Redox status regulates autophagy in thymic stromal cells and promotes T cell tolerance.

Thymic stromal cells (TSCs) are critical regulators of T cell tolerance, but their basic biology has remained under-characterized because they are relatively rare and difficult to isolate. Recent work has revealed that constitutive autophagy in TSCs is required for self-antigen presentation and central T cell tolerance induction; however, the mechanisms regulating constitutive autophagy in TSCs are not well understood. Hydrogen peroxide has been shown to increase autophagy flux in other tissues, and we previously identified conspicuously low expression of the hydrogen peroxide-quenching enzyme catalase in TSCs. We investigated whether the redox status of TSCs established by low catalase expression regulates their basal autophagy levels and their capacity to impose central T cell tolerance. Transgenic overexpression of catalase diminished autophagy in TSCs and impaired thymocyte clonal deletion, concomitant with increased frequencies of spontaneous lymphocytic infiltrates in lung and liver and of serum antinuclear antigen reactivity. Effects on clonal deletion and autoimmune indicators were diminished in catalase transgenic mice when autophagy was rescued by expression of the Becn1F121A/F121A knock-in allele. These results suggest a metabolic mechanism by which the redox status of TSCs may regulate central T cell tolerance.

Delta-like 4-Derived Notch Signals Differentially Regulate Thymic Generation of Skin-Homing CCR10+NK1.1+ Innate Lymphoid Cells at Neonatal and Adult Stages.

The thymus is a primary lymphoid organ for T cell development. Increasing evidence found that the thymus is also an important site for development of innate lymphoid cells (ILCs). ILCs generated in thymi acquire unique homing properties that direct their localization into barrier tissues such as the skin and intestine, where they help local homeostasis. Mechanisms underlying the developmental programming of unique tissue-homing properties of ILCs are poorly understood. We report in this article that thymic stroma-derived Notch signaling is differentially involved in thymic generation of a population of NK1.1+ group 1 ILCs (ILC1s) with the CCR10+ skin-homing property in adult and neonatal mice. We found that thymic generation of CCR10+NK1.1+ ILC1s is increased in T cell-deficient mice at adult, but not neonatal, stages, supporting the notion that a large number of developing T cells interfere with signals required for generation of CCR10+NK1.1+ ILC1s. In an in vitro differentiation assay, increasing Notch signals promotes generation of CCR10+NK1.1+ ILC1s from hematopoietic progenitors. Knockout of the Notch ligand Delta-like 4 in thymic stroma impairs generation of CCR10+NK1.1+ ILC1s in adult thymi, but development of CCR10+NK1.1+ ILC1s in neonatal thymi is less dependent on Delta-like 4-derived Notch signals. Mechanistically, the Notch signaling is required for proper expression of the IL-7R CD127 on thymic NK1.1+ ILC1s, and deficiency of CD127 also impairs thymic generation of CCR10+NK1.1+ ILC1s at adult, but not perinatal, stages. Our findings advanced understanding of regulatory mechanisms of thymic innate lymphocyte development.

Redox regulation of age-associated defects in generation and maintenance of T cell self-tolerance and immunity to foreign antigens.

Thymic atrophy reduces naive T cell production and contributes to increased susceptibility to viral infection with age. Expression of tissue-restricted antigen (TRA) genes also declines with age and has been thought to increase autoimmune disease susceptibility. We find that diminished expression of a model TRA gene in aged thymic stromal cells correlates with impaired clonal deletion of cognate T cells recognizing an autoantigen involved in atherosclerosis. Clonal deletion in the polyclonal thymocyte population is also perturbed. Distinct age-associated defects in the generation of antigen-specific T cells include a conspicuous decline in generation of T cells recognizing an immunodominant influenza epitope. Increased catalase activity delays thymic atrophy, and here, we show that it mitigates declining production of influenza-specific T cells and their frequency in lung after infection, but does not reverse declines in TRA expression or efficient negative selection. These results reveal important considerations for strategies to restore thymic function.

Metabolic Regulation of Thymic Epithelial Cell Function.

The thymus is the primary site of T lymphocyte development, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells directs the progenitors along a well-characterized program of differentiation. Although thymic stromal cells, including thymic epithelial cells (TECs) are critical for the development of T cell-mediated immunity, many aspects of their basic biology have been difficult to resolve because they represent a small fraction of thymus cellularity, and because their isolation requires enzymatic digestion that induces broad physiological changes. These obstacles are especially relevant to the study of metabolic regulation of cell function, since isolation procedures necessarily disrupt metabolic homeostasis. In contrast to the well-characterized relationships between metabolism and intracellular signaling in T cell function during an immune response, metabolic regulation of thymic stromal cell function represents an emerging area of study. Here, we review recent advances in three distinct, but interconnected areas: regulation of mTOR signaling, reactive oxygen species (ROS), and autophagy, with respect to their roles in the establishment and maintenance of the thymic stromal microenvironment.

Dynamic changes in epithelial cell morphology control thymic organ size during atrophy and regeneration.

T lymphocytes must be produced throughout life, yet the thymus, where T lymphocytes are made, exhibits accelerated atrophy with age. Even in advanced atrophy, however, the thymus remains plastic, and can be regenerated by appropriate stimuli. Logically, thymic atrophy is thought to reflect senescent cell death, while regeneration requires proliferation of stem or progenitor cells, although evidence is scarce. Here we use conditional reporters to show that accelerated thymic atrophy reflects contraction of complex cell projections unique to cortical epithelial cells, while regeneration requires their regrowth. Both atrophy and regeneration are independent of changes in epithelial cell number, suggesting that the size of the thymus is regulated primarily by rate-limiting morphological changes in cortical stroma, rather than by their cell death or proliferation. Our data also suggest that cortical epithelial morphology is under the control of medullary stromal signals, revealing a previously unrecognized endocrine-paracrine signaling axis in the thymus.

Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens.

Although autoimmune disorders are a significant source of morbidity and mortality in older individuals, the mechanisms governing age-associated increases in susceptibility remain incompletely understood. Central T cell tolerance is mediated through presentation of self-antigens by cells constituting the thymic microenvironment, including epithelial cells, dendritic cells, and B cells. Medullary thymic epithelial cells (mTECs) and B cells express distinct cohorts of self-antigens, including tissue-restricted self-antigens (TRAs), such that developing T cells are tolerized to antigens from peripheral tissues. We find that expression of the TRA transcriptional regulator Aire, as well as Aire-dependent genes, declines with age in thymic B cells in mice and humans and that cell-intrinsic and cell-extrinsic mechanisms contribute to the diminished capacity of peripheral B cells to express Aire within the thymus. Our findings indicate that aging may diminish the ability of thymic B cells to tolerize T cells, revealing a potential mechanistic link between aging and autoimmunity.

Thymic stromal cells: Roles in atrophy and age-associated dysfunction of the thymus.

Atrophy of the thymus, the primary site of T lymphocyte generation, is a hallmark of the aging immune system. Age-associated thymic atrophy results in diminished output of new, naïve T cells, with immune sequelae that include diminished responses to novel pathogenic challenge and vaccines, as well as diminished tumor surveillance. Although a variety of stimuli are known to regulate transient thymic atrophy, mechanisms governing progressive age-associated atrophy have been difficult to resolve. This has been due in part to the fact that one of the primary targets of age-associated thymic atrophy is a relatively rare population, thymic stromal cells. This review focuses on changes in thymic stromal cells during aging and on the contributions of periodic, stochastic, and progressive causes of thymic atrophy.

Metabolic Damage and Premature Thymus Aging Caused by Stromal Catalase Deficiency.

T lymphocytes are essential mediators of immunity that are produced by the thymus in proportion to its size. The thymus atrophies rapidly with age, resulting in progressive diminution of new T cell production. This decreased output is compensated by duplication of existing T cells, but it results in gradual dominance by memory T cells and decreased ability to respond to new pathogens or vaccines. Here, we show that accelerated and irreversible thymic atrophy results from stromal deficiency in the reducing enzyme catalase, leading to increased damage by hydrogen peroxide generated by aerobic metabolism. Genetic complementation of catalase in stromal cells diminished atrophy, as did chemical antioxidants, thus providing a mechanistic link between antioxidants, metabolism, and normal immune function. We propose that irreversible thymic atrophy represents a conventional aging process that is accelerated by stromal catalase deficiency in the context of an intensely anabolic (lymphoid) environment.

Cell-autonomous defects in thymic epithelial cells disrupt endothelial-perivascular cell interactions in the mouse thymus.

The thymus is composed of multiple stromal elements comprising specialized stromal microenvironments responsible for the development of self-tolerant and self-restricted T cells. Here, we investigated the ontogeny and maturation of the thymic vasculature. We show that endothelial cells initially enter the thymus at E13.5, with PDGFR-ß(+) mesenchymal cells following at E14.5. Using an allelic series of the thymic epithelial cell (TEC) specific transcription factor Foxn1, we showed that these events are delayed by 1-2 days in Foxn1 (Δ/Δ) mice, and this phenotype was exacerbated with reduced Foxn1 dosage. At subsequent stages there were fewer capillaries, leaky blood vessels, disrupted endothelium - perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization. The expression of both VEGF-A and PDGF-B, which are both primarily expressed in vasculature-associated mesenchyme or endothelium in the thymus, were reduced at E13.5 and E15.5 in Foxn1 (Δ/Δ) mice compared with controls. These data suggest that Foxn1 is required in TECs both to recruit endothelial cells and for endothelial cells to communicate with thymic mesenchyme, and for the differentiation of vascular-associated mesenchymal cells. These data show that Foxn1 function in TECs is required for normal thymus size and to generate the cellular and molecular environment needed for normal thymic vascularization. These data further demonstrate a novel TEC-mesenchyme-endothelial interaction required for proper fetal thymus organogenesis.

Persistent degenerative changes in thymic organ function revealed by an inducible model of organ regrowth.

The thymus is the most rapidly aging tissue in the body, with progressive atrophy beginning as early as birth and not later than adolescence. Latent regenerative potential exists in the atrophic thymus, because certain stimuli can induce quantitative regrowth, but qualitative function of T lymphocytes produced by the regenerated organ has not been fully assessed. Using a genome-wide computational approach, we show that accelerated thymic aging is primarily a function of stromal cells, and that while overall cellularity of the thymus can be restored, many other aspects of thymic function cannot. Medullary islet complexity and tissue-restricted antigen expression decrease with age, representing potential mechanisms for age-related increases in autoimmune disease, but neither of these is restored by induced regrowth, suggesting that new T cells produced by the regrown thymus will probably include more autoreactive cells. Global analysis of stromal gene expression profiles implicates widespread changes in Wnt signaling as the most significant hallmark of degeneration, changes that once again persist even at peak regrowth. Consistent with the permanent nature of age-related molecular changes in stromal cells, induced thymic regrowth is not durable, with the regrown organ returning to an atrophic state within 2 weeks of reaching peak size. Our findings indicate that while quantitative regrowth of the thymus is achievable, the changes associated with aging persist, including potential negative implications for autoimmunity.

Increased thymus- and decreased parathyroid-fated organ domains in Splotch mutant embryos.

Embryos that are homozygous for Splotch, a null allele of Pax3, have a severe neural crest cell (NCC) deficiency that generates a complex phenotype including spina bifida, exencephaly and cardiac outflow tract abnormalities. Contrary to the widely held perception that thymus aplasia or hypoplasia is a characteristic feature of Pax3(Sp/Sp) embryos, we find that thymic rudiments are larger and parathyroid rudiments are smaller in E11.5-12.5 Pax3(Sp/Sp) compared to Pax3(+/+) embryos. The thymus originates from bilateral third pharyngeal pouch primordia containing endodermal progenitors of both thymus and parathyroid glands. Analyses of Foxn1 and Gcm2 expression revealed a dorsal shift in the border between parathyroid- and thymus-fated domains at E11.5, with no change in the overall cellularity or volume of each shared primordium. The border shift increases the allocation of third pouch progenitors to the thymus domain and correspondingly decreases allocation to the parathyroid domain. Initial patterning in the E10.5 pouch was normal suggesting that the observed change in the location of the organ domain interface arises during border refinement between E10.5 and E11.5. Given the well-characterized NCC defects in Splotch mutants, these findings implicate NCCs in regulating patterning of third pouch endoderm into thymus- versus parathyroid-specified domains, and suggest that organ size is determined in part by the number of progenitor cells specified to a given fate.

Spatial mapping of thymic stromal microenvironments reveals unique features influencing T lymphoid differentiation.

Interaction of hematopoietic progenitors with the thymic microenvironment induces them to proliferate, adopt the T lineage fate, and asymmetrically diverge into multiple functional lineages. Progenitors at various developmental stages are stratified within the thymus, implying that the corresponding microenvironments provide distinct sets of signals to progenitors migrating between them. These differences remain largely undefined. Here we used physical and computational approaches to generate a comprehensive spatial map of stromal gene expression in the thymus. Although most stromal regions were characterized by a unique gene expression signature, the central cortex lacked distinctive features. Instead, a key function of this region appears to be the sequestration of unique microenvironments found at the cortical extremities, thus modulating the relative proximity of progenitors moving between them. Our findings compel reexamination of how cell migration, lineage specification, and proliferation are controlled by thymic architecture and provide an in-depth resource for global characterization of this control.