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.     

IL-7 gene therapy in aging restores early thymopoiesis without reversing involution

Thymic involution begins early in life and continues throughout adulthood, resulting in a decreased population of naive T cells in the periphery and a reduced ability to fight off newly encountered infectious diseases. We have previously shown that the first step of thymopoiesis is specifically blocked in aging. This block at the DN1 to DN2 transition and the subsequent loss of thymic output in old age mirrors the changes seen in IL-7-deficient mice, and it is hypothesized that decreased intrathymic IL-7 is involved in age-related thymic involution. To separate the effect of IL-7 on thymic involution from its function as a peripheral lymphocyte growth cofactor, we injected IL-7-secreting stromal cells into the thymi of recipient mice. The increased local concentration of IL-7 maintained the first step of thymopoiesis at a level far higher than was seen in age-matched controls. However, despite this success, there was no decrease in thymic involution or increase in T cell output. The inability of IL-7 to prevent involution led us to the discovery of an additional age-sensitive step in thymopoiesis, proliferation of the DN4 population, which is unaffected by IL-7 expression.     

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.     

Reversible disruption of thymic function by steroid treatment

The effect of steroid treatment on the thymic output of T cells was examined in an avian model. Recent thymic emigrants in chickens transiently express the chicken T cell Ag 1 thymocyte marker, and thymic function can be monitored indirectly by measuring the levels of TCR gene rearrangement excision circles in peripheral T cells. Both parameters were used to show that intensive steroid treatment induces thymic involution and a profound reduction in the supply of naive T cells to the periphery. Conversely, resident T cells in the peripheral lymphocyte pool were relatively spared. Thymopoiesis immediately recovered following cessation of steroid treatment, concurrent with restoration of the thymic output of newly formed T cells. Repopulation of the peripheral T cell pool recapitulated the ontogenetic pattern of gamma delta T cell replenishment before alpha beta T cell reseeding, thereby indicating the complete recovery of thymic function after a course of steroid treatment.     

Impacto de la función tímica en el deterioro inmunológico asociado a la edad

Age-related biological deterioration also includes immune system deterioration and, in consequence, a rise in the incidence and prevalence of infections and cancers, as well as low responses to vaccination strategies. Out of all immune cell subsets, T-lymphocytes seem to be involved in most of the age-related defects. Since T-lymphocytes mature during their passage through the thymus, and the thymus shows an age-related process of atrophy, thymic regression has been proposed as the triggering event of this immune deterioration in elderly people. Historically, it has been accepted that the young thymus sets the T-lymphocyte repertoire during the childhood, whereupon atrophy begins until the elderly thymus is a non-functional evolutionary trace. However, a rising body of knowledge points toward the thymus functioning during adulthood. In the elderly, higher thymic function is associated with a younger immune system, while thymic function failure is associated with all-cause mortality. Therefore, any new strategy focused on the improvement of the elderly quality of life, especially those trying to influence the immune system, should take into account, together with peripheral homeostasis, thymus function as a key element in slowing down age-related decline.     

Thymic generation and regeneration: a new paradigm for establishing clinical tolerance of stem cell-based therapies

Tolerance to tissue-engineering products is a major obstacle hindering the clinical application of this rapidly advancing technology. Manipulation of central tolerance, by establishing thymus chimerism of both donor and host-derived haemopoietic cells (haemopoietic stem cell transplant--HSCT), should purge any T cells reactive to potential donor organ or tissue transplant. A functional thymus, however, is required to induce chimerism and repopulate the peripheral T cell pool, but age-related thymic atrophy and damage caused by ablative conditioning regimes significantly reduce thymic function and increase incident of infection-dependent morbidity and mortality. Thus rejuvenation of the thymus alongside HSCT may potentiate the use of this strategy in the clinic. In addition, the use of thymic epithelial progenitor cell technology may allow growth of ex vivo thymic tissue for use in clinical situations of immunodeficiency as well as in establishing tolerance to tissue/organ products derived from the same source.     

Lymph nodes as barriers to T‐cell rejuvenation in aging mice and nonhuman primates

In youth, thymic involution curtails production of new naïve T cells, placing the onus of T‐cell maintenance upon secondary lymphoid organs (SLO). This peripheral maintenance preserves the size of the T‐cell pool for much of the lifespan, but wanes in the last third of life, leading to a dearth of naïve T cells in blood and SLO, and contributing to suboptimal immune defense. Both keratinocyte growth factor (KGF) and sex steroid ablation (SSA) have been shown to transiently increase the size and cellularity of the old thymus. It is less clear whether this increase can improve protection of old animals from infectious challenge. Here, we directly measured the extent to which thymic rejuvenation benefits the peripheral T‐cell compartment of old mice and nonhuman primates. Following treatment of old animals with either KGF or SSA, we observed robust rejuvenation of thymic size and cellularity, and, in a reporter mouse model, an increase in recent thymic emigrants (RTE) in the blood. However, few RTE were found in the spleen and even fewer in the lymph nodes, and SSA‐treated mice showed no improvement in immune defense against West Nile virus. In parallel, we found increased disorganization and fibrosis in old LN of both mice and nonhuman primates. These results suggest that SLO defects with aging can negate the effects of successful thymic rejuvenation in immune defense.     

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.     

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.     

Architectural changes in the thymus of aging mice

Age-associated thymic involution is one of the most dramatic and ubiquitous changes in the immune system, although the precise mechanisms involved still remain obscured. Several hypotheses have been proposed incorporating extrinsic and intrinsic factors, however, changes in the thymic microenvironment itself is one of the least investigated. We therefore decided to undertake a detailed histological examination of the aging thymus in order to elucidate possible mechanisms of thymic atrophy. This investigation provides insight into the changes within the murine thymus with age, demonstrating a new approach to quantify protein expressional differences while preserving the thymic architecture. There is a decline in expression of thymic epithelial cell-specific makers and an increase in fibroblast content in the aging mouse thymus. This is concurrent with a disorganization of the thymic compartments, a morphological transformation within the epithelial cells and alterations of their archetypal staining patterns. Furthermore, this is linked to a rise in apoptotic cells and the novel finding of increased senescence in the thymus of older mice that appears to be colocalized in the epithelial compartment. These changes within the thymic epithelial cells may be in part accountable for thymic atrophy and responsible for the decline in T-cell output.     

Nerve growth factor stimulates proliferation, adhesion and thymopoietic cytokine expression in mouse thymic epithelial cells in vitro

Thymic epithelial cells, which constitute a major component of the thymic microenvironment, provide a crucial signal for intrathymic T cell development and selection. Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. NGF is increasingly recognized as a potent immunomodulator, promoting “cross-talk” between various types of immune system cells. The present study clearly shows that NGF stimulates mouse thymic epithelial cell activities in vitro including cell proliferation, thymocyte adhesion to thymic epithelial cells, and the expression of cell adhesion molecules such as ICAM-1 and VCAM-1, and thymopoietic factors including IL-7, GM-CSF, SDF-1, TARC and TECK. Thus, our data are of considerable clinical importance showing that trophic NGF activity could be used to enhance the thymus regeneration and develop methods to improve host immunity when the immune function is depressed due to thymic involution.     

Effects of castration on thymocyte development in two different models of thymic involution

Age-associated thymic involution is accompanied by decreased thymic output. This adversely affects general immune competence and T cell recovery following cytoreductive treatments such as chemotherapy. A causal link between increasing sex steroids and age-related thymic atrophy is well established. Although castration has been demonstrated to regenerate the atrophied thymus, little is known about how this is initiated or the kinetics of thymocyte regeneration. The present study shows that although castration impacts globally across thymocyte development in middle-aged mice, the regenerative effects are initiated in the immature triple-negative compartment and early T lineage progenitors (ETP). Specifically, there was a reduction in number of ETP with age, which was restored following castration. There was, however, no change in ETP reconstitution potential in ETP at this age or following castration. Furthermore, in a chemotherapy-induced model of thymic involution, we demonstrate castration enhances intrathymic proliferation and promotes differentiation through the triple-negative program. Clinically, reversible sex steroid ablation is achieved hormonally, and thus presents a means of ameliorating immune inadequacies, for example, following chemotherapy for bone marrow transplantation. By improving our understanding of the kinetics of thymic recovery, this study will allow more appropriate timing of therapy to achieve maximal reconstitution, especially in the elderly.     

Reduction in the developmental potential of intrathymic T cell progenitors with age

Current models of thymic involution propose that intrinsic developmental defects in intrathymic T cell precursors do not contribute to age-related declines in thymopoiesis. This premise was reassessed in a murine model in light of the recent definition of the early T lineage progenitor (ETP), which appears to be the earliest intrathymic precursor defined to date. The results demonstrate that the frequency of ETP declines with age and their potential to reconstitute the thymus is diminished. These findings are consistent with the fact that ETP from aged mice proliferate less and have a higher rate of apoptosis than their counterparts from young animals. Taken together, these data suggest that age-associated changes in T cell precursors should be considered when attempts to rejuvenate the involuted thymus are made.     

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.     

Early thymic T cell development in young transgenic mice overexpressing human Cu/Zn superoxide dismutase, a model of Down syndrome

Previous studies have shown that transgenic mice overexpressing Cu/Zn superoxide dismutase, a model of Down syndrome, exhibit premature thymic involution. We have performed a flow cytometry analysis of the developing thymus in these homozygous transgenic mice (hSOD1/hSOD1: Tg-SOD). Longitudinal follow-up analysis from day 3 to day 280 showed an early thymic development in Tg-SOD mice compared with controls. This early thymic development was associated with an increased migration of mature T cells to peripheral lymphoid organs. BrdU labeling showed no difference between Tg-SOD and control mice, confirming that the greater number of peripheral T cells in Tg-SOD mice was not due to extensive proliferation of these cells but rather to a greater pool of emigrant T cells in Tg-SOD.     

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 effects of age, thymectomy, and HIV Infection on alpha and beta TCR excision circles in naive T cells

Due to homeostasis total naive T cell numbers remain fairly constant over life despite a gradual involution of the thymus. The contribution of the thymus to maintaining naive T cell pools is typically measured with TCR excision circles (TRECs) that are formed in thymocytes. The mechanisms underlying thymic involution are poorly understood. Some data suggest that thymocytes undergo fewer divisions in old (small) than young (large) thymi, and other data suggest that the number of TRECs per thymocyte is independent of age. If thymic involution were associated with a decreased number of divisions of the thymocytes, this would markedly complicate the interpretation of TREC data. To study this we develop a mathematical model in which the division rate of thymocytes decreases with increasing age. We describe the dilution of TRECs formed during the arrangement of both chains of the TCR by division of thymocytes, recent thymic emigrants, and mature naive T cells. The model behavior is complicated as TREC contents in naive T cells can increase with age due to decreased dilution in the thymus. Because our model is consistent with current data on the effects of age and thymectomy on TRECs in peripheral T cells, we conclude that aging may well affect thymocyte division, which markedly complicates the interpretation of TREC data. It is possible, but more difficult, to let the model be consistent with the rapid changes in alpha and beta TRECs observed shortly after HIV infection.     

Thymic regeneration: teaching an old immune system new tricks

Recent studies in mice and humans show that the importance of the thymus extends well beyond the initial seeding of the peripheral T-cell pool. Although peripheral homeostasis can maintain T-cell numbers, the thymus is the major, if not the exclusive, source of new T-cell specificities. With age, thymus atrophy dramatically reduces the export of new T cells and predisposes an individual to impaired T-cell function, reduced T-cell immunity, and increased autoimmunity. Thymus atrophy is also the primary obstacle to restoration of the T-cell pool in the aftermath of HIV treatment or lymphoablative therapies. Here, we review thymus T-cell production, with particular attention to the factors that influence thymocyte export, and examine the impact that recent thymic emigrants have on the peripheral pool. In the future, thymic regeneration might become a feasible and potentially powerful approach to rejuvenating a depleted peripheral T-cell pool.