Embryonic Thymus Research Articles

John Joseph Thomas Hayes Owen. 7 January 1934—5 January 2023

John Owen was an anatomist and immunologist who conducted pioneering work on the origins and development of the two major classes of lymphocytes, the cells that are the central players in the adaptive immune systems of vertebrates. He was a meticulous biological experimenter who liked to keep questions simple and precise. His observations contributed to two key discoveries: that mouse and avian stem cells migrate into the embryonic thymus and give rise to T lymphocytes; and that in birds the bursa of Fabricius is the tissue in which stem cells develop into B lymphocytes. He also helped to establish that in mammals the earliest B lymphocytes develop in the liver of the fetus. He and his colleagues went on to define how combinations of several cell populations in the thymus provide the settings in which mammals first generate and then select from the variety of T lymphocyte lineages and immune specificities that will equip them with healthy immune surveillance and responses. His wry Liverpudlian sense of humour let him engage easily with everyone, from any walk of life, and he was an inspiration to young colleagues, many of whom would go on to flourish in the research areas that he pioneered. John died on 5 January 2023, from prostate cancer and other ailments: but he was in the gym only a couple of weeks before he died. He is buried in Kirkdale cemetery, Lancashire, under a family tombstone that also celebrates his grandfather, his father and his mother: his epitaph reads ‘One push-up too many’.

Abstract 4188: Kyinno PBMC humanized mouse platform: Pioneering service in biomedical research

Abstract Tumor immunotherapy is the use of the human immune system to kill tumors. Compared with traditional chemotherapy and radiotherapy, immunotherapy has significant advantages such as significant efficacy, long-lasting effects, and low toxicity. Wild-type mice are widely used as preclinical models for immunotherapy due to their normal immune system, but they have many shortcomings, such as only being able to inoculate mouse tumor cells, differences between mouse and human immune systems, etc., which cannot accurately simulate the interaction between drugs and tumor cells in the human body. Humanized mouse models include PBMCs humanized mouse models, HSC humanized mouse models, and human embryonic bone, liver, and thymus tissue (BLT) transplantation models. Among them, the PBMCs humanized mouse model is widely used due to its simple preparation, short cycle, and high levels of hCD3+ T cells in the peripheral blood of mice. To assist in the development of immunotherapy, KYINNO has established a PBMC humanized mouse platform that can perform in vivo drug efficacy verification of various immunotherapeutic drugs such as CD3 bispecific antibodies or multi-specific antibodies, immune checkpoint inhibitors, etc. We screen many PBMC donors and select PBMC donors with light GvHD and good hCD3+ T cell reconstruction effects. The drug administration window of the PBMC humanized model has been extended to 6 weeks. Platform verification has been performed on subcutaneous and intravenous injection models of various tumors. Overall, we successfully established the PBMC humanized mouse platform, which is a powerful tool for immunotherapy. Citation Format: Tongtong Liu, Guojin Wu, Feng He, Longtan Luo, Zhengcheng Guo, Shuliang Li, Jinying Ning, Feng Hao. Kyinno PBMC humanized mouse platform: Pioneering service in biomedical research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4188.

Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here, we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny in mice. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium.

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here, we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny in mice. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

Embryonic keratin19+ progenitors generate multiple functionally distinct progeny to maintain epithelial diversity in the adult thymus medulla

The thymus medulla is a key site for immunoregulation and tolerance, and its functional specialisation is achieved through the complexity of medullary thymic epithelial cells (mTEC). While the importance of the medulla for thymus function is clear, the production and maintenance of mTEC diversity remains poorly understood. Here, using ontogenetic and inducible fate-mapping approaches, we identify mTEC-restricted progenitors as a cytokeratin19+ (K19+) TEC subset that emerges in the embryonic thymus. Importantly, labelling of a single cohort of K19+ TEC during embryogenesis sustains the production of multiple mTEC subsets into adulthood, including CCL21+ mTEClo, Aire+ mTEChi and thymic tuft cells. We show K19+ progenitors arise prior to the acquisition of multiple mTEC-defining features including RANK and CCL21 and are generated independently of the key mTEC regulator, Relb. In conclusion, we identify and define a multipotent mTEC progenitor that emerges during embryogenesis to support mTEC diversity into adult life.

Thymic macrophages consist of two populations with distinct localization and origin.

Tissue-resident macrophages are essential to protect from pathogen invasion and maintain organ homeostasis. The ability of thymic macrophages to engulf apoptotic thymocytes is well appreciated, but little is known about their ontogeny, maintenance, and diversity. Here, we characterized the surface phenotype and transcriptional profile of these cells and defined their expression signature. Thymic macrophages were most closely related to spleen red pulp macrophages and Kupffer cells and shared the expression of the transcription factor SpiC with these cells. Single-cell RNA sequencing showed that the macrophages in the adult thymus are composed of two populations distinguished by the expression of Timd4 and Cx3cr1. Remarkably, Timd4+ cells were located in the cortex, while Cx3cr1+ macrophages were restricted to the medulla and the cortico-medullary junction. Using shield chimeras, transplantation of embryonic thymuses, and genetic fate mapping, we found that the two populations have distinct origins. Timd4+ thymic macrophages are of embryonic origin, while Cx3cr1+ macrophages are derived from adult hematopoietic stem cells. Aging has a profound effect on the macrophages in the thymus. Timd4+ cells underwent gradual attrition, while Cx3cr1+ cells slowly accumulated with age and, in older mice, were the dominant macrophage population in the thymus. Altogether, our work defines the phenotype, origin, and diversity of thymic macrophages.

Mesenchymal cell replacement corrects thymic hypoplasia in murine models of 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11.2DS) is the most common human chromosomal microdeletion, causing developmentally linked congenital malformations, thymic hypoplasia, hypoparathyroidism, and/or cardiac defects. Thymic hypoplasia leads to T cell lymphopenia, which most often results in mild SCID. Despite decades of research, the molecular underpinnings leading to thymic hypoplasia in 22q11.2DS remain unknown. Comparison of embryonic thymuses from mouse models of 22q11.2DS (Tbx1neo2/neo2) revealed proportions of mesenchymal, epithelial, and hematopoietic cell types similar to those of control thymuses. Yet, the small thymuses were growth restricted in fetal organ cultures. Replacement of Tbx1neo2/neo2 thymic mesenchymal cells with normal ones restored tissue growth. Comparative single-cell RNA-Seq of embryonic thymuses uncovered 17 distinct cell subsets, with transcriptome differences predominant in the 5 mesenchymal subsets from the Tbx1neo2/neo2 cell line. The transcripts affected included those for extracellular matrix proteins, consistent with the increased collagen deposition we observed in the small thymuses. Attenuating collagen cross-links with minoxidil restored thymic tissue expansion for hypoplastic lobes. In colony-forming assays, the Tbx1neo2/neo2-derived mesenchymal cells had reduced expansion potential, in contrast to the normal growth of thymic epithelial cells. These findings suggest that mesenchymal cells were causal to the small embryonic thymuses in the 22q11.2DS mouse models, which was correctable by substitution with normal mesenchyme.

Waves of layered immunity over innate lymphoid cells.

Innate lymphoid cells (ILCs) harbor tissue-resident properties in border zones, such as the mucosal membranes and the skin. ILCs exert a wide range of biological functions, including inflammatory response, maintenance of tissue homeostasis, and metabolism. Since its discovery, tremendous effort has been made to clarify the nature of ILCs, and scientific progress revealed that progenitor cells of ILC can produce ILC subsets that are functionally reminiscent of T-cell subsets such as Th1, Th2, and Th17. Thus, now it comes to the notion that ILC progenitors are considered an innate version of naïve T cells. Another important discovery was that ILC progenitors in the different tissues undergo different modes of differentiation pathways. Furthermore, during the embryonic phase, progenitor cells in different developmental chronologies give rise to the unique spectra of immune cells and cause a wave to replenish the immune cells in tissues. This observation leads to the concept of layered immunity, which explains the ontology of some cell populations, such as B-1a cells, γδ T cells, and tissue-resident macrophages. Thus, recent reports in ILC biology posed a possibility that the concept of layered immunity might disentangle the complexity of ILC heterogeneity. In this review, we compare ILC ontogeny in the bone marrow with those of embryonic tissues, such as the fetal liver and embryonic thymus, to disentangle ILC heterogeneity in light of layered immunity.

Identification of fibroblast progenitors in the developing mouse thymus.

ABSTRACTThe thymus stroma constitutes a fundamental microenvironment for T-cell generation. Despite the chief contribution of thymic epithelial cells, recent studies emphasize the regulatory role of mesenchymal cells in thymic function. Mesenchymal progenitors are suggested to exist in the postnatal thymus; nonetheless, an understanding of their nature and the mechanism controlling their homeostasis in vivo remains elusive. We resolved two new thymic fibroblast subsets with distinct developmental features. Whereas CD140αβ+GP38+SCA-1− cells prevailed in the embryonic thymus and declined thereafter, CD140αβ+GP38+SCA-1+ cells emerged in the late embryonic period and predominated in postnatal life. The fibroblastic-associated transcriptional programme was upregulated in CD140αβ+GP38+SCA-1+ cells, suggesting that they represent a mature subset. Lineage analysis showed that CD140αβ+GP38+SCA-1+ maintained their phenotype in thymic organoids. Strikingly, CD140αβ+GP38+SCA-1− generated CD140αβ+GP38+SCA-1+, inferring that this subset harboured progenitor cell activity. Moreover, the abundance of CD140αβ+GP38+SCA-1+ fibroblasts was gradually reduced in Rag2−/− and Rag2−/−Il2rg−/− thymi, indicating that fibroblast maturation depends on thymic crosstalk. Our findings identify CD140αβ+GP38+SCA-1− as a source of fibroblast progenitors and define SCA-1 as a marker for developmental stages of thymic fibroblast differentiation.

P63 targeted deletion under the FOXN1 promoter disrupts pre-and post-natal thymus development, function and maintenance as well as induces severe hair loss.

Progressive immune deficiency of aging is characterized by severe thymic atrophy, contracted T cell repertoire, and poor immune function. p63 is critical for the proliferative potential of embryonic and adult stem cells, as well as thymic epithelial cells (TECs). Because p63 null mice experience rapid post-natal lethality due to epidermal and limb morphogenesis defects, studies to define a role for p63 expression in TEC biology focused on embryonic thymus development and in vitro experiments. Since post-natal thymic stromal development and function differs from that of the embryo, we assessed the impact of lineage-restricted p63 loss on pre- and post-natal murine TEC function by generating mice with a loss of p63 function targeted to TEC, termed p63TECko mice. In adult p63TECko mice, severe thymic hypoplasia was observed with a lack in a discernable segregation into medullary and cortical compartments and peripheral T cell lymphopenia. This profound thymic defect was seen in both neonatal as well as embryonic p63TECko mice. In addition to TECs, p63 also plays in important role in the development of stratified epithelium of the skin; lack of p63 results in defects in skin epidermal stratification and differentiation. Interestingly, all adult p63TECko mice lacked hair follicles despite having normal p63 expression in the skin. Together our results show a critical role of TEC p63 in thymic development and maintenance and show that p63 expression is critical for hair follicle formation.

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.

Vγ5Vδ1 TCR signaling is required to different extents for embryonic versus postnatal development of DETCs.

γδ T cells expressing Vγ5Vδ1 TCR originally develop in the embryonic thymus and migrate to the epidermis, forming dendritic epidermal T cells (DETCs) throughout life. It is thought that a TCR signal is essential for their development; e.g., lack of TCR signal-transducer ZAP70 significantly decreases DETC numbers. On the other hand, lack of ZAP70 does not affect Vγ5Vδ1+ T cells in the embryonic thymus; thus, the involvement of TCR signaling remains elusive. Here, we used SKG mice with attenuated TCR signaling rather than gene-knockout mice. In SKG mice, Vγ5+ T cells showed a marked decrease [10% of wild-type (WT)] in adult epidermis; however, there was just a moderate decrease (50% of WT) in the embryonic thymus. In early postnatal epidermis in SKG mice, substantial numbers of Vγ5+ T cells were observed (50% of WT). Their activation markers including CD122, a component of the IL-15 receptor indispensable for DETC proliferation, were comparable to those of WT. However, the Vγ5+ T cells in SKG mice did not proliferate and form DETCs thereafter. Furthermore, in SKG/+ mice, the number of thymic Vγ5Vδ1+ T cells increased, compared to SKG mice; however, the number of DETCs remained significantly lower than in WT, similar to SKG mice. Our results suggest that signaling via Vγ5Vδ1 TCR is indispensable for DETC development, with distinct contributions to embryonic development and postnatal proliferation.

Rare presentation of ectopic thymoma

Thymoma is the most common tumor of the anterior mediastinum with an incidence of 0.15 cases per 100,000. Ninety percent of all thymoma occur in the anterior mediastinum, with the remainder occurring in the neck or other mediastinal areas. Ectopic cervical thymoma (ECT) is an extremely rare tumor that originates from ectopic tissue and is caused by the aberrant migration of the embryonic thymus. ECTs are often confused with thyroid or parathyroid swellings due to their anatomical positioning. Intrathyroid epithelial thymoma is a rare tumour comprising about 0.08% of all primary thyroid malignancies. It is low-grade thyroid carcinoma with squamous cell differentiation whose overall survival rate was found to be 71%. Here we presented a case of 43 year old female, came with swelling in front of the neck since many years. Ultrasound neck and fine needle aspiration cytology (FNAC) was performed. Patient underwent total thyroidectomy and specimen obtained was sent for histopathological examination. It revealed ectopic intrathyroid thymoma.

DL4-\u03bcbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system

T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-μbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αβ T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-μbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources.

Delayed maturation of thymic epithelium in mice with specific deletion of \u03b2-catenin gene in FoxN1 positive cells

Wnt signalling pathways have been reported to be involved in thymus development but their precise role in the development of both thymic epithelium (TE) and thymocytes is controversial. Herein, we examined embryonic, postnatal and adult thymi of mice with a specific deletion of β-catenin gene in FoxN1+ thymic epithelial cells (TECs). Together with a high postnatal mouse mortality, the analysis showed severe thymic hypocellularity, largely due an important reduction in numbers of developing thymocytes, and delayed, partially blocked maturation of mutant TECs. Affected TECs included largely cortical (c) TEC subsets, such as immature MTS20+ TECs, Ly51+ cTECs and a remarkable, rare Ly51+MTS20+MHCIIhi cell subpopulation previously reported to contain thymic epithelial progenitor cells (TEPCs) (Ulyanchenko et al., Cell Rep 14:2819–2832, 2016). In addition, altered postnatal organization of mutant thymic medulla failed to organize a unique, central epithelial area. This delayed maturation of TE cell components correlated with low transcript production of some molecules reported to be masters for TEC maturation, such as EphB2, EphB3 and RANK. Changes in the thymic lymphoid component became particularly evident after birth, when molecules expressed by TECs and involved in early T-cell maturation, such as CCL25, CXCL12 and Dll4, exhibited minimal values. This represented a partial blockade of the progression of DN to DP cells and reduced proportions of this last thymocyte subset. At 1 month, in correlation with a significant increase in transcript production, the DP cell percentage increased in correlation with a significant fall in the number of mature TCRαβhi thymocytes and peripheral T lymphocytes.

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.

RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus.

Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 can arise in the embryonic thymus, from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 migrated to mucosal tissues with colonization of the intestinal lamina propria. RORα expression repressed T cell development, while promoting ILC2 in the thymus. From RNA-seq, ATAC-seq and ChIP-seq data we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 from common progenitors in the thymus. When Notch signaling is present, Bcl11b dampens Nfil3/Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the Nfil3/Id2 repression, allowing Id2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.

Erratum to: Expression and the Role of Type 1A Serotonin Receptor in Rats’ Embryonic Thymus

Erratum to: Expression and the Role of Type 1A Serotonin Receptor in Rats’ Embryonic Thymus

Expression and the Role of Type 1A Serotonin Receptor in Rats’ Embryonic Thymus

Expression and the Role of Type 1A Serotonin Receptor in Rats’ Embryonic Thymus

Deciphering the regulatory landscape of fetal and adult γδ T‐cell development at single‐cell resolution

γδ T cells with distinct properties develop in the embryonic and adult thymus and have been identified as critical players in a broad range of infections, antitumor surveillance, autoimmune diseases, and tissue homeostasis. Despite their potential value for immunotherapy, differentiation of γδ T cells in the thymus is incompletely understood. Here, we establish a high‐resolution map of γδ T‐cell differentiation from the fetal and adult thymus using single‐cell RNA sequencing. We reveal novel sub‐types of immature and mature γδ T cells and identify an unpolarized thymic population which is expanded in the blood and lymph nodes. Our detailed comparative analysis reveals remarkable similarities between the gene networks active during fetal and adult γδ T‐cell differentiation. By performing a combined single‐cell analysis of Sox13, Maf, and Rorc knockout mice, we demonstrate sequential activation of these factors during IL‐17‐producing γδ T‐cell (γδT17) differentiation. These findings substantially expand our understanding of γδ T‐cell ontogeny in fetal and adult life. Our experimental and computational strategy provides a blueprint for comparing immune cell differentiation across developmental stages.