Thymic Rejuvenation Research Articles

Thymic rejuvenation via FOXN1-reprogrammed embryonic fibroblasts (FREFs) to counteract age-related inflammation.

Age-associated systemic, chronic inflammation is partially attributed to increased self-autoreactivity, resulting from disruption of central tolerance in the aged, involuted thymus. This involution causally results from gradually decreased expression of the transcription factor FOXN1 in thymic epithelial cells (TECs), whereas exogenous FOXN1 in TECs can partially rescue age-related thymic involution. TECs induced from FOXN1-overexpressing embryonic fibroblasts can generate an ectopic de novo thymus under the kidney capsule, and intrathymic injection of naturally young TECs can lead to middle-aged thymus regrowth. Therefore, as a thymic rejuvenation strategy, we extended these 2 findings by combining them with 2 types of promoter-driven (Rosa26CreERT and FoxN1Cre) Cre-mediated FOXN1-reprogrammed embryonic fibroblasts (FREFs). We engrafted these FREFs directly into the aged murine thymus. We found substantial regrowth of the native aged thymus with rejuvenated architecture and function in both males and females, exhibiting increased thymopoiesis and reinforced thymocyte negative selection, along with reduced senescent T cells and autoreactive T cell–mediated inflammation in old mice. Therefore, this approach has preclinical significance and presents a strategy to potentially rescue decreased thymopoiesis and perturbed negative selection to substantially, albeit partially, restore defective central tolerance and reduce subclinical autoimmune symptoms in elderly people.

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.

Restoring T Cell Homeostasis After Allogeneic Stem Cell Transplantation; Principal Limitations and Future Challenges

For several leukemia patients, allogeneic stem cell transplantation (allogeneic-SCT) is the unique therapeutic modality that could potentially cure their disease. Despite significant progress made in clinical management of allogeneic-SCT, acute graft-versus-host disease (aGVHD) and infectious complications remain the second and third cause of death after disease recurrence. Clinical options to restore immunocompetence after allogeneic-SCT are very limited as studies have raised awareness about the safety with regards to graft-versus-host disease (GVHD). Preclinical works are now focusing on strategies to improve thymic functions and to restore the peripheral niche that have been damaged by alloreactive T cells. In this mini review, we will provide a brief overview about the adverse effects of GVHD on the thymus and the peripheral niche and the resulting negative outcome on peripheral T cell homeostasis. Finally, we will discuss the potential relevance of coordinating our studies on thymic rejuvenation and improvement of the peripheral lymphoid niche to achieve optimal T cell regeneration in GVHD patients.

Measurement of fat fraction in the human thymus by localized NMR and three-point Dixon MRI techniques

PurposeTo develop a protocol to non-invasively measure and map fat fraction, fat/(fat+water), as a function of age in the adult thymus for future studies monitoring the effects of interventions aimed at promoting thymic rejuvenation and preservation of immunity in older adults. Materials and methodsThree-dimensional spoiled gradient echo 3T MRI with 3-point Dixon fat-water separation was performed at full inspiration for thymus conspicuity in 36 volunteers 19 to 56 years old. Reproducible breath-holding was facilitated by real-time pressure recording external to the console. The MRI method was validated against localized spectroscopy in vivo, with ECG triggering to compensate for stretching during the cardiac cycle. Fat fractions were corrected for T1 and T2 bias using relaxation times measured using inversion recovery-prepared PRESS with incremented echo time. ResultsIn thymus at 3 T, T1water = 978 ± 75 ms, T1fat = 323 ± 37 ms, T2water = 43.4 ± 9.7 ms and T2fat = 52.1 ± 7.6 ms were measured. Mean T1-corrected MRI fat fractions varied from 0.2 to 0.8 and were positively correlated with age, weight and body mass index (BMI). In subjects with matching MRI and MRS fat fraction measurements, the difference between these measurements exhibited a mean of −0.008 with a 95% confidence interval of (0.123, −0.138). Conclusions3-point Dixon MRI of the thymus with T1 bias correction produces quantitative fat fraction maps that correlate with T2-corrected MRS measurements and show age trends consistent with thymic involution.

Genetically Enhanced Pluripotent Stem Cell-Derived T Lymphocytes for Off-the-Shelf Cellular Immunotherapy

Cellular immunotherapies are poised to transform the treatment of cancer and immunological disorders. In the most promising setting to date, genetic modification to the T lymphocytes in the form of chimeric antigen receptors (CAR) has dramatically increased therapeutic efficacy with reported initial complete remission rates in acute lymphoblastic leukemia ranging between 80-100%. However, pressing challenges remain to be solved to ensure that engineered T-cell immunotherapies can be cost-effectively and consistently manufactured, and safely and reliably delivered at the scale necessary to support wide patient base commercialization.Human induced pluripotent stem cell (hiPSC) derived T lymphocytes represent a unique, renewable source of genetically engineered T cells for "off-the-shelf" immunotherapy. Through the precise genetic engineering at the hiPSC stage, clonal and uniform populations of modified cell lines can be banked and reliably tapped into on demand to generate highly efficacious T cells for therapeutic applications. Although great progress has been made, several challenges need to be addressed including the ability to enhance effector function through genome-engineering of persistence, targeting, histocompatibility and controlled safety mechanisms at the hiPSC juncture while retaining the capacity to efficiently and reproducibly generate the intricate stages of lymphocyte development in an accurate and scalable process. We have previously demonstrated that our proprietary reprogramming platform supports efficient and rapid derivation of clonal hiPSC lines with properties indicative of the naïve state of pluripotency. In addition to maintaining a homogeneous population of hiPSCs, our platform enables efficient multi-gene and multi-loci targeted engineering at a single cell level resulting in clonal population of pluripotent cell lines with desired genetic attributes.Here we will provide an update on our "off-the-shelf" T-cell immunotherapy preclinical program where engineered hiPSC lines are uniquely used as the renewable starting material. We will also highlight our novel differentiation platform to derive definitive hematopoietic progenitor cells termed hemogenic endothelium (HE); a well-defined, small molecule-driven, staged process that is currently being translated into cGMP (current good manufacturing practice) settings. The highly efficient differentiation system (on average >65% hiPSC to CD34 conversion) delivers approximately 100 CD34+ HE cells per each input hiPSC, representing a highly scalable process that is further expanded during lymphocyte differentiation and maturation. To validate that the iCD34+ HE is definitive in nature we demonstrate that during further hematopoietic differentiation the emerging CD43+ hematopoietic cells exhibit Notch dependency and high expression of key genes such as MYB and the HOXA cluster, found only in definitive hematopoietic progenitors. The hiPSC-derived HE exhibits multi-lineage potential and can be successfully cryopreserved and banked, serving as a highly-stable cell bank for subsequent therapeutic use. Through genetic modifications at the single cell hiPSC stage, we confer antigen-specificity via the expression of temporally inducible CARs as premature expression of CAR proteins during in vitro differentiation has been found to skew development towards innate-lymphoid like lineages. Utilizing our stage-specific hematopoietic differentiation platform we have identified the optimal developmental window to induce the expression of CAR proteins to maintain optimal differentiation towards functional effector lymphocytes. The hiPSC-derived engineered T lymphocytes are currently under preclinical investigation for in vitro and in vivo effector function including thymic rejuvenation, T cell repertoire repopulation, target specific recognition and enhanced killing potential. Preliminary data suggests that hiPSC-derived lymphocytes are functional and can home to their respective niche to support initial repopulation in vivo. Our study continues to support that naïve hiPSCs are an ideal renewal source for "off-the-shelf" hematopoietic cell-based immunotherapies and represent a potentially exponential advancement in adoptive T cell therapy. DisclosuresClarke:Fate Therapeutics: Employment. Groff:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics: Employment. Bauer:Fate Therapeutics: Employment. Lee:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Burrascano:Fate Therapeutics: Employment. Abujarour:Fate Therapeutics: Employment. Bonello:Fate Therapeutics: Employment. Robinson:Fate Therapeutics: Employment. Foster:Fate Therapeutics: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Wolchko:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Valamehr:Fate Therapeutics, Inc: Employment.

Role of Bone Marrow Maturity, Insulin-Like Growth Factor 1 Receptor, and Forkhead BoxProtein N1 in Thymic Involution and Rejuvenation.

Thymic involution is associated with age-related changes of the immune system. Utilizing our innovative technique of transplantation of a thymus as an isolated vascularized graft in MHC-inbred miniature swine, we have previously demonstrated that aged thymi are rejuvenated after transplantation into juvenile swine. Here we have studied the role of insulin-like growth factor (IGF) and forkhead-box protein-N1 (FOXN1) as well as bone marrow (BM) in thymic rejuvenation and involution. We examined thymic rejuvenation and involution by means of histology and flow cytometry. Thymic function was assessed by the ability to induce tolerance of allogeneic kidneys. Aged thymi were rejuvenated in a juvenile environment, and successfully induced organ tolerance, while juvenile thymi in aged recipients involuted and had a limited ability to induce tolerance. However, juvenile BM inhibited the involution process of juvenile thymi in aged recipients. An elevated expression of both FOXN1 and IGF1 receptors (IGF-1R) was observed in juvenile thymi and rejuvenated thymi. Juvenile BM plays a role in promoting the local thymic milieu as indicated by its ability to inhibit thymic involution in aged animals. The expression of FOXN1 and IGF-1R was noted to increase under conditions that stimulated rejuvenation, suggesting that these factors are involved in thymic recovery.

The rejuvenating effects of leuprolide acetate on the aged baboon's thymus

BackgroundWe have previously demonstrated that the juvenile thymus plays an essential role in tolerance induced by both renal transplantation and a short course of calcineurin inhibitors. Aged thymi have a decreased ability to induce tolerance. Luteinizing hormone-releasing hormone (LHRH) is known to pharmacologically rejuvenate the thymus in rodents. In order to develop a clinically applicable regimen of transplantation tolerance in adults, we sought to determine if thymic rejuvenation would occur with LHRH agonism in non-human primates. Methods and resultsThymic rejuvenation was evaluated by magnetic resonance imaging (MRI), histology, as well as in-vitro cellular and molecular tests. Four aged male hamadryas baboons underwent subcutaneous injection of a 3-month depot of Lupron (11.25mg; LI) and were followed for 3months. Thymi increased volumetrically by MRI. After LI, thymic cellularity markedly increased within the cortical and medullary thymus. Additionally, a significant increase in the CD4+/CD45RAhi+ population in the peripheral blood occurred for 50days after LI, and flow cytometry of thymic tissue revealed a large increase in the percentage of CD4+/CD8+ cells. TREC assay corroborated enhancement in thymic function. ConclusionThese data indicate that LI is associated with thymic rejuvenation in baboons, and further confirm that extrinsic factors play an important role in thymic rejuvenation in a non-human primate model.

Diversity and clonal selection in the human T-cell repertoire.

T-cell receptor (TCR) diversity, a prerequisite for immune system recognition of the universe of foreign antigens, is generated in the first two decades of life in the thymus and then persists to an unknown extent through life via homeostatic proliferation of naïve T cells. We have used next-generation sequencing and nonparametric statistical analysis to estimate a lower bound for the total number of different TCR beta (TCRB) sequences in human repertoires. We arrived at surprisingly high minimal estimates of 100 million unique TCRB sequences in naïve CD4 and CD8 T-cell repertoires of young adults. Naïve repertoire richness modestly declined two- to fivefold in healthy elderly. Repertoire richness contraction with age was even less pronounced for memory CD4 and CD8 T cells. In contrast, age had a major impact on the inequality of clonal sizes, as estimated by a modified Gini-Simpson index clonality score. In particular, large naïve T-cell clones that were distinct from memory clones were found in the repertoires of elderly individuals, indicating uneven homeostatic proliferation without development of a memory cell phenotype. Our results suggest that a highly diverse repertoire is maintained despite thymic involution; however, peripheral fitness selection of T cells leads to repertoire perturbations that can influence the immune response in the elderly.

Mechanisms shaping the naïve T cell repertoire in the elderly — Thymic involution or peripheral homeostatic proliferation?

The ability of the human immune system to repel infections is drastically diminished with age. Elderly individuals are more susceptible to new threats and are less able to control endogenous infections. The thymus, which is the sole source of new T cells, has been proposed as a target for regenerative efforts to improve immune competence, as thymic activity is dramatically reduced after puberty. In this review, we review the role of the thymus in the maintenance of T cell homeostasis throughout life and contrast the differences in mice and humans. We propose that in humans, lack of thymic T cell generation does not explain a decline in T cell receptor diversity nor would thymic rejuvenation restore diversity. Initial studies using next generation sequencing are beginning to establish lower boundaries of T cell receptor diversity. With increasing sequencing depth and the development of new statistical models, we are now in the position to test this model and to assess the impact of age on T cell diversity and clonality.

Reversal of age-related thymic involution by an LHRH agonist in miniature swine

Background and aims of study We have previously demonstrated a requirement for the presence of a juvenile thymus for the induction of transplantation tolerance to renal allografts by a short-course of calcineurin inhibition in miniature swine. We have also shown that aged, involuted thymi can be rejuvenated when transplanted as vascularized thymic lobes into juvenile swine recipients. The present studies were aimed at elucidating the extrinsic factors facilitating this restoration of function in the aged thymus. In particular, we tested the impact of sex steroid blockade by Luteinizing Hormone-Releasing Hormone (LHRH). Materials and methods 30 naive animals (25 males and 5 females) were used for measurement of serum testosterone levels. 3 mature male pigs (aged at 22, 22 and 29 months old) were used to test the effects of Lupron (LHRH analog) injection at 45 mg (per 70–80 kg body weight) as a 3-month depot on testosterone levels and thymic rejuvenation. Thymic rejuvenation was assessed by histology, flow cytometric analysis, morphometric analysis and TREC assays. Results Hormonal alterations were induced by Lupron and resulted in macroscopic and histologic regeneration of the thymus of aged animals within 2 months, as evidenced by restoration of juvenile thymus architecture and increased cellularity. Two animals that were evaluated for TREC both showed increased levels in the periphery following Lupron treatment. Conclusion Treatment of aged animals with Lupron leads to thymic rejuventaion in adult miniature swine. This result could expand the applicability of thymus-dependent tolerance-inducing regimens to adult recipients.

Deficient Ghrelin Receptor-mediated Signaling Compromises Thymic Stromal Cell Microenvironment by Accelerating Thymic Adiposity

With progressive aging, adipocytes are the major cell types that constitute the bulk of thymic microenvironment. Understanding the origin of thymic adipocytes and mechanisms responsible for age-related thymic adiposity is thus germane for the design of long lasting thymic rejuvenation strategies. We have recently identified that ghrelin, an orexigenic anti-inflammatory peptide, can partially reverse age-related thymic involution. Here we demonstrate that Ghrl and ghrelin receptor (growth hormone secretagogue receptor (GHSR)) are expressed in thymic stromal cells and that their expression declines with physiological aging. Genetic ablation of ghrelin and GHSR leads to loss of thymic epithelial cells (TEC) and an increase in adipogenic fibroblasts in the thymus, suggesting potential cellular transitions. Using FoxN1Cre;R26RstopLacZ double transgenic mice, we provide qualitative evidence that thymic epithelial cells can transition to mesenchymal cells that express proadipogenic regulators in the thymus. We found that loss of functional Ghrl-GHSR interactions facilitates EMT and induces thymic adipogenesis with age. In addition, the compromised thymic stromal microenvironment due to lack of Ghrl-GHSR interactions is associated with reduced number of naive T cells. These data suggest that Ghrl may be a novel regulator of EMT and preserves thymic stromal cell microenvironment by controlling age-related adipocyte development within the thymus.

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.

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.

Centenarians as a model for healthy aging.

For over 10 years we have studied centenarians as a model to address the biological basis of aging and longevity, with particular attention to immunology and genetics. The most important findings can be summarized as follows. (i) Human immunosenescence represents a complex remodelling, whereby clonotypical immunity deteriorates, while ancestral, innate immunity is largely preserved. (ii) Continuous exposure to antigens causes a lifelong, chronic antigenic stress, which is responsible, together with the involution of the thymus, for the accumulation of memory/effector T cells and the exhaustion of naïve T cells. (iii) Aging is characterized by a peculiar chronic inflammatory status that we propose to call 'inflammaging', which appears to be under genetic control, is detrimental for longevity and is more evident in men than in women. Inflammaging, i.e. the up-regulation of a variety of anti-stress responses at the cellular and molecular level, is the consequence of the ability of the body to adapt to and counteract the effects of a variety of stressors, which causes the accumulation of molecular and cellular scars. Inflammaging is considered the common and most important driving force of age-related pathologies, such as neurodegeneration, atherosclerosis, diabetes and sarcopenia, among others, all of which share an inflammatory pathogenesis. (iv) Possible strategies to counteract the major effects of immunosenescence and inflammaging, such as the systematic reduction of the lifelong antigenic load, the elimination of chronic infections, thymic rejuvenation and preventative treatment with anti-inflammatory drugs in people with a pro-inflammatory genotype, are envisaged.