Thymus Organogenesis Research Articles

European Society for Immunodeficiencies guidelines for the management of patients with congenital athymia

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-B+NK+ 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 (SCID) and T lymphocytopaenia 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 of Immunodeficiencies provide a comprehensive review for clinicians who manage patients with inborn thymic stromal cell defects, and offer clinical practice recommendations focused on the diagnosis, investigation, risk stratification and management of congenital athymia, with the aim of improving patient outcomes.

Accelerating Immune Reconstitution in HSCT Patients through iPSC-Derived Thymic Epithelial Cells

T cells are central to host defense and tolerance, yet the critical role of the thymus in facilitating T cell development and TCR repertoire selection is often overlooked. Thymic function declines in the second decade of life and is susceptible to a wide range of pathogenic insults. Compromise of thymic function is a frequent complication after HSCT as a result of radiation, anti-thymocyte globulin, CMV infection and acute GVHD. Thymic injury can lead to delayed immune reconstitution, opportunistic infections, autoimmunity and chronic GVHD. Reconstituting thymic function, e.g. through induced pluripotent stem cell (iPSC)-derived thymic epithelial cells (TECs), holds great therapeutic promise. However, the regenerative potential of the thymus remains clinically untapped as the signals that drive thymic ontogeny beyond the anterior foregut are incompletely understood. In this study, we elucidated pathways instructing commitment and specialization of the human thymic epithelial stroma through orthogonal single cell transcriptomic analysis strategies. First, we inferred inductive cues that contribute to the initiation of thymic organogenesis by comparing gene regulatory networks expressed in human fetal epithelial cells of the thymus and other anterior foregut-derived organs. Then we defined lineage trajectories within the thymic epithelium by evaluating human thymus samples sequentially across embryonic, fetal, and early postnatal stages. Our data revealed that responses to types I and II interferon distinguish epithelial cells of the thymus from other anterior foregut-derived organs. We further found that the downstream effects of interferon extend differentially within the cortical and medullary lineages, reflected in characteristic NFκB and IRF signatures (Figure 1A). We have translated the core set of gene regulatory networks identified in our transcriptomic study into a morphogen-based approach to advance the differentiation of iPSCs into TECs. TECs derived from this protocol demonstrate high transcriptional fidelity with primary human fetal intertypic TECs that can give rise to cortical and medullary lineages (Fig. 1B). To test the functional capacity of iPSC-derived TECs to give rise to human T cells, and specifically to execute positive and negative selection, we transplanted iPSC-derived TECs into humanized athymic NSG-nude mice (NSG-Foxn1 Null) (Figure 1C). 12 weeks after transplantation, flowcytometric analysis of dissociated TEC grafts showed robust human T cell development recapitulating the physiologic progression from double-negative to double-positive to TCRab-rearranged single CD4 and CD8 positive T cells observed in primary human thymus. (Figure 1D) These results provide proof of concept that transplantable iPSC-derived TECs can promote T cell development in vivo and have potential as a regenerative thymic replacement therapy for patients suffering from complications of defective T cell immune reconstitution after HSCT.

Thymic epithelial cell fate and potency in early organogenesis assessed by single cell transcriptional and functional analysis.

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.

Neural crest-derived mesenchymal cells support thymic reconstitution after lethal irradiation.

Abstract The thymus is composed of thymocytes and stromal cells including thymic epithelial and mesenchymal cells. Thymic mesenchymal cells are originated from Neural Crest (NC) cells and the mesoderm. Although it is known that NC-derived (NCd) cells contribute to thymus organogenesis and thymus development, their specific role in T cell regeneration is unclear. To evaluate the regenerative ability of NCd mesenchymal cells in the thymus, we used lethally irradiated mice to examine the effect of ionizing irradiation (IR) on the thymus size, thymocytes and the thymic stromal cells (thymic epithelial cells (TECs) and thymic mesenchymal cells). Additionally, we ablated NC-derived mesenchymal cells via in vitro diphtheria toxin (DT) administration in double transgenic mice (Wnt1-Cre/+; Rosa YFP/DTRmice) that express NC-specific Cre and Cre-driven DT receptor (DTR) and transplanted the thymus depleted of NC-derived mesenchyme under the renal capsules of non-irradiated C57BL/6 (CD45.1) mice. IR reduced the size of the thymus, the absolute numbers and percentages of thymocytes, primarily (CD4 +CD8 +) T cells and TECs. Moreover, IR reduced the number of thymic mesenchymal cells but not that of NC-derived mesenchymal cells. NC-derived mesenchymal cells upregulated several hematopoietic factors such as Flt3l, Periostin, Scf, and Bmp4after irradiation and supported thymic reconstitution. Taken together, our findings indicate that NC-derived mesenchymal cells are not only radio-resistant, but also produce molecules that may support early T-cell development for thymic reconstitution after irradiation .

Analyses of thymocyte commitment to regulatory T cell lineage in thymus of healthy subjects and patients with 22q11.2 deletion syndrome.

The Chromosome 22q11.2 deletion syndrome (22q11.2DS) results in an inborn error of immunity due to defective thymic organogenesis. Immunological abnormalities in 22q11.2DS patients are thymic hypoplasia, reduced output of T lymphocytes by the thymus, immunodeficiency and increased incidence of autoimmunity. While the precise mechanism responsible for increased incidence of autoimmunity is not completely understood, a previous study suggested a defect in regulatory T cells (Treg) cell lineage commitment during T cell development in thymus. Here, we aimed to analyze this defect in more detail. Since Treg development in human is still ill-defined, we first analyzed where Treg lineage commitment occurs. We performed systematic epigenetic analyses of the Treg specific demethylation region (TSDR) of the FOXP3 gene in sorted thymocytes at different developmental stages. We defined CD3+CD4+CD8+ FOXP3+CD25+ as the T cell developmental stage in human where TSDR demethylation first occurs. Using this knowledge, we analyzed the intrathymic defect in Treg development in 22q11.2DS patients by combination of TSDR, CD3, CD4, CD8 locus epigenetics and multicolor flow cytometry. Our data showed no significant differences in Treg cell frequencies nor in their basic phenotype. Collectively, these data suggest that although 22q11.2DS patients present with reduced thymic size and T cell output, the frequencies and the phenotype of Treg cell at each developmental stage are surprisingly well preserved.

Dental niche cells directly contribute to tooth reconstitution and morphogenesis.

Mammalian teeth develop from the inductive epithelial-mesenchymal interaction, an important mechanism shared by many organs. The cellular basis for such interaction remains elusive. Here, we generate a dual-fluorescence model to track and analyze dental cells from embryonic to postnatal stages, in which Pitx2+ epithelium and Msx1+ mesenchyme are sufficient for tooth reconstitution. Single-cell RNA sequencing and spatial mapping further revealed critical cellular dynamics during molar development, where tooth germs are organized by Msx1+Sdc1+ dental papilla and surrounding dental niche. Surprisingly, niche cells are more efficient in tooth reconstitution and can directly regenerate papilla cells through interaction with dental epithelium. Finally, from the dental niche, we identify a group of previously unappreciated migratory Msx1+ Sox9+ cells as the potential cell origin for dental papilla. Our results indicate that the dental niche cells directly contribute to tooth organogenesis and provide critical insights into the essential cell composition for tooth engineering.

Differentiation of Pluripotent Stem Cells Into Thymic Epithelial Cells and Generation of Thymic Organoids: Applications for Therapeutic Strategies Against APECED

The thymus is a primary lymphoid organ essential for the induction of central immune tolerance. Maturing T cells undergo several steps of expansion and selection mediated by thymic epithelial cells (TECs). In APECED and other congenital pathologies, a deficiency in genes that regulate TEC development or their ability to select non auto-reactive thymocytes results in a defective immune balance, and consequently in a general autoimmune syndrome. Restoration of thymic function is thus crucial for the emergence of curative treatments. The last decade has seen remarkable progress in both gene editing and pluripotent stem cell differentiation, with the emergence of CRISPR-based gene correction, the trivialization of reprogramming of somatic cells to induced pluripotent stem cells (iPSc) and their subsequent differentiation into multiple cellular fates. The combination of these two approaches has paved the way to the generation of genetically corrected thymic organoids and their use to control thymic genetic pathologies affecting self-tolerance. Here we review the recent advances in differentiation of iPSc into TECs and the ability of the latter to support a proper and efficient maturation of thymocytes into functional and non-autoreactive T cells. A special focus is given on thymus organogenesis and pathway modulation during iPSc differentiation, on the impact of the 2/3D structure on the generated TECs, and on perspectives for therapeutic strategies in APECED based on patient-derived iPSc corrected for AIRE gene mutations.

Signaling Crosstalks Drive Generation and Regeneration of the Thymus.

Optimal recovery of immune competence after periods of hematopoietic insults or stress is crucial to re-establish patient response to vaccines, pathogens and tumor antigens. This is particularly relevant for patients receiving high doses of chemotherapy or radiotherapy, who experience prolonged periods of lymphopenia, which can be associated with an increased risk of infections, malignant relapse, and adverse clinical outcome. While the thymus represents the primary organ responsible for the generation of a diverse pool of T cells, its function is profoundly impaired by a range of acute insults (including those caused by cytoreductive chemo/radiation therapy, infections and graft-versus-host disease) and by the chronic physiological deterioration associated with aging. Impaired thymic function increases the risk of infections and tumor antigen escape due to a restriction in T-cell receptor diversity and suboptimal immune response. Therapeutic approaches that can promote the renewal of the thymus have the potential to restore immune competence in patients. Previous work has documented the importance of the crosstalk between thymocytes and thymic epithelial cells in establishing correct architecture and function of thymic epithelium. This crosstalk is relevant not only during thymus organogenesis, but also to promote the recovery of its function after injuries. In this review, we will analyze the signals involved in the crosstalk between TECs and hematopoietic cells. We will focus in particular on how signals from T-cells can regulate TEC function and discuss the relevance of these pathways in restoring thymic function and T-cell immunity in experimental models, as well as in the clinical setting.

Developmental dynamics of the neural crest-mesenchymal axis in creating the thymic microenvironment.

The thymic stroma is composed of epithelial and nonepithelial cells providing separate microenvironments controlling homing, differentiation, and selection of hematopoietic precursor cells to functional T cells. Here, we explore at single-cell resolution the complex composition and dynamic changes of the nonepithelial stromal compartment across different developmental stages in the human and mouse thymus, and in an experimental model of the DiGeorge syndrome, the most common form of human thymic hypoplasia. The detected gene expression signatures identify previously unknown stromal subtypes and relate their individual molecular profiles to separate differentiation trajectories and functions, revealing an unprecedented heterogeneity of different cell types that emerge at discrete developmental stages and vary in their expression of key regulatory signaling circuits and extracellular matrix components. Together, these findings highlight the dynamic complexity of the nonepithelial thymus stroma and link this to separate instructive roles essential for normal thymus organogenesis and tissue maintenance.

Depletion of Ift88 in thymic epithelial cells affects thymic synapse and T-cell differentiation in aged mice.

Primary cilia are ubiquitous hair-like organelles, usually projecting from the cell surface. They are essential for the organogenesis and homeostasis of various physiological functions, and their dysfunction leads to a plethora of human diseases. However, there are few reports on the role of primary cilia in the immune system; therefore, we focused on their role in the thymus that nurtures immature lymphocytes to full-fledged T cells. We detected primary cilia on the thymic epithelial cell (TEC) expressing transforming growth factor β (TGF-β) receptor in the basal body, and established a line of an intraflagellar transport protein 88 (Ift88) knockout mice lacking primary cilia in TECs (Ift88-TEC null mutant) to clarify their precise role in thymic organogenesis and T-cell differentiation. The Ift88-TEC null mutant mice showed stunted cilia or lack of cilia in TECs. The intercellular contact between T cells and the "thymic synapse" of medullary TECs was slightly disorganized in Ift88-TEC null mutants. Notably, the CD4- and CD8-single positive thymocyte subsets increased significantly. The absence or disorganization of thymic cilia downregulated the TGF-β signaling cascade, increasing the number of single positive thymocytes. To our knowledge, this is the first study reporting the physiological role of primary cilia and Ift88 in regulating the differentiation of the thymus and T cells.

The Lineage Differentiation and Dynamic Heterogeneity of Thymic Epithelial Cells During Thymus Organogenesis.

Although much progress has been made recently in revealing the heterogeneity of the thymic stromal components, the molecular programs of cell lineage divergency and temporal dynamics of thymic epithelial cell (TEC) development are largely elusive. Here, we constructed a single-cell transcriptional landscape of non-hematopoietic cells from mouse thymus spanning embryonic to adult stages, producing transcriptomes of 30,959 TECs. We resolved the transcriptional heterogeneity of developing TECs and highlighted the molecular nature of early TEC lineage determination and cortico-medullary thymic epithelial cell lineage divergency. We further characterized the differentiation dynamics of TECs by clarification of molecularly distinct cell states in the thymus developing trajectory. We also identified a population of Bpifa1+ Plet1+ mTECs that was preserved during thymus organogenesis and highly expressed tissue-resident adult stem cell markers. Finally, we highlighted the expression of Aire-dependent tissue-restricted antigens mainly in Aire+ Csn2+ mTECs and Spink5+ Dmkn+ mTECs in postnatal thymus. Overall, our data provided a comprehensive characterization of cell lineage differentiation, maturation, and temporal dynamics of thymic epithelial cells during thymus organogenesis.

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

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

The fibroblast: An emerging key player in thymic T cell selection.

Fibroblasts have recently attracted attention as a key stromal component that controls the immune responses in lymphoid tissues. The thymus has a unique microenvironment comprised of a variety of stromal cells, including fibroblasts and thymic epithelial cells (TECs), the latter of which is known to be important for T cell development because of their ability to express self‐antigens. Thymic fibroblasts contribute to thymus organogenesis during embryogenesis and form the capsule and medullary reticular network in the adult thymus. However, the immunological significance of thymic fibroblasts has thus far only been poorly elucidated. In this review, we will summarize the current views on the development and functions of thymic fibroblasts as revealed by new technologies such as multicolor flow cytometry and single cell–based transcriptome profiling. Furthermore, the recently discovered role of medullary fibroblasts in the establishment of T cell tolerance by producing a unique set of self‐antigens will be highlighted.

Recent Advancements in Regenerative Approaches for Thymus Rejuvenation.

The thymus plays a key role in adaptive immunity by generating a diverse population of T cells that defend the body against pathogens. Various factors from disease and toxic insults contribute to the degeneration of the thymus resulting in a fewer output of T cells. Consequently, the body is prone to a wide host of diseases and infections. In this review, first, the relevance of the thymus is discussed, followed by thymic embryological organogenesis and anatomy as well as the development and functionality of T cells. Attempts to regenerate the thymus include in vitro methods, such as forming thymic organoids aided by biofabrication techniques that are transplantable. Ex vivo methods that have shown promise in enhancing thymic regeneration are also discussed. Current regenerative technologies have not yet matched the complexity and functionality of the thymus. Therefore, emerging techniques that have shown promise and the challenges that lie ahead are explored.

A wave of bipotent T/ILC-restricted progenitors shapes the embryonic thymus microenvironment in a time-dependent manner

AbstractDuring embryonic development, multiple waves of hematopoietic progenitors with distinct lineage potential are differentially regulated in time and space. Two different waves of thymic progenitors colonize the fetal thymus where they contribute to thymic organogenesis and homeostasis. The origin, the lineage differentiation potential of the first wave, and their relative contribution in shaping the thymus architecture, remained, however, unclear. Here, we show that the first wave of thymic progenitors comprises a unique population of bipotent T and innatel lymphoid cells (T/ILC), generating a lymphoid tissue inducer cells (LTi's), in addition to invariant Vγ5+ T cells. Transcriptional analysis revealed that innate lymphoid gene signatures and, more precisely, the LTi-associated transcripts were expressed in the first, but not in the second, wave of thymic progenitors. Depletion of early thymic progenitors in a temporally controlled manner showed that the progeny of the first wave is indispensable for the differentiation of autoimmune regulator–expressing medullary thymic epithelial cells (mTECs). We further show that these progenitors are of strict hematopoietic stem cell origin, despite the overlap between lymphopoiesis initiation and the transient expression of lymphoid-associated transcripts in yolk sac (YS) erythromyeloid-restricted precursors. Our work highlights the relevance of the developmental timing on the emergence of different lymphoid subsets, required for the establishment of a functionally diverse immune system.

Leaving no one behind: tracing every human thymocyte by single-cell RNA-sequencing.

The thymus is the primary organ for T-cell development, providing an essential microenvironment consisting of the appropriate cytokine milieu and specialized stromal cells. Thymus-seeding progenitors from circulation immigrate into the thymus and undergo the stepwise T-cell specification, commitment, and selection processes. The transcriptional factors, epigenetic regulators, and signaling pathways involved in the T-cell development have been intensively studied using mouse models. Despite our growing knowledge of T-cell development, major questions remain unanswered regarding the ontogeny and early events of T-cell development at the fetal stage, especially in humans. The recently developed single-cell RNA-sequencing technique provides an ideal tool to investigate the heterogeneity of T-cell precursors and the molecular mechanisms underlying the divergent fates of certain T-cell precursors at the single-cell level. In this review, we aim to summarize the current progress of the study on human thymus organogenesis and thymocyte and thymic epithelial cell development, which is to shed new lights on developing novel strategies for in vitro T-cell regeneration and thymus rejuvenation.

Non-Epithelial Thymic Stromal Cells: Unsung Heroes in Thymus Organogenesis and T Cell Development.

The stromal microenvironment in the thymus is essential for generating a functional T cell repertoire. Thymic epithelial cells (TECs) are numerically and phenotypically one of the most prominent stromal cell types in the thymus, and have been recognized as one of most unusual cell types in the body by virtue of their unique functions in the course of the positive and negative selection of developing T cells. In addition to TECs, there are other stromal cell types of mesenchymal origin, such as fibroblasts and endothelial cells. These mesenchymal stromal cells are not only components of the parenchymal and vascular architecture, but also have a pivotal role in controlling TEC development, although their functions have been less extensively explored than TECs. Here, we review both the historical studies on and recent advances in our understanding of the contribution of such non-TEC stromal cells to thymic organogenesis and T cell development. In particular, we highlight the recently discovered functional effect of thymic fibroblasts on T cell repertoire selection.

Heparan sulfate is essential for thymus growth

Thymus organogenesis and T cell development are coordinated by various soluble and cell-bound molecules. Heparan sulfate (HS) proteoglycans can interact with and immobilize many soluble mediators, creating fields or gradients of secreted ligands. While the role of HS in the development of many organs has been studied extensively, little is known about its function in the thymus. Here, we examined the distribution of HS in the thymus and the effect of its absence on thymus organogenesis and T cell development. We found that HS was expressed most abundantly on the thymic fibroblasts and at lower levels on endothelial, epithelial, and hematopoietic cells. To study the function of HS in the thymus, we eliminated most of HS in this organ by genetically disrupting the glycosyltransferase Ext1 that is essential for its synthesis. The absence of HS greatly reduced the size of the thymus in fetal thymic organ cultures and in vivo, in mice, and decreased the production of T cells. However, no specific blocks in T cell development were observed. Wild-type thymic fibroblasts were able to physically bind the homeostatic chemokines CCL19, CCL21, and CXCL12 ex vivo. However, this binding was abolished upon HS degradation, disrupting the CCL19/CCL21 chemokine gradients and causing impaired migration of dendritic cells in thymic slices. Thus, our results show that HS plays an essential role in the development and growth of the thymus and in regulating interstitial cell migration.

T-Cell Immunodeficiencies With Congenital Alterations of Thymic Development: Genes Implicated and Differential Immunological and Clinical Features.

Combined Immunodeficiencies (CID) are rare congenital disorders characterized by defective T-cell development that may be associated with B- and NK-cell deficiency. They are usually due to alterations in genes expressed in hematopoietic precursors but in few cases, they are caused by impaired thymic development. Athymia was classically associated with DiGeorge Syndrome due to TBX1 gene haploinsufficiency. Other genes, implicated in thymic organogenesis include FOXN1, associated with Nude SCID syndrome, PAX1, associated with Otofaciocervical Syndrome type 2, and CHD7, one of the genes implicated in CHARGE syndrome. More recently, chromosome 2p11.2 microdeletion, causing FOXI3 haploinsufficiency, has been identified in 5 families with impaired thymus development. In this review, we will summarize the main genetic, clinical, and immunological features related to the abovementioned gene mutations. We will also focus on different therapeutic approaches to treat SCID in these patients.

Thymus Inception: Molecular Network in the Early Stages of Thymus Organogenesis.

The thymus generates central immune tolerance by producing self-restricted and self-tolerant T-cells as a result of interactions between the developing thymocytes and the stromal microenvironment, mainly formed by the thymic epithelial cells. The thymic epithelium derives from the endoderm of the pharyngeal pouches, embryonic structures that rely on environmental cues from the surrounding mesenchyme for its development. Here, we review the most recent advances in our understanding of the molecular mechanisms involved in early thymic organogenesis at stages preceding the expression of the transcription factor Foxn1, the early marker of thymic epithelial cells identity. Foxn1-independent developmental stages, such as the specification of the pharyngeal endoderm, patterning of the pouches, and thymus fate commitment are discussed, with a special focus on epithelial–mesenchymal interactions.