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Seminars in Immunology 19 (2007) 331–340

Review

Impact of niche aging on thymic regeneration andimmune reconstitution

Ann Chidgey ∗, Jarrod Dudakov, Natalie Seach, Richard BoydMonash Immunology and Stem Cell Laboratories (MISCL), Level 3, Bldg-75, Monash University,

Wellington Road, Clayton, Melbourne 3800, Australia

bstract

The immune system undergoes dramatic changes with age—the thymus involutes, particularly from puberty, with the gradual loss of newlyroduced naı̈ve T cells resulting in a restricted T cell receptor repertoire, skewed towards memory cells. Coupled with a similar, though lessramatic age-linked decline in bone marrow function, this translates to a reduction in immune responsiveness and has important clinical implicationsarticularly in immune reconstitution following cytoablation regimes for cancer treatment or following severe viral infections such as HIV. Givenhat long-term reconstitution of the immune system is dependent on the bi-directional interplay between primary lymphoid organ stromal cells

nd the progenitors whose downstream differentiation they direct, regeneration of the thymus is fundamental to developing new strategies for thelinical management of many major diseases of immunological origin. This review will discuss the impact of aging on primary lymphoid organiches and current approaches for thymic regeneration and immune reconstitution.

2007 Elsevier Ltd. All rights reserved.

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eywords: Thymus; Involution; Aging; Regeneration; Sex steroids

. Introduction

Central to age related changes in the immune system is therofound atrophy of thymic tissue, most significant from pubertyhen sex steroids increase in production. The thymus under-oes a loss of total cellularity with a disproportionate loss inhymic epithelial cells (TEC) forming the stromal microenvi-onment crucial to thymopoiesis [1]. Co-incident with this ishe reduced export of newly derived naı̈ve T cells and even-ually a skewing towards memory T cells in the peripheryriven by progressive contact with environmental pathogens.hilst the residual thymic tissue remains active in adults,

he reduced production and export of T cells can also facili-ate the clonal expansion of memory T cells in the peripherys a result of vacant niches – further limiting the complex-ty of the T cell receptor repertoire. The collective impact ofhis loss of thymus function and T cell repertoire skewing is

subsequent decline in immune responsiveness. The clinicalmplications typically include delayed immune recovery frommmunodeficiency states induced by cytotoxic antineoplastic

∗ Corresponding author. Tel.: +61 3 9905 0628; fax: +61 3 9905 0780.E-mail address: ann.chidgey@med.monash.edu.au (A. Chidgey).

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044-5323/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.smim.2007.10.006

reatment and after chronic infection such as by the humanmmunodeficiency virus (HIV), where prolonged immunod-ficiency can lead to opportunistic infections and contributeo increased morbidity and mortality. Other clinical indica-ions include a reduced response to vaccinations in adults andncreased incidence of major diseases such as cancer and autoim-

unity.Normalisation of T cell numbers in the periphery solely

hrough the expansion of the pre-existing T cell pool, doesot replenish the T cell receptor repertoire and ability to gen-rate new immune responses. As such, more recent researchas involved identifying methods to improve immune recon-titution by regeneration of the thymic tissue itself to increaseroduction of naı̈ve T cells or by passive T cell transfers, toeplenish the diminishing T cell numbers and repertoire. Theone marrow (BM) produces the progenitor cells that ulti-ately develop into T cells and so can influence the quantity

nd quality of T precursors seeding the thymus. Hence, mini-al damage by clinical conditioning treatments to both the BM

nd thymic niche should be considered in all immune recovery

trategies. This review will highlight the importance of both theone marrow and thymic niches in repairing/regenerating a dys-unctional immune system and current strategies for immuneeconstitution.

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32 A. Chidgey et al. / Seminars i

. Aging of the immune system

.1. The thymic niche

The adult thymus is a three-dimensional epithelial network ofiscrete medullary and cortical regions with contributions fromesenchyme-derived fibroblasts, resident macrophages and

ells of neuroendocrine origin [2]. The thymic microenviron-ent provides specialized ‘niche’ space where haematopoieticcell progenitors undergo expansion and progress through pro-

rammed stages of T cell development, critically dependentpon interactions with specific thymic stromal compartments.n return, thymocyte subsets also play a role in the develop-ent and maintenance of the thymic stroma in a bi-directional

nterplay deemed ‘crosstalk’ [3].The thymic primordium forms from an outpocketing of the

hird pharyngeal pouch endoderm at around embryonic day0.5 (E10.5) and whilst cell lineage analysis has establishedhe physical contribution of neural-crest-derived mesenchymeo the thymic capsule [4] and perivascular connective tissue4,5], current theories exclude ectodermal contribution to thehymic anlage [6–8]. At around E12 of gestation, the thymicudiment is first colonized by a wave of haematopoietic pro-enitors [9]. At this stage TEC are still immature and containi-potent progenitors for subsequent generation of segregatededullary and cortical regions typical of the adult thymus [10].arly thymocytes are not required for the initial patterning of

he thymic epithelium [11], however, contribution from cellsf mesenchymal origin is essential for their proliferation andxpansion, and ablation of this neural-crest-derived contributionesults in severe thymic aplasia [12,13]. Thymic mesenchyme-ependent production of fibroblast growth factor (FGF) familyembers, FGF10 and receptor FGFR2IIIb, were found to be

ritical for TEC proliferation in the embryo [14–17], however,recent study of the E12 thymus suggests a critical role for

nsulin-like growth factor (IGF)-1 and -2 [18], in place of FGF.n these studies mesenchymal–epithelial interactions were foundo be essential for the expansion but not differentiation of embry-nic TEC, as T cell development was able to proceeds normally,lbeit at severely reduced levels due to limiting intrathymic nichepace. Beyond this stage, interactions with thymocytes are abso-utely required for proper organization of adult thymic stromalompartments, with blocks at specific stages of thymocyte devel-pment corresponding with defined blocks in thymic epithelialevelopment [1,19].

Proper development of thymic medulla is of particular impor-ance due to the role in negative selection of autoreactive Tells by medullary epithelium (mTEC) and dendritic cells (DC).olecular control of medullary formation has centred pre-

ominately on signalling through nuclear factor-kB (NF-kB)embers, a family of transcription factors implicated in the

evelopment and maintenance of immune organs. To this end,ice deficient or mutant for NF-kB members or upstream sig-

alling components demonstrate various degrees of perturbededullary formation (reviewed in [20]). Specifically, promiscu-

us gene expression (PGE) of ectopic self-antigens is a uniqueeature of mTEC and is essential for tolerance of T cells to

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unology 19 (2007) 331–340

eripheral organs and tissue. So far, the only known mediatorf PGE is Aire (autoimmune regulator), a transcription factorhose monogenic loss results in severe multi-organ autoimmuneisease [21]. Control of Aire-expressing mTEC has been contro-ersially linked to lymphotoxin signalling via NF-kB [22,23],owever recent reports demonstrate convincing evidence for reg-lation via RANK ligand signals (receptor activator of NF-kB)ound on a population of thymic CD4+ CD3− inducer cells.onsistent with this, RANK deficiency in TECs promotes thenset of autoimmunity [24].

Research on T cells and thymic stromal cells that constitutehe specific inductive niche through which T cells develop, hasetter defined our understanding of the aging process. Whilsthe aged thymus is severely atrophic and infiltrated with adiposeells, all T cell subsets are present—albeit reduced in number.he architectural structure and integrity of the thymic microenvi-

onment, however, are severely disrupted [25]. The proportionf CD45− stromal cells does not change dramatically in theost-natal mouse thymus, that is, both thymocytes and stro-al cells are proportionally reduced with age. However, the

pithelial to fibroblast cell ratio within the stromal populationecreases, resulting in a disproportionate loss of the epithelialells crucial to the development and selection of thymocytes,ncluding the MHC II high-expressing mTECs [1]. The latterlso express Aire and hence Aire-dependent self-tissue restrictedntigens (TRAs), important in maintaining self-tolerance. Thelear delineation of functionally distinct microenvironmentsresent in the young thymus is also severely disrupted in theged. Therefore, not only is there a loss in immune respon-iveness with age, but also an increased risk of autoimmunityhrough escape of potentially autoreactive cells from the dis-upted thymic microenvironment. A reduction in the productionf regulatory T cells may also contribute to the latter.

In humans, reduction in TEC mass begins as early as 2 yearsn age with some acceleration associated with puberty, suchhat by 40–50 years in age, the thymus is producing less than0% of its maximal capacity [26]. Thymic involution has beeninked with increased sex steroid production from puberty inoth males and females, and loss of cytokines and/or growthactors such as interleukin-7 (IL7) and growth hormone (GH)reviewed in [27]). However, there is still some controversy aso whether these are the direct causes for atrophy or whethert is due to intrinsic haematopoietic defects that affect theifferentiation and proliferative potential of T cell precursors28].

While the residual atrophic thymus may be sufficient to main-ain some level of immune competence, by 30–40 years of agehis is severely compromised with minimal recovery likely fromamaging immunodepletive regimes such as chemotherapy andrradiation in cancer treatment or severe viral infections.

.2. Haematopoietic progenitor cells

The BM, and its progenitors are critical for maintainingaematopoiesis throughout adult life. Despite an overall increasen the frequency of haematopoietic stem cells (HSCs) with age,t is becoming increasingly clear that intrinsic changes within

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SCs or their immediate progenitor progeny, contribute towardshe degeneration in both B- and T-lymphopoiesis.

There are five times the numbers of HSCs in the aged, com-ared to the young mouse [29–31], however, they exhibit roughlyne quarter the homing and engraftment efficiency [32]. Stud-es examining the function of aged HSCs have found that whenransplanted into an unablated young congenic recipient, totalonor reconstitution was consistently diminished. Furthermore,n a competitive transplant setting aged BM was severely com-romised in the ability to repopulate the immune system whenompeted with young [30,31,33]. A change in HSC phenotypes also evident with reduced expression of fms-like tyrosineinase 3 (Flt3) with age, corresponding to a decrease in the mul-ipotent progenitor (MPP) population of Lin−Sca1+ckit+ cells[30]; Dudakov, unpublished observations). Within the commit-ed lymphoid progenitor populations, there is a large reduction inhe number and frequency of the common lymphoid precursor-

(CLP1), a decline in their responsiveness to IL7 as well as aecrease in their proliferative potential [34,35].

The significant decline in ability to give rise to peripheral B-ymphocytes while exhibiting an increased propensity to gener-te CD11b+ (Mac-1) myeloid cells was further confirmed by thebservation that granulocyte/macrophage precursors (GMPs)ncrease with age [30]. This appears to be lineage specific witho corresponding change in the common myeloid progenitorr megakaryocyte/erythroid precursor. Furthermore, increasedyeloid engraftment with age has led to the postulation that

opulations of phenotypically indistinct myeloid-biased stemells progressively accumulate [31,36]. These findings wereeflected in gene profiles of young and aged long-term HSCsLT-HSCs), particularly with regard to an upregulation ofyeloid-associated genes with a corresponding downregulation

f lymphoid-associated genes with age [30]. In turn this led tohe clinical hypothesis that it is these myeloid-restricted HSCshat may be the transforming site in myelogenous leukaemias37,38]. Intrinsic changes such as increases in p53 have beenmplicated in reducing the repopulation potential of HSCs [39]nd accumulation of mitochondrial DNA mutations with age isssociated with a loss of function [40].

The exact mechanism of the aging of HSCs is unclear, how-ver, changes in the cellularity of the niche and hence signallingotential are likely to play a major role, in parallel with intrinsichanges in the HSCs themselves.

.3. Thymic seeding

Throughout life, progenitors continually leave the BM andeed the thymus to maintain thymopoiesis. Indeed, there appearso be a cyclic mobilization and gated importation of T cell pro-enitors into the thymus, suggesting a thymus-BM feedbackoop (reviewed in [41]). The exact nature of this feedback loopas yet to be fully elucidated but it appears that intrathymic nichevailability influences the opening of intrathymic microvascular

IMV) gates that allow the importation of blood-borne progen-tors [42–44]. Blood-borne progenitors which seed the thymusxpress CD44 [45], CCR9 [46–48], Flt3 [49,50] and PSGL1 [51]n HSCs and expression of their ligands by thymic stromal cells

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unology 19 (2007) 331–340 333

ave been implicated in their entry into the thymus. It has alsoeen proposed that diffusible chemokines secreted by the tripleegative-2 (TN2) subset of immature thymocytes may influencehe egress of HSCs from the BM by acting in a mobilizing way,n molecules such as VLA-4 [52], CXCR4 and possibly CD44reviewed in [41]).

.4. Bone marrow niche

Alterations in the BM niche leading to a reduction in BM-erived T cell lineage precursors being produced and emigratingo the thymus may also be a contributing factor to the overallecline in the immune system. The post-natal BM niche rep-esents a complex specialised microenvironment that provideshe signals required for the maintenance, proliferation, differen-iation and mobilization of HSCs, essential for the continuousroduction of circulating blood and immune cells—includingymphocytes and myeloid cells, throughout life (Fig. 1). Thendosteal niche is comprised of spindle shaped N-cadherin+

steoblasts (SNO) and their interaction with LT-HSCs includeD44-hyaluronic acid/osteopontin, CXCR4-SDF1, ckit-mSCF,LA4-VCAM1 and N-cadherin (reviewed in [53]). Adminis-

ration of such exogenous ligands has profound implicationsor modifying haematopoiesis. While osteopontin, which is pro-uced by SNO cells, is considered to be a negative regulator ofhe HSC niche by inhibiting HSC proliferation, it is also impor-ant for transmarrow migration towards the endosteal regionfter transplant [54]. Disruption of the molecules involved inhe tethering of HSCs to the niche components results in anncrease in mobilization of HSCs into the periphery. These find-ngs have led to the widespread clinical use of mobilizing agentsuch as the CXCR4 antagonist AMD3100 [55], as well as gran-locyte colony stimulating factor (G-CSF) and even low doseyclophosphamide, which promote the release of neutrophil pro-eases which cleave receptors from the surface of haematopoieticrogenitor cells such as the c-kit receptor, CD117 [56,57]. Com-atible with reduced engraftment with age, is the finding thatSC mobilization from the bone marrow to peripheral blood

n response to G-CSF is increased in aged mice and correlatesith reduced adhesion of HSCs to BM stroma [58]. Mesenchy-al stem cells (MSCs) are also an important component of theM niche—not only for the generation of niche stromal cells,ut also they have been proposed to promote engraftment andavour immune reconstitution following allogeneic transplanta-ion [59,60]. Hence BM niche dysfunction can have far reachinglinical consequences.

Within the BM vascular niche stroma, both a decrease inroduction of IL7 [61,62], and a decline in the responsivenesso IL7 with age [63] have been reported, as well as a differentialesponsiveness of developing B cells to the effects of TGF�.his leads to the development of fewer new B cells and hence aignificant decline in the number of recent BM emigrants exitingnto the periphery. The cumulative effect of reduced production

nd output of B cells from the BM, and subsequent maintenancef peripheral B cell numbers, leads to a severely decreased B celleceptor repertoire with age [64–66]. Furthermore, the recoveryf the B cell receptor repertoire following cyclophosphamide-

334 A. Chidgey et al. / Seminars in Immunology 19 (2007) 331–340

Fig. 1. Model of BM homing, engraftment, retention and mobilization. (1) Peripheral haematopoietic stem cells (HSCs), either intravenously transferred or endoge-nously mobilised, can home into and engraft the BM, a process mediated by expression of molecules such as VLA-4, E-selectin, P-selectin, CXCL12 on BM stromaand CXCR4, CD44 and PSGL1 on HSCs. (2) Once in the BM, HSCs migrate across the marrow to lodge in either endosteal or sinusoidal niches in a processknown as transmarrow migration which is mediated by interactions between CXCR4 and CXCL12, calcium-sensing receptor and hyaluronic acid. (3) Once lodgedin the endosteal niche interactions between HSCs and specialised niche cells, such as CAR (CXCL12 abundant reticulocytes) and SNO (spindle-shaped, N-cadherin+

osteoblasts) cells determine quiescence or differentiation of the HSC. (4) Differentiation of MPPs into either myeloid or lymphoid committed progenitors is dependenton upregulation of lineage specific transcription factors as well as growth factors provided by the supporting microenvironment. Factors such as G-CSF, GM-CSFand EPO drive MPPs down the myeloid lineage while IL7, Flt3L and CXCL12 have all been implicated in lymphoid commitment. (5) HSCs can be mobilised out ofthe BM and into the periphery in either an inducible (using agents such as G-CSF, cyclophosphamide and CXCR4 antagonists) or an endogenous fashion for thymics tionsO ell dee ly imp

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eeding or extra-medullary haematopoiesis. Mobilisation is mediated by interacnce out in the periphery, HSCs are able to home to and seed the thymus for T-c

xpression of molecules such as PSGL1, CCR9 and CD44 on HPCs are critical

nduced immunodepletion is severely decreased in aged mice61]. It has also been observed that, in humans, the antibodyesponse to an initial hepatitis B vaccine is markedly decreasedhen given for the first time to aged patients [67], although thisay also entail defective T-helper cell responsiveness.

. Challenges facing haematopoietic stem cellransplantation (HSCT)

While changes in an aging immune system may not impactramatically on healthy individuals, recovery from immun-depletion states, such as chemo- and radiotherapy, is severelyompromised. High morbidity and mortality are associated with

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between molecules such as ckit-mSCF, VLA4-VCAM1, CXCR4-CXCL12. (6)velopment and, while the identity of the thymus seeding precursor is unknown,ortant for entry of these cells.

blation of the haematopoietic immune system of the recipientrior to bone marrow or HSC transplantation and delay inmmune reconstitution post-transplantation. Opportunisticnfections, inflammation and reactivation of latent viral andarasitic infections are associated with this period of immuneeficiency.

Furthermore, there is a reduction in successful HSCransplantation (HSCT) due to the toxicity of the chemother-py/conditioning regimen on the BM. This causes unacceptable

orbidity in older patients and a two to threefold reduction in

uccessful homing and engraftment of HSC, compared to theoung. This deterioration of the BM is most likely due to aombination of alterations in both extrinsic and intrinsic fac-

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ors effecting HSC status, very likely influenced by the damagend degeneration of the microenvironments in which these cellsevelop.

Consistent with this, we have found an alteration in BM stro-al cellularity with age and after chemotherapy (Chidgey et

l., in preparation)—both in the endosteal niche, important forong-term HSC survival and maintenance, and in the vasculariche, that supports B lymphopoiesis. Given the importance ofoth osteoblasts and endothelial cells in the regulation of stemell adhesion, homing and engraftment following BMT, this lossn cellularity would impact directly on the extent of long-termmmune reconstitution. Chemotherapy has also been found toisrupt translation of regulatory proteins in the bone marrowiche, such as matrix metalloproteinase 2 [68] and alter stromallycosaminoglycans (GAGs)-disrupting supportive propertiesf stromal fibroblasts [69]. Total body irradiation induces thexpression of P16ink4a, which has been implicated in establish-ent of senescence in HSCs and in mediating growth arrest and

oss of self-renewal capacity, which is reflected in the functionf these cells both in vivo and in vitro [70].

Cytotoxic antineoplastic regimes also have a profound impactn the thymic niche (Goldberg, paper in preparation). In partic-lar, a specific subset of medullary epithelial cells, the MHCIIigh-expressing TECs that include the Aire-expressing subset,re selectively depleted (Fletcher et al, in preparation), havingmplications in the temporary disruption of immune tolerance.he thymic damage imposed is further compounded by graftersus host disease (GvHD) in allogeneic bone marrow trans-lantation (BMT) and the rate of thymic recovery is inverselyelated to the age of the patient and level of thymic involution.

hile the inclusion of alloreactive donor T cells in allogeneicMT provides anti-leukaemic effects, GvHD is also initiated byonor T cells, with donor repopulation resulting from thymic-ndependent peripheral expansion of mature donor cells andhymic-dependent generation from donor HSCs (reviewed in71]). More recently, donor T cell activation to host alloanti-ens in acute GvHD was found to induce IFN-g secretion,ignal transducer and activator of transcription (STAT)-1 acti-ation in thymic epithelial cells (TECs) and caspase-dependentpoptosis of TECs [72], disrupting the integrity of the thymicicroenvironment. Haploidentical haematopoietic cell trans-

lant protocols to minimize GvHD have accordingly led to anmproved induction of allograft tolerance.

Protection of the bone marrow and thymus niche from dam-ge by antineoplastic conditioning regimes and reduction invHD are fundamental to enabling the de novo regenerationf T cells with a broad T-cell repertoire and hence crucial in theevelopment of strategies for thymic regeneration and improvedmmune reconstitution.

. Restoring the immune system and primary lymphoidrgan niches

A number of strategies have been applied to replenish theand B cell pool after cytotoxic conditioning treatments andore recently, a focus in regenerating the aged or damaged

iches to ensure long-term reconstitution of the immune sys-

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unology 19 (2007) 331–340 335

em. Treatments involving the endocrine-immune axis, such asHRH inhibitors, growth hormone and parathyroid hormone, orrowth factors and cytokines such as KGF and IL7, address bothestoration of niche and lymphoid development.

.1. Regenerating the bone marrow niche to restoremmune competence

Experiments in aged mice have shown that the number of B-ineage cells in the BM decreases during aging at a similar rateo thymic involution [73]. Furthermore with age there is a sig-ificant decline in the frequency and number of ETPs and theirbility to develop T cells in vitro is significantly impaired [74].hese effects of aging on HPCs in the murine model translate

o the human: using purified human CD34+ HSCs in an in vitroTOC, cells derived from aged BM have lower T-cell potential

han young BM and may be severely affected by chemotherapy75]. Donor age has also been implicated in survival of BMTecipients [76].

Analogous to the thymic niche, it is apparent that differ-nt functional subsets exist within the major classes of stromalells in the BM, suggesting a limited number of true niches oricroniches are able to support quiescent HSCs. Osteoblasts, an

mportant component of the BM endosteal niche, express recep-ors important in anchoring LT-HSCs to the endosteal niche,roduce haematopoietic growth factors and are activated byarathyroid hormone (PTH) or PTH-related protein (PTHrP)hrough the PTH/PTHrP receptor (PTR).

Since osteoblast depletion also prevents the earliestetectable transitions from the HSC compartment [77–79], itay also lead to a decline in the number of T cell progenitors

vailable for emigration to the thymus—thereby directly con-ributing to age-related thymic involution. Improving osteoblastunction thus may enhance immune reconstitution.

Activation of the PTH-1 receptor in osteoblastic cellsncreases the Notch ligand Jagged-1 and results in an expansionf HSCs and furthermore, ligand-dependent activation of PPRith PTH increases the number of osteoblasts [77], partly due

o the activation of survival signalling in osteoblasts such as theunx2-dependent expression of anti-apoptotic genes like Bcl-2

80]. In mice, the stimulation of osteoblast cells with recombi-ant human PTH-(1–34) resulted in an enlargement of the HSCool, promotion of BMT engraftment and growth and survival ofethally irradiated mice [77]. Whether improved engraftment cane translated into pre-clinical non-human primate models andltimately in humans is yet to be determined, however in animalodels, it does show therapeutic potential for treatment of bonearrow depletion-inducing regimes such as ionizing radiation

nd chemotherapy [81,82]. PTH is currently in clinical trials foratients that are poor in mobilising autologous stem cells and tomprove engraftment and immune reconstitution following cordlood transplantation.

.2. LHRH agonists reverse age-induced thymic atrophy

nd restores T and B cell lymphopoiesis

A number of factors contribute to the decline in immuneunction with age, with sex steroids, which are strong immune

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uppressants, having a profound impact. We have shown that sexteroid inhibition can dramatically reverse this degenerative statend induce recovery in both the thymic and bone marrow cellu-arity and function [25,83,84] and return immune competence tooung levels. An increase in thymic-derived naı̈ve T cells result-ng in a rejuvenation of the naı̈ve:memory peripheral T cell poolnd improved peripheral T cell response in aged mice, alongith an enhanced production of IL7 responsive progenitors in

he BM and subsequent increase in B-cell export, contribute tohe increased immunocompetence and reflect an improved qual-ty in thymus and BM niche. Clinically, the reversible reductionn sex steroids can be achieved through agonist variants of lutein-sing hormone releasing hormone (LHRH/GnRH)—this is thestandard of care” for now millions of patients with many dis-ases exacerbated by sex steroids such as prostate cancer, someorms of breast cancer, endometriosis and precocious puberty.

Sex steroid ablation also improves immune recovery afterhemotherapy in young mice compared to sham castrate con-rols (Goldberg et al., in preparation). Importantly, in termsf niche recovery, TEC numbers and phenotype (cortical toedulla ratio) are similar to that of untreated controls by 4eeks post-castration/chemotherapy treatment, with a tendency

owards hypertrophy before returning to homeostatic levels byweeks, whilst sham-castrate/chemotherapy-treated controls

emained atrophic beyond the 6-week timepoint.We have also recently translated these observations in a pilot

linical trial, by treating cancer patients undergoing myeloabla-ive chemotherapy and HSCT, with LHRH. Preliminary analysisrovides good evidence for thymic reconstitution and productionf naı̈ve CD4+ T cells (Sutherland et al., submitted manuscript;nterim data analysis presented at ASH 2003). However whileon-LHRH control allogeneic-HSCT patients showed virtuallyo naı̈ve T cell recovery and ∼60% of the treated patients did,t still required almost 6–9 months. Hence combination therapy

ay be required to enhance the rate of reconstitution.

.3. Keratinocyte growth factor (KGF) protects TEC andnduces thymic regeneration

KGF or FGF7 is a paracrine-acting epithelial mitogen pro-uced by mesenchymal cells. KGF acts through a subsetf fibroblast growth factor receptor isoforms expressed bypithelial cells, in particular FGFR2IIIb. KGF treatment haseen found to protect thymic epithelial cells from GvHDn the allogeneic BMT setting and enhance thymic recoveryrom radiation, cyclophosphamide and dexamethasone treat-ent [85–87], illustrating clear therapeutic potential to increase

mmune reconstitution after BMT. The mode of action of sys-emically administered KGF on thymic regeneration appears toe a direct affect on TEC by inducing a transient expansion ofoth mature and immature subsets which in turn initiate androgress thymopoiesis, evident by an early expansion of imma-ure triple negative thymocytes and culminating in an enhanced

xport of mature cells into the periphery [88]. One area thatemains unresolved is whether KGF induces recovery and mat-ration of all TEC subsets, because initial evaluation of Airexpression in the chronic GvHD model of TEC destruction,

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unology 19 (2007) 331–340

uggested it was not recovered [86]. In Rhesus Macaques con-itioned with myeloablative total body irradiation followed byutologous HSCT, KGF treatment resulted in improved thymic-ependent reconstitution evidenced by an improved thymicrchitecture 1 year post-transplantation compared to controlsnd increased T cell receptor excision circles (TRECs) at 5onths through to 1 year, although only after multiple doseGF treatment. There was also a broader TCR repertoire at oneear compared to control animals [89]. The only anomaly is thathere was an increase in naı̈ve T cells after 3 months but this didot correlate with TREC levels, suggesting KGF may facilitateelease into the peripheral circulation of naı̈ve cells harbouredn epithelial tissues. Overall, however, while KGF appears toold great promise, these primate studies using short term KGFreatment warrant further evaluation using larger animal num-ers and in an allogeneic HSCT setting to ascertain its clinicalmpact.

.4. Growth hormone and insulin-like growth factor1

There is a steady decline with age in production of GH andGF1, which is induced by GH and mediates some GH actions,n rodents and humans and as such, has been linked with thymicnvolution. Administration of exogenous GH and IGFs (IGF1nd IGF2) does increase thymic cellularity, even though it did notegenerate the atrophic thymus back to young levels (reviewed27]). However, it is not known to what degree of thymic regen-ration is required for sufficient T cell production for clinicalelevance. Napolitano et al., 2002 showed that GH treatment ofIV-1 infected adults over a 6–12-month period was associatedith an increase in thymic tissue and circulating naı̈ve CD4+ T

ells from 3 to 9 months [90]. However, some patients did experi-nce significant drug toxicity and ceased treatment, necessitatingurther assessment of GH for immune reconstitution.

.5. Creating an ex vivo thymus

The field of stem cell therapy has fuelled the developmentf biomatrices and scaffolds as three-dimensional structures toacilitate tissue regeneration. Certainly with regards to the thy-us, it is well known that TECs, an essential component of

he thymic microenvironment, require a three-dimensional con-guration to enable migration of T cell precursors through thetroma and lose their capacity to sustain T cell lymphopoiesishen in two dimensions [19]. This is thought to be associatedith the loss in expression of important factors such as Foxn1,transcription factor of the forkhead winged helix class, impor-

ant for crosstalk-dependent TEC differentiation—mutations ofhich are associated with congenital athymia and hairlessness

n mouse and rat. This has been overcome to some degree byhe transfection of Notch ligands into cell lines such as the OP9-elta like-1 cell line (OP9-DL1) [91]. However, reconstruction

f an ex vivo adult thymus that is able to induce both posi-ive selection of developing MHC classes I and II restrictedhymocytes and delete self-reactive clones, is yet to be fullyealised.

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The existence of thymic resident epithelial precursors haseen demonstrated in the mouse embryo, with a single thymicpithelial precursor cell able to differentiate into both corticalnd medullary TEC phenotypes and sustain full T cell develop-ent [10]. MTS24+ TEC, while preferentially able to form a

hymus if isolated mid-gestation [92], lose this ability from fullerm. It is not clear if this is a technical problem or a true loss ofunction [106]. In this regard, there is evidence for potentiallyhree TEC progenitor cells to exist in the post-natal thymus; ai-potent progenitor that can differentiate into both cortex andedulla TECs, and unipotent cells that can differentiate into

ither cortex or medulla TECs [93]. Clark et al. (2005) usingpreviously published solid matrix [94] showed that human

kin keratinocytes, together with mesenchymal fibroblasts andxogenous growth factors, can support the development of func-ional “self”-tolerant T cells with a broad TCR repertoire. Asromising as this system is, it is currently very inefficient andnlikely in its present form to be readily translated into thelinic [95]. Furthermore, whether they have the capacity to rig-rously apply both positive and negative selection is unclear.ollectively, this research provides the possibility that an exivo thymus can be produced. However for any clinical rele-ance, the isolation of thymic epithelial cell precursors from auman thymus will be needed.

.6. T cell reconstitution

Early expansion of pre-existing T cells may be useful in thearly phases of immune deficiencies after cytotoxic condition-ng regimes, however, such cells will by necessity be of limitedepertoire diversity and will not substitute the generation of aew naı̈ve T cell pool for long-term repopulation of the immuneystem.

.6.1. Cytokine administration: interleukin 7The stromal cell-derived cytokine, IL7, is required for T cell

evelopment and plays a major role in lymphopenic-inducedxpansion of peripheral T cells. Hence recombinant humanL7 administration has been considered as having potential inmproving immune reconstitution. A significant improvementn reconstitution was found following administration of IL7 inn allogeneic model of HSCT without aggravating GvHD [96].L7 selectively expanded de novo generated T cells and non-lloreactive peripheral T cells—hence promoting peripheral

cell reconstitution through selective proliferative and anti-poptotic effects [97]. IL7 has also been shown to enhance CD4cell reconstitution after lethal irradiation and anti-thymocyte

lobulin administration in a non-human primate model [98]. InIV-1 patients, high levels of IL7 are associated with high viral

oad and increased CD4+ depletion—the increased productionf IL7 by stromal cells likely responding to the virus-inducedymphopenic state [99]. However, contrary to the positive rolef IL7 in T cell homeostasis, this increased IL7 production

as more recently found to increase Fas expression on naı̈ve

nd memory T cells and render T cells more sensitive to Fas-ediated apoptosis in HIV-1 infected patients [100]. Increased

irculating levels of IL7 have also been found to associate with

ambr

unology 19 (2007) 331–340 337

ncreased viral load. Since IL7 has been shown to accelerateIV-1 infection both in vitro and in vivo, the increase in circu-

ating levels of IL7 may drive viral replication; render T cellsore sensitive to apoptosis and result in an accelerated T cell

oss [99,100]. Thus abrogation of viral load in HIV-1 infectedatients and general immune activation prior to IL7 treatmenthould be an essential consideration for IL7 treatment to restoremmune competence.

.6.2. Flt 3 ligandFlt3L has been shown to enhance T cell reconstitution how-

ver this effect is not dependent on the thymus. In athymicice, Flt3L promotes the expansion of peripheral T-cells while

n a wildtype mouse enhances thymic reconstitution followingMT [101]. Despite these effects on peripheral expansion, there

s also a direct effect of Flt3L administration on thymopoieticeconstitution after HSCT, which is mediated by an expansion ofymphoid progenitors [102]. However, administration of Flt3Lauses reduced B-lymphopoiesis by its effects on a recentlyescribed early progenitor with lymphoid and myeloid potentialEPLM) and its ability to produce B cells [103,104].

.6.3. Passive T cell transferTransferring HSCs that have undergone ex vivo differentia-

ion into pro-T cells on the OP9-DL1 culture system still hasome way to go before this technique can be applied clinically,owever, Zakrzewski et al. showed that in animal models, thymiceconstitution could be significantly improved and donor T-cellhimerism enhanced compared to use of BM or HSCs alone105]. These treatments are useful due to the short seeding andngraftment time compared to BM or purified HSCs, which firsteed to seed the BM before migrating to the thymus. As a result,hese therapies may prove to be useful in rapidly reconstitutinghymopoiesis after HSCT but will not be feasible for long-termngraftment and reconstitution.

. Concluding comments

A vast literature has confirmed that the status of the thymicicroenvironment, primarily encompassed within the epithe-

ial subsets, dictates the immune capacity of the individual.hile the thymus is often the target of collateral damage caused

y chemo- and radiotherapy as standard of care for treatingany cancer patients, paradoxically it also undergoes natural

trophy as a consequence of normal aging. In instances wherehymic function is compromised, there is an inevitable transla-ion into immune dysregulation ranging from immunodeficiencynd opportunistic infections, through to increased incidence ofutoimmune disease and cancer. The treatment of such clinicalroblems is thus best approached by a rejuvenation of thy-us function, which in turn is fundamentally dependent on a

onstant source of appropriate bone marrow-derived progeni-or cells. Both of these require knowledge of the thymic and

lso bone marrow stromal cell niches and any damage or abnor-alities therein. Now that the molecular basis to thymic and

one marrow stromal cell function are being elucidated, moreational approaches to clinical management of immune disorders

3 n Imm

aIt

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re evolving. The use of sex steroid inhibitors, GH, KGF andL7 represent the first candidates in a new age in thymus-basedherapies.

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