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.

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.

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.

Tolerance strategies for stem-cell-based therapies.

There is much interest in using embryonic stem cells to regenerate tissues and organs. For this approach to succeed, these stem cells or their derivatives must engraft in patients over the long term. Unless a cell transplant is derived from the patient's own cells, however, the cells will be targeted for rejection by the immune system. Although standard methods for suppressing the immune system achieve some success, rejection of the transplant is inevitable. Emerging approaches to address this issue include 're-educating' the immune system to induce tolerance to foreign cells and reducing the immune targeting of the transplant by administering 'self stem cells' instead of foreign cells, but each of these approaches has associated challenges.

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.

The activity of early-life gene regulatory elements is hijacked in aging through pervasive AP-1-linked chromatin opening.

A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening.

Feeding the fire: the role of defective bone marrow function in exacerbating thymic involution.

Most of the steps of lymphopoiesis have been elucidated but contentious issues remain, particularly regarding the identity and function of the earliest lymphoid progenitors that leave the bone marrow and seed the thymus. Hematopoiesis is effectively continuous throughout life, but there is a profound decline in immune function with increasing age, driven by thymus involution and severely curtailed B cell development. A key question is whether defects in bone marrow progenitors, such as reduced differentiation and repopulation potential, are the common denominator. While thymic involution temporally precedes overt HSC functional decline, a logical supposition is that the latter exacerbates the former. This review explores this possible link, and concludes that improving bone marrow function is fundamental to sustained thymic regeneration.

Thymic involution and immune reconstitution.

Chronic thymus involution associated with aging results in less efficient T-cell development and decreased emigration of naïve T cells to the periphery. Thymic decline in the aged is linked to increased morbidity and mortality in a wide range of clinical settings. Negative consequences of these effects on global health make it of paramount importance to understand the mechanisms driving thymic involution and homeostatic processes across the lifespan. There is growing evidence that thymus tissue is plastic and that the involution process might be therapeutically halted or reversed. We present here progress on the exploitation of thymosuppressive and thymostimulatory pathways using factors such as keratinocyte growth factor, interleukin 7 or sex steroid ablation for therapeutic thymus restoration and peripheral immune reconstitution in adults.

The contribution of thymic stromal abnormalities to autoimmune disease.

In essence, normal thymus function involves the production of a broad repertoire of αßT cells capable of responding to foreign antigens with low risk of autoreactivity. Thymic epithelial cells are an essential component of the thymic stromal microenvironment, promoting the growth and export of self-tolerant thymocytes. Autoimmune disease, resulting from a loss of self-tolerance, is clinically and genetically complex, and accordingly has many potential etiological origins. However, it is commonly linked to defects in the thymic epithelial microenvironment. The study of autoimmune-linked thymic stromal dysfunction has indisputably advanced our understanding of T cell tolerance; notably, a field-wide paradigm shift occurred when autoimmune regulator (Aire) was found to drive expression of a multitude of peripheral tissue-restricted antigens in medullary thymic epithelial cells. Many other associations with polygenically controlled autoimmune diseases have been reported but are more difficult to definitively dissect. Paradoxically, immunodeficiency and age-related immunosenescence are also linked with increased autoimmunity. Here we discuss the theoretical basis and the evidence gathered thus far to support these associations.

Sex steroid ablation enhances immune reconstitution following cytotoxic antineoplastic therapy in young mice.

Cytotoxic antineoplastic therapy is used to treat malignant disease but results in long-term immunosuppression in postpubertal and adult individuals, leading to increased incidence and severity of opportunistic infections. We have previously shown that sex steroid ablation (SSA) reverses immunodeficiencies associated with age and hematopoietic stem cell transplantation in both autologous and allogeneic settings. In this study, we have assessed the effects of SSA by surgical castration on T cell recovery of young male mice following cyclophosphamide treatment as a model for the impact of chemotherapy. SSA increased thymic cellularity, involving all of the thymocyte subsets and early T lineage progenitors. It also induced early repair of damage to the thymic stromal microenvironment, which is crucial to the recovery of a fully functional T cell-based immune system. These functional changes in thymic stromal subsets included enhanced production of growth factors and chemokines important for thymopoiesis, which preceded increases in both thymocyte and stromal cellularity. These effects collectively translated to an increase in peripheral and splenic naive T cells. In conclusion, SSA enhances T cell recovery following cyclophosphamide treatment of mice, at the level of the thymocytes and their stromal niches. This provides a new approach to immune reconstitution following antineoplastic therapy.

Characterization of the thymic IL-7 niche in vivo.

The thymus represents the "cradle" for T cell development, with thymic stroma providing multiple soluble and membrane cues to developing thymocytes. Although IL-7 is recognized as an essential factor for thymopoiesis, the "environmental niche" of thymic IL-7 activity remains poorly characterized in vivo. Using bacterial artificial chromosome transgenic mice in which YFP is under control of IL-7 promoter, we identify a subset of thymic epithelial cells (TECs) that co-express YFP and high levels of Il7 transcripts (IL-7(hi) cells). IL-7(hi) TECs arise during early fetal development, persist throughout life, and co-express homeostatic chemokines (Ccl19, Ccl25, Cxcl12) and cytokines (Il15) that are critical for normal thymopoiesis. In the adult thymus, IL-7(hi) cells localize to the cortico-medullary junction and display traits of both cortical and medullary TECs. Interestingly, the frequency of IL-7(hi) cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining IL-7 levels. Our temporal-spatial analysis of IL-7-producing cells in the thymus in vivo suggests that thymic IL-7 levels are dynamically regulated under distinct physiological conditions. This IL-7 reporter mouse provides a valuable tool to further dissect the mechanisms that govern thymic IL-7 expression in vivo.

Androgen depletion increases the efficacy of bone marrow transplantation in ameliorating experimental autoimmune encephalomyelitis.

Bone marrow transplantation (BMT) potentially represents a novel therapy for the amelioration and even cure for multiple sclerosis (MS). It has important advantages over immunosuppressive drug treatments because, while effecting broad-based ablation of the immune system and autoreactive cells, it provides an important means for overcoming the resultant immunodeficiency, while possibly restoring self-tolerance. However, both of these benefits are predicated on a functional thymus that undergoes profound age-induced atrophy from puberty. Reversal of thymic atrophy has been achieved by several procedures, including removal of sex steroids by surgical or chemical (LHRH agonist) castration. Using a murine model of MS, experimental autoimmune encephalomyelitis (EAE), we combined BMT with androgen depletion to induce immune regeneration, and investigated the kinetics of increased thymic function on immune reconstitution and disease reduction. We show that androgen depletion significantly increased the efficacy of BMT to ameliorate the clinical signs of EAE while concurrently restoring the periphery with increased naive and regulatory lymphocytic populations. Upon rechallenge, mice with a regenerated thymus had a slower onset of clinical symptoms compared with mice undergoing BMT only. These results suggest that thymic regeneration strategies may be used as a complement to conventional BMT protocols for the treatment of MS.

VEGF-mediated cross-talk within the neonatal murine thymus.

Although the mechanisms of cross-talk that regulate the hematopoietic and epithelial compartments of the thymus are well established, the interactions of these compartments with the thymic endothelium have been largely ignored. Current understanding of the thymic vasculature is based on studies of adult thymus. We show that the neonatal period represents a unique phase of thymic growth and differentiation, marked by endothelium that is organized as primitive, dense networks of capillaries dependent on vascular endothelial growth factor (VEGF). VEGF dependence in neonates is mediated by significantly higher levels of both VEGF production and endothelial VEGF receptor 2 (VEGF-R2) expression than in the adult thymus. VEGF is expressed locally in the neonatal thymus by immature, CD4(-)CD8(-) "double negative" (DN) thymocytes and thymic epithelium. Relative to adult thymus, the neonatal thymus has greater thymocyte proliferation, and a predominance of immature thymocytes and cortical thymic epithelial cells (cTECs). Inhibition of VEGF signaling during the neonatal period results in rapid loss of the dense capillaries in the thymus and a marked reduction in the number of thymocytes. These data demonstrate that, during the early postnatal period, VEGF mediates cross-talk between the thymocyte and endothelial compartments of the thymus.

Thymic involution: where endocrinology meets immunology.

The decline in immune function with aging represents a major clinical challenge in many disease conditions. It is manifest in many parameters but is essentially linked to the adaptive immune responses. The prediction would be that abnormalities in both T and B lymphocytes underlie the loss of cellular and humoral capacity, respectively. Somewhat surprisingly, this is not reflected in numerical losses but more in alterations at the population and single cell levels. There is a major reduction in naïve T cells with a proportional increase in memory cells, and also a generally reduced function of these cells. While bone marrow function reduces with age, the most obvious reason for the T cell defects is the severe atrophy of the thymus. This is closely aligned with puberty, thereby implicating a major aetiological role for sex steroids in both thymus and immune system deterioration with age. Accordingly surgical or chemical castration (utilizing luteinizing hormone-releasing hormone) blocks sex steroids resulting in profound rejuvenation of the immune system.

Withdrawal of sex steroids reverses age- and chemotherapy-related defects in bone marrow lymphopoiesis.

A significant decline in immune function is characteristic of aging. Along with the involution of the thymus and associated impaired architecture, which contributes to profound loss of naive T cell production, there are also significant declines in B cell development and the progenitors that support lymphopoiesis. These collectively lead to a reduced peripheral immune repertoire, increase in opportunistic infections, and limited recovery following cytoablation through chemo- or radiotherapy. We have previously shown that sex steroid ablation (SSA) causes a major reversal of age-related thymic atrophy and improves recovery from hematopoietic stem cell transplant. This study focused on the impact of SSA on the B cell compartment and their progenitors in middle-aged and cyclophosphamide-treated mice. In both models, SSA enhanced the number of lymphoid progenitors and developing B cells in the bone marrow (BM) as well as reversing age-related defects in the cycling kinetics of these cells. Enhanced BM lymphopoiesis was reflected in the periphery by an increase in recent BM emigrants as well as immature and mature plasma cells, leading to an enhanced humoral response to challenge by hepatitis B vaccine. In conclusion, SSA improves lymphoid progenitor and B cell recovery from age- and chemotherapy-induced immunodepletion, complimenting the effects on T cells. Since SSA has been achieved clinically for over 25 years, this provides a novel, rational basis for approaching the need for immune recovery in many clinical conditions.

Sex steroid ablation enhances hematopoietic recovery following cytotoxic antineoplastic therapy in aged mice.

Cytotoxic antineoplastic therapy is widely used in the clinic as a treatment for malignant diseases. The treatment itself, however, leads to long-term depletion of the adaptive immune system, which is more pronounced in older patients, predominantly due to thymic atrophy. We and others have previously shown that withdrawal of sex steroids is able to regenerate the aged thymus and enhance recovery from autologous and allogeneic hematopoietic stem cell transplant. In this study we have examined the effects of sex steroid ablation (SSA) on the recovery of lymphopoiesis in the bone marrow (BM) and thymus following treatment with the chemotherapeutic agent cyclophosphamide (Cy) in middle-aged and old mice. Furthermore, we have also examined the impact of this regeneration on peripheral immunity. SSA enhanced the recovery of BM resident hematopoietic stem cells and lymphoid progenitors and promoted lymphopoiesis. Interestingly, Cy alone caused a profound increase in the recently described common lymphoid progenitor 2 (CLP-2) population in the BM. In the thymus, SSA caused a profound increase in cellularity as well as all intrathymic T-lineage progenitors including early T-lineage progenitors (ETPs) and non-canonical T cell progenitors such as the CLP-2. We also found that these transferred into numerical increases in the periphery with enhanced B and T cell numbers. Furthermore, these lymphocytes were found to have an enhanced functional capacity with no perturbation of the TCR repertoire. Taken together, these results provide the basis for the use of SSA in the clinic to enhance treatment outcomes from cytotoxic antineoplastic therapy.

Ablation and regeneration of tolerance-inducing medullary thymic epithelial cells after cyclosporine, cyclophosphamide, and dexamethasone treatment.

Immunosuppressive drugs and cytotoxic chemotherapy agents are designed to kill or suppress autoreactive, alloaggressive, or hyperinflammatory T cells, or disseminated malignancies. However, they also cause severe immunological side effects ranging from interrupted thymopoiesis and general immunodeficiency to, paradoxically, autoimmunity. Consistent with the cross-talk between thymocytes and stromal cells, we now show that these common therapeutic agents have major effects on murine thymic epithelial cells (TEC), crucially required to rebuild immunity posttreatment. We show that the immunosuppressant cyclosporine A, which has been linked to a thymus-dependent autoimmune syndrome in some patients, causes extensive loss of autoimmune regulator (Aire(+)) tolerance-inducing MHC class II(high) medullary TEC (mTEC(high)). Post-cyclosporine A, Aire expression was restored within 7 days. Full recovery of the mTEC(high) subset occurred within 10 days and was linked to a decrease in a relatively resistant MHC class II(low) mTEC subset (mTEC(low)), consistent with a previously described precursor-product relationship. Cyclophosphamide and dexamethasone caused more extensive ablation of thymocytes and stromal cells but again severely depleted tolerance-inducing mTEC(high). Together, these data show that Aire(+) mTECs are highly sensitive to damage and that mTEC regeneration follows a conserved pattern regardless of the treatment regimen used.

Luteinizing hormone-releasing hormone enhances T cell recovery following allogeneic bone marrow transplantation.

Posttransplant immunodeficiency, specifically a lack of T cell reconstitution, is a major complication of allogeneic bone marrow transplantation. This immunosuppression results in an increase in morbidity and mortality from infections and very likely contributes to relapse. In this study, we demonstrate that sex steroid ablation using leuprolide acetate, a luteinizing hormone-releasing hormone agonist (LHRHa), increases the number of lymphoid and myeloid progenitor cells in the bone marrow and developing thymocytes in the thymus. Although few differences are observed in the peripheral myeloid compartments, the enhanced thymic reconstitution following LHRHa treatment and allogeneic bone marrow transplantation leads to enhanced peripheral T cell recovery, predominantly in the naive T cell compartment. This results in an increase in T cell function in vivo and in vitro. Graft-versus-host-disease is not exacerbated by LHRHa treatment and graft-versus-tumor activity is maintained. Because LHRHa allows for reversible (and temporary) sex steroid ablation, has a strong safety profile, and has been clinically approved for diseases such as prostate and breast cancer, this drug treatment represents a novel therapeutic approach to reversal of thymic atrophy and enhancement of immunity following immunosuppression.

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.

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.