Age-associated Thymic Atrophy Research Articles

Role of paracrine mTOR signaling in regulating thymus size and function.

Abstract The thymus is the primary site of T cell generation, where the unique thymic stromal microenvironment governs T cell differentiation. However, the size of the thymus declines precipitously relatively early in life, resulting in declining production of naïve T cells. Homeostatic mechanisms drive a shift toward an oligoclonal T cell memory, leaving the elderly less responsive to vaccines and new infections. Preventing or reversing age-associated thymic atrophy therefore holds potential for extending the health span. Mechanisms governing thymic atrophy have been difficult to identify because the primary targets of atrophy, cortical thymic epithelial cells (cTECs), are rare and difficult to isolate. Characterization of the transcriptional response of thymic stromal cells during age-related atrophy and experimental regeneration suggested that paracrine mTOR signaling in cTECs promoted by ligands expressed in medullary TECs (mTECs) may be required to maintain the cellular architecture of the cortical stroma and preserve thymus size. To test these hypotheses, we generated two new tissue-specific transgenic mouse lines overexpressing mTOR-activating growth factors (IGF1 and FGF21) in most mTECs. Ongoing flow cytometry and confocal microscopy experiments indicate faithful transgene expression in mTECs, and suggest that mTEC-derived growth factors promote temporally distinct patterns of mTORC1 and mTORC2 signaling in cTECs to promote thymus growth and maintenance with age. This work was supported by NIH R21AI154109 and T32 AI138944-05. Supported by grants from NIH (R21 AI154109, T32 AI138944-05)

Role of paracrine mTOR signaling in regulating thymus size and function.

Abstract The thymus is the primary site of T cell generation, and the unique thymic stromal microenvironment directs T cell differentiation, self-tolerance and self-restriction. However, the size of the thymus declines precipitously relatively early in life, resulting in declining production of naïve T cells. Homeostatic mechanisms drive a shift toward an oligoclonal T cell memory, leaving the elderly less responsive to vaccines and new infections, especially viral infections. Preventing or reversing age-associated thymic atrophy therefore holds potential for extending the healthspan. Mechanisms governing thymic atrophy have been difficult to identify because the primary targets of atrophy, cortical thymic stromal cells, are rare and difficult to isolate. Using an informatic approach to characterize the transcriptional response of thymic stromal cells during age-related atrophy or experimental regeneration, we previously showed that cortical thymic epithelial cells (cTECs) display a unique morphology characterized by extensive looping projections. Atrophy is associated with contraction of these projections, which are renewed during induced regeneration. Further informatic analysis suggested that paracrine mTOR signaling in cTECs promoted by ligands expressed in medullary TECs (mTECs) may be required to maintain the cellular architecture of the cortical stroma and preserve thymus size. To test these hypotheses, we generated two new tissue-specific transgenic mouse lines overexpressing mTOR-activating ligands (IGF1 and FGF21) in most mTECs. Ongoing experiments aim to characterize the impact of increased paracrine mTOR signaling on age-associated thymic dysfunction. This work was supported by N.I.H. R21AI154109. Supported by grant from NIH (R21 AI154109)

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.

A GMCSF and IL7 fusion cytokine leads to functional thymic-dependent T-cell regeneration in age-associated immune deficiency.

The competence of cellular immunity depends on a diverse T-cell receptor (TCR) repertoire arising from thymic output. Normal thymopoiesis arises from marrow-derived CD3−CD4−CD8− triple-negative T-cell progenitors (TN), which develop into mature single-positive (SP) CD4 or CD8 T cells after expressing both CD4 and CD8 (double-positive, DP) transiently, leading to de novo T-cell production. Interleukin-7 (IL7) is a singularly important common γ-chain IL involved in normal thymic development. Our previous work has demonstrated that γc cytokines fused with granulocyte-macrophage colony stimulating factor (GMCSF) at the N-terminus acquire unheralded biological properties. Therefore, to enhance thymopoiesis, we developed a novel biopharmaceutical based on the fusion of GMCSF and IL7, hereafter GIFT7. Systemic administration of GIFT7 leads to cortical thymic hyperplasia including the specific expansion of CD44intCD25− double-negative 1 (DN1) thymic progenitors. During murine cytomegalovirus (mCMV) infection of aged animals, GIFT7-mediated neo-thymopoiesis led to increased absolute numbers of viral-specific CD8+ T cell. Our work demonstrated that thymic precursors can be therapeutically repopulated and its reconstitution leads to meaningful central and peripheral T-cell neogenesis, correcting immune dysfunction arising from age-associated thymic atrophy.

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levels and impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo.

A Novel Synthetic GMCSF and IL7 Fusion Cytokine (GIFT7) Leads to T Cell Neogenesis by Reversing Age-Related Thymic Atrophy and Overcoming PD-1-Assocaited CD8 Exhaustion

AbstractAbstract 3289Functional T cell immunity depends on the diversity of T cell receptor (TCR) repertoire. Numerous cytolytic assaults can perturb effector cell fitness by inhibiting thymic de novo T cell production or predisposing peripheral CD8+ T cells to exhaustion. In fact, age-associated thymic atrophy and PD-1 upregulation remain the critical components of immune dysfunction.Normal thymopoiesis arises from marrow-derived CD3−CD4−CD8− triple-negative T cell progenitors (TN) which seed the thymic cortex. Thereafter, TN develop into mature single-positive (SP) CD4 or CD8 T cells after expressing both CD4 and CD8 (double-positive-DP) transiently, leading to de novo T cell production. Interleukin-7 (IL7) is a singularly important common γ-chain interleukin involved in this process. In order to pharmacologically enhance thymopoiesis, we report the development of a novel biopharmaceutical based on the fusion of GMCSF and IL7, hereafter GIFT7. Systemic administration of GIFT7 compared to IL7 in young immune competent mice leads to a transient increase in the number of DN1 (5.48±1.01 v.s. 2.74±1.07 × 106, p=0.046) by day 7. Total thymic cellularity significantly increases in GIFT7-treated group by day 14 (237±51.3 v.s. 123±25.6 × 106, p=0.026). GIFT7-mediated hypertrophic effect is significantly more pronounced in aged thymi: 111±21.8, 67.8±13.9, 60.3±4.6 × 106 for 7-dose of (5ug kg−1) GIFT7, IL7, or PBS treatment respectively. We observed a significant increase in DN1 and DN4 subsets with the greatest fold difference in the CD44int DN1 subset. Histologically, GIFT7 administration leads to significant cortical thymic hyperplasia (cortex: medulla ratio 2.67:1). Functionally, GIFT7 enhances viral-specific CTL responsiveness as modeled by murine cytomegalovirus (MCMV) peptide MHC+ tetramer enumeration (10.4±3.8 v.s. 2.9±1.4 × 106 for GIFT7 and untreated respectively, p<0.05).In the periphery, GIFT7 selectively expand a CD8+ subset with a Central Memory (CM) phenotype defined as CD8+CD44+CD62L+CCR7+KLRG−CD27+PD1−. This unique expansive effect of GIFT7 is also demonstrated on peripheral blood mononuclear cells (PBMC) derived from non-human primates (NHP), in that GIFT7 stimulation of pre-activated NHP-PBMC leads to a two-fold increase in total cell number after 3 days and >80% of Ki67+ expression in both CD4+ and CD8+ compartments. Interestingly, GIFT7-mediated proliferation in CD8+ T cells is accompanied by a 40% decrease in the mean fluorescent intensity (MFI) of PD1 expression.Our work identifies a CD44intCD25− subset of DN thymocytes most responsive to IL7R-mediated STAT5 hypersignalling and their function as T cell progenitors in GIFT7-driven thymic reconstitution. This points to the translational potential of GIFT7 or GIFT7-primed T lineage cells as treatment of immune malfunction. Disclosures:No relevant conflicts of interest to declare.

Age-associated alterations of lipid metabolism and oxidative stress in the murine thymus are attenuated by in vivo growth hormone administration (104.13)

Abstract The mechanisms by which GH facilitates the recovery of thymopoiesis during age-associated thymic atrophy are unknown. Herein, we analyzed the age-related lipid alterations in total thymus and thymocyte preparations derived from 04 to 18 month-old mice, and in thymi of GH-treated aged mice. Electrospray tandem mass spectrometry permitted us to detect increased amounts of tryglicerides (TG), free-cholesterol and C24:1 sphingomyelin in the total thymus of old mice as compared to those of young. Moreover, we verified a decrease in the sulfatide C24:1 and in unknown long-chain ceramides, and an increase in 4-HNE and 8-epi-prostaglandin F2α (PGF) in the thymi of older animals. Using immunohistochemistry, we verified an increase in the expression of TNFα and CD204 within the aged thymus sections. In analyzing thymocytes, we found a decrease in TG, free cholesterol and in sphingomyelin species with age. Interestingly, we detected a significant amount of ceramide C24:1 in thymocytes derived from old mice but not young. Following GH treatment, we observed a significant decrease in TG, free-cholesterol and PGF in the thymus of older animals. GH treatment significantly increased the amount of dihydroceramides and promoted a trend to decrease 4-HNE levels compared to thymi of age-matched denatured GH-treated control mice. These results suggest that increased oxidative stress associated with perturbed lipid metabolism within aged thymus can be attenuated after GH treatment.

Tracing thymic output in older individuals

As a result of age-associated thymic atrophy, T cell production declines with age. Some studies suggest that production undergoes an exponential decline starting at birth, while others consider the decline to be in a biphasic manner with a rapid reduction in output occurring before middle age followed by a phase in which output declines at a regular, albeit much slower, rate. Both approaches provide estimations of the time of termination of thymic output, but on the basis of limited amounts of data. We have analysed blood from more than 200 individuals between the ages of 58 and 104 years to determine changes in thymic output using signal-joint T cell receptor excision circles (sjTREC)/T cells as our measure. To reduce any potential geographical or nutritional bias we have obtained samples from five different European countries. Our results reveal that while the absolute number of T cells per microlitre of blood does not change significantly across the age range we tested, the values of sjTREC per microlitre show wide variation and reveal an age-associated decline in thymic output. In addition we show gender differences, with notably higher thymic output in females than males at each decade. More importantly, we noted a significant decline in sjTREC/T cell levels in those more than 90 years of age in both males and females. Our results provide information about the potential end-point for thymic output and suggest that sjTREC analysis may be a biomarker of effective ageing.

Clonotypic structure of human memory alpha-T cell repertoires to influenza in elderly individuals (45.20)

Abstract The age-associated thymic atrophy and reduced repertoires of naïve T cells could be confounding factors of high morbidity and mortality of older populations due to the seasonal influenza infections. Given that influenza A viral epitope M158-66 induces robust CD8 T cell responses among middle-aged populations, we examined clonotypical α-TCR repertoires among older blood donors. Previously we reported that α-TCR repertoires in middle-age individuals are polyclonal where M158-66 specific T cells were selected as memory cells due to expression JA42 α -chains with poly-G/S/A runs in the CDR3. In the present study we compare α-TCR repertoires between middle-aged and older individuals in respect to Vα family usage and clonotypical diversities. Using CDR3α- spectratyping and sequencing, we found that middle-aged adults possess stable polyclonal response to influenza where diverse JA42 CD8 T cells are distributed among eleven Vα families and VA27 family was dominated in vitro response. In contrary, with aging such dominance was still present in some but not all individuals. Two subjects responses to M158-66 was mediated by VA12.1 family, which represents minor component in the middle-aged cohort. In addition to reduced diversity of VA families, the older subjects posses reduced diversity of JA42 clonotypes. Therefore, we concluded that influenza re-exposure during lifetime induces clonotypical exhaustion of T cells leading to reshaping of T cell repertoire.

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.

Reversal of age-associated thymic atrophy: Treatments, delivery, and side effects

Our ability to survive infectious agents depends on making adequate immune responses, but as we get older our thymus atrophies. Production and export of T cells bearing new antigen receptor specificities to the peripheral T cell pool declines and results in shrinkage of the repertoire. Other changes in the peripheral T cell pool include an increase in cells moving closer to their replicative limit. Age related immune dysfunction, evident through the increased susceptibility to infection, follows these changes. Improvement in immune function in the elderly may require us to rejuvenate the immune system starting first with reversing the atrophy seen in the thymus. This has been achieved experimentally with interleukin 7, growth hormone, growth hormone secretagogues, keratinocyte growth factor or through chemical or surgical castration. The widespread use of one or more of these treatments will depend upon their effectiveness, their ease of delivery and the extent of any side effects.

Reversal of thymic atrophy

Age-associated thymic atrophy is a key event preceding the inefficient functioning of the immune system, resulting in a diminished capacity to generate new T-cells. This thymic involution has been proposed to be due to changes in the thymic microenvironment resulting in its failure to support thymopoiesis. A key cytokine in the early stages of thymocyte development is IL-7 and expression levels are greatly reduced with age. The ability of IL-7 to restore the immune system by enhancing thymic output remains controversial. In this review, we highlight the advances in molecular approaches used to evaluate recent thymic emigrants and assess the success of these strategies in determining whether IL-7 can lead to immune reconstitution.

Age-associated thymic atrophy is linked to a decline in IL-7 production

Age-associated thymic atrophy results in a decline in T lymphocyte output and has been identified as one of the key events that precede inefficient functioning of the immune system in later life. Thymic atrophy is thought to result from a failure of the thymic microenvironment to support thymopoiesis in old age and recent evidence suggests that a decline in interleukin-7 (IL-7) expression may limit thymocyte development by restricting combinations of survival, proliferation and rearrangement of the TCRβ chain. Using RT-PCR and the RNase protection assay, we show that the expression of IL-7 declines with age. Analysis of Connexin 43 expression, a component molecule of gap junctions, whose function is to connect epithelial cells, does not markedly decline with age. These observations suggest that a decline in IL-7 expression is not matched by a similar loss of epithelial cells. These results in conjunction with other studies lead us to speculate that IL-7 producing MHC class II positive TECs are being replaced by cells that do not have this capacity.

Age-Associated Thymic Atrophy Is Not Associated with a Deficiency in the CD44+CD25−CD3−CD4−CD8− Thymocyte Population

Age-associated thymic atrophy has been proposed to be due to changes in both the thymic microenvironment and in the intrinsic properties of the early T cell progenitors, the CD44+CD25−CD3−CD4−CD8− cells. We have purified these cells from the thymus of both old and young mice and demonstrate no age-associated defect in their ability to differentiate into their progeny in vitro when used to reconstitute fetal thymic organ cultures. We also demonstrate that in the presence of anti-IL-7, CD44+CD25−CD3−CD4−CD8− cells from young mice show reduced thymocyte development in fetal thymic organ cultures compared with controls. Finally we have shownthat old mice treated with IL-7 show improved thymopoiesis compared with control groups. The increased thymopoiesis seen in the old animals occurs in the sequential manner which would be anticipated for an agent working directly on the early stages, including the CD44+CD25−CD3−CD4−CD8− cells.

Gender-related differences in the rates of age associated thymic atrophy.

Age associated thymic atrophy has been shown to be linked to problems with rearrangement of the β chain of the T cell receptor (TCR) in male mice during the early phases of the intrathymic T cell developmental pathway. In this study, thymic atrophy in female mice was found to occur at a different rate than in male mice. At 9 months of age there was a significantly greater number of cells in the thymus of female mice compared with male mice, with the major difference found in the CD4+CD8+ populations. The thymii of female mice at 9 months of age contained double the number of these cells compared with male mice. Analysis of the CD4+CD8+ cells at 9 months of age demonstrated increased numbers of cells expressing higher levels of CD3 in females compared with males indicating that in females more of these cells were producing successful αβTCR pairings. In F5 transgenic mice comparison of the CD4+CD8+ population revealed no significant difference in their absolute numbers at 9 months of age. These results indicate that the gender differences at this time point were due to fewer permitted divisions prior to the expression of a selectable TCR α chain within the CD4+CD8+ populations in male compared with female mice. This gender difference was not due to the action of testosterone and unlikely to be due to differences in the level of oestrogen. The potential mechanisms of this difference may be related to a regulatory feedback of peripheral T cells on the developing thymocyte populations. Such age related changes in the numbers of cells within distinct thymic subpopulations leads to the possibility that the potential repertoire in females is greater than in males later in life.

Age-associated thymic atrophy in the mouse is due to a deficiency affecting rearrangement of the TCR during intrathymic T cell development.

Involution of the thymus is a feature of age and precedes inefficient functioning of the immune system. C57BL/10 mice show an 83% reduction in number of thymocytes between 3 and 20 mo of age, with a significant decline in each of the thymic subsets defined by their expression of CD4 and CD8. The similar percentage contribution of each subset to the whole at 3, 12, and 20 mo suggests a lesion in the T cell developmental pathway within an early subset. The CD3- CD4- CD8- subset showed a significant decline in number by 20 mo of age, but despite this reduction, no significant difference was noted in the number of CD44+ CD25- cells, the earliest stage of this subset between 3 and 20 mo of age. A significant decline in the number of their progeny, the CD44+ CD25+, and the progeny of these cells, the CD44- CD25+ cells, was noted by 12 mo of age. Expression of CD25 within this subset is associated with rearrangement of TCR beta-chain genes. F5 transgenic mice, carrying a complete TCR-alphabeta transgene under the control of a CD2 minigene cassette on a C57BL/10 background, showed no age-associated thymic atrophy in any of the defined thymic subsets over the same period as the normal C57BL/10 mice. Similar results were noted with mice carrying the same transgene but which in addition were also RAG-1-. The results indicate that age-associated thymic involution was associated with problems with rearrangement of the TCR beta-chain genes affecting the production of thymocytes.