Aged Thymus Research Articles

Thymopoiesis is Enhanced by Inhibition of gp130 Signaling in Young and Aged Mice (85.15)

Abstract Normal aging is characterized by attenuation of cellular immune function and progressive decline in ability to respond to pathogens. Understanding mechanisms of immunosenescence with aging will enable development of targeted therapies for enhancing immune response. We previously reported that both human and mouse thymus aging is characterized by increased expression of IL-6 family cytokines. Further we demonstrated that these cytokines are directly suppressive and involute the thymus. IL-6 family cytokines activate the gp130 cell surface receptor on thymocytes, thus we hypothesized inhibition of gp130 receptor signaling will stimulate thymopoiesis in aged mice. To test this hypothesis we infused aged BALB/c mice (20 mo) or young mice (2 mo) with neutralizing gp130 Ab and monitored thymus function over 8 weeks. Inhibition of gp130 signaling significantly elevated thymus weight and TCR gene rearrangement in the aged thymus. In young mice we also observed with gp130 inhibition significant increases in thymus weight, cellularity, TCR gene rearrangement, and naïve export to the periphery. Overall, these data demonstrate release of thymosuppressive cytokine signaling stimulates thymopoiesis by both young and aged thymus tissue. Furthermore, gp130 signaling is an important thymoregulatory pathway to explore in the pursuit of human T cell immune reconstitution therapies. Supported by NIH HL67314, AG025150.

Thymic rejuvenation and the induction of tolerance by adult thymic grafts

The thymus, the site of origin of T cell immunity, shapes the repertoire of T cell reactivity through positive selection of developing T cells and prevents autoimmunity through negative selection of autoreactive T cells. Previous studies have demonstrated an important role for the thymus not only in central deletional tolerance, but also in the induction of peripheral tolerance by vascularized renal allografts in juvenile miniature swine recipients. The same protocol did not induce tolerance in thymectomized recipients nor in recipients beyond the age of thymic involution. We subsequently reported that vascularized thymic lobe grafts from juvenile donors were capable of inducing tolerance in thymectomized juvenile hosts. However, the important question remained whether aged, involuted thymus could also induce tolerance if transplanted into thymectomized hosts, which, if true, would imply that thymic involution is not an intrinsic property of thymic tissue but is rather determined by host factors extrinsic to the thymus. We report here that aged, involuted thymus transplanted as a vascularized graft into juvenile recipients leads to rejuvenation of both thymic structure and function, suggesting that factors extrinsic to the thymus are capable of restoring juvenile thymic function to aged recipients. We show furthermore that rejuvenated aged thymus has the ability to induce transplant tolerance across class I MHC barriers. These findings indicate that it may be possible to manipulate thymic function in adults to induce transplantation tolerance after the age of thymic involution.

Morphometrical Characteristics of Age‐Associated Changes in the Thymus of Old Male Wistar Rats

In order to provide a morphometrical description of the changes in the aged rat thymus and to relate them to apoptotic and proliferative activity of thymocytes, the thymuses from 3- and 18-month-old male Wistar rats and the percentages of bromodeoxyuridine-incorporating and apoptotic cells in cultures of thymocytes were assessed by stereological analysis and flow cytometry, respectively. In old rats the volume of lymphoepithelial thymic tissue is markedly reduced, reflecting a sharp decrease in the total number of thymocytes. A reduction in the proliferative capacity of thymocytes and increase in their susceptibility to apoptosis are, most likely, primarily responsible for a 7-fold reduction in thymic cellularity in old animals. Furthermore, only the volume of cortical compartment was affected by aging, while that of medulla, despite of reduced cellularity, was not significantly altered. The loss of functional tissue in aged thymus is compensated by a substantial increase in the volume of inter-lobular connective and adipose tissue, so the thymic weight remained unaltered in old rats. These results suggest that thymus of aged Wistar rats exhibits morphological characteristics similar to those found in aged human thymus and thus may serve as an animal model for further investigations of thymus-related changes in immunological aging.

ABH Histo-Blood Group Antigens in Human Thymus Involution

Human histo-blood group antigens (HBGA) are genetically determined glycoproteins supposed to participate in cell differentiation, adhesion, cancer metastasis and angiogenesis. In tissues, HBGA are mostly expressed in epithelial cells (EC). The EC comprising the thymocyte microenvironment play an important role in the ontogeny of the thymus. The aim of the present work is to investigate ABH HBGA in senile thymus and to characterize their expression pattern related to the process of aging. Routine histology and immunohistochemical techniques were applied on thymus glands from senile and young individuals. Involuted thymus exhibited large areas of adipose tissue containing scattered EC, all positive for HBGA. Stromal EC revealed different morphology and intrathymic localization but uniform cytoplasmic staining for ABH antigens. Endothelial cells of blood vessels and red blood cells were intensely stained for HBGA. Only single scattered lymphocytes possessed HBGA. In contrast with senile thymus, most lymphocyte populations in the gland of young individuals, as well as the Hassall's corpuscules, expressed HBGA. The epithelial framework reorganization during age-related thymus involution involves modulation in ABH antigen expression in EC. These molecules are required by thymic EC to maintain the reduced but important crosstalk with lymphocytes during involution. The diminished reactivity for ABH antigens in the lymphocytes of aged thymus might reflect the impaired communication between these two cell types. We present novel evidence for permanent presence and modulation of ABH antigen reactivity in senile thymus, supporting the view that these molecules might be developmentally regulated.

Thymic output in aged mice

Using GFP to mark recent thymic emigrants (RTEs) in mice carrying a GFP transgene driven by the recombination-activating gene 2 promoter, we demonstrate that RTEs are readily detectable even in 2-year-old mice, despite the fact that the proportion of the peripheral T cell pool comprised of RTEs declines with age. Although the number of RTEs decreases after reaching a peak at 6 weeks of age, thymic output as a function of thymic size is surprisingly age-independent. The CD4:CD8 ratio of RTEs declines with age, partly because of a striking decrease in steady-state proliferation of CD4+ RTEs in older mice. RTEs in aged mice undergo phenotypic maturation in the lymphoid periphery with delayed kinetics compared with young mice. RTEs from aged mice secrete less IL-2, proliferate less well, and achieve only weak expression of early-activation markers compared with more mature naïve peripheral T cells from the same mice. The proportion of GFP- cells in the CD4+ and CD8+ thymic compartments increases with age, partly as a result of leakiness in the aged thymus, allowing reentry of naïve peripheral T cells.

A Novel Approach to Thymic Rejuvenation in the Aged

With advancing age, the mammalian thymus undergoes involution, a progressive loss of architectural integrity and lymphoid cellularity that results in reduced T lymphopoiesis. Thymic involution also is associated with extreme malnutrition and states of immune deficiency, such as active HIV infection, after chemotherapy, or during pregnancy. Immune recovery appears to require restoration of normal thymopoiesis. Although several means are known to increase thymic cellularity in the aged, including systemic administration of hormones, androgen ablation, and thymic tissue transplantation, each suffers from specific limitations that prevent widespread application. This paper presents a novel approach to rejuvenate T cell differentiation in the aged that employs intrathymic implantation of engineered stromal cells. Two different proteins have been examined for their impact on thymopoiesis after delivery by somatic cell implantation. Intrathymic injection of IL-7-producing stromal cells enhances the earliest specification steps of T cell development, resulting in the increased representation of pro-T cells in the aged thymus. In contrast, increasing the intrathymic levels of sonic hedgehog diminishes this aspect of T cell poiesis.

Activation of Thymic Regeneration in Mice and Humans following Androgen Blockade

The thymus undergoes age-related atrophy, coincident with increased circulating sex steroids from puberty. The impact of thymic atrophy is most profound in clinical conditions that cause a severe loss in peripheral T cells with the ability to regenerate adequate numbers of naive CD4+ T cells indirectly correlating with patient age. The present study demonstrates that androgen ablation results in the complete regeneration of the aged male mouse thymus, restoration of peripheral T cell phenotype and function and enhanced thymus regeneration following bone marrow transplantation. Importantly, this technique is also applicable to humans, with analysis of elderly males undergoing sex steroid ablation therapy for prostatic carcinoma, demonstrating an increase in circulating T cell numbers, particularly naive (TREC+) T cells. Collectively these studies represent a fundamentally new approach to treating immunodeficiency states in humans.

Epithelial Framework Reorganization during Human Thymus Involution

Background: The thymus undergoes age-related (physiological) involution in the course of normal ontogenetic development. In addition to this chronic involution, the thymus can also undergo an acute (age-independent) regression, defined as spontaneous, transient involution. This process is induced by either exogenous or endogenous factors, including some infections (infection-type involution). Objective: The purpose of the present work was to undertake a comparative study of the epithelial framework organization and cytokeratin immunoreactivity of human thymic epithelial cells during age-related and infection-induced involution. Methods: Routine methods for light and transmission electron microscopy, as well as indirect immunoperoxidase staining and immunogold electron microscopy, were applied. Results: The epithelial thymocyte microenvironment was of a generally similar cellular composition in both chronically and acutely involuted thymus. Structurally, aged thymus glands and infection-affected thymus glands displayed a large mass of adipose tissue containing scattered islands composed of epithelial cells, lymphocytes and reticular connective tissue. Correlation between thymus involution and regional peculiarities in the presence and distribution of cytokeratin-immunopositive cells and their intermediate filaments was investigated. Conclusion: The epithelial framework of the thymus undergoes reorganization during both age-related and infection-induced thymus involution. The involutionary processes demonstrated essential regional and intracellular (structural and immunocytochemical) differences. The epithelial cell rearrangement and cytokeratin modulation that we observed might be involved in thymic microenvironment plasticity and reorganization in the course of these processes.

Essential microenvironment for thymopoiesis is preserved in human adult and aged thymus.

Normal human thymuses at various ages were immunohistologically examined in order to determine whether adult or aged thymus maintained the microenvironment for the T cell development and thymopoiesis was really ongoing. To analyze the thymic microenvironment, two monoclonal antibodies (MoAb) were employed. One is MoAb to IL-1 receptor (IL-1R) recognizing medullary and subcapsular cortical epithelial cells of normal infant human thymus. The other is UH-1 MoAb recognizing thymic epithelial cells within the cortex, which are negative with IL-1R-MoAb. Thymus of subjects over 20 years of age was split into many fragments and dispersed in the fatty tissue. However, the microenvironment of each fragment was composed of both IL-1R positive and UH-1 positive epithelial cells, and the UH-1 positive portion was populated with lymphocytes showing a follicle-like appearance. Lymphocytes in these follicle-like portions were mostly CD4+CD8+ double positive cells and contained many proliferating cells as well as apoptotic cells. Thus these follicle-like portions in adult and aged thymus were considered to be functioning as cortex as in infant thymus. Proliferative activity of thymocytes in the thymic cortex and the follicle-like portions definitely declined with advance of age, while incidence of apoptotic thymocytes increased with aging.

Relationships between monoaminergic and cholinergic innervation of the rat thymus during aging

The present study has been undertaken in order to investigate whether aging is accompanied by alterations in the thymic autonomic innervation. The results showed that in aged rats compared to young adult rats the density of monoaminergic histofluorescent nerve profiles decreased remarkably, while their pattern of intrathymic distribution remained unchanged. The thymic concentrations of noradrenaline (NA) and dopamine (DA) also significantly decreased between the age of 12 and 18 months. However, the density of thymic autofluorescent cells (afc) markedly increased over the same period, as well as the concentration of 5-hydroxytryptamine (5-HT). The aged rat thymus seemed to be able to maintain its cholinergic innervation in terms of density and pattern of distribution, while the density of cells with intracytoplasmic acetylcholinesterase (AChE) staining even increased. The neurochemical measurement showed an increase in the activity of AChE between the age of 9 to 18 months. The results indicate an altered relation between the components of thymic autonomic innervation of aged rats that might be related to the reduced immunocompetence of their T cells.

The thymic epithelial reticulum and interdigitating cells in SIV-induced thymus atrophy and its comparison with other forms of thymus involution

Alterations in the thymus were investigated in the early course of SIV infection of rhesus monkeys and compared with age-related and acute accidental thymus atrophy. The SIV-induced pathology was characterized by shrinkage of the thymic parenchyma and capsule, whereas in age-related thymus atrophy, the size of the capsule remained unaltered and the emerging space was filled by fatty tissue. Acute accidental thymus involution is characterized by massive cell death of the thymocytes, but there was no increase in pycnotic thymocytes either in SIV-induced or in age-related thymus atrophy. Ultrastructural analysis revealed no major differences between the juvenile control and the aged thymus. In contrast, SIV-induced thymus atrophy exhibited severe alterations of the epithelial cells of the cortex and the interdigitating dendritic cells, which were not found in the aged thymus nor in the juvenile control cortex.

Aging in the T lymphocyte compartment. A developmental view.

A decline in the capacity of bone marrow cells to differentiate to T lymphocytes was found when cells from young and old donors were seeded onto an alymphoid fetal thymus. A step-by-step analysis of cell-cell interactions of the lymphohemopoietic cells and the thymic stroma indicated an effect of age on a variety of cell differentiation parameters. These included a decrease in the affinity of bone marrow cells to the stroma, and in their capacity to compete with the thymic lymphoid resident cells on colonization of the thymus. There was a significant decrease in the ability of cells of old donors to replicate sequentially within the thymic microenvironment. There was a reduced capacity of bone marrow cells from aging mice to express a developmental preference after seeding onto a syngeneic fetal thymus in a mixture with cells from allogeneic donors. We addressed the question whether the aging thymus contains increased levels of immature cells that fail to differentiate in the involuted thymic microenvironment by seeding thymocytes from young and old donors onto the fetal thymic stroma. The values of T cells that developed from the old donor inoculum were lower under these conditions. Our studies suggest that at least some of the manifestations of aging in the T cell compartment are related to developmentally programmed events in the lymphohemopoietic cell compartment.

Calcitonin Gene‐Related Peptide in the Developing and Aging Thymus

Calcitonin gene-related peptide (CGRP) is known to block Con A and PHA induced T cell proliferation. As a first step in determining the role of this peptide in T cell education and function we have studied the distribution of CGRP within the developing mouse thymus using immunocytochemistry. CGRP-like immunoreactivity (CGRP-IR) was found in the thymic nerves in close proximity to blood vessels in the 17-day-old embryonic mouse thymus. A discrete population of small cells at the cortico-medullary junction also stained intensely for CGRP. As the mouse thymus reached maturity (three to eight weeks) CGRP innervation became more dense, with fibers running along the vasculature at the cortico-medullary boundary, then branching into the cortical and medullary regions. Some fibers were invested in the blood vessels while a large portion formed varicosities among the cells of the thymus. In the mature thymus, the small CGRP-IR cortico-medullary cells were more numerous, and CGRP-IR was also found in subcapsular and trabecular mast cells. The pattern of innervation remained the same in the aging mouse thymus (six months), but there appeared to be somewhat fewer cortico-medullary cells and an increase in mast cell number. In the aged (eighteen months) thymus, the small CGRP-IR cortico-medullary cells were rarely seen, but mast cells were more numerous, most of which stained positively for CGRP, in the connective tissue. Nerves containing CGRP-IR generally had the same distribution as in the younger mice but appeared somewhat truncated. The distribution of CGRP-IR nerves in the mouse thymus at different stages of development was similar to that reported for cholinergic (AChE-positive) nerves. Since the brain-stem vagal nuclei have been shown by retrograde transport studies to project to the thymus as well as to contain CGRP-IR neurons, our findings suggest that CGRP-IR thymic nerves may be derived from the vagus complex.

Age influence on the thymic capacity to promote differentiation of T cells: Induction of different composition of T cell subsets by aging thymus

Three kinds of experiments were performed to see the differential effect of aging thymus on T cell differentiation in nude mice and thymectomized mice. In the experiment of thymus grafting into nude mice, the thymic capacity to promote T cell differentiation was the highest at newborn stage, and declined to 80% of the peak level at as early as 1 week of age. The level at 4 weeks of age was 50–60% of the peak level and did not greatly change thereafter with advancing age of thymus donors, up to 24 months of age. However, composition of T cell subsets differed with age of thymus graft; i.e. L3T4(CD4) + T cells were more easily induced than Lyt-2(CD8) + T cells by aging thymus, resulting in an increase of the ratio of L3T4 +/Lyt-2 + T cells with advancing age of thymus donors. The decreased number of T cells and their subsets in the mice thymectomized at 4 weeks of age could be almost totally recovered by the grafting of newborn thymus, but less efficiently by the grafting of 24-month-old thymus. In the latter case again, L3T4 + T cells were more easily induced than Lyt-2 + T cells, resulting in an increase of the ratio of L3T4 +/Lyt-2 + T cells by the grafting of the old thymus. In neonatal mice thymectomized 3 days after the birth, Lyt-2 + T cells were more severely affected than L3T4 + cells, resulting in high ratio of L3T4 +/Lyt-2 + T cells. It was suggested that the capacity of the thymus to induce T cells started to decline as early as 1 week of age and did not greatly change between 4 weeks and 24 months of age. However, the composition of T Cell subsets induced by the thymus changed with age, with preference for L3T4 + T cells over Lyt-2 + T cells.

Influence of age of thymic grafts on the differentiation of T cells in nude mice

Age-related changes in the capacity of thymic grafts to promote T-cell differentiation were studied in congenic nude BALB/c mice. The results revealed that the cytolytic T-cell activity and T-cell-dependent anti-SRBC response declined exponentially with age, the former starting its decline at birth, and the latter after adulthood. In contrast, the T-cell-dependent mitogenic response to PHA and Con A stimulation and the number of Thy-1 + cells declined minimally with age of the thymic graft. These findings suggest that aging decreases the capacity of the thymus to synthesize the various factors needed for the generation of T-cell subpopulations differently with respect to the time of onset and rate of decline. In addition, results suggest that the aging thymus is also losing its ability to regulate natural killer cells.