Thymic rejuvenation and the induction of toleranceby adult thymic graftsShuji Nobori, Akira Shimizu, Masayoshi Okumi, Emma Samelson-Jones, Adam Griesemer, Atsushi Hirakata,David H. Sachs, and Kazuhiko Yamada*

Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02129

Edited by Ralph M. Steinman, The Rockefeller University, New York, NY, and approved October 25, 2006 (received for review June 22, 2006)

The thymus, the site of origin of T cell immunity, shapes the repertoireof T cell reactivity through positive selection of developing T cells andprevents autoimmunity through negative selection of autoreactive Tcells. Previous studies have demonstrated an important role for thethymus not only in central deletional tolerance, but also in theinduction of peripheral tolerance by vascularized renal allografts injuvenile miniature swine recipients. The same protocol did not inducetolerance in thymectomized recipients nor in recipients beyond theage of thymic involution. We subsequently reported that vascularizedthymic lobe grafts from juvenile donors were capable of inducingtolerance in thymectomized juvenile hosts. However, the importantquestion remained whether aged, involuted thymus could also in-duce tolerance if transplanted into thymectomized hosts, which, iftrue, would imply that thymic involution is not an intrinsic propertyof thymic tissue but is rather determined by host factors extrinsic tothe thymus. We report here that aged, involuted thymus transplantedas a vascularized graft into juvenile recipients leads to rejuvenation ofboth thymic structure and function, suggesting that factors extrinsicto the thymus are capable of restoring juvenile thymic function toaged recipients. We show furthermore that rejuvenated aged thymushas the ability to induce transplant tolerance across class I MHCbarriers. These findings indicate that it may be possible to manipulatethymic function in adults to induce transplantation tolerance after theage of thymic involution.

aging � miniature swine � thymus

The thymus plays a central role in the development of immuno-logic tolerance. Thymic epithelial cells and dendritic cells in the

thymus are responsible for the induction of central tolerance to selfamong developing T cells through negative selection. A similarprocess has been shown to lead to the development of centraltransplantation tolerance to alloantigens after allogeneic hemato-poietic stem cell (HSC) transplantation, because of negative selec-tion by HSC-derived donor dendritic cells that migrate to the hostthymus (1–4). Finally, the thymus has been shown to be requiredfor the rapid induction of the stable peripheral tolerance that occursunder the influence of a short course of calcineurin inhibitors afterallogeneic renal transplantation in miniature swine (5).

Thymic structure and function decline with age, however, leadingby adolescence to a significant reduction in thymic mass and animpaired ability to regenerate normal T cell numbers after T celldepletion (6, 7). It is not clear whether this thymic involution is anintrinsic property of thymic tissue or is due to the effects of factorsextrinsic to the thymus. Likewise, it remains unclear whichtolerance-inducing properties of the thymus are affected by itsinvolution.

We have recently established a procedure for vascularized thymictransplantation in MHC inbred miniature swine that permits atransplanted thymus to function immediately after revasculariza-tion (8). We have now used this technique to study the basis of theimmunologic consequences of aging on the thymus. For thispurpose, we transplanted vascularized thymic lobe (VTL) graftsfrom aged miniature swine to young animals of the same swineleukocyte antigen (SLA) inbred line (9). As we report here, theseaged, involuted VTL grafts underwent rejuvenation after this

procedure and then facilitated the induction of tolerance, suggest-ing that thymic function is largely controlled by the host environ-ment rather than by the local thymic milieu.

ResultsThymic Involution with Age In Miniature Swine. We first examinedthe natural course of thymic involution with aging in naive minia-ture swine (9). Right cervical thymic lobes were biopsied from 53naive Massachusetts General Hospital (MGH) miniature swine ofdifferent ages. The cortex and medulla in whole biopsy specimenscontaining at least 10 lobules were subjected to morphometricanalysis by using NIH Image (Scion Image) software (10).

Fig. 1A shows a plot of the ratio of cortical to medullary areas(c/m ratio) as a function of age in miniature swine. Fig. 1 B and Cshows representative histologic findings of naive thymic tissue at 4months and 20 months of age, respectively. As shown in this figure,thymi from 4-month-old miniature swine had well defined, thickcortical thymic areas, whereas thymi from 20-month-old animalswere consistently involuted. At 4 months of age, at which pointperipheral tolerance can readily be induced (11), pigs had a c/mratio between 3 and 5, whereas pigs older than 20 months, whensuch peripheral tolerance could no longer be induced (12), had c/mratios of �0.8 (Fig. 1A). The correlation coefficient between c/mratio and age (�0.82) was highly significant (P � 0.0004). Nodifference was seen between the thymi of female and male animals.

Rejuvenation of Aged Thymus Grafts in Juvenile Recipients. We nextassessed the ability of involuted thymus grafts to rejuvenate and tosupport thymopoiesis after transplantation into juvenile animals.VTL grafts were harvested from aged pigs (20–21 months of age)and transplanted into MHC-matched, 4-month-old juvenile ani-mals (recipients 1–3) that had been thymectomized 3 weeks earlier.A 28-day course of FK506 (tacrolimus) was administered to assurethat the grafts would not be rejected because of minor antigendifferences. FK506 has been found previously not to have measur-able effects on thymus structure or involution in miniature swine(13). The morphometric analysis of aged thymus grafts transplantedinto juvenile recipients revealed marked rejuvenation of thesegrafts within two months of transplantation (Fig. 2). Aged thymicgrafts with an initial c/m ratio of 1 � 0.3 showed c/m ratios of 3.1 �0.8 by 60 days after transplant and 4.1 � 0.5 by 100 days aftertransplant. After day 180, the thymic architecture began to rein-

Author contributions: K.Y. designed research; S.N., A.S., and K.Y. performed research; S.N.,M.O., E.S.-J., A.G., A.H., D.H.S., and K.Y. analyzed data; and K.Y. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: CML, cell-mediated lympholysis; CTL, cytotoxic T lymphocyte; CyA, cyclo-sporine A; DP, double positive; FK506, tacrolimus; MLR, mixed lymphocyte response; PAA,porcine allele-specific antibody; SLA, swine leukocyte antigen; PBMC, peripheral bloodmononuclear cells; Treg; regulatory T cell; VTL, vascularized thymic lobe; PCNA, prolifer-ating cell nuclear antigen; c/m, cortical/medullary.

*To whom correspondence should be addressed. E-mail: kaz.yamada@tbrc.mgh.harvard.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0605159103/DC1.

© 2006 by The National Academy of Sciences of the USA

www.pnas.org�cgi�doi�10.1073�pnas.0605159103 PNAS � December 12, 2006 � vol. 103 � no. 50 � 19081–19086

MED

ICA

LSC

IEN

CES

Dow

nloa

ded

by g

uest

on

Oct

ober

16,

202

0

volute, with the c/m ratios on day 240 resembling those of recipient-age-matched naive animals at these time points (Fig. 2). Fig. 3 showsthe histology of biopsy specimens from one representative thymec-tomized juvenile animal (recipient 1) that received an aged thymicgraft (c/m ratio of 0.79). By 60 days after transplant, the corticalthymus had markedly expanded with larger lobular structures andwell defined cortical and medullary regions (Fig. 3B). The animalmaintained a thymic architecture that resembled that of a juvenileanimal until day 180 [Fig. 3C and supporting information (SI) Fig.6A]. By day 240 (SI Fig. 6B), the cortical thymus of the graftsseemed to be thinner. This reinvolution was likely due to therecipient’s age, not to immunologic causes, because the thymi ofnaive pigs also involute at this age (Fig. 1).

We stained the thymus biopsy tissues for proliferating cell nuclearantigen (PCNA, a marker of proliferation) and cytokeratin (amarker of thymic epithelial cells) (SI Fig. 6 C–F), to assess theproliferation of epithelial cells in the VTL graft. The expression of

PCNA on cytokeratin-positive cells in both the cortical and med-ullary regions increased to a maximum on day 100 (SI Fig. 6 D andF), indicating that active proliferation of thymic epithelial cellsoccurred. After day 100, the expression of PCNA decreased in apattern similar to the expression of PCNA in a naive, age-matchedthymus.

Cells dispersed from thymic biopsy tissues were also analyzedby flow cytometry by using allele-specific antibodies. Whereas allof the thymocytes on day 0 were donor-type cells [negative forpig allele-specific antigen (PAA)], most of the thymocytes in theday 60 VTL graft were recipient-type cells (PAA-positive) (SIFig. 7a). All thymocytes in the VTL graft were recipient type byday 100. The day 100 analysis revealed a phenotypic patternsimilar to that of a naive 4-month-old thymus (SI Fig. 7a).Furthermore, the FACS profile of the thymocytes from agedthymic grafts on day 60 and thereafter revealed that a majorityof the cells were CD4/CD8 double-positive, CD1 highly positivecells that expressed CD3 poorly (SI Fig. 7 b–d). This profileindicated that the cells in the VTL on day 60 and thereafter werehost-type thymocytes derived by thymopoiesis, and not graftinfiltrating cells.

Fig. 1. Morphometric analysis and histology of naive thymus at variousstages. (A) Morphometric analysis showing c/m areas of porcine thymus atvarious times during the aging process. Histologic findings of porcine thymusat 4 months (H&E, �100) (B) and 20 months (H&E, �100) (C) of age. Corticalregions contain small, dense thymocytes showing strong hematophilic stain-ing (c) compared with medullary regions (m).

Fig. 2. Morphometric analysis showing comparison of c/m ratios of aged porcine thymus grafts after transplantation to juvenile recipients versus recipientage-matched naive thymuses. Shown are results for recipient-age-matched naive thymus (average) (�) and for recipients 1 (‚), 2 (�), and 3 (�).

Fig. 3. Representative histology of VTL grafts. H&E staining (�40) of biopsysamples from an aged VTL graft (from a 20-month-old donor) in a juvenile4-month-old recipient pig on day 0 (c/m ratio 0.79) (A), day 60 (c/m ratio 2.68)(B), and day 100 (c/m ratio 3.62) (C).

19082 � www.pnas.org�cgi�doi�10.1073�pnas.0605159103 Nobori et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

16,

202

0

Aged VTLs Induce Transplant Tolerance Across MHC Disparate Barriersbut Require a Longer Period of Immunosuppression than JuvenileVTLs. We subsequently assessed the ability of older thymus grafts toinduce tolerance across MHC disparate barriers with the same28-day course of FK506 that was used across MHC-matchedbarriers. Based on our previous experience with mouse and porcinethymic transplantations (13, 14), recipients (recipients 4–6) werecompletely thymectomized before the transplantation of two hap-lotype, class I-mismatched VTL grafts. Frequent VTL biopsieswere performed on recipient 4 to examine the development ofthymopoiesis in the VTL graft. The other two animals receiveddonor-matched kidney grafts on day 100 and third-party kidneygrafts �250 days after VTL transplantation to test in vivo tolerance.Because of previous evidence (15) that thymic biopsies during theinduction period may interfere with the induction of transplanttolerance, we did not perform VTL biopsies on these recipientsafter kidney transplantation until the end of the experimentalperiod.Thymopoiesis. Thymopoiesis was markedly delayed in MHC-mismatched VTL grafts as compared with either MHC-matched grafts (see Figs. 2 and 3 and SI Fig. 7) or juvenileMHC-mismatched VTLs (13). Aged MHC-mismatched VTLgrafts were still hypocellular on day 60, but thymic stromal cellswere present without vasculitis (Fig. 4 Aa and Ab). However,by day 100, the thymus seemed to have essentially normal,juvenile architecture. The cortex was well developed withdense mononuclear cell migration. The medullary thymus wasnot as well defined as the cortex, probably because of the stageof thymic development at this time point (Fig. 4Ac). Immu-nohistochemistry revealed a small number of recipient-typeclass I-positive cells in the medullary area (dull brown stainedcells), indicating that thymopoiesis occurred between day 60and 100 (Fig. 4Ba).

To assess thymic function at later time points, one animal

(recipient 4) that did not receive a renal transplant, underwentserial thymic biopsies on days 150, 180, and 260. The VTL graftbiopsies at day 150 and 180 showed markedly expanded thymiclobules with a thick cortical thymic area and well defined medullarstructure (Fig. 4Ad). Recipient-type MHC class I staining showedthat the majority of thymocytes in the medullary region of the VTLgrafts were of recipient-type (dull brown cells) (Fig. 4Bb). SI Fig. 8shows the FACS profile of the VTL graft biopsy on day 180.Consistent with the immunohistochemistry, the majority of cells inthe VTL grafts were recipient-type cells as assessed by porcineallele-specific antibody (PAA) (SI Fig. 8b). FSC/SCC, CD4/CD8,and CD1/CD3 profiles confirmed that these cells were thymocytes,indicating that rejuvenation with active thymopoiesis occurred inthe aged VTL graft (SI Fig. 8 a and c–e). However, in a mannersimilar to that seen in MHC-matched grafts, the thymus beganreinvoluting without evidence of vasculitis after day 260 (Fig. 4Ae).VTL grafts in the two animals that received kidney transplants wereexamined when either the third-party kidney was removed or theexperiment was terminated (see next section). The VTL graft onday 259 in recipient 5 maintained thymic structure with slightinvolution, comparable with the animal that did not receive akidney transplant (recipient 4). Biopsies of VTL grafts on day 310(recipient 6) and 315 (recipient 5) revealed more obvious involutionwith fatty degeneration.In vivo donor-specific tolerance. Two animals, including the animalthat had anti-donor CTL responses at day 60 (see below), receiveddonor-matched kidneys without immunosuppression on days 95(recipient 5) and 105 (recipient 6), respectively. Both animalsmaintained their grafts for longer than 5 months without any signsof rejection (Fig. 5A, solid lines). There was no endothelialitis,hemorrhage, or mononuclear cell infiltrate in the kidney grafts atthe time of biopsies (days 30, 60, and 153/163 after kidney trans-plantation) (Fig. 5B). These two animals received third-partykidneys 163 (recipient 5) and 153 (recipient 6) days after donor-

Fig. 4. Histology of aged VTL grafts transplanted across a class I-mismatched barrier with a 28-day course of FK506. (A) Results for day 60 (H&E, �100) (Aa),day 60 (cytokeratin, �100) (Ab), day 100 (H&E, �100) (Ac), day 180 (H&E, �100) (Ad), and day 260 (H&E, �100) (Ae). (B) Immunohistochemistry of VTL graftsstained with anti-recipient MHC class I at day 100 (�400) (Ba) and day 180 (�400) (Bb) and with anti-donor MHC class I antibody at day 180 (�400) (Bc). Arrowsin Bc indicate donor-type cells with dendritic cell morphology at the corticomedullary junction. (Bd and Be) Double-staining with donor-type class Iphycoerythrin-conjugated antibody and class II pig-specific FITC-conjugated antibody (�800) at day 100 (Bd) and day 180 (Be). Double-stained cells (donor classI-positive/pig class II-positive) appear yellow. Morphologically dendritic-like cells double-positive for donor class I and pig class II were detected at days 100 and180 (white arrows in Bd and Be). Class II-positive recipient-type cells with dendritic cell-like morphology (green cells in Bd and Be) increased between days 100and 180.

Nobori et al. PNAS � December 12, 2006 � vol. 103 � no. 50 � 19083

MED

ICA

LSC

IEN

CES

Dow

nloa

ded

by g

uest

on

Oct

ober

16,

202

0

matched kidney transplantation (days 259 and 258 after VTL),respectively, to evaluate immunocompetence. The ureter of thetolerated kidney was tied to permit evaluation of function of thesecond kidney. Third-party grafts were rejected within 10 days (Fig.5A, dashed line) with histologic evidence of both cellular andhumoral rejection (Fig. 5C), whereas no rejection was noted in thetolerated kidneys (Fig. 5B), indicating that aged VTL and 28 daysof FK506 had donor-specific tolerance. Both animals were keptalive by untying the ureter of the tolerated kidney when thethird-party graft was nephrectomized for in vitro study. The creat-inine levels immediately returned to normal, where they remaineduntil euthanization on days 315 and 310 after VTL transplantation.In vitro assays to determine mechanisms of tolerance. All animals hadanti-donor CTL responses �40% at a 100:1 effector to targetratio before VTL transplantation. Two (recipients 4 and 5) of thethree animals developed donor-specific unresponsiveness by day60 (SI Fig. 9 a and b). The other animal (recipient 6) maintainedanti-donor responses at day 60 (SI Fig. 9c), thereafter becomingdonor-unresponsive by day 120 (SI Fig. 9d). We further assessedthe mechanisms of tolerance in this model by using cocultureCTL assays to examine the regulatory mechanisms. Cocultureassays were set up on day 60 in recipient 5 and on days �250 inall three recipients. Donor-specific suppression of the naive Tcell response was seen in all of these assays, indicating thepresence of regulatory cells in the peripheral blood (SI Fig. 9efrom recipient 5 at day 315). In addition, we assessed whetheranti-donor CTLs were restored by removal of CD25-positivecells from PBLs from a long-term acceptor on day 315 (recipient5). The anti-donor CTL response was restored only minimally inthe CD25-depleted CML culture (blue solid line compared withblue dashed line in SI Fig. 9f). Because the CD25 depletion wasconfirmed by FACS (see Materials and Methods), these datasuggest that deletional tolerance mechanisms are also likely to beoperative.

To further characterize deletional mechanisms, we performeddouble-staining with donor-type class I phycoerythrin (PE)-conjugated antibody and class II pig-specific FITC-conjugatedantibody. At day 100, donor-type class I-positive/class II-positive

double-stained morphologically dendritic-like cells (Fig. 4Bd,yellow cells indicated by white arrows) were seen at the corti-comedullary junction. In addition, recipient-type morphologi-cally dendritic cells (Fig. 4Bd, green cells that were donor classI-negative but pig class II-positive) migrated to this area by thistime point. Donor-type class I-positive, morphologically den-dritic-like cells remained at the corticomedullary junction at day180 (Fig. 4Bc, dense brown cells with arrows). These cells wereconfirmed to be class II-positive (Fig. 4Be, yellow cells indicatedby white arrows). Class II-positive recipient-type cells withdendritic cell-like morphology (green cells in Fig. 4 Bd and Be)increased between day 100 and day 180. The coexistence of thesedonor and recipient cells suggests the potential for host anddonor negative selection of T cells in the VTL graft (16).Inferior ability of aged VTL relative to juvenile VTL to induce tolerance. Wehave previously shown that VTL grafts from juvenile donorsinduce tolerance with a 12-day course of FK506 across MHC-mismatched barriers in miniature swine. In contrast to a 28-dayFK506 regimen, biopsies on day 60 of the VTLs of three animals(recipients 7–9) receiving only 12 days of FK506 revealedmononuclear cells with vasculitis (SI Fig. 10a). FACS dataindicated that a majority of these cells were CD3-positive withvery few CD1-positive cells, a phenotypic pattern similar to thatof peripheral blood and indicating the presence of graft infil-trating cells (SI Fig. 10 b and c). Two of the animals had very fewcytokeratin-positive cells in the VTL (SI Fig. 10d); the VTL inthe remaining animal (recipient 9) had slightly more cytokeratin-positive epithelial cells (SI Fig. 10e). All recipients maintainedanti-donor CTL responses after VTL transplants (SI Fig. 10f).

To evaluate the in vivo immunologic status of these recipients,we transplanted donor-matched kidney grafts without immuno-suppression to all three animals on day 100. Two animalsrejected their renal grafts acutely on days 7 and 15, respectively(SI Fig. 10g). The third animal, which had a slightly highernumber of cytokeratin-positive epithelial cells on day 60, main-tained its graft with unstable renal function (Crea 3–6 mg/dl) for102 days, at which point it was euthanized because of weight lossand renal dysfunction (SI Fig. 10g). This animal showed signs ofacute rejection in the day 30 renal graft biopsy specimen, withdiffuse mononuclear cell infiltrates, vasculitis and glomerulitis.A renal biopsy on day 60 revealed chronic glomerulopathy,moderate mononuclear cell infiltrate, and mild fibrosis (SI Fig.10h). At the time of euthanization, the renal graft showedmoderate interstitial fibrosis, with increased chronic glomeru-lopathy. The VTL graft at this time point was totally fibrotic.Thus, VTL grafts transplanted from an aged donor to a juvenilerecipient with a 12-day course of FK506 prolonged the survivalof donor-matched kidney grafts but failed to induce stabletolerance.

DiscussionThe thymus plays a central role in the development of T cells andthe induction of immunologic tolerance. However, thymic struc-ture and function decline with age, leading by adolescence to asignificant reduction in thymic mass and an impaired ability toregenerate normal T cell numbers after T cell depletion (6, 7).Whereas the age related changes of the thymus are well estab-lished, it is not clear whether the involution and decline infunction is more dependent on the host environment (extrinsicfactors) or on the local thymic milieu (intrinsic factors). Usingour model of vascularized thymic lobe transplantation, wetransplanted aged thymus into juvenile animals to study theeffect of the juvenile milieu on aged thymus (13). The datapresented demonstrate the rejuvenation of aged VTL graftswithin juvenile recipients, suggesting that thymic structure andfunction are more dependent on extrinsic factors than onintrinsic ones.

Fig. 5. Plasma creatinine levels after donor-matched kidney transplant inrecipients of aged VTLs with 28 days of FK506 across a class I-mismatched barrier(A), donor-matched kidney biopsy at day 153 after kidney transplant (H&E, �100)(B), and third-party kidney biopsy rejected on day 7 (H&E, �100) (C).

19084 � www.pnas.org�cgi�doi�10.1073�pnas.0605159103 Nobori et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

16,

202

0

Age-related changes that may affect T cell development occursimultaneously within the bone marrow, the extrathymic milieu,and the thymus (17, 18). Mackall et al. (19) have reported thatthe administration of young T cell-depleted bone marrow tolethally irradiated, aged mice failed to reverse thymic involutionor histologic abnormalities (including a loss of cortical epithe-lium and a disruption of the normal cortical architecture)associated with the aged recipient thymus. Although youngmarrow restored the ability to negatively select autoreactive Tcell clones, it did not rejuvenate the aged thymi. These studies areconsistent with our conclusion that host factors extrinsic to thethymus control its involution and suggest that these factors arenot provided by the bone marrow.

The effects of the young vs. old milieu on liver and muscle stemcells and on progenitor cells has recently been studied by usingparabiotic pairings (20). The normal decline in tissue regener-ative potential with age was shown to be reversed in the olderpaired mice through exposure to serum factors from young mice.The authors did not, however, examine the involution of thethymi in these studies, possibly because of the potentially con-founding effects of having old and young thymi present in thesame system. Our thymus transplantation model between MHC-matched recipients provides a model that eliminates this variablewhile exposing aged thymus to a young milieu.

There have been several studies attempting to characterize thefactors in the microenvironment that cause thymic involution.Surgical castration and chemical castration using luteinizinghormone-releasing hormone (LH-RH) have been shown to havea profound rejuvenating effect on the thymus in mammalianmodels (17, 21–23). Additionally, IL-7, produced by MHC classII-positive thymic epithelial cells, has been linked to the survivaland proliferation of thymocytes (24). IL-7 levels decrease withage (25), but whereas thymopoiesis increased in young mice thatreceived IL-7, treatment of aged mice did not augment thymo-poiesis (26, 27). Although the present study demonstrates thatextrinsic factors can facilitate thymic regeneration, thus far, nofactor has definitively been shown to be directly involved in theinvolution or regeneration of the thymus.

Data in the present study have also demonstrated that reju-venated aged VTLs could induce tolerance but require a longercourse of immunosuppression to establish tolerance than dorecipients of juvenile VTLs. We have previously shown that a12-day course of FK506 facilitated the induction of tolerance byjuvenile VTL grafts across even a full MHC mismatch (13). AgedVTL grafts required a 28-day course of FK506 to inducetolerance in the class I MHC disparate model studied here.Plausible explanations for the requirement of prolonged immu-nosuppression include: (i) in aged VTL grafts, there are fewernatural regulatory cells to inhibit alloreactive T cells in anonspecific manner during the induction period, and (ii) theaged thymic grafts are slower in generating donor-specificregulatory cells and in deleting/anergizing new alloreactivethymocytes. Because T cells were not depleted in the presentstudy, preexisting alloreactive precursor T cells circulating in theblood could enter the VTL graft (28, 29) during the inductionperiod and damage the thymic epithelial and stromal cells. Thisdamage could further inhibit the graft’s ability to generatedonor-specific antigen-dependent regulatory cells and to deleteor anergize alloreactive cells in the graft. Aged VTL grafts thatwere protected by a 28-day course of FK506 in the inductionperiod had no evidence of vasculitis, indicating that this immu-nosuppression was sufficient to protect the aged, involuted VTLgraft from alloreactive circulating T cells. Thymopoiesis wasfurther delayed by transplanting aged VTLs across a class Imismatch barrier (thymopoiesis occurred after day 60) as com-pared with across a minor MHC mismatch (near completeconversion to recipient-type thymopoiesis by day 60). The in-creased immunological response caused by the class I disparity

during the induction period may have damaged the thymicepithelial and stromal cells either in number or function, inhib-iting thymopoiesis at early time points despite the prolongedcourse of immunosuppression.

The present study, involving the transplantation of old thymi intoyoung recipients, may provide a model for isolating and examiningindividual external factors that control the regeneration of thymiccapacity. Further decreases in the number of thymic stromal cellsby more formidable immunologic barriers (i.e., full MHC mismatchor xenotransplantation) or aged VTL into older recipients, couldpotentially result in a negative cytokine milieu for the aged VTLsand limit the ability of the VTLs to induce T cell tolerance to donorantigens. We are therefore currently investigating ways to acceler-ate the regeneration process of aged thymus to facilitate more rapidengraftment and function in these grafts. Preliminary data usingLupron, an LH-RH agonist, suggest that the regeneration of theaged thymus may be possible (K.Y., unpublished data). Medicalthymic regeneration, combined with VTL transplantation, couldmove our tolerance induction regimen one step closer to the clinicby regenerating aged thymus either before or after VTL transplan-tation into an aged recipient. This strategy could have practicalimplications for extending thymus-dependent tolerance-inducingprotocols (5, 12, 13, 30–32) to adult patient populations.

Materials and MethodsAnimals. Animals were cared for according to the guidelines ofMassachusetts General Hospital Institutional Animal Care andUse Committee. MGH miniature swine represent one of the fewlarge animal species in which breeding characteristics makegenetic experiments possible (9). Fifty-three naive animals eu-thanized as organ donors for other experiments were used for amorphometric study to assess natural thymic involution. Sixjuveniles (�6 months of age) and six adults (20–21 months ofage) served as donors of VTL transplants in either MHC-matched or class I MHC-mismatched combinations.

Surgical Procedures. Complete thymectomy in the recipient. As described(5), the pretracheal muscles were retracted, exposing the cervicalthymus and trachea from the cervicothoracic junction to themandibular area. The cervical thymus was excised, after whichthe mediastinal thymus was removed through a sternotomy.VTL transplantation. The procedure for VTL graft transplantationhas been reported (13). Briefly, the right cervical thymic lobewas carefully harvested, perfused with cold Eurocollins solution,and transplanted into the retroperitoneum of recipients, by usingthe abdominal aorta and the inferior vena cava. This techniqueled to perfusion of the VTL grafts with an immediate bloodsupply. Six animals received a 28-day course of FK506, and threeanimals received a 12-day course of FK506, starting at 0.15 mg/kgper day and adjusted to maintain blood levels between 30 and 40ng/ml (31).Thymus biopsy. VTL biopsies were carried out at 0, 60, 100, 180,and �240 days after VTL transplantation. Renal transplantrecipients had thymus biopsies when the experiment was termi-nated. Approximately 1 � 0.5 � 0.5 cm of thymic tissue wasbiopsied for histology (formalin and frozen samples) and flowcytometric analysis. The sections of naive thymus used formorphometric analysis were taken from naive pigs that served asorgan donors for other studies. Tissue samples were fixed in 1%formaldehyde solution (Denison Pharmaceuticals, Pawtucket,RI), embedded in paraffin, and subsequently sectioned. Tissueswere stained by using hematoxylin/eosin (H&E).Kidney transplantation. The kidney transplantation procedure hasbeen reported (5). Both native kidneys were removed on the dayof kidney transplantation.Catheter placement. Two semipermanent, indwelling, Hickmansilastic central venous catheters were placed in the externaljugular veins to facilitate drug administration and frequent blood

Nobori et al. PNAS � December 12, 2006 � vol. 103 � no. 50 � 19085

MED

ICA

LSC

IEN

CES

Dow

nloa

ded

by g

uest

on

Oct

ober

16,

202

0

sampling for in vitro assays and laboratory tests including com-plete blood count and blood chemistry, and for monitoring ofwhole-blood FK506 levels.

Evaluation of Thymic Rejuvenation/Involution. Preparation of thymo-cytes. Biopsied tissue from thymic grafts (100–200 mg) was finelyminced in Hanks’ balanced salt solution (HBSS) buffer; the cellsuspension was then filtered twice through a 200-�m nylon mesh.Flow cytometry. FACS analysis of PBMCs was performed by usinga Becton Dickinson FACScan (San Jose, CA) with CellQuestFACStation software (Becton Dickinson) as reported (13).Phenotypic analysis of cells was attained by three-color stainingwith directly conjugated murine anti-swine mAbs.Monoclonal antibodies (mAbs) used for phenotypic characterization of cellpopulations in VTL grafts. Thymocyte development was assessed byimmunohistochemistry and FACS analyses by using the murineanti-swine mAbs 74-12-4 (IgG2b, anti-swine CD4), 76-2-11 (IgG2a,anti-swine CD8), 76-7-4 (IgG2a, anti-swine CD1), BB23-8E6(IgG2b, anti-swine CD3), 231.3B2 (IgG1, anti-swine CD25), 10-9(anti-PAA), 2.12.3a (IgM, class Id), 16.7.E4.2 (IgM, class Ic), andTH16 (IgG2a, anti-swine class II DQ) as reported (8).Histological analysis. Formalin-embedded tissue was stained by usingH&E. Coded samples were examined through a light microscope bya pathologist. Frozen samples were prepared for immunohisto-chemical analysis with the avidin–biotin horseradish-peroxidasecomplex technique (30). Thymopoiesis was assessed by immuno-histochemistry by using the swine-specific murine mAbs describedabove. Double immunostaining with PCNA and cytokeratin (Z622;Dako, Carpinteria, CA) for the identification of the proliferatingthymic epithelial cells was performed in formalin-fixed paraffinsections by using a combination of immunoalkaline phosphataseand standard avidin–biotin complex (ABC) technique.

Morphometric analysis. Morphometric studies were performed onthymic biopsy samples by using NIH Image (Scion Image,Frederick, MD) software (10). Biopsy samples with at least 10lobules were stained with H&E, and the areas of the cortex andmedulla were measured to obtain a c/m ratio in the cross-sectionof the thymus. Increases in this ratio were taken as a measure ofrejuvenation of the thymus.Statistical analysis. Student’s t test was used to compare c/m ratios.

In Vitro Cellular Assays. Cell-mediated lymphocytotoxicity (CML) assays.CML assays were set up before VTL transplantation, and days30, 60, 90, 150, 180, and �250. Methods have been described (5).CD25 depletion CML assays. To test whether anti-donor CTL activitywas restored by removal of CD25-positive cells from responders,a CD25 depletion CML was set up with PBL from a long-termacceptor (recipient 6). CD25-positive cells were depleted bymagnetic cell separation (MACS; Miltenyi Biotec, Auburn, CA).CD25-negative cells were used as responders and cultured withstimulators in the same manner as in a primary CML. Comple-tion of CD25 depletion was confirmed by FACS analysis.Coculture assays. Peripheral regulatory mechanisms were investi-gated by in vitro coculture assays (33). The primary culture wasset up as in CML assays. The primed responder cells were thenharvested and rested overnight at 4°C, after which they werecoincubated with naive SLA-matched PBMCs and irradiateddonor-type or third-party PBMCs for 5 additional days.

We thank Norwood Immunology (Victoria, Australia) for partial sup-port of these studies, Fujisawa Healthcare, Inc. (Deerfield, IL) forgenerously providing FK506, Dr. Isabel McMorrow for helpful review ofthe manuscript, and Maria Doherty for editorial assistance. This workwas supported by National Institutes of Health Program Project Grants5P01-A145897 and 5P01-A139755 and by Fujisawa Health Care, Inc.

1. Sharabi Y, Sachs DH (1989) J Exp Med 169:493–502.2. Sachs DH, Sharabi Y, Sykes M (1990) Progress in Immunology: Proceedings of

the 7th International Congress of Immunology Berlin 1989, eds Melchers F,Nicklin L (Springer, Berlin), Vol VII, pp 1171–1176.

3. Khan A, Tomita Y, Sykes M (1996) Transplantation 62:380–387.4. Manilay JO, Pearson DA, Sergio JJ, Swenson KG, Sykes M (1998) Transplan-

tation 66:96–102.5. Yamada K, Gianello PR, Ierino FL, Lorf T, Shimizu A, Meehan S, Colvin RB,

Sachs DH (1997) J Exp Med 186:497–506.6. Tyan ML (1977) J Immunol 118:846–851.7. Mackall CL, Gress RE (1997) Immunol Rev 160:91–102.8. LaMattina JC, Kumagai N, Barth RN, Yamamoto S, Kitamura H, Moran SG,

Mezrich JD, Sachs DH, Yamada K (2002) Transplantation 73:826–831.9. Sachs DH (1992) in Swine as Models in Biomed Res, eds Swindle MM, Moody

DC, Phillips LD (Iowa State Univ Press, Ames, IA), pp 3–15.10. Rasband W, Bright D (1995) Microbeam Analysis 4:137–149.11. Rosengard BR, Ojikutu CA, Guzzetta PC, Smith CV, Sundt TM, III, Nakajima

K, Boorstein SM, Hill GS, Fishbein JM, Sachs DH (1992) Transplantation54:490–497.

12. Yamada K, Gianello PR, Ierino FL, Fishbein J, Lorf T, Shimizu A, Colvin RB,Sachs DH (1999) Transplantation 67:458–467.

13. Kamano C, Vagefi PA, Kumagai N, Yamamoto S, Barth RN, LaMattina JC,Moran SG, Sachs DH, Yamada K (2004) Proc Natl Acad Sci USA 101:3827–3832.

14. Rodriguez-Barbosa JI, Haller GW, Zhao G, Sachs DH, Sykes M (2005) TransplImmunol 15:25–33.

15. Yamada K, Ierino FL, Gianello PR, Shimizu A, Colvin RB, Sachs DH (1999)Transplantation 67:1112–1119.

16. Zhao Y, Sergio JJ, Swenson K, Arn JS, Sachs DH, Sykes M (1997) J Immunol159:2100–2107.

17. Kappler JW, Roehm N, Marrack P (1987) Cell 49:273–280.18. Marchetti B, Guarcello V, Morale MC, Bartoloni G, Raiti F, Palumbo G, Jr,

Farinella Z, Cordaro S, Scapagnini U (1989) Endocrinology 125:1037–1045.19. Mackall CL, Punt JA, Morgan P, Farr AG, Gress RE (1998) Eur J Immunol

28:1886–1893.20. Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA

(2005) Nature 433:760–764.21. Oner H, Ozan E (2002) Arch Androl 48:115–126.22. Azad N, LaPaglia N, Agrawal L, Steiner J, Uddin S, Williams DW, Lawrence

AM, Emanuele NV (1998) J Endocrinol 158:229–235.23. Aspinall R, Andrew D (2000) Vaccine 18:1629–1637.24. Moore TA, Freeden-Jeffry U, Murray R, Zlotnik A (1996) J Immunol

157:2366–2373.25. Aspinall R, Andrew D, Pido-Lopez J (2002) Springer Semin Immunopathol

24:87–101.26. Fry TJ, Mackall CL (2002) Springer Semin Immunopathol 24:7–22.27. Sempowski GD, Gooding ME, Liao HX, Le PT, Haynes BF (2002) Mol

Immunol 38:841–848.28. Gopinathan R, DePaz HA, Oluwole OO, Ali AO, Garrovillo M, Engelstad K,

Hardy MA, Oluwole SF (2001) Transplantation 72:1533–1541.29. Agus DB, Surh CD, Sprent J (1991) J Exp Med 173:1039–1046.30. Yamada K, Shimizu A, Utsugi R, Ierino FL, Gargollo P, Haller GW, Colvin

RB, Sachs DH (2000) J Immunol 164:3079–3086.31. Utsugi R, Barth RN, Lee RS, Kitamura H, LaMattina JC, Ambroz J, Sachs H,

Yamada K (2001) Transplantation 71:1368–1379.32. Yamada K, Yazawa K, Shimizu A, Iwanaga T, Hisashi Y, Nuhn M, O’Malley

P, Nobori S, Vagefi PA, Patience, C, et al. (2005) Nat Med 11:32–34.33. Ierino FL, Yamada K, Hatch T, Rembert J, Sachs DH (1999) J Immunol

162:550–559.

19086 � www.pnas.org�cgi�doi�10.1073�pnas.0605159103 Nobori et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

16,

202

0