Age-associated Thymic Involution Research Articles

The potential role of the thymus in immunotherapies for acute myeloid leukemia.

Understanding the factors which shape T-lymphocyte immunity is critical for the development and application of future immunotherapeutic strategies in treating hematological malignancies. The thymus, a specialized central lymphoid organ, plays important roles in generating a diverse T lymphocyte repertoire during the infantile and juvenile stages of humans. However, age-associated thymic involution and diseases or treatment associated injury result in a decline in its continuous role in the maintenance of T cell-mediated anti-tumor/virus immunity. Acute myeloid leukemia (AML) is an aggressive hematologic malignancy that mainly affects older adults, and the disease's progression is known to consist of an impaired immune surveillance including a reduction in naïve T cell output, a restriction in T cell receptor repertoire, and an increase in frequencies of regulatory T cells. As one of the most successful immunotherapies thus far developed for malignancy, T-cell-based adoptive cell therapies could be essential for the development of a durable effective treatment to eliminate residue leukemic cells (blasts) and prevent AML relapse. Thus, a detailed cellular and molecular landscape of how the adult thymus functions within the context of the AML microenvironment will provide new insights into both the immune-related pathogenesis and the regeneration of a functional immune system against leukemia in AML patients. Herein, we review the available evidence supporting the potential correlation between thymic dysfunction and T-lymphocyte impairment with the ontogeny of AML (II-VI). We then discuss how the thymus could impact current and future therapeutic approaches in AML (VII). Finally, we review various strategies to rejuvenate thymic function to improve the precision and efficacy of cancer immunotherapy (VIII).

High-Oxygen Submersion Fetal Thymus Organ Cultures Enable FOXN1-Dependent and -Independent Support of T Lymphopoiesis.

T cell development is effectively supported in fetal thymus organ cultures (FTOCs), which places thymus lobes atop an air-liquid interface (ALI) culture system. The direct exposure to air is critical for its success, as fetal thymus lobes placed in low oxygen submersion (LOS)-FTOCs fail to support thymocyte development. However, submersion cultures performed in the presence of high concentration of ambient oxygen (60~80%) allow for normal thymocyte development, but the underlying mechanism for this rescue has remained elusive. Here, we show that FOXN1 expression in thymic epithelial cells (TECs) from LOS-FTOCs was greatly reduced compared to conventional ALI-FTOCs. Consequently, the expression of important FOXN1 target genes, including Dll4 and Ccl25, in TECs was extinguished. The loss of DLL4 and CCL25 interrupted thymocyte differentiation and led to CD4+CD8+ cells exiting the lobes, respectively. High oxygen submersion (HOS)-FTOCs restored the expression of FOXN1 and its target genes, as well as maintained high levels of MHCII expression in TECs. In addition, HOS-FTOCs promoted the self-renewal of CD4−CD8−CD44−CD25+ cells, allowing for the continuous generation of later stage thymocytes. Forced FOXN1 expression in TECs rescued thymocyte developmental progression, but not cellularity, in LOS-FTOCs. Given that oxidative stress has been reported to accelerate the onset of age-associated thymic involution, we postulate that regulation of FOXN1 by oxygen and antioxidants may underpin this biological process.

Neem leaf glycoprotein reverses tumor-induced and age-associated thymic involution to maintain peripheral CD8+ T cell pool.

Aim: As tumor causes atrophy inthethymus to target effector-T cells, this study is aimed to decipher the efficacy of neem leaf glycoprotein (NLGP) in tumor-and age-associated thymic atrophy. Materials &methods: Different thymus parameters were studied using flow cytometry, reversetranscriptase PCR andimmunocyto-/histochemistry in murine melanoma and sarcoma models. Results: Longitudinal NLGP therapy in tumor hosts show tumor-reduction along with significant normalization of thymic alterations. NLGP downregulates intrathymic IL-10, which eventually promotes Notch1 to rescue blockade in CD25+CD44+c-Kit+DN2 to CD25+CD44-c-Kit-DN3 transition in T cell maturation and suppress Ikaros/IRF8/Pu.1 to prevent DN2-T to DC differentiation in tumor hosts.TheCD5intTCRαβhigh DP3 population was also increased to endorse CD8+ Tcell generation. Conclusion: NLGP rescues tumor-induced altered thymic events to generate more effector T cells to restrain tumor.

Gallic acid attenuates thymic involution in the d-galactose induced accelerated aging mice.

Senescence is an inevitable and complicated phenomenon. Age-associated thymic involution increases the risk of infectious diseases, which results in the immunosenescence and leads to a poor immune function. d-galactose (d-gal) can cause damages that resemble accelerated aging in mice. Gallic acid (GA), as one of the natural phenolic compounds, has been demonstrated to act in antioxidant and anti-tumor effects. In this study, we explored the effects of GA in preventing the age-related thymic involution and the alterations of the forkhead box protein N1 (FoxN1) in d-gal induced accelerated aging mice. The accelerated aging mice model was established by intraperitoneal injection d-gal for eight weeks and given GA with 200, 250, 500 mg/kg body weight per day, respectively, for six weeks. It showed that the d-gal-treated mice developed structural changes in the thymi compared to normal control mice. With supplement of GA, the mice restored the normal thymic anatomy, including the thickening cortex compartment and clearer cortico-medullary junction. The d-gal-treated mice showed a severe reduction in the number of thymocytes, GA mice also displayed the increased numbers of CD4 + T cells through flow cytometric analysis. GA treatment increased the proliferative cells by BrdU incorporation assay and reduced the numbers of apoptotic cells with FITC-12-dUTP labeling (TUNEL). The expression of FoxN1 was also found increased in GA treated mice by immunohistochemistry and quantitative reverse transcriptase PCR (qRT-PCR). Taken together, our results suggested that the administration of GA opposed the involution of thymus via stimulation of FoxN1 expression and proliferation of cells in a dose-dependent manner.

Time-course transcriptome analysis of medullary thymic epithelial cells in the early phase of thymic involution

Degeneration of the thymic epithelium is believed to be the primary cause of age-associated thymic involution. In order to investigate the molecular events during the early phase of thymic involution, RNA-seq was performed to gain the transcriptional profiles of medullary thymic epithelial cells (mTEC) from mice of 2, 6 and 10 weeks of age. We confirmed and extended the previous observation of declined expression of cell cycle-related genes and diminished E2F3 activity during thymic involution, showing that it occurred as early as 2–6 weeks after birth. Moreover, we demonstrated that mTEC aging was coupled with augmented expression of inflammatory chemokines and cytokines, reminiscent of the senescence-associated secretory phenotype. Impaired cell cycling and proinflammatoty response therefore represent two predominant transcriptional signatures during the very early phase of thymic involution. Taken together, the present study provides not only complimentary information about, but also new insight into the molecular mechanisms underlying age-related degeneration of thymic epithelial cells.

Polymorphisms in the canine IL7R 3′UTR are associated with thymic output in Labrador retriever dogs and influence post-transcriptional regulation by microRNA 185

Interleukin-7 (IL-7) and its receptor (IL-7R) are essential for T cell development in the thymus, and changes in the IL-7/IL-7R pathway have been implicated in age-associated thymic involution which results in a reduction of naïve T cell output. The aim of this study was to investigate the relationship between IL7 and IL7R genetic variation and thymic output in dogs. No single nucleotide polymorphisms (SNPs) were identified in the canine IL7 gene, but a number were present in the canine IL7R gene. Polymorphisms in the IL7R exon 8 and 3′UTR were found to be associated with signal joint T cell receptor excision circle (sj-TREC) values (a biomarker of thymic output) in young and geriatric Labrador retrievers. Additionally, one of the SNPs in the IL7R 3′UTR (SNP 14 c.1371 + 446 A > C) was found to cause a change in the seed-binding site for microRNA 185 which, a luciferase reporter assay demonstrated, caused changes in post-transcriptional regulation, and therefore might be capable of influencing IL-7R expression. The research findings suggest a genetic link between IL7R genotype and thymic output in dogs, which might impact on immune function as these animals age and provide further evidence of the involvement of IL-7/IL-7R pathway in age-associated thymic involution.

Age-associated alterations in the levels of cytotoxic lipid molecular species and oxidative stress in the murine thymus are reduced by growth hormone treatment

During age-associated thymic involution, thymocytes decrease and lipid-laden cells accumulate. However, if and how aging affects the thymic lipid profile is not well understood, nor is it known if the hormonal milieu modifies this process. Here we demonstrate a correlation between reduced thymocyte numbers and markers of inflammation and oxidative stress with age. Evaluating the lipidomics profile of the whole thymus, between the ages of 4 (young) and 18 months (old), we found increased amounts of triacylglycerides, free cholesterol, cholesterol ester and 4-hydroxynonenal (4-HNE) with age. Moreover, levels of C24:0 and C24:1 sphingomyelins and ceramide C16:0 were elevated in 12–14 month-old (middle-aged) mice while the levels of sulfatide ceramide and ganglioside GD1a increased in the old thymus. Evaluating isolated thymocytes, we found increased levels of cholesterol ester and 4-HNE adducts, as compared to young mice. Next, we treated middle-aged mice with growth hormone (GH), which has been considered a potent immunomodulator. GH reduced thymic levels of TNF-α and 4-HNE and increased the number of thymocytes as well as the thymic levels of dihydroceramide, a ceramide precursor and autophagic stimuli for cell survival. In conclusion, GH treatment attenuated inflammation and age-related increases in oxidative stress and lipotoxicity in the thymus.

Human naïve regulatory T-cells feature high steady-state turnover and are maintained by IL-7.

Naïve FoxP3-expressing regulatory T-cells (Tregs) are essential to control immune responses via continuous replenishment of the activated-Treg pool with thymus-committed suppressor cells. The mechanisms underlying naïve-Treg maintenance throughout life in face of the age-associated thymic involution remain unclear. We found that in adults thymectomized early in infancy the naïve-Treg pool is remarkably well preserved, in contrast to conventional naïve CD4 T-cells. Naïve-Tregs featured high levels of cycling and pro-survival markers, even in healthy individuals, and contrasted with other circulating naïve/memory CD4 T-cell subsets in terms of their strong γc-cytokine-dependent signaling, particularly in response to IL-7. Accordingly, ex-vivo stimulation of naïve-Tregs with IL-7 induced robust cytokine-dependent signaling, Bcl-2 expression, and phosphatidylinositol 3-kinase (PI3K)-dependent proliferation, whilst preserving naïve phenotype and suppressive capacity. Altogether, our data strongly implicate IL-7 in the thymus-independent long-term survival of functional naïve-Tregs, and highlight the potential of targeting the IL-7 pathway to modulate Tregs in different clinical settings.

덱사메타손에 의해 유발된 흉선 T세포사멸에 대한 그렐린의 세포사멸억제효과

28개의 아미노산으로 이루어져 있는 Ghrelin은 위 기저부의 X/A-유사 신경내분비 세포에서 주로 합성 분비되는 물질로 음식의 섭취나 비만, 에너지 항상성 등을 조절하는 역할을 하며, 이러한 Ghrelin 활성화는 수용체인 G-protein coupled growth hormone secretagogue receptor-1a (GHS-R1a)와 결합을 통해 일어난다. 최근 보고된 자료에 따르면, ghrelin과 수용체는 위나 시상하부, 뇌하수체 등뿐만 아니라 T 세포나 단핵구 및 대식세포 등 면역 세포에서도 생성되며, 염증반응을 유도하는 사이토카인의 생성을 억제하는 역할을 한다. 또한 흉선의 퇴화 등 면역기관에 있어서도 중요한 호르몬으로 보고되고 있지만 그 기전이나 기능에 대한 연구가 아직 미미한 실정이다. 본 연구에서는 흉선에서 T 세포의 성숙이나 세포활성을 억제하는 물질로 알려져 있는 덱사메타손(Dexamethasone; DEX)으로 세포사멸을 유도시킨 흉선세포에 ghrelin을 처리하여 세포사멸 억제효과를 알아보았다. 그 결과 Ghrelin은 세포사멸에 중요 단백질인 Caspase-3와 PARP 및 Bim의 활성화가 in vivo 및 in vitro 모두에서 효과적으로 저해됨을 확인할 수 있었으며, 이러한 세포사멸의 억제효과는 ghrelin을 처리할 경우 DEX에 의해 활성화된 Glucocorticoid 수용체(GR)의 인산화의 억제와 HSP90 등과 복합체를 이루고 있는 GR이 활성화되면서 분해되는 과정을 억제시켜 결과적으로 핵 안으로 이동하는 과정을 억제하는 기전을 통해 나타남을 알 수 있었다. 이러한 결과 등을 통해 볼 때, ghrelin은 약물이나 체내 생리적 스트레스 등으로 인해 발생하는 흉선 내 면역세포들의 세포사멸에 도움을 줄 것으로 사료되며, 나아가 이로 인한 흉선위축을 보호할 수 있는 치료 후보물질로서의 연구를 기대할 수 있으리라 사료된다. Ghrelin is a 28 amino acid orexigenic peptide hormone that is secreted predominantly by tX/A cells in the stomach, and it plays a major role in energy homeostasis. Activated ghrelin has an n-octanoyl group covalently linked to the hydroxyl group of the Ser3 residue, which is critical for its binding to the G-protein coupled growth hormone secretagogue receptor-1a (GHS-R1a). According to recent reports, both ghrelin and its receptor, GHS-R1a, are expressed by a variety of immune cells, including T- and B-lymphocytes, monocytes, and dendritic cells, and ghrelin stimulation of leukocytes provides a potent immunomodulatory signal controlling systemic and age-associated inflammation and thymic involution. Here, we report that ghrelin protected murine thymocytes from dexamethasone (DEX)-induced cell death both in vivo and in vitro. Subsequently, we explored the molecular mechanisms of the antiapoptotic effect of ghrelin. According to our experiments, ghrelin inhibited the expression of proapoptotic proteins via the regulation of glucocorticoid receptor (GR) phosphorylation. As a result, ghrelin inhibited the proapoptotic activation of proteins, such as Caspase-3, PARP, and Bim. These data suggest that ghrelin, through GHS-R, inhibits the pathway to apoptosis by regulation of the proapoptotic protein activation signal pathway. They provide evidence that blocking apoptosis is an essential function of ghrelin during the development of thymocytes.

Global Transcriptional Profiling Reveals Distinct Functions of Thymic Stromal Subsets and Age-Related Changes during Thymic Involution

Age-associated thymic involution results in diminished T cell output and function in aged individuals. However, molecular mediators contributing to the decline in thymic function during early thymic involution remain largely unknown. Here, we present transcriptional profiling of purified thymic stromal subsets from mice 1, 3, and 6 months of age spanning early thymic involution. The data implicate unanticipated biological functions for a subset of thymic epithelial cells. The predominant transcriptional signature of early thymic involution is decreased expression of cell-cycle-associated genes and E2F3 transcriptional targets in thymic epithelial subsets. Also, expression of proinflammatory genes increases with age in thymic dendritic cells. Many genes previously implicated in late involution are already deregulated by 3-6 months of age. We provide these thymic stromal data sets, along with thymocyte data sets, in a readily searchable web-based platform, as a resource for investigations into thymocyte:stromal interactions and mechanisms of thymic involution.

Thymus and aging: morphological, radiological, and functional overview

Aging is a continuous process that induces many alterations in the cytoarchitecture of different organs and systems both in humans and animals. Moreover, it is associated with increased susceptibility to infectious, autoimmune, and neoplastic processes. The thymus is a primary lymphoid organ responsible for the production of immunocompetent T cells and, with aging, it atrophies and declines in functions. Universality of thymic involution in all species possessing thymus, including human, indicates it as a long-standing evolutionary event. Although it is accepted that many factors contribute to age-associated thymic involution, little is known about the mechanisms involved in the process. The exact time point of the initiation is not well defined. To address the issue, we report the exact age of thymus throughout the review so that readers can have a nicely pictured synoptic view of the process. Focusing our attention on the different stages of the development of the thymus gland (natal, postnatal, adult, and old), we describe chronologically the morphological changes of the gland. We report that the thymic morphology and cell types are evolutionarily preserved in several vertebrate species. This finding is important in understanding the similar problems caused by senescence and other diseases. Another point that we considered very important is to indicate the assessment of the thymus through radiological images to highlight its variability in shape, size, and anatomical conformation.

Overexpression of Foxn1 attenuates age-associated thymic involution and prevents the expansion of peripheral CD4 memory T cells

AbstractThe forkhead box n1 (Foxn1) transcription factor is essential for thymic organogenesis during embryonic development; however, a functional role of Foxn1 in the postnatal thymus is less well understood. We developed Foxn1 transgenic mice (Foxn1Tg), in which overexpression of Foxn1 is driven by the human keratin-14 promoter. Expression of the Foxn1 transgene increased the endogenous Foxn1 levels. In aged mice, overexpression of Foxn1 in the thymus attenuated the decline in thymocyte numbers, prevented the decline in frequency of early thymic progenitors, and generated a higher number of signal joint TCR excised circle. Histologic studies revealed that structural alterations associated with thymic involution were diminished in aged Foxn1 Tg. Total numbers of EpCAM+ MHC II+ and MHC IIhi thymic epithelial cells were higher in young and old Foxn1Tg and more EpCAM+ MHC IIhi TEC expressed Ki-67 in aged Foxn1Tg compared with WT. Furthermore, Foxn1Tg displayed a significant reduction in the expansion of splenic CD4+ memory compartments and attenuated the decline in CD4+ and CD8+ naive compartments. Our data indicate that manipulation of Foxn1 expression in the thymus ameliorates thymopoiesis in aged mice and offer a strategy to combat the age-associated decline in naive T-cell production and CD4 naive/memory ratios in the elderly.

Stem Cell Factor (SCF) Improves Thymopoiesis After Experimental Hematopoietic Stem Cell Transplantation In Both a Murine BMT Model and In “human Immune system” (HIS) Mice, Receiving a Human Stem Cell Graft.

AbstractAbstract 3725Deficient thymopoiesis, due to epithelial injury by chemo- and/or radiotherapy, age-associated thymic involution and by graft-versus-host disease, is an important determinant of the impaired immune competence following allogeneic transplantation. Therefore, strategies to improve thymopoiesis are considered pivotal to improve T cell recovery and immune competence after transplantation. As SCF is a cytokine produced by thymic stroma and its receptor, c-kit, is expressed by the earliest thymocytes, we evaluated whether SCF administration would improve thymic recovery following stem cell transplantation in immuno-deficient mice receiving: (1) a T-cell depleted bone marrow (BM) graft of congenic mice, or (2) a CD34+CD38low-selected xenogenic hematopoietic stem cell (HSC) graft of human fetal liver origin (HIS) mice model). In the mouse-mouse model, 10–12 week old rag-1−/− mice were 3 Gy irradiated (137Cs -source) and received 2×105 T-cell depleted C57Bl/6 (CD45.1) congeneic bone marrow cells intravenously. Recipient mice received either PBS or recombinat rat SCF (Amgen, USA, 100μg/kg per injection) by subcutaneous injection 3 times a week from day 1 until the end of the experiment. In this model, SCF enhanced thymopoiesis and peripheral T-cell recovery. BM lymphoid progenitor recovery was not affected. Median thymic cellularity increased from 0.9 in PBS- to 266 × 104/thymus in SCF-treated mice (p=0.05), which increase was similarly distributed over the thymocyte subsets of double negative (DN), double positive (DP), and CD4+, CD8+ single positive thymocytes. Next, we assessed whether SCF-induced improved thymic recovery also translated into improved T-cell recovery in the periphery. Absolute numbers of donor-derived newly developed CD4+ and CD8+ T-cells were quantified in peripheral blood, spleen and lymph nodes at weeks 4 and 6 post-transplantation. T-cell numbers were low at 4 weeks after transplantation and did not differ between PBS or SCF treated animals. However, at 6 weeks T-cell numbers were significantly increased in spleen and lymph nodes of SCF treated animals (p<0.05).Next, we studied the effect of recombinant human (rh) SCF in our HIS mouse model. In short, newborn (days 3–7) Rag-2−/−gc−/− mice were 3.5 Gy irradiated and transplanted with 5–10 × 104 CD34+CD38low human fetal liver (FL) cells intra-hepatically. CD34+CD38low cells were isolated via a two step procedure. CD34+− FL cells were isolated using a CD34 human progenitor cell-isolation kit and further sorted as CD38low using a FACS Aria (BD biosciences). In HIS mice, PBS or rhSCF (Amgen, USA) 100μg/kg per injection) was administered intraperitoneally (i.p.) 3 times weekly as of day 14 following transplantation.Similar to the murine BMT model, a higher thymic cellularity was observed in SCF treated mice (Fig. 1) in the HIS model. DN and early DP thymocyte subsets were enhanced, albeit not significantly. In contrast, lateDP, CD4SP and CD8SP thymocyte subset recovery was significantly enhanced in thymi of SCF-treated HIS mice. As the HIS model remains a hybrid human–mouse system with limited cytokine cross reactivity and in which MHC-HLA mismatch compromises peripheral T-cell survival (Legrand et al J IMMUNOL 2009), the model did not allow us to study the effect of SCF on T-cells in the peripheral lymphoid organs. [Display omitted] Collectively, these results show that SCF may significantly improve post-transplant thymopoiesis after both experimental murine BMT and human fetal liver HSC transplantation in HIS mice. While peripheral T-cell recovery was significantly enhanced in the murine BMT model, it remains to be established whether improved human thymopoiesis will also translate into better peripheral T-cell recovery and enhanced immune competence towards opportunistic infections as well as a better recovery of regulatory T cells. These studies now set the stage for studying SCF, either alone or combined with other thymopoietic cytokines, in a larger pre-clinical animal model. Disclosures:No relevant conflicts of interest to declare.

Rejuvenation of the aging thymus: growth hormone-mediated and ghrelin-mediated signaling pathways

One of the major fundamental causes for the aging of the immune system is the structural and functional involution of the thymus, and the associated decline in de novo naïve T-lymphocyte output. This loss of naïve T-cell production weakens the ability of the adaptive immune system to respond to new antigenic stimuli and eventually leads to a peripheral T-cell bias to the memory phenotype. While the precise mechanisms responsible for age-associated thymic involution remain unknown, a variety of theories have been forwarded including the loss of expression of various growth factors and hormones that influence the lymphoid compartment and promote thymic function. Extensive studies examining two hormones, namely growth hormone (GH) and ghrelin (GRL), have demonstrated their contributions to thymus biology. In the current review, we discuss the literature supporting a role for these hormones in thymic physiology and age-associated thymic involution and their potential use in the restoration of thymic function in aged and immunocompromised individuals.

Wnt4 and LAP2alpha as Pacemakers of Thymic Epithelial Senescence

Age-associated thymic involution has considerable physiological impact by inhibiting de novo T-cell selection. This impaired T-cell production leads to weakened immune responses. Yet the molecular mechanisms of thymic stromal adipose involution are not clear. Age-related alterations also occur in the murine thymus providing an excellent model system. In the present work structural and molecular changes of the murine thymic stroma were investigated during aging. We show that thymic epithelial senescence correlates with significant destruction of epithelial network followed by adipose involution. We also show in purified thymic epithelial cells the age-related down-regulation of Wnt4 (and subsequently FoxN1), and the prominent increase in LAP2α expression. These senescence-related changes of gene expression are strikingly similar to those observed during mesenchymal to pre-adipocyte differentiation of fibroblast cells suggesting similar molecular background in epithelial cells. For molecular level proof-of-principle stable LAP2α and Wnt4-over-expressing thymic epithelial cell lines were established. LAP2α over-expression provoked a surge of PPARγ expression, a transcription factor expressed in pre-adipocytes. In contrast, additional Wnt4 decreased the mRNA level of ADRP, a target gene of PPARγ. Murine embryonic thymic lobes have also been transfected with LAP2α- or Wnt4-encoding lentiviral vectors. As expected LAP2α over-expression increased, while additional Wnt4 secretion suppressed PPARγ expression. Based on these pioneer experiments we propose that decreased Wnt activity and increased LAP2α expression provide the molecular basis during thymic senescence. We suggest that these molecular changes trigger thymic epithelial senescence accompanied by adipose involution. This process may either occur directly where epithelium can trans-differentiate into pre-adipocytes; or indirectly where first epithelial to mesenchymal transition (EMT) occurs followed by subsequent pre-adipocyte differentiation. The latter version fits better with literature data and is supported by the observed histological and molecular level changes.

A Quantitative Trait Locus on chr.4 Regulates Thymic Involution

The mechanisms underlying age-associated thymic involution are unknown. In mice, thymic involution shows mouse strain-dependent genetic variation. Identification of the underlying genes would provide mechanistic insight into this elusive process. We previously showed that responsiveness of hematopoietic stem and progenitor cells (HSPCs) to transforming growth factor-beta 2, a positive regulator of HSPC proliferation, is regulated by a quantitative trait locus (QTL) on chr. 4, Tb2r1. Interestingly, Tgfb2(+/-) mice have delayed thymic involution. Therefore, we tested the hypothesis that a QTL on chr. 4 might regulate thymic involution. Aged, but not young, B6.D2-chr.4 congenic mice, where the telomeric region of chr. 4 was introgressed from DBA/2 to C57BL/6 mice, had larger thymi, and better maintenance of early thymic precursors than C57BL/6 control mice. These observations unequivocally demonstrate that the telomeric region of chr. 4 contains a QTL, Ti1 (thymic involution 1) that regulates thymic involution, and suggest the possibility that Ti1 may be identical to Tb2r1.

The Effect of Age on the Phenotype and Function of Developing Thymocytes

The immune system declines with age leading to a progressive deterioration in the ability to respond to infection and vaccination. Age-associated thymic involution is one of the most recognized changes in the ageing immune system and is believed to be a major contributor towards immunosenescence; however, the precise mechanisms involved in age-associated thymic involution remain unclear. In order to gain further insight into the effect of ageing on T-cell development, steady-state thymopoiesis was studied in mice ranging from 1 to 18 months of age. There was a decrease in thymic cellularity with age, but the most dramatic loss occurred early in life. Although there were no alterations in the proportion of the major thymocyte subsets, there was a significant decline in the expression of other key molecules including CD3 and CD24. There was a decline in the ability of thymocytes from older mice to respond to mitogens, which was demonstrated by a failure to up-regulate expression of the activation marker CD69 and to enter the G(2)--M phase of the cell cycle. This was concurrent with an increased resistance to apoptosis in thymocytes from aged animals. Together, these results suggest that T cells may be flawed even before exiting to the periphery and that this could contribute to the age-associated decline in immune function.

Fat-Storing Multilocular Cells Expressing CCR5 Increase in the Thymus with Advancing Age: Potential Role for CCR5 Ligands on the Differentiation and Migration of Preadipocytes

Age-associated thymic involution is characterized by decreased thymopoiesis, adipocyte deposition and changes in the expression of various thymic microenvironmental factors. In this work, we characterized the distribution of fat-storing cells within the aging thymus. We found an increase of unilocular adipocytes, ERTR7(+) and CCR5(+ )fat-storing multilocular cells in the thymic septa and parenchymal regions, thus suggesting that mesenchymal cells could be immigrating and differentiating in the aging thymus. We verified that the expression of CCR5 and its ligands, CCL3, CCL4 and CCL5, were increased in the thymus with age. Hypothesizing that the increased expression of chemokines and the CCR5 receptor may play a role in adipocyte recruitment and/or differentiation within the aging thymus, we examined the potential role for CCR5 signaling on adipocyte physiology using 3T3-L1 pre-adipocyte cell line. Increased expression of the adipocyte differentiation markers, PPARgamma2 and aP2 in 3T3-L1 cells was observed under treatment with CCR5 ligands. Moreover, 3T3-L1 cells demonstrated an ability to migrate in vitro in response to CCR5 ligands. We believe that the increased presence of fat-storing cells expressing CCR5 within the aging thymus strongly suggests that these cells may be an active component of the thymic stromal cell compartment in the physiology of thymic aging. Moreover, we found that adipocyte differentiation appear to be influenced by the proinflammatory chemokines, CCL3, CCL4 and CCL5.

Role of ovarian hormones in age-associated thymic involution revisited

A commonly held view that ovarian hormones are causally involved in age-associated thymic involution has been recently challenged. In particular, their relevance in the progression of thymic involution has been disputed. To reassess this issue 10-month-old rats with well advanced thymic involutive changes were ovariectomized (Ovx), and after 1 month thymic cellularity, thymocyte development and levels of recent thymic emigrants (RTEs) were examined in peripheral blood and spleen. In addition, the distribution of major conventional and regulatory T-cell subsets was analyzed in the same peripheral lymphocyte compartments. Ovariectomy increased thymic weight and cellularity above the levels in both 10-month-old and age-matched controls indicating that ovarian hormone ablation not only prevented further progression of thymic involution, but also reversed it. The increased thymic cellularity was accompanied by altered thymocyte differentiation/maturation culminating in increased thymic output of naïve T cells as indicated by elevated levels of both CD4+ and CD8+ RTEs in peripheral blood and spleen. The changes in T-cell development produced: (i) a disproportional increase in cellularity across thymocyte subsets, so that relative proportions of cells at all maturational stages preceding the CD4+CD8+ T cell receptor (TCR)alphabeta(low) stage were reduced; the relative numbers of CD4+CD8+ TCRalphabeta(low) cells entering positive selection and their immediate CD4+CD8+ TCRalphabeta(high) descendents were increased, while those of the most mature CD4+CD8- and CD4-CD8+ TCRalphabeta(high) cells remained unaltered; (ii) enhanced cell proliferation across all thymocyte subsets and (iii) reduced apoptosis of cells within the CD4+CD8+ thymocyte subset. The augmented thymic output of naïve T cells in Ovx rats most likely reflected an early disinhibition of thymocyte development followed by increased positive/reduced negative selection, at least partly, due to raised thymocyte surface Thy-1 expression. The greater number of CD4+CD25+Foxp3+ cells in both thymus and peripheral blood suggested augmented thymic production of these cells. In addition, an increased CD4+/CD8+ cell ratio was found in the spleen of Ovx rats. Thus, ovarian hormone ablation led not only to increased diversity of the T-cell repertoire, but also to a new balance among distinct T-cell subsets in the periphery.

Peripubertal ovariectomy provides long-term postponement of age-associated decline in thymic cellularity and T-cell output

The present study was undertaken to reassess the recentlychallenged role of ovarian hormones in age-associated thymicinvolution. For this purpose, in eleven-month-old peripubertallyovariectomized(Ox)ratsweanalyzed:i)thymicweightandcellularity,ii)size of CD4+CD8+ double-positive (DP) thymocyte population, whichis believed to correlate to the thymic capacity to export mature T cells,iii) number of recent thymic emigrants (RTEs), and iv) number ofperipheral blood CD4+ and CD8+ lymphocytes. It was found that boththymic weight and cellularity were greater in Ox than in control rats. Inaddition, in Ox rats the numbers of DP thymocytes and both CD4+ andCD8+ RTEs, were significantly greater than in controls, indicating amore efficient generation of T cells in these rats. Furthermore, thesefindings, coupled with data indicating that the number of neither CD4+nor CD8+ peripheral blood lymphocytes was affected by ovariectomy,most likely, suggest a reduced homeostatic proliferation of memorycells in Ox rats, i.e. broadening of TCR peripheral repertoire withoutchanges in the overall number of T cells leading to a more efficientresponse to newly encountered antigens. The results indicate that theovarian steroid deprivation from early peripubertal period leads to along lasting postponement/alleviation of age-associated decline in T-cell mediated immune response.Key words: ovariectomy, ageing, thymic cellularity, recent thymicemigrants