Gender Disparity Impacts on Thymus Aging and LHRH Receptor Antagonist-Induced Thymic Reconstitution Following Chemotherapeutic Damage.

One of the main consequences of thymus aging is the decrease in naïve T cell output. This condition accelerates at the onset of puberty, and presents as a major clinical complication for cancer patients who require cytoablative therapy. Specifically, the extensive use of chemotherapeutics, such as cyclophosphamide, in such treatments damage thymic structure and eliminate the existing naïve T cell repertoire. The resulting immunodeficiency can lead to increased incidence of opportunistic infections, tumor growth relapse and/or autoimmune diseases, particularly in older patients. Thus, strategies aimed at rejuvenating the aged thymus following chemotherapeutic damage are required. Previous studies have revealed that sex hormone deprivation in male mice is capable of regenerating the thymic microenvironment following chemotherapy treatment, however, further investigation is crucial to identify gender-based differences, and the molecular mechanisms involved during thymus regeneration. Through phenotypic analyzes, we identified gender-specific alterations in thymocytes and thymic epithelial cell (TEC) subsets from the onset of puberty. By middle-age, females presented with a higher number of thymocytes in comparison to males, yet a decrease in their Aire+ medullary TEC/thymocyte ratio was observed. This reduction could be associated with an increased risk of autoimmune disease in middle-aged women. Given the concurrent increase in female Aire+ cTEC/thymocyte ratio, we proposed that there may be an impediment in Aire+ mTEChi differentiation, and Aire+ cTEChi as its upstream precursor. The regenerative effects of LHRH receptor antagonist, degarelix, on TEC subsets was also less pronounced in middle-aged females compared to males, possibly due to slower progression of thymic involution in the former, which presented with greater TEChi proportions. Furthermore, following cyclophosphamide treatment, degarelix enhanced thymocyte and mature TEC subset recovery, with faster recovery kinetics observed in females. These events were found to involve both reactivation and proliferation of thymic epithelial progenitor cells. Taken together, the findings from this study portray a relationship between gender disparity and thymus aging, and highlight the potential benefits of LHRH receptor antagonist treatment for thymic regeneration. Further research is required, however, to determine how gender may impact on the mechanisms underpinning these events.

Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids.

Defining the ontogeny of the human adaptive immune system during embryogenesis has implications for understanding childhood diseases including leukaemias and autoimmune conditions. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cell genesis from pluripotent-stem-cell-derived haematopoietic organoids. Under conditions favouring T-cell development, RAG1+ cells progressively upregulated a cohort of recognized T-cell-associated genes, arresting development at the CD4+CD8+ stage. Sort and re-culture experiments showed that early RAG1+ cells also possessed B-cell, myeloid and erythroid potential. Flow cytometry and single-cell-RNA-sequencing data showed that early RAG1+ cells co-expressed the endothelial/haematopoietic progenitor markers CD34, VECAD and CD90, whereas imaging studies identified RAG1+ cells within CD31+ endothelial structures that co-expressed SOX17+ or the endothelial marker CAV1. Collectively, these observations provide evidence for a wave of human T-cell development that originates directly from haemogenic endothelium via a RAG1+ intermediate with multilineage potential.

Gelatin-Based 3D Microgels for In Vitro T Lineage Cell Generation.

T cells are predominantly produced by the thymus and play a significant role in maintaining our adaptive immune system. Physiological involution of the thymus occurs gradually with age, compromising naive T cell output, which can have severe clinical complications. Also, T cells are utilized as therapeutic agents in cancer immunotherapies. Therefore, there is an increasing need for strategies aimed at generating naive T cells. The majority of in vitro T cell generation studies are performed in two-dimensional (2D) cultures, which ignore the physiological thymic microenvironment and are not scalable; therefore, we applied a new three-dimensional (3D) approach. Here, we use a gelatin-based 3D microgel system for T lineage induction by co-culturing OP9-DL4 cells and mouse fetal-liver-derived hematopoietic stem cells (HSCs). Flow cytometric analysis revealed that microgel co-cultures supported T lineage induction similar to 2D cultures while providing a 3D environment. We also encapsulated mouse embryonic thymic epithelial cells (TECs) within the microgels to provide a defined 3D culture platform. The microgel system supported TEC maintenance and retained their phenotype. Together, these data show that our microgel system has the capacity for TEC maintenance and induction of in vitro T lineage differentiation with potential for scalability.

Decreased maternal serum acetate and impaired fetal thymic and regulatory T cell development in preeclampsia.

Maternal immune dysregulation seems to affect fetal or postnatal immune development. Preeclampsia is a pregnancy-associated disorder with an immune basis and is linked to atopic disorders in offspring. Here we show reduction of fetal thymic size, altered thymic architecture and reduced fetal thymic regulatory T (Treg) cell output in preeclamptic pregnancies, which persists up to 4 years of age in human offspring. In germ-free mice, fetal thymic CD4+ T cell and Treg cell development are compromised, but rescued by maternal supplementation with the intestinal bacterial metabolite short chain fatty acid (SCFA) acetate, which induces upregulation of the autoimmune regulator (AIRE), known to contribute to Treg cell generation. In our human cohorts, low maternal serum acetate is associated with subsequent preeclampsia, and correlates with serum acetate in the fetus. These findings suggest a potential role of acetate in the pathogenesis of preeclampsia and immune development in offspring.

Interplay between Follistatin, Activin A, and BMP4 Signaling Regulates Postnatal Thymic Epithelial Progenitor Cell Differentiation during Aging.

A key feature of immune functional impairment with age is the progressive involution of thymic tissue responsible for naive T cell production. In this study, we identify two major phases of thymic epithelial cell (TEC) loss during aging: a block in mature TEC differentiation from the pool of immature precursors, occurring at the onset of puberty, followed by impaired bipotent TEC progenitor differentiation and depletion of Sca-1lo cTEC and mTEC lineage-specific precursors. We reveal that an increase in follistatin production by aging TECs contributes to their own demise. TEC loss occurs primarily through the antagonism of activin A signaling, which we show is required for TEC maturation and acts in dissonance to BMP4, which promotes the maintenance of TEC progenitors. These results support a model in which an imbalance of activin A and BMP4 signaling underpins the degeneration of postnatal TEC maintenance during aging, and its reversal enables the transient replenishment of mature TECs.

Autoimmune-Mediated Thymic Atrophy Is Accelerated but Reversible in RelB-Deficient Mice.

Polymorphisms impacting thymic function may decrease peripheral tolerance and hasten autoimmune disease. The NF-κB transcription factor subunit, RelB, is essential for the development and differentiation of medullary thymic epithelial cells (mTECs): RelB-deficient mice have reduced thymic cellularity and markedly fewer mTECs, lacking AIRE. The precise mechanism of this mTEC reduction in the absence of RelB is unclear. To address this, we studied mTECs and dendritic cells (DCs), which critically regulate negative selection, and thymic regulatory T-cells (tTreg) in RelB-/- mice, which have spontaneous multiorgan autoimmune disease. RelB-/- thymi were organized, with medullary structures containing AIRE- mTECs, DCs, and CD4+ thymocytes, but fewer tTreg. Granulocytes infiltrated the RelB-/- thymic cortex, capsule, and medulla, producing inflammatory thymic medullary atrophy, which could be treated by granulocyte depletion or RelB+ DC immunotherapy, with concomitant recovery of mTEC and tTreg numbers. These data indicate that central tolerance defects may be accelerated by autoimmune thymic inflammation where impaired RelB signaling impairs the medullary niche, and may be reversible by therapies enhancing peripheral Treg or suppressing inflammation.

Inflammation and Thymus Ageing.

The thymus is primarily responsible for T cell production. However, it begins to recede in size and function, from early in life. This decreased generation of naive T cells during normal thymus ageing, or linked with pathology (i.e. chronic inflammation), leads to reduced T cell specificities, peripheral T cell imbalances, and higher susceptibilities to infections. Various clinical strategies for thymus and T cell recovery have been investigated, although no effective clinical treatments for the reconstitution of peripheral T cell diversity in severe immune deficiencies are available. The recent identification of thymic epithelial progenitor cells (TEPC) in the adult thymus will enable investigations into a new generation of therapies focused on regenerating the thymic microenvironment for diverse specificity T cell reconstitution in the elderly. The specific mechanisms underlying TEPC activation are still being investigated. Recent data point to an important role of the intrathymic transforming growth factor-ß (TGF-ß) circuitry. Although dual actions of this cytokine have been reported in the immune system, TGF-ß signaling is transiently activated in hematopoietic stem and progenitor cells during hematopoietic regeneration. This review investigates the current strategies for thymus reactivation to replenish the peripheral T cell repertoire and potentially reverse the age-related inflammatory milieu.

Native thymic extracellular matrix improves in vivo thymic organoid T cell output, and drives in vitro thymic epithelial cell differentiation.

Although the thymus is a primary lymphoid organ, its function is compromised by an age-induced loss of resident epithelial cells, which results in reduced naïve T cell output. This has important implications for immune recovery in aged and elderly patients following damage from cytoablative therapies. As thymic architecture plays a crucial role in naïve T cell development, a tissue specific scaffold that provides essential supporting matrix may assist in stem cell-based thymus regeneration to recreate complex organoids. Here we investigate thymus decellularization approaches that preserve major extracellular matrix components and support thymic epithelial cells for the generation of a functional thymic microenvironment with improved T cell output. We also established an in vitro, serum-free culture system that both maintains a progenitor thymic epithelial cell pool and drives their differentiation in the presence of decellularized thymic matrix. This approach enables further dissection of key cellular and niche components involved in thymic epithelial stem cell maintenance and T cell production.

A novel Foxn1eGFP/+ mouse model identifies Bmp4-induced maintenance of Foxn1 expression and thymic epithelial progenitor populations.

Although forkhead-box n1 (Foxn1) is a critical thymic epithelial cell regulator in thymus organogenesis, its association with epithelial differentiation and homeostasis in the postnatal and aged thymic microenvironment remains conflicting. Consequently, we have generated a Foxn1eGFP/+ knock-in mouse model that allows for refined investigation of the aging thymic epithelium. This reporter line differs from those previously published in that concomitant expression of enhanced green fluorescent protein enables live cell sorting of Foxn1+ cell populations. Our heterozygotes did not exhibit haploinsufficiency, with Foxn1 expression resembling that of wild-type mice. Comparative analysis between Foxn1 and enhanced green fluorescent protein at both the transcriptional and translational levels revealed co-localization, with progressive down-regulation observed predominantly in the aging cortical epithelium. Supplementation with bone morphogenetic protein (Bmp)-4 enhanced Foxn1 expression and colony forming efficiency in both embryonic and adult progenitor 3D cultures. Strikingly, selective maintenance of immature cortical and medullary epithelial cells was observed which is consistent with the higher Bmp receptor 2 expression levels seen in these progenitor populations. This study demonstrates the significance of our mouse model in unraveling the role of this master regulator in thymus development, homeostasis and aging, providing a faithful reporter system for phenotypic and functional investigations.

In situ-forming click-crosslinked gelatin based hydrogels for 3D culture of thymic epithelial cells.

Hydrogels prepared from naturally derived gelatin can provide a suitable environment for cell attachment and growth, making them favourable materials in tissue engineering. However, physically crosslinked gelatin hydrogels are not stable under physiological conditions while chemical crosslinking of gelatin by radical polymerization may be harmful to cells. In this study, we attached the norbornene functional group to gelatin, which was subsequently crosslinked with a polyethylene glycol (PEG) linker via the nitrile oxide-norbornene click reaction. The rapid crosslinking process allows the hydrogel to be formed within minutes of mixing the polymer solutions under physiological conditions, allowing the gels to be used as injectable materials. The hydrogels properties including mechanical strength, swelling and degradation, can be tuned by changing either the ratio of the reacting groups or the total concentration of the polymer precursors. Murine embryonic fibroblastic cells cultured in soft gels (2 wt% of gelatin and 1 wt% of PEG linker) demonstrated high cell viability as well as similar phenotypic profiles (PDGFRα and MTS15) to Matrigel cultures over 5 days. Thymic epithelial cell and fibroblast co-cultures produced epithelial colonies in these gels following 7 days incubation. These studies demonstrate that gelatin based hydrogels, prepared using "click" crosslinking, provide a robust cell culture platform with retained benefits of the gelatin material, and are therefore suitable for use in various tissue engineering applications.

New role for the (pro)renin receptor in T-cell development.

The (pro)renin receptor (PRR) was originally thought to be important for regulating blood pressure via the renin-angiotensin system. However, it is now emerging that PRR has instead a generic role in cellular development. Here, we have specifically deleted PRR from T cells. T-cell-specific PRR-knockout mice had a significant decrease in thymic cellularity, corresponding with a 100-fold decrease in the number of CD4(+) and CD8(+) thymocytes, and a large increase in double-negative (DN) precursors. Gene expression analysis on sorted DN3 thymocytes indicated that PRR-deficient thymocytes have perturbations in key cellular pathways essential at the DN3 stage, including transcription and translation. Further characterization of DN T-cell progenitors leads us to propose that PRR deletion affects thymocyte survival and development at multiple stages; from DN3 through to DN4, double-positive, and single-positive CD4 and CD8. Our study thus identifies a new role for PRR in T-cell development.

Enhanced hematopoietic stem cell function mediates immune regeneration following sex steroid blockade.

Mechanisms underlying age-related defects within lymphoid-lineages remain poorly understood. We previously reported that sex steroid ablation (SSA) induced lymphoid rejuvenation and enhanced recovery from hematopoietic stem cell (HSC) transplantation (HSCT). We herein show that, mechanistically, SSA induces hematopoietic and lymphoid recovery by functionally enhancing both HSC self-renewal and propensity for lymphoid differentiation through intrinsic molecular changes. Our transcriptome analysis revealed further hematopoietic support through rejuvenation of the bone marrow (BM) microenvironment, with upregulation of key hematopoietic factors and master regulatory factors associated with aging such as Foxo1. These studies provide important cellular and molecular insights into understanding how SSA-induced regeneration of the hematopoietic compartment can underpin recovery of the immune system following damaging cytoablative treatments. These findings support a short-term strategy for clinical use of SSA to enhance the production of lymphoid cells and HSC engraftment, leading to improved outcomes in adult patients undergoing HSCT and immune depletion in general.

Perspectives for Improvement of the Thymic Microenvironment through Manipulation of Thymic Epithelial Cells: A Mini-Review.

Thymic involution during aging is a major reason for the decreased production of naive T cells and reduced immunity. Alterations within the thymic microenvironment, characterized by the loss of function of thymic epithelial cells (TECs) and fibro-adipogenetic transformation, seem to underlie this process, mainly through declining communication between thymic stromal cells and developing thymocytes. Specifically, the signaling mediated by cytokines and hormones secreted by TECs declines during aging. Many therapies based on the manipulation of growth factors and hormones have succeeded in partially recovering the lymphoid compartment and promoting thymic function. However, considering that aging-induced thymic involution is multifactorial, the thymic reestablishment achieved with treatments that target isolated pathways is incomplete and transitory. Here, we discuss the development of three novel approaches for potentially sustained thymic recovery: the induction of sustained forkhead box N1 expression, the activation of endogenous thymic epithelial progenitor cells (TEPCs), and the generation of TEPCs from pluripotent stem cells. Combined approaches targeting both TECs and lymphoid cells will provide a potentially more effective strategy for sustained rejuvenation of the thymus.

Multilineage potential and self-renewal define an epithelial progenitor cell population in the adult thymus.

Thymic epithelial cells (TECs) are critical for T cell development and self-tolerance but are gradually lost with age. The existence of thymic epithelial progenitors (TEPCs) in the postnatal thymus has been inferred, but their identity has remained enigmatic. Here, we assessed the entire adult TEC compartment in order to reveal progenitor capacity is retained exclusively within a subset of immature thymic epithelium displaying several hallmark features of stem/progenitor function. These adult TEPCs generate mature cortical and medullary lineages in a stepwise fashion, including Aire+ TEC, within fetal thymus reaggregate grafts. Although relatively quiescent in vivo, adult TEPCs demonstrate significant in vitro colony formation and self-renewal. Importantly, 3D-cultured TEPCs retain their capacity to differentiate into cortical and medullary TEC lineages when returned to an in vivo thymic microenvironment. No other postnatal TEC subset exhibits this combination of properties. The characterization of adult TEPC will enable progress in understanding TEC biology in aging and regeneration.

FOXN1 (GFP/w) reporter hESCs enable identification of integrin-ß4, HLA-DR, and EpCAM as markers of human PSC-derived FOXN1(+) thymic epithelial progenitors.

Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1 (GFP/w) line as a readout, we developed a reproducible protocol for generating FOXN1-GFP(+) thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-ß4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1(+) TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1(+) TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.

The role of Tenascin C in the lymphoid progenitor cell niche.

Hemopoietic stem cells (HSCs) are extrinsically controlled by the bone marrow (BM) microenvironment. Mice devoid of the extracellular matrix molecule Tenascin-C (TNC) were reported to develop normally. The current study explores the relationship between TNC and hemopoiesis, from HSCs within their niche to maturing progenitors in alternate niches. Although the absence of TNC did not alter the size of the BM stem cell pool, we report decreased thymic T cell progenitors with redistribution to other lymphoid organs, suggesting an anchoring role for TNC. TNC did not play an essential role in stem and progenitor cell homing to BM, but significantly altered lymphoid primed progenitor cell homing. These cells express the TNC receptor, integrin α9ß1, with the same reduced homing evident in the absence of this integrin. The absence of TNC also resulted in an increased proportion and number of mature circulating T cells. In addition, the absence of TNC significantly impaired hemopoietic reconstitution after transplant and increased stem and progenitor cell mobilization. In summary, our analysis revealed unidentified roles for TNC in hemopoiesis: in lineage commitment of thymic T cell progenitors, peripheral T cell migration, and hemopoietic reconstitution.

Rewiring immunity: generating a functional thymus from hESCs are we there yet?

Two papers in this issue of Cell Stem Cell have made a significant advance in solving one of the great challenges of modern immunology-resurrecting thymus function through the induction of thymus epithelial cells (TECs) by directed differentiation of human embryonic stem cells (hESCs).

Thymic deletion and regulatory T cells prevent antimyeloperoxidase GN.

Loss of tolerance to neutrophil myeloperoxidase (MPO) underlies the development of ANCA-associated vasculitis and GN, but the mechanisms underlying this loss of tolerance are poorly understood. Here, we assessed the role of the thymus in deletion of autoreactive anti-MPO T cells and the importance of peripheral regulatory T cells in maintaining tolerance to MPO and protecting from GN. Thymic expression of MPO mRNA predominantly localized to medullary thymic epithelial cells. To assess the role of MPO in forming the T cell repertoire and the role of the autoimmune regulator Aire in thymic MPO expression, we compared the effects of immunizing Mpo(-/-) mice, Aire(-/-) mice, and control littermates with MPO. Immunized Mpo(-/-) and Aire(-/-) mice developed significantly more proinflammatory cytokine-producing anti-MPO T cells and higher ANCA titers than control mice. When we triggered GN with a subnephritogenic dose of anti-glomerular basement membrane antibody, Aire(-/-) mice had more severe renal disease than Aire(+/+) mice, consistent with a role for Aire-dependent central deletion in establishing tolerance to MPO. Furthermore, depleting peripheral regulatory T cells in wild-type mice also led to more anti-MPO T cells, higher ANCA titers, and more severe GN after immunization with MPO. Taken together, these results suggest that Aire-dependent central deletion and regulatory T cell-mediated peripheral tolerance both play major roles in establishing and maintaining tolerance to MPO, thereby protecting against the development of anti-MPO GN.

Isolation, characterization, and reaggregate culture of thymic epithelial cells.

The thymus organ is composed of a three-dimensional (3D) network of adjoining epithelium and stromal cells. Bone marrow-derived T cell precursors, upon entering the thymus, interact with and migrate through this cellular network as they differentiate and mature. An essential component of the stroma is the thymic epithelial cells (TEC), which play a vital role in T cell development and induction of self-tolerance for adaptive immunity. TEC can be isolated from the embryonic and adult thymus by a series of gentle enzymatic digestions and characterized into discrete subpopulations based on their expression of surface markers by flow cytometry. Enrichment of adult TEC can be achieved by depletion of hematopoietic cells, allowing sufficient numbers to be purified for subsequent functional and molecular analysis. Although monolayer cultures have been used to study TEC phenotype and T cell interaction, methods that mimic the 3D thymic microenvironment, such as fetal and reaggregate thymic organ cultures, are more accurate for the analysis of TEC function and support more complete T cell development. Herein, we describe methods for the efficient isolation and enrichment of TEC for downstream analyses as well as the reaggregation of embryonic progenitor epithelium to form a functional thymus graft under the kidney capsule.