of the thymus, and their degeneration is the primary cause of age-induced thymic devolution. Thymosin β4 (Tβ4) serves as a significant important G-actin sequestering peptide. The objective of this study was to explore whether Tβ4 influences thymocyte differentiation by regulating the cytoskeletal rearrangement and mitochondrial transfer of TECs. A combination of H&E staining, immunofluorescence, transmission electron microscopy, RT-qPCR, flow cytometry, cytoskeletal immunolabeling, and mitochondrial immunolabeling were employed to observe the effects of Tβ4 on TECs' skeleton rearrangement, mitochondrial transfer, and thymocyte differentiation. The study revealed that the Tβ4 primarily regulates the formation of microfilaments and the mitochondrial transfer of TECs, along with the formation and maturation of double-negative cells (CD4-CD8-) and CD4 single-positive cells (CD3+TCRβ+CD4+CD8-) thymocytes. This study suggests that Tβ4 plays a crucial role in thymocyte differentiation by influencing the cytoskeletal rearrangement and mitochondrial transfer of TECs. These effects may be associated with Tβ4's impact on the aggregation of F-actin. This finding opens up new avenues for research in the field of immune aging.

Thymus is an important primary lymphoid organ for T cell development. After T-lineage commitment, the early thymic progenitors (ETPs) develop into CD4-CD8- (DN), CD4+CD8+ (DP) and further CD4+ SP or CD8+ SP T cells. Under the help of thymic epithelial cells (TEC), dendritic cell (DC), macrophage, and B cells, ETPs undergo proliferation, T cell receptor (TCR) rearrangement, β-selection, positive selection, and negative selection, and thus leading to the generation of T cells that are diverse repertoire immunocompetent but not self-reactive. Additionally, some self-reactive thymocytes give rise to Treg under the help of TEC and DC. The regulation of T cell development is complicated. As a post-translational modification, ubiquitination regulates signal transduction in diverse biological processes. Ubiquitination functions in T cell development through regulating key signal pathway or maturation and function of related cells. In this review, the regulation of T cell development by ubiquitination is summarized and discussed.

Allogeneic T cell platforms utilizing induced pluripotent stem cell (iPSC) technology exhibit significant promise for the facilitation of adoptive immunotherapies. While mature T cell receptor (TCR) signaling plays a crucial role in generating T cells from iPSCs, the introduction of exogenous mature TCR genes carries a potential risk of causing graft-versus-host disease (GvHD). In this study, we present the development of truncated TCRα and TCRβ chains, termed mini-TCRs, which lack variable domains responsible for recognizing human leukocyte antigen (HLA)-peptide complexes. We successfully induced cytotoxic T lymphocytes (CTLs) from iPSCs by employing mini-TCRs. Combinations of TCRα and TCRβ fragments were screened from mini-TCR libraries based on the surface localization of CD3 proteins and their ability to transduce T cell signaling. Consequently, mini-TCR-expressing iPSCs underwent physiological T cell development, progressing from the CD4 and CD8 double-positive stage to the CD8 single-positive stage. The resulting iPSC-derived CTLs exhibited comparable cytokine production and cytotoxicity in comparison to that of full-length TCR-expressing T lymphocytes when chimeric antigen receptors (CARs) were expressed. These findings demonstrate the potential of mini-TCR-carrying iPSCs as a versatile platform for CAR T cell therapy, offering a promising avenue for advancing adoptive immunotherapies.

Myasthenia gravis (MG) is a type of autoimmune disease caused by the blockage of neuromuscular junction transmission owing to the attack of autoantibodies on transmission-related proteins. Related antibodies, such as anti-AChR, anti-MuSK and anti-LRP4 antibodies, can be detected in most patients with MG. Although traditional therapies can control most symptoms, several challenges remain to be addressed, necessitating the development of more effective and safe treatment strategies for MG. With the in-depth exploration on the mechanism and immune targets of MG, effective therapies, especially therapies using biologicals, have been reported recently. Given the important roles of immune cells, cytokines and intercellular interactions in the pathological process of MG, B-cell targeted therapy, T-cell targeted therapy, proteasome inhibitors targeting plasma cell, complement inhibitors, FcRn inhibitors have been developed for the treatment of MG. Although these novel therapies exert good therapeutic effects, they may weaken the immunity and increase the risk of infection in MG patients. This review elaborates on the pathogenesis of MG and discusses the advantages and disadvantages of the strategies of traditional treatment and biologicals. In addition, this review emphasises that combined therapy may have better therapeutic effects and reducing the risk of side effects of treatments, which has great prospects for the treatment of MG. With the deepening of research on immunotherapy targets in MG, novel opportunities and challenges in the treatment of MG will be introduced.

As a population of homogeneous cells with both self-renewal and differentiation potential, stem cell pools are highly compartmentalized and contain distinct subsets that exhibit stable but limited heterogeneity during homeostasis. However, their striking plasticity is showcased under natural or artificial stress, such as injury, transplantation, cancer, and aging, leading to changes in their phenotype, constitution, metabolism, and function. The complex and diverse network of cell-extrinsic niches and signaling pathways, together with cell-intrinsic genetic and epigenetic regulators, tightly regulate both the heterogeneity during homeostasis and the plasticity under perturbation. Manipulating these factors offers better control of stem cell behavior and a potential revolution in the current state of regenerative medicine. However, disruptions of normal regulation by genetic mutation or excessive plasticity acquisition may contribute to the formation of tumors. By harnessing innovative techniques that enhance our understanding of stem cell heterogeneity and employing novel approaches to maximize the utilization of stem cell plasticity, stem cell therapy holds immense promise for revolutionizing the future of medicine.

Distinct pathways and molecules may support embryonic versus postnatal thymic epithelial cell (TEC) development and maintenance. Here, we identify a mechanism by which TEC numbers and function are maintained postnatally. A viable missense allele (C120Y) of Ovol2, expressed ubiquitously or specifically in TECs, results in lymphopenia, in which T cell development is compromised by loss of medullary TECs and dysfunction of cortical TECs. We show that the epithelial identity of TECs is aberrantly subverted towards a mesenchymal state in OVOL2-deficient mice. We demonstrate that OVOL2 inhibits the epigenetic regulatory BRAF-HDAC complex, specifically disrupting RCOR1-LSD1 interaction. This causes inhibition of LSD1-mediated H3K4me2 demethylation, resulting in chromatin accessibility and transcriptional activation of epithelial genes. Thus, OVOL2 controls the epigenetic landscape of TECs to enforce TEC identity. The identification of a non-redundant postnatal mechanism for TEC maintenance offers an entry point to understanding thymic involution, which normally begins in early adulthood.

Thymus implantation is a recently FDA-approved therapy for congenital athymia. Patients receiving thymus implantation develop a functional but incomplete T cell compartment. Our objective was to develop a mouse model to study clinical thymus implantation in congenital athymia and to optimise implantation procedures to maximise T cell education and expansion of naïve T cells.

Using Foxn1 nu athymic mice as recipients, we tested MHC-matched and -mismatched donor thymi that were implanted as fresh tissue or cultured to remove donor T cells. We first implanted thymus under the kidney capsule and then optimised intramuscular implantation. Using competitive adoptive transfer assays, we investigated whether the failure of newly developed T cells to expand into a complete T cell compartment was because of intrinsic deficits or whether there were deficits in engaging MHC molecules in the periphery. Finally, we tested whether recombinant IL-7 would promote the expansion of host naïve T cells educated by the implanted thymus.

We determined that thymus implants in Foxn1 nu athymic mice mimic many aspects of clinical thymus implants in patients with congenital athymia. When we implanted cultured, MHC-mismatched donor thymus into Foxn1 nu athymic mice, mice developed a limited T cell compartment with notably underdeveloped naïve populations and overrepresented memory-like T cells. Newly generated T cells were predominantly educated by MHC molecules expressed by the donor thymus, thus potentially undergoing another round of selection once in the peripheral circulation. Using competitive adoptive transfer assays, we compared expansion rates of T cells educated on donor thymus versus T cells educated during typical thymopoiesis in MHC-matched and -mismatched environments. Once in the circulation, regardless of the MHC haplotypes, T cells educated on a donor thymus underwent abnormal expansion with initially more robust proliferation coupled with greater cell death, resembling IL-7 independent spontaneous expansion. Treating implanted mice with recombinant interleukin (IL-7) promoted homeostatic expansion that improved T cell development, expanded the T cell receptor repertoire, and normalised the naïve T cell compartment.

We conclude that implanting cultured thymus into the muscle of Foxn1 nu athymic mice is an appropriate system to study thymus implantation for congenital athymia and immunodeficiencies. T cells are educated by the donor thymus, yet naïve T cells have deficits in expansion. IL-7 greatly improves T cell development after thymus implantation and may offer a novel strategy to improve outcomes of clinical thymus implantation.

Thymus is a primary lymphoid organ essential for the development of T lymphocytes. Age-related thymic involution is a prominent feature of immune senescence. The thymus undergoes rapid growth during fetal and neonatal development, peaks in size before puberty and then begins to undergo a decrease in cellularity with age. Dramatic changes occur with age-associated thymic involution. The most prominent features of thymic involution include: (i) epithelial structure disruption, (ii) adipogenesis, and (iii) thymocyte development arrest. There is a sex disparity in thymus aging. It is a multifactorial process controlled and regulated by a series of molecules, including the transcription factor FOXN1, fibroblast and keratinocyte growth factors (FGF and KGF, respectively), sex steroids, Notch signaling, WNT signaling, and microRNAs. Nevertheless, there is still no satisfactory evolutionary or physiological explanation for age-associated thymic involution, and understanding the precise mechanism(s) for thymus aging remains challenging. Sustained thymic regeneration has yet to be achieved by sex steroid ablation. Recent preclinical studies indicate that long-term thymic reconstitution can be achieved via adoptive transfer of in vitro-generated progenitor T (proT) cells, and improvements in the methods for the generation of human proT cells make this an attractive approach. Future clinical applications may rely on new applications integrating proT cells, cytokine support and sex-steroid inhibition treatments.

Autoimmune hepatitis (AIH) is a chronic autoimmune liver disease that can lead to hepatocyte destruction, inflammation, liver fibrosis, cirrhosis, and liver failure. The diagnosis of AIH requires the identification of lymphoblast cell interface hepatitis and serum biochemical abnormalities, as well as the exclusion of related diseases. According to different specific autoantibodies, AIH can be divided into AIH-1 and AIH-2. The first-line treatment for AIH is a corticosteroid and azathioprine regimen, and patients with liver failure require liver transplantation. However, the long-term use of corticosteroids has obvious side effects, and patients are prone to relapse after drug withdrawal. Autoimmune diseases are characterized by an imbalance in immune tolerance of self-antigens, activation of autoreactive T cells, overactivity of B cells, and increased production of autoantibodies. CD4+ T cells are key players in adaptive immunity and can secrete cytokines, activate B cells to produce antibodies, and influence the cytotoxicity of CD8+ T cells. According to their characteristics, CD4+ T cells can be divided into different subsets. In this review, we discuss the changes in T helper (Th)1, Th2, Th17, Th9, Th22, regulatory T cell, T follicular helper, and T peripheral helper cells and their related factors in AIH and discuss the therapeutic potential of targeting CD4+ T-cell subsets in AIH.

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 (Aβ) plaques and neurofibrillary tangles. Immune cells play an important role in the clearance of Aβ 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.

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.

Administration of rFOXN1 into AD mice improves cognitive performance and reduces Aβ 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-Aβ antibodies in the serum and the brain, and the macrophage phagocytosis of Aβ are enhanced in rFOXN1-treated AD mice.

Our results suggest that rFOXN1 protein has the potential to provide a novel approach to treat AD patients.

Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Mature donor T cells within the graft contribute to severe damage of thymic epithelial cells (TECs), which are known as key mediators in the continuum of acute GVHD (aGVHD) and cGVHD pathology. Mesenchymal stromal cells (MSCs) are reportedly effective in the prevention and treatment of cGVHD. In our previous pilot clinical trial in patients with refractory aGVHD, the incidence and severity of cGVHD were decreased, along with an increase in levels of blood signal joint T-cell receptor excision DNA circles after MSCs treatment, which indicated an improvement in thymus function of patients with GVHD, but the mechanisms leading to these effects remain unknown. Here, we show in a murine GVHD model that MSCs promoted the quantity and maturity of TECs as well as elevated the proportion of Aire-positive medullary TECs, improving both CD4+CD8+ double-positive thymocytes and thymic regulatory T cells, balancing the CD4:CD8 ratio in the blood. In addition, CCL25-CCR9 signaling axis was found to play an important role in guiding MSC homing to the thymus. These studies reveal mechanisms through which MSCs ameliorate cGVHD by boosting thymic regeneration and offer innovative strategies for improving thymus function in patients with GVHD.

The thymus is required for T cell development and the formation of the adaptive immunity. Stromal cells, which include thymic epithelial cells (TECs) and mesenchymal stromal cells (MSCs), are essential for thymic function. However, the immunomodulatory function of thymus-derived MSCs (T-MSCs) has not been fully explored.

MSCs were isolated from mouse thymus and their general characteristics including surface markers and multi-differentiation potential were characterized. The immunomodulatory function of T-MSCs stimulated by IFN-γ and TNF-α was evaluated in vitro and in vivo. Furthermore, the spatial distribution of MSCs in the thymus was interrogated by using tdTomato-flox mice corssed to various MSC lineage Cre recombinase lines.

A subset of T-MSCs express Nestin, and are mainly distributed in the thymic medulla region and cortical-medulla junction, but not in the capsule. The Nestin-positive T-MSCs exhibit typical immunophenotypic characteristics and differentiation potential. Additionally, when stimulated with IFN-γ and TNF-α, they can inhibit activated T lymphocytes as efficiently as BM-MSCs, and this function is dependent on the production of nitric oxide (NO). Additionally, the T-MSCs exhibit a remarkable therapeutic efficacy in acute liver injury and inflammatory bowel disease (IBD).

Nestin-positive MSCs are mainly distributed in medulla and cortical-medulla junction in thymus and possess immunosuppressive ability upon stimulation by inflammatory cytokines. The findings have implications in understanding the physiological function of MSCs in thymus.

Declining thymic functions associated either with old age (i.e., age-related thymic involution), or with acute involution as a result of stress, infectious disease, or cytoreductive therapies (e.g., chemotherapy/radiotherapy), have been associated with cancer development. A key mechanism underlying such increased cancer risk is the thymus-dependent debilitation of adaptive immunity, which is responsible for orchestrating immunoediting mechanisms and tumor immune surveillance. In the past few years, a blooming set of evidence has intriguingly linked obesity with cancer development and progression. The majority of such studies has focused on obesity-driven chronic inflammation, steroid/sex hormone and adipokine production, and hyperinsulinemia, as principal factors affecting the tumor microenvironment and driving the development of primary malignancy. However, experimental observations about the negative impact of obesity on T cell development and maturation have existed for more than half a century. Here, we critically discuss the molecular and cellular mechanisms of obesity-driven thymic involution as a previously underrepresented intermediary pathology leading to cancer development and progression. This knowledge could be especially relevant in the context of childhood obesity, because impaired thymic function in young individuals leads to immune system abnormalities, and predisposes to various pediatric cancers. A thorough understanding behind the molecular and cellular circuitries governing obesity-induced thymic involution could therefore help towards the rationalized development of targeted thymic regeneration strategies for obese individuals at high risk of cancer development.

During development, cortical (c) and medullary (m) thymic epithelial cells (TEC) arise from the third pharyngeal pouch endoderm. Current models suggest that within the thymic primordium most TEC exist in a bipotent/common thymic epithelial progenitor cell (TEPC) state able to generate both cTEC and mTEC, at least until embryonic day 12.5 (E12.5) in the mouse. This view, however, is challenged by recent transcriptomics and genetic evidence. We therefore set out to investigate the fate and potency of TEC in the early thymus. Here using single cell (sc) RNAseq we identify a candidate mTEC progenitor population at E12.5, consistent with recent reports. Via lineage-tracing we demonstrate this population as mTEC fate-restricted, validating our bioinformatics prediction. Using potency analyses we also establish that most E11.5 and E12.5 progenitor TEC are cTEC-fated. Finally we show that overnight culture causes most if not all E12.5 cTEC-fated TEPC to acquire functional bipotency, and provide a likely molecular mechanism for this changed differentiation potential. Collectively, our data overturn the widely held view that a common TEPC predominates in the E12.5 thymus, showing instead that sublineage-primed progenitors are present from the earliest stages of thymus organogenesis but that these early fetal TEPC exhibit cell-fate plasticity in response to extrinsic factors. Our data provide a significant advance in the understanding of fetal thymic epithelial development and thus have implications for thymus-related clinical research, in particular research focussed on generating TEC from pluripotent stem cells.

The network of thymic stromal cells provides essential niches with unique molecular cues controlling T cell development and selection. Recent single-cell RNA sequencing studies have uncovered previously unappreciated transcriptional heterogeneity among thymic epithelial cells (TEC). However, there are only very few cell markers that allow a comparable phenotypic identification of TEC. Here, using massively parallel flow cytometry and machine learning, we deconvoluted known TEC phenotypes into novel subpopulations. Using CITEseq, these phenotypes were related to corresponding TEC subtypes defined by the cells' RNA profiles. This approach allowed the phenotypic identification of perinatal cTEC and their physical localisation within the cortical stromal scaffold. In addition, we demonstrate the dynamic change in the frequency of perinatal cTEC in response to developing thymocytes and reveal their exceptional efficiency in positive selection. Collectively, our study identifies markers that allow for an unprecedented dissection of the thymus stromal complexity, as well as physical isolation of TEC populations and assignment of specific functions to individual TEC subtypes.

Immunotherapy has emerged to play a rapidly expanding role in the treatment of cancers. Currently, many clinical trials of therapeutic agents are on ongoing with majority of immune checkpoint inhibitors (ICIs) especially programmed death receptor 1 (PD-1) and its ligand 1 (PD-L1) inhibitors. PD-1 and PD-L1, two main immune checkpoints, are expressed at high levels in thymic epithelial tumors (TETs) and could be predictors of the progression and immunotherapeutic efficacy of TETs. However, despite inspiring efficacy reported in clinical trials and clinical practice, significantly higher incidence of immune-related adverse events (irAEs) than other tumors bring challenges to the administration of ICIs in TETs. To develop safe and effective immunotherapeutic patterns in TETs, understanding the clinical properties of patients, the cellular and molecular mechanisms of immunotherapy and irAEs occurrence are crucial. In this review, the progress of both basic and clinical research on immune checkpoints in TETs, the evidence of therapeutic efficacy and irAEs based on PD-1 /PD-L1 inhibitors in TETs treatment are discussed. Additionally, we highlighted the possible mechanisms underlying irAEs, prevention and management strategies, the insufficiency of current research and some worthy research insights. High PD-1/PD-L1 expression in TETs provides a rationale for ICI use. Completed clinical trials have shown an encouraging efficacy of ICIs, despite the high rate of irAEs. A deeper mechanism understanding at molecular level how ICIs function in TETs and why irAEs occur will help maximize the immunotherapeutic efficacy while minimizing irAEs risks in TET treatment to improve patient prognosis.

Although the thymus continues to export T cells throughout life, it undergoes a profound involution/atrophy with age, resulting in decreased numbers of T cells in the older adult, which has direct etiological linkages with many diseases. T cell development in the thymus is dependent on the thymic microenvironment, in which thymic epithelial cells (TECs) are the major component. However, TECs undergo both a qualitative and quantitative loss during aging, which is believed to be the major factor responsible for age-dependent thymic atrophy. FOXN1 plays a critical role in TEC development and adult TECs maintenance. We have previously reported that intrathymic injection of a recombinant (r) protein containing FOXN1 and a protein transduction domain increases the number of TECs in mice, leading to enhanced thymopoiesis. However, intrathymic injection may not be an ideal choice for clinical applications. In this study, we produce a rFOXN1 fusion protein containing the N-terminal of CCR9, FOXN1 and a protein transduction domain.

We show here that, when injected intravenously into aged mice, the rFOXN1 fusion protein migrates into the thymus and enhances thymopoiesis, resulting in increased T cell generation in the thymus and increased number of T cells in peripheral lymphoid organ.

Our results suggest that the rFOXN1 fusion protein has the potential to be used in preventing and treating T cell immunodeficiency in the older adult.