Effect of age on T cell differentiation.

A brief overview of the area of T cell aging is presented by first discussing the age-related changes in T cell activities, and then by focusing attention on the possible mechanisms that may be responsible for the decline. Present evidence indicates that thymic involution precedes and therefore may be responsible for the age-dependent decline in the ability of the immune system to generate functional T cells. At this time, it appears that the primary effect of thymic involution is on a T cell differentiation pathway affecting the more mature T cells first with time, and then the less mature T cells. Thus, the thymus may be the aging clock for the immune system. Further studies should be centered around processes regulating growth and atrophy of the thymus.     

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.     

The role of sex steroids and gonadectomy in the control of thymic involution

A major underlying cause for aging of the immune system is the structural and functional atrophy of the thymus, and associated decline in T cell genesis. This loss of na?ve T cells reduces adaptive immunity to new stimuli and precipitates a peripheral bias to memory cells against prior antigens. Whilst multiple mechanisms may contribute to this process, the temporal alliance of thymic decline with puberty has implicated a causative role for sex steroids. Accordingly ablation of sex steroids induces profound thymic rejuvenation. Although the thymus retains some, albeit highly limited, function in healthy adults, this is insufficient for resurrecting the T cell pool following cytoablative treatments such as chemo- and radiation-therapy and AIDS. Increased risk of opportunistic infections and cancer relapse or appearance, are a direct consequence. Temporary sex steroid ablation may thus provide a clinically effective means to regenerate the thymus and immune system in immunodeficiency states.     

Declining expression of a single epithelial cell‐autonomous gene accelerates age‐related thymic involution

Age‐related thymic involution may be triggered by gene expression changes in lymphohematopoietic and/or nonhematopoietic thymic epithelial cells (TECs). The role of epithelial cell‐autonomous gene FoxN1 may be involved in the process, but it is still a puzzle because of the shortage of evidence from gradual loss‐of‐function and exogenous gain‐of‐function studies. Using our recently generated loxP‐floxed‐FoxN1(fx) mouse carrying the ubiquitous CreER^T (uCreER^T) transgene with a low dose of spontaneous activation, which causes gradual FoxN1 deletion with age, we found that the uCreER^T‐fx/fx mice showed an accelerated age‐related thymic involution owing to progressive loss of FoxN1⁺ TECs. The thymic aging phenotypes were clearly observable as early as at 3–6 months of age, resembling the naturally aged (18–22‐month‐old) murine thymus. By intrathymically supplying aged wild‐type mice with exogenous FoxN1‐cDNA, thymic involution and defective peripheral CD4⁺ T‐cell function could be partially rescued. The results support the notion that decline of a single epithelial cell‐autonomous gene FoxN1 levels with age causes primary deterioration in TECs followed by impairment of the total postnatal thymic microenvironment, and potentially triggers age‐related thymic involution in mice.     

Development of functional thymic epithelial cells occurs independently of lymphostromal interactions

The thymus provides a specialised microenvironment for the development of T-cell precursors. This developmental programme depends upon interactions with stromal cells such as thymic epithelial cells, which provide signals for proliferation, survival and differentiation. In turn, it has been proposed that development of thymic epithelial cells themselves is regulated by signals produced by developing thymocytes. Evidence in support of this symbiotic relationship, termed thymic crosstalk, comes from studies analysing the thymus of adult mice harbouring blocks at specific stages of thymocyte development, where it is difficult to separate mechanisms regulating the initial development of thymic epithelial cells from those regulating their maintenance. To distinguish between these processes, we have analysed the initial developmental programme of thymic epithelial cells within the embryonic thymus, in either the presence or absence of normal T-cell development. We show that keratin 5+8+ precursor epithelial cells present in the early thymic rudiment differentiate into discrete cortical and medullary epithelial subsets displaying normal gene expression profiles, and acquire functional competence, independently of signals from T-cell precursors. Thus, our findings redefine current models of thymus development and argue against a role for thymocyte-epithelial cell crosstalk in the development of thymic epithelial progenitors.     

Interferons mediate terminal differentiation of human cortical thymic epithelial cells

In the thymus, epithelial cells comprise a heterogeneous population required for the generation of functional T lymphocytes, suggesting that thymic epithelium disruption by viruses may compromise T-cell lymphopoiesis in this organ. In a previous report, we demonstrated that in vitro, measles virus induced differentiation of cortical thymic epithelial cells as characterized by (i) cell growth arrest, (ii) morphological and phenotypic changes, and (iii) apoptotis as a final step of this process. In the present report, we have analyzed the mechanisms involved. First, measles virus-induced differentiation of thymic epithelial cells is shown to be strictly dependent on beta interferon (IFN-beta) secretion. In addition, transfection with double-stranded RNA, a common intermediate of replication for a broad spectrum of viruses, is reported to similarly mediate thymic epithelial cell differentiation through IFN-beta induction. Finally, we demonstrated that recombinant IFN-alpha, IFN-beta, or IFN-gamma was sufficient to induce differentiation and apoptosis of uninfected thymic epithelial cells. These observations suggested that interferon secretion by either infected cells or activated leukocytes, such as plasmacytoid dendritic cells or lymphocytes, may induce thymic epithelium disruption in a pathological context. Thus, we have identified a new mechanism that may contribute to thymic atrophy and altered T-cell lymphopoiesis associated with many infections.     

Resveratrol ameliorates thymus senescence changes in D-galactose induced mice

The thymic microenvironment plays an important role in the development of T cells. A decrease of thymic epithelial cells is the main cause of age-related thymic atrophy or degeneration. Resveratrol (RSV), a phytoalexin produced from plants, has been shown to inhibit the adverse effects of dietary obesity on the structure and function of the thymus. D-Galactose (D-gal) can induce accelerated aging in mice. In the present study, young mice (2 months old) were injected with D-gal (120 mg/kg/day) for 8 consecutive weeks to construct an accelerated aging model. Compared with normal control mice, the thymus epithelium of the D-gal treated mice had structural changes, the number of senescent cells increased, the number of CD4+ T cells decreased, and CD8+ T cells increased. After RSV administration by gavage for 6 weeks, it was found that RSV improved the surface phenotypes of D-gal treated mice, and recovered thymus function by maintaining the ratio of CD4+ to CD8+ cells. It also indicated that RSV enhanced the cell proliferation and inhibited cell senescence. Increased autoimmune regulator (Aire) expression was present in the RSV treated mice. The lymphotoxin-beta receptor (LTβR) expression also increased. These findings suggested that RSV intake could restore the alterations caused by D-gal treatment in the thymus via stimulation of Aire expression.     

Implantation of cultured thymic fragments in congenitally athymic (nude) rats

Summary Cultured thymic fragments correspond to the thymic microenvironment depleted of lymphocytes and dendritic cells. When these fragments are implanted under the kidney capsule of congenitally athymic rats, lymphocytes and dendritic cells of host origin enter the graft and induce thymus-dependent immunity in the recipient. This paper describes the ultrastructure of the fragments and the changes that occur during the restoration of normal thymic architecture. At the end of the culture period of 6–9 days and in the early stages after implantation, the grafts consist of keratin-containing epithelial cells of unusual morphology that can be labelled with antibodies raised against the epithelium of the mid/deep cortex and the subcapsule/medulla. Normal thymic architecture develops, including nerves and blood vessels, as lymphocytes populate the environment, and by 4–6 weeks the epithelial cells are the same phenotypically and ultrastructurally as those found in normal rat thymus. However, some areas without lymphocytes still contain the atypical epithelial cells seen before implantation. Large multinucleated giant cells are also present with a few associated epithelial cells of subcapsular/medullary phenotype. In conclusion, the cultured thymic fragments contain a hitherto unknown precursor epithelial cell with an atypical ultrastructure and phenotype that is not seen in normal development.     

Central tolerance is impaired in the middle‐aged thymic environment

One of the earliest hallmarks of immune aging is thymus involution, which not only reduces the number of newly generated and exported T cells, but also alters the composition and organization of the thymus microenvironment. Thymic T‐cell export continues into adulthood, yet the impact of thymus involution on the quality of newly generated T‐cell clones is not well established. Notably, the number and proportion of medullary thymic epithelial cells (mTECs) and expression of tissue‐restricted antigens (TRAs) decline with age, suggesting the involuting thymus may not promote efficient central tolerance. Here, we demonstrate that the middle‐aged thymic environment does not support rapid motility of medullary thymocytes, potentially diminishing their ability to scan antigen presenting cells (APCs) that display the diverse self‐antigens that induce central tolerance. Consistent with this possibility, thymic slice assays reveal that the middle‐aged thymic environment does not support efficient negative selection or regulatory T‐cell (Treg) induction of thymocytes responsive to either TRAs or ubiquitous self‐antigens. This decline in central tolerance is not universal, but instead impacts lower‐avidity self‐antigens that are either less abundant or bind to TCRs with moderate affinities. Additionally, the decline in thymic tolerance by middle age is accompanied by both a reduction in mTECs and hematopoietic APC subsets that cooperate to drive central tolerance. Thus, age‐associated changes in the thymic environment result in impaired central tolerance against moderate‐avidity self‐antigens, potentially resulting in export of increasingly autoreactive naive T cells, with a deficit of Treg counterparts by middle age.     

Establishment and characterization of a thymic medullary epithelial cell clone

Summary Thymic stromal cells were cultured in conditions which select for epithelial cells. These were then transformed in vitro by contact with N-methyl-N′-nitro-N-nitrosoguanidine and cloned at limit dilution. One of the clones was characterized as being of medullary origin on the basis of its reactivity with a battery of antibodies previously shown to distinguish cortical from medullary thymic epithelial cells. The importance of this clone lies in the potential it offers to delineate how various T cell subpopulations acquire their distinct markers and function within the thymus. This work was supported by the Medical Research Council of Canada and the National Cancer Institute of Canada Editor's Statement Thymocyte-thymic epithelial cell interaction and its effect on T cell maturation has been postulated by a number of people. The studies on specific homing proteins and adherence molecules provide improtant information on the maturation and migration of T lymphocytes. This article provides clean and convincing evidence for the role of such specific glycoprotein TMF.     

The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner

Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^ 8^ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.     

Thymic epithelial progenitor cells and thymus regeneration： an update

The thymus provides the essential microenvironment for T-cell development and maturation. Thymic epithelial cells （TECs）, which are composed of thymic cortical epithelial cells （cTECs） and thymic medullary epithelial cells （mTECs）, have been well documented to be critical for these tightly regulated processes. It has long been controversial whether the common progenitor cells of TECs could give rise to both cTECs and mTECs. Great progress has been made to characterize the common TEC progenitor cells in recent years. We herein discuss the sole origin paradigm with regard to TEC differentiation as well as these progenitor cells in thymus regeneration.     

Lymphohematopoietic progenitors do not have a synchronized defect with age-related thymic involution

It has been speculated that aging lymphohematopoietic progenitor cells (LPC) including hematopoietic stem cells (HSC) and early T-cell progenitors (ETP) have intrinsic defects that trigger age-related thymic involution. However, using a different approach, we suggest that that is not the case. We provided a young thymic microenvironment to aged mice by transplanting a fetal thymus into the kidney capsule of aged animals, and demonstrated that old mouse-derived LPCs could re-establish normal thymic lymphopoiesis and all thymocyte subpopulations, including ETPs, double negative subsets, double positive, and CD4(+) and CD8(+) single positive T cells. LPCs derived from aged mice could turn over young RAG(-/-) thymic architecture by interactions, as well as elevate percentage of peripheral CD4(+)IL-2(+) T cells in response to costimulator in aged mice. Conversely, intrathymic injection of ETPs sorted from young animals into old mice did not restore normal thymic lymphopoiesis, implying that a shortage and/or defect of ETPs in aged thymus do not account for age-related thymic involution. Together, our findings suggest that the underlying cause of age-related thymic involution results primarily from changes in the thymic microenvironment, causing extrinsic, rather than intrinsic, defects in T-lymphocyte progenitors.     

Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Infection Induced Apoptosis and Autophagy in Thymi of Infected Piglets

Previously, we demonstrated that the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) HuN4 strain causes obvious thymic atrophy and thymocytes apoptosis in infected piglets after birth, which is more severe than that induced by classical PRRSV. In this study, we investigated apoptosis and autophagy in the thymus of piglets infected with the HP-PRRSV HuN4 strain, and found that both apoptosis and autophagy occurred in the thymus of piglets infected with HP-PRRSV. In addition to a few virus-infected cells, CD14⁺ cells, the main autophagic cells in the thymus were thymic epithelial cells. These findings demonstrated that HP-PRRSV induces apoptosis in bystander cells, and induces autophagy in both infected and bystander cells in the thymus of infected piglets. Herein, we first present new data on the thymic lesions induced by HP-PRRSV, and show that apoptosis and autophagy are key mechanisms involved in cell survival and determinants of the severity of thymic atrophy in infected piglets. Finally, future studies of the mechanism underlying immune responses are proposed based on our current understanding of PRRSV-host interactions.     

An epithelial progenitor pool regulates thymus growth

Thymic epithelium provides an essential cellular substrate for T cell development and selection. Gradual age-associated thymic atrophy leads to a reduction in functional thymic tissue and a decline in de novo T cell generation. Development of strategies tailored toward regeneration of thymic tissue provides an important possibility to improve immune function in elderly individuals and increase the capacity for immune recovery in patients having undergone bone marrow transfer following immunoablative therapies. In this study we show that restriction of the size of the functional thymic epithelial progenitor pool affects the number of mature thymic epithelial cells. Using an embryo fusion chimera-based approach, we demonstrate a reduction in the total number of both embryonic and adult thymic epithelium, which relates to the initial size of the progenitor cell pool. The inability of thymic epithelial progenitor cells to undergo sufficient compensatory proliferation to rescue the deficit in progenitor numbers suggests that in addition to extrinsic regulation of thymus growth by provision of growth factors, intrinsic factors such as a proliferative restriction of thymic epithelial progenitors and availability of progenitor cell niches may limit thymic epithelial recovery. Collectively, our data demonstrate an important level of regulation of thymic growth and recovery at the thymic epithelial progenitor level, providing an important consideration for developing methods targeted toward inducing thymic regeneration.     

Reevaluation of T cell receptor excision circles as a measure of human recent thymic emigrants

The human thymus exports newly generated T cells to the periphery. As no markers have been identified for these recent thymic emigrants (RTE), it is presently impossible to measure human thymic output. T cell receptor excision circles (TREC) have been recently used to assess thymic output during both health and disease. Using a mathematical model, we quantify age-dependent changes both in the number of RTE generated per day and in TREC concentration during an 80-year lifespan. Through analyses, we demonstrate that RTE and peripheral T cell division have the same potential to affect TREC concentration at any age in healthy people. T cell death also influences TREC concentration, but to a lesser extent. During aging, our results indicate that thymic involution primarily induces an age-dependent decline in TREC concentrations within both CD4(+) and CD8(+) T cell populations. We further apply this model for studying TREC concentration during HIV-1 infection. Our analyses reveal that a decrease in thymic output is the major contributor to the decline in TREC concentration within CD4(+) T cells, whereas both increased peripheral T cell division and decreased thymic output induce the decline in TREC concentration within CD8(+) T cells. Therefore, we suggest that T cell turnover should be examined together with TREC concentration as a measure of RTE. If peripheral T cell division remains relatively unchanged, then TREC concentration indeed reflects thymic output.     

The thymus in fish: Development and possible function in the immune response

With the exception of Agnatha, fish possess the functional equivalent of the thymus gland found in higher vertebrates. As in other vertebrates, this gland originates from the pharyngeal pouches and ontogenically is the first lymphoid organ to be infiltrated with lymphoid cells. Histology of the structure may differ from one species to another but the cellular component is basically similar. The (paired) gland is surrounded by an epithelial capsule. Within the gland a framework of reticulo-epithelial cells supports the lymphocytes. The age-related involution process, which characterizes the thymus of higher vertebrates, does not necessarily occur in fish. Nevertheless, thymus growth and function may be modulated by those factors that induce its involution such as aging, season, sexual maturity, and stress. The major role played by the thymus in the immune response of higher vertebrates is presumed to occur in fish. Thymus-derived cell dependent immune reactions have been demonstrated in fish. The cells that mediate these functions are designated as T-like cells. So far, cell surface markers equivalent to those of mammalian T lymphocytes have not been characterized. The T lymphocyte specificities are supposed to be acquired within or via the thymic microenvironment. Unfortunately, there is limited data concerned with the cytological and physiological basis of the maturation of thymus-derived cells. Direct involvement of the fish thymus in defense mechanisms has not been investigated extensively. The gland appears to be weakly protected because of its superficial location and is easily exposed to pathogens. Neoplasia is the main pathologic condition reported in the thymus of fish, with little else having been published regarding thymic pathology.