Thymic Transplantation in Miniature Swine. II. Induction of … · 2014-04-18 · Thymic Transplantation in Miniature Swine. II. Induction of Tolerance by Transplantation of Composite

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Thymectomized Recipientsof Composite Thymokidneys to

TransplantationII. Induction of Tolerance by Thymic Transplantation in Miniature Swine.

and David H. SachsIerino, Patricio Gargollo, Gary W. Haller, Robert B. Colvin Kazuhiko Yamada, Akira Shimizu, Ryu Utsugi, Francesco L.

http://www.jimmunol.org/content/164/6/3079doi: 10.4049/jimmunol.164.6.3079

2000; 164:3079-3086; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Thymic Transplantation in Miniature Swine. II. Induction ofTolerance by Transplantation of Composite Thymokidneys toThymectomized Recipients1

Kazuhiko Yamada,* Akira Shimizu, † Ryu Utsugi,* Francesco L. Ierino,* Patricio Gargollo,*Gary W. Haller,* Robert B. Colvin, † and David H. Sachs2*

Previous studies in our laboratory have demonstrated that the presence of the thymus is essential for rapid and stable toleranceinduction in allotransplant models. We now report an attempt to induce tolerance to kidney allografts by transplanting donorthymic grafts simultaneously with the kidney in thymectomized recipients. Recipients were thymectomized 3 wk before receivingan organ and/or tissues from a class I-mismatched donor. Recipients received 1) a kidney allograft alone, 2) a composite allogeneicthymokidney (kidney with vascularized autologous thymic tissue under its capsule), or 3) separate kidney and thymic grafts fromthe same donor. All recipients received a 12-day course of cyclosporine. Thymectomized animals receiving a kidney allograft aloneor receiving separate thymic and kidney grafts had unstable renal function due to severe rejection with the persistence ofanti-donor cytotoxic T cell reactivity. In contrast, recipients of composite thymokidney grafts had stable renal function with noevidence of rejection histologically and donor-specific unresponsiveness. By postoperative day 14, the thymic tissue in the thy-mokidney contained recipient-type dendritic cells. By postoperative day 60, recipient-type class I positive thymocytes appeared inthe thymic medulla, indicating thymopoiesis. T cells were both recipient and donor MHC-restricted. These data demonstrate thatthe presence of vascularized-donor thymic tissue induces rapid and stable tolerance to class I-disparate kidney allografts inthymectomized recipients. To our knowledge, this is the first evidence of functional vascularized thymic grafts permitting trans-plantation tolerance to be induced in a large animal model. The Journal of Immunology,2000, 164: 3079–3086.

T he thymus plays an important role in the development oftolerance to alloantigens and is critical for tolerance toself-Ags (1–5). Recent studies from our research center

have demonstrated the successful induction of tolerance to xeno-geneic swine Ags by transplanting fetal swine thymuses intothymectomized, T and NK cell-depleted mice (6, 7). If such meth-odology could be applied to thymic transplantation in large animalmodels, it could potentially be applicable to clinical discordantxenotransplantation.

As a prelude to studies of pig-to-primate discordant thymic xe-notransplantation, we have begun to evaluate the function of thy-mic grafts across an allogeneic barrier in our miniature swinemodel. We have previously demonstrated that the presence of anintact thymus is required for the development of rapid and stabletolerance in this model (8). In the present study, we have investi-gated the ability of an allogeneic thymic graft to induce toleranceto a class I-mismatched kidney allograft in thymectomized recip-ients. Allogeneic thymic grafts were transplanted either as a com-posite “thymokidney” allograft or as separate thymic and kidneygrafts from the same donor. To transplant donor thymus as a com-posite thymokidney graft, we used a method described recently for

creating a vascularized thymic graft by implanting autologous thy-mic tissue under the renal capsule (9, 45). Such thymokidneyswere able to reconstitute T cells and restore immunocompetency(45). Transplanting donor thymus as part of a vascularized organgraft allows it to function immediately after transplantation. Theresults of the present study show that a composite thymokidneyinduces transplantation tolerance, whereas separate thymic andkidney grafts from the same donor do not, indicating the impor-tance of prior vascularization of the thymic graft.

Materials and Methods

Animals

Animals were selected from our herd of MHC-inbred miniature swine (10,11) at 6–9 wk of age (juvenile animals) to create composite thymokidneygrafts. Except in one case, pigs of 5–9 mo of age were used as recipientsof allogeneic kidney or thymokidney transplants. One of the thymokidneytransplant recipients was a 4.4-year-old pig that had previously undergonethymectomy 3.7 years before. We have used SLAdd animals (swine leu-kocyte antigen, SLA3) as the recipients and SLAgg animals as the donorsfor class I-mismatched transplant in all cases to provide a class II matchand class I mismatch (10).

Experimental groups

Recipients of class I-mismatched grafts were divided into the followingthree groups: the recipients receiving a kidney alone (group 1), separatethymic and kidney grafts from the same donor (group 2), or a compositethymokidney (group 3).

*Transplantation Biology Research Center and†Department of Pathology, Massa-chusetts General Hospital/Harvard Medical School, Boston, MA 02129

Received for publication September 22, 1999. Accepted for publication January5, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by National Institutes of Health Grants 2RO1-AI31046 and 2PO1-HL18646.2 Address correspondence and reprint requests to Dr. David H. Sachs, TransplantationBiology Research Center, Massachusetts General Hospital, MGH-East, Building 149-9019, 13th Street, Boston, MA 02129. E-mail address: [email protected]

3 Abbreviations used in this paper: SLA, swine leukocyte antigen; CyA, cyclosporine;POD, postoperative day; H&E, hematoxylin and eosin; CML, cell-mediated lym-pholysis; TNP, trinitrophenyl; SP, single positive.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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Operative procedures

Creation of thymokidney in the donor. A partial thymectomy (ap-proximately three-fourths of the cervical thymus) was performed to pro-vide autologous thymic tissue, which was minced into 2–3 mm3 pieces andgrafted under the renal capsule. The thymokidney was allowed to developfor 3 mo before excision and transplantation into the allogeneic recipient.Complete thymectomy in the recipient.Complete thymectomy wasperformed before allogeneic transplantation as previously described (8).Briefly, the pretracheal muscles were retracted, exposing the cervical thy-mus and trachea from the cervico-thoracic junction to the mandibular area.The cervical thymus was excised, after which the mediastinal thymus wasremoved through a sternotomy.Allogeneic kidney or thymokidney transplantation.The transplantwas performed as previously established for allogeneic kidney transplan-tation (8, 12). In both the kidney and thymokidney transplants, the renalartery was implanted end-to-side into the recipient’s aorta using a Carrelpatch, and the renal vein was anastomosed end-to-side to the recipient’sinferior vena cava. Urinary drainage was accomplished via a ureterove-sicular anastomosis. The kidney or thymokidney was transplanted intoclass I-disparate recipients. Both native kidneys were excised. One recip-ient (#11196) had been thymectomized and had received a life-supportingclass I-mismatched kidney allograft (with a 12-day course of cyclosporine(CyA)) 3.7 years before; the kidney allograft had been accepted after ashort rejection crisis. In this pig, the kidney allograft was removed at thetime of composite thymokidney allografting from a donor SLA-matched tothe original kidney allograft donor.Separate allogeneic thymic and kidney transplantation from thesame donor. The thymic grafts were obtained in the same manner as theautologous thymic grafts (described above) on the day of kidney harvestfrom the same donor. More than 30 pieces of allogeneic thymus (equivalentto that in a composite thymokidney graft) were transplanted into the ster-nomastoid muscles of the recipient. Previous studies in our laboratory havedemonstrated that autologous thymic tissue engrafts equally well in thissite and under the kidney capsule (our unpublished observations).Biopsy of the composite thymokidney allografts.Biopsies wereconducted through a flank abdominal incision on postoperative days (POD)0, 30, 60, 80 (#11196 only), and.100 (#12750 and #12827). The thy-mokidney was exposed for macroscopic evaluation, and wedge kidney bi-opsies were taken (which included both thymic and renal tissue) for his-tological examination and FACS.

Immunosuppressive therapy

CyA (Sandimmune) was generously provided by Novartis Pharmaceuticals(East Hanover, NJ) and was administered as an i.v. suspension accordingto the manufacturer’s specifications. CyA was given daily as a single in-fusion at a dose of 10–13 mg/kg (adjusted to maintain a blood trough levelof 400–800 ng/ml) for 12 consecutive days, beginning on the day of trans-plantation. CyA levels were determined by a fluorescence polarization im-munoassay (Abbott Laboratories, Dallas, Texas), which measured the par-ent compound but not metabolites.

Histological examination

Formaldehyde-processed specimens were stained using hematoxylin andeosin (H&E) and periodic acid-Schiff, and frozen tissues were used forimmunohistochemistry analysis with the avidin-biotin-HRP complex tech-nique. Thymocyte development was assessed using the murine anti-pigmAbs 74-12-4 (IgG2b, anti-swine CD4), 76-2-11 (IgG2a, anti-swine CD8),76-7-4 (IgG2a, anti-swine CD1) (13), MSA4 (IgG2a, anti-swine CD2)(14), BB23-8E6 (IgG2b, anti-swine CD3), 2-27-3a (IgG2a, anti-swineclass I), and ISCR-3 (IgG2a, anti-swine DR) (15). Four-micron sectionswere incubated with 1% normal horse serum and avidin (100mg/ml in PBSPBS) to inhibit nonspecific binding of horse IgG and endogenous biotin,respectively. After 20 min, the tissue was covered with optimally dilutedprimary mAb and incubated for 60 min at room temperature. Sections wererinsed in PBS and incubated in a solution of biotin (10mg/ml in PBS) with0.3% hydrogen peroxide for 30 min to block endogenous peroxidase. Thebiotinylated secondary Ab (horse anti-mouse IgG) was added and incu-bated for 45 min. After a further PBS wash, sections were incubated in anoptimal dilution of avidin-biotin-peroxidase complex for 60 min, rinsed inPBS, and visualized by staining with hydrogen peroxide (H2O2) containing3,39-diaminobenzidine in 0.05 M Tris buffer. Staining was stopped by dip-ping the slides into distilled water. Sections were then counterstained withGill’s single-strength hematoxylin. Controls included omission of primaryAb, horse anti-mouse Ab, and an irrelevant primary mAb (36.7.5, murineanti-mouse KK) (16). Double immunostaning with class II (ISCR-3), class

Ic, or class Id and cytokeratin (Z622, Dako, Carpinteria, CA) for the iden-tification of the proliferating thymic epithelial cells was performed in for-malin-fixed paraffin sections using a combination of immunoalkaline phos-phatase and standard ABC (avidin/biotin complex) technique (17). Thesections were stained with anti-class II (ISCR-3), class Ic, or class Id (im-munoalkaline phosphatase with a blue reaction product) and then with arabbit polyclonal cytokeratin (avidin/biotin complex, H2O2 containing3,39-diaminobenzidine with a brown reaction product).

Preparation of PBL

Blood was drawn from the external jugular vein. For separation of PBL,freshly heparinized whole blood was diluted;1:2 with HBSS (Life Tech-nologies, Grand Island, NY), and the mononuclear cells were obtained bygradient centrifugation using lymphocyte separation medium (Organon,Teknika, Durham, NC). The mononuclear cells were washed once withHBSS, and contaminating red cells were lysed with ammonium chloridepotassium buffer (B & B Research, Friskeville, RI). Cells were thenwashed with HBSS and resuspended in tissue culture medium. All cellsuspensions were kept at 4°C until they were used in cellular assays.

Preparation of thymocytes

Biopsies from thymic grafts (100–200 mg) were finely minced with ascalpel blade and then dispersed with the tip of a syringe plunger in HBSSbuffer. The cell suspension was then filtered through 200-mm nylon mesh,pelleted by centrifugation, and resuspended in flow cytometry medium.

Flow cytometry

Flow cytometry of PBL and thymocytes was performed using a BectonDickinson (San Jose, CA) FACScan. Cells were stained using directlyconjugated murine mAbs which were the same as those used for immu-nohistochemistry (see above). Phenotype was analyzed by three-colorstaining. The staining procedure was performed as follows. A total of 13106 cells were resuspended in flow cytometry buffer (HBSS containing0.1% BSA and 0.1% NaN3) and incubated for 30 min at 4°C with satu-rating concentrations of a FITC-labeled mAb. After a single wash, thesecondary PE-conjugated Ab was added, and cells were incubated for 30min at 4°C. After a further wash, the final biotinylated Ab was added andincubated for 30 min. The cells were washed, cytochrome was added, andthe cells were incubated for 8 min to stain the biotinylated Ab. Cells werethen washed twice and analyzed by FACScan.

Cell-mediated lymphocytotoxicity (CML) assay

Tissue culture media used for CML assays consisted of RPMI 1640 (LifeTechnologies) supplemented with 6% FCS (Sigma, St. Louis, MO), 100U/ml penicillin, and 135mg/ml streptomycin (Life Technologies); 50mg/ml gentamicin (Life Technologies); 10 mM HEPES (Fisher Scientific,Pittsburgh, PA); 2 mML-glutamine (Life Technologies); 1 mM sodiumpyruvate (BioWhittaker, Walkersville, MD); nonessential amino acids(BioWhittaker); and 53 1025 M 2-ME (Sigma). The effector phase of theCML assay was performed using Basal Medium Eagle (Life Technologies)supplemented with 6% controlled processed serum replacement-3 (Sigma)and 10 mM HEPES.

CML assays were performed as previously described (12, 18, 19).Briefly, lymphocyte cultures containing 43 106 responder and 43 106

stimulator PBL (irradiated with 2500 cGy) per ml were incubated for 6days at 37°C in 7.5% CO2 and 100% humidity. Bulk cultures were har-vested and effectors were tested for cytotoxic activity on51Cr-labeled (Am-ersham, Arlington Heights, IL) lymphoblast targets. Effector cells wereincubated for 5.5 h with target cells at E:T ratios of 100:1, 50:1, 25:1, and12.5:1. The following three target cells were tested in each assay: SLA-matched PBL to the effectors (negative control), donor-matched PBL(SLAgg, class Icc and class IIdd), and third-party PBL. Supernatants werethen harvested using the Skatron (Sterling, VA) collection system and51Crrelease was determined on a gamma counter (Micromedic Systems, Hunts-ville, AL). The results were expressed as percent specific lysis (PSL) andwere calculated as: PSL5 {[experimental release (cpm)2 spontaneousrelease (cpm)]/[maximum release (cpm)2 spontaneous release (cpm)]}3100%.

Sensitization cultures (Trinitrophenyl (TNP) CML assay)

MHC restriction of T cells was examined by in vitro TNP CML assay asmodified from previously published studies in rodents (20). PBL for invitro sensitization (stimulators and targets) were incubated with 10 mMtrinitrobenzenesulfonic acid (Sigma) in CML medium for 10 min at 37°C.After three washes in medium supplemented with 5% FBS, the in vitro-sensitized stimulator cells were irradiated with 2500 cGy, 43 106 cells

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were added to an equal number of responding cells per well and culturedfor 6 days, and the in vitro-sensitized target cells were incubated with51Crfor 1.5 h.

ResultsClinical course, histological findings, and immunologic statusafter class I-disparate kidney transplantation

Thymectomy before kidney transplant (day221) interferes withthe induction of tolerance. We have previously demonstratedthat thymectomy performed 3 wk before kidney transplantationinterferes with the induction of tolerance (group 1: historic con-trols) (8). Four of five thymectomized animals developed severerejection crises (Fig. 1a) manifested by markedly elevated creati-nine levels, the development of anti-donor CML (Fig. 2, filledbars), histologic evidence during the second to fourth postopera-tive weeks (Fig. 3a), and chronic rejection (Fig. 3b) at later timeperiods. One animal eventually accepted its kidney allograft butshowed a prolonged period of unstable creatinine levels with per-sistence of anti-donor CTL throughout the experimental period;this has never been observed in euthymic recipients of a classI-mismatched kidney transplant.

A nonvascularized thymic graft did not induce tolerance (group 2).Two thymectomized animals (#12834 and #12858) received sep-

FIGURE 1. Clinical course, as indicatedby plasma creatinine level, of class I-mis-matched kidney transplants in thymecto-mized recipients of kidney alone (a), kid-ney and thymus separately (b), andcomposite thymokidney transplants (c), alltreated by standard CyA protocol (a 12-daycourse of CyA i.v. to maintain blood levelsat 400–800 ng/ml).

FIGURE 2. Anti-donor CTL responses on POD 30 in thymectomizedrecipients of kidney alone (f), kidney and thymus separately (M), and thecomposite thymokidney (u). Percent specific lysis at E:T ratio of 100:1 isshown.

FIGURE 3. Histology (H&E) of kidney allografts on POD 30 in a recipientof kidney alone (#11809) (a), a recipient of kidney and thymus separately(#12858) (c), and a recipient of the composite thymokidney (#12750) (e).Histology (H&E) of a kidney allograft on POD 60 in a recipient of kidneyalone (#11809) (b), a kidney allograft on POD 100 in a recipient of kidney andthymus separately (#12858) (d), and a kidney allograft on POD 150 in a re-cipient of the composite thymokidney (#12750) (f).

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arate thymic and kidney grafts on the same day from the sameclass I-mismatched donor. Both recipients had a marked rejectioncrisis early (before POD 30), and both grafts developed chronicchanges similar to the clinical course in thymectomized recipientsof a kidney alone (Fig. 1b). Histologically, diffuse mononuclearcell infiltration as well as acute glomerulitis and vasculitis wereseen in the kidney graft on POD 30 (Fig. 3c), and chronic glomer-opathy developed later (Fig. 3d). The thymic grafts were rejectedby POD 30 with evidence of mononuclear cell infiltration (Fig.4a). Immunologically, both animals maintained the same level ofanti-donor CTL reactivity as a thymectomized recipient of a kid-ney alone (Fig. 2, open bars) and developed anti-donor alloanti-bodies (IgG and IgM). The clinical courses and histological find-ings as well as immunological statuses of these animals weresimilar to those of thymectomized control animals.

A vascularized thymic graft (composite thymokidney) inducedrapid and stable tolerance (group 3).Three thymectomized re-cipients (#12750, #12827, and #11196) received class I-mis-matched composite thymokidney grafts. Fig. 4b shows a trans-planted composite thymokidney graft in the recipient. The thymicgrafts were vascularized by vessels from both kidney parenchymaand kidney capsule. All recipients accepted their grafts and hadstable renal function indefinitely (Fig. 1c). Plasma creatinine levelswere 1.1 mg/dl on POD 314 in #12750, 1.5 mg/dl on POD 225 in#12827, and 1.0 mg/dl on POD 165 (#11196). Histologically, therewas minimal cell infiltration, and no vascular changes were ob-served in the kidney allograft on POD 30 (Fig. 3e). No chronicchanges developed in later biopsies (Fig. 3f, POD 150 kidney bi-opsy). The thymic portion of the thymokidney also showed anintact thymic structure without any cell infiltration (Fig. 4c). Allrecipients had donor-specific unresponsiveness on day 30 andthereafter (Fig. 2, gray bars), and no measurable anti-donor Abs(IgM or IgG) developed.

These results indicated that a vascularized composite thymokid-ney graft but not a neovascularized thymic graft induced rapid andstable tolerance to a class I-mismatched kidney allograft in athymectomized recipient in a manner similar to that reported ineuthymic recipients receiving kidneys alone (21).

Mechanisms of tolerance in the composite thymokidneytransplant

Nonvascularized donor thymic tissue failed to induce tolerance,indicating that donor thymic emigrants are unlikely to be entirelyresponsible for this process. To further elucidate the mechanismsunderlying this phenomenon, we examined 1) changes in thymicstroma and thymopoiesis in the transplanted vascularized thymus,and 2) T cell restriction after a composite thymokidney transplant,as well as the presence of peripheral macrochimerism.Thymopoiesis in vascularized thymic grafts.Fig. 5 shows im-munohistochemistry demonstrating 1) migration of recipient-typedendritic cells and 2) thymopoiesis in the thymic graft. To differ-entiate donor and recipient cells, class Ic (donor-type) mAb andclass Id (recipient-type) mAb were used.

The specimen from POD 14 showed recipient-type class I-pos-itive cells that morphologically appeared to be dendritic cells mi-grating to the cortico-medullary junction of the thymic graft (Fig.5a, brown cells). Double-staining demonstrated that the putativedendritic cells were recipient-type class I-positive (blue)/class II-positive (brown) (Fig. 5d). The cells were cytokeratine-negative(not stained brown) (Fig. 5e), indicating that these recipient-typecells were likely to be dendritic cells.

Although recipient-type dendritic cells had migrated into thethymic graft by POD 14, there were few recipient-type cells seenin the medulla on POD 14 and 30. However, by POD 60, recipient-type class I-positive cells were observed (Fig. 5b, brown cells), andtheir numbers increased thereafter (after POD 60) in the medullabut not in the cortical zone, indicating that thymopoiesis was oc-curring without rejection.

We also examined changes in donor-type cells. Class I donor-type staining demonstrated that the number of donor-type thymo-cytes had decreased markedly in the POD 60 specimen and there-after. However, donor-type class I-positive stromal cells anddonor-type cells having dendritic morphology were still present atPOD 100 (Fig. 5c).

Thymopoiesis was also examined by three-color FACS analysisto determine whether it involved both CD4 and CD8 T cells. The

FIGURE 4. Histology (H&E) of thymic graft in neck muscle on POD 30 in a recipient of kidney and thymus separately (#12858) (a), and of a thymicgraft under the renal capsule on POD 30 in a recipient of composite thymokidney (#12750)(c). The thymic graft ina shows fibrosis and a mononuclearcell infiltrate with no residual thymic structure or Hassall’s corpuscles, indicating a rejected thymic graft. In contrast, the thymic graft in the compositethymokidney (c) shows that thymic structure is well-preserved without evidence of rejection. The macroscopic appearance of the composite thymokidneygraft (b) demonstrates the autologous thymic graft (arrow) under the kidney capsule 3 mo after thymic transplantation.

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phenotypic analysis of donor-type and recipient-type thymocytesis shown in Fig. 6. Recipient-type CD4-positive cells and CD8-positive cells were detected with FACS by POD 30 (Fig. 6a), andthe number of recipient-type cells markedly increased by POD 100(Fig. 6b), indicating that both CD4 and CD8 T cells developed inthe donor thymic tissue. In addition,;30% of the total thymocytesexpressed both CD3 and class I, indicating that they were maturethymocytes. These levels were similar to those seen in a thymokid-ney before it was transplanted as well as in a native thymus. Im-munohistochemistry and FACS data confirmed that rapid thymo-poiesis without any rejection crisis occurred in the thymic graft ofthe vascularized composite thymokidney.

Peripheral macrochimerism. Macrochimerism, defined as chi-merism detectable by FACS, was evaluated in the recipients ofcomposite thymokidney grafts and of thymic grafts transplantedsimultaneously with kidney allografts but in separate locations.Both animals receiving the thymokidney and animals receiving thekidney and thymus separately showed donor cell macrochimerism(0.07–0.18% in lymphocyte population) by POD 8 (during CyAtreatment period). This percentage of chimerism was higher thanthat seen in nonthymectomized recipients of class I-mismatchedkidney transplants (22), suggesting that donor thymic emigrantswere involved. However, after cessation of CyA treatment, mac-rochimerism disappeared in the recipients of separate kidney andthymus grafts (probably due to rejection), whereas a small per-centage (0.02–0.03% in lymphocyte population) of macrochimer-ism persisted at POD 30 in thymokidney recipients (Fig. 7a). Do-nor cells (donor-type class I-positive cells) were also detected inthe T cell area of mesenteric lymph nodes on POD 30 by FACSand immunohistochemistry (Fig. 7b) in recipients of thymokid-neys. Three percent of cells in the lymphocyte gate were donor-type class I1/CD31 T cells in mesenteric lymph node on POD 30;however, donor cells were not detected by either FACS or immu-nohistochemistry by POD 100. No recipients of kidney and thymusgrafted separately showed macrochimerism in mesenteric lymphnodes on POD 30.

Recipients of composite grafts and separate thymus and kidneytransplants both demonstrated similar levels of circulating donorleukocytes postoperatively, including recent donor thymic emi-grants. These results suggested that comparable populations of do-nor leukocytes were not sufficient alone to induce tolerance in thismodel.

MHC restriction after the composite thymokidney graft.We re-cently demonstrated that 1) the number of T cells, particularly ofCD4 single positive (SP) cells, decreased after thymectomy over aperiod of 2 years, and 2) CD45RA1/CD4 SP cells increased aftera thymokidney transplant in a long-term thymectomized animal,indicating that T cell development was occurring in the thymokid-ney (45).

We have now studied MHC restriction after transplantation ofthe composite thymokidney graft in the same long-term animal(#11196), which had been thymectomized and received a life-sup-porting class I-mismatched kidney allograft 3.7 years earlier. Be-fore the thymokidney transplant, this animal had few CD4 SPcells, and its general unresponsiveness was demonstrated by MLR.The animal accepted the composite thymokidney allograft. CD4SP cells, including CD45RA1/CD4 SP cells, increased markedlyfrom 30 days after the transplant, indicating that the compositethymokidney was capable of reconstituting T cells (45). Becausenew T cells developed from the thymic graft and the animal be-came immunocompetent, as shown by MLR 3 mo after the com-posite thymokidney transplant, we examined the MHC restriction

FIGURE 5. Immunohistochemical study of thymic grafts in the com-posite thymokidney grafts. Recipient-type class I-positive cells (brown) areseen at corticomedular junction of thymic graft on POD 14 (a); recipient-type class I-positive cells (brown) in medulla on POD 60 (b); and donortype class I-positive cells (brown) in thymic graft on POD 100 (c). Doublestaining of thymic graft in the composite thymokidney on POD 14: arrowsindicate recipient-type class I-positive (blue)/class II-positive (brown) cells(d) and recipient-type class I-positive (blue)/cytokeratine negative (notstained as brown) cells (e).

FIGURE 6. Thymopoiesis in the thymic graft of a composite thymokid-ney as assessed by three-color FACS analysis with staining to determinewhether thymopoiesis involved both CD4 and CD8 T cells. Recipient-typeCD4-positive cells and CD8-positive cells were detected on POD 30 (a),and the number of recipient-type cells markedly increased on POD 100 (b).

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of T cells in vitro by TNP CML assays 3 mo after the compositethymokidney transplant in this animal (Fig. 8). Although the ani-mal had no anti-donor response (donor-specific unresponsiveness(Fig. 8, top left, open bar)), a positive response was seen if bothanti-donor effector cells and donor target cells were incubated withTNP (Fig. 8,top left, filled bar). This result indicated that donorMHC-restricted CTL were present. In addition, a positive responsewas seen if anti-self effector cells and self target cells were incu-bated with TNP, indicating that self MHC-restricted CTL werealso present (Fig. 8,bottom left, filled bar). No killing was seen ifeither stimulator or target cells were incubated without TNP (Fig.8, bottom left, open or gray bar). These data demonstrated thatthere were both donor and recipient MHC restrictions of CTL re-activity after composite thymokidney transplantation.

DiscussionIt has previously been demonstrated that fetal porcine thymic tis-sue transplanted under the kidney capsule of thymectomized, Tcell-depleted mice can induce tolerance to swine Ags (6, 7). Wehope to eventually assess this strategy in a large animal xenogeneictransplant model. In preparation for such studies, the ability of thethymic grafts to induce tolerance across a full allogeneic barrier inminiature swine was evaluated. We have previously demonstratedthat the presence of an intact thymus is required for the develop-ment of rapid and stable tolerance in miniature swine (8, 23, 46).Thus, if the recipient thymus was removed 3 wk before a classI-disparate kidney or heart transplant, tolerance was not obtained(8, 24). In the present study, thymectomized recipients were usedto determine whether a thymic graft is capable of replacing theeffect of the host thymus in permitting the induction of tolerance toa class I-mismatched kidney transplant. We have found that a vas-cularized thymic graft induces tolerance, whereas the transplanta-tion of nonvascularized thymic tissue does not.

It is generally accepted that vascularized organs are more tol-eragenic than nonvascularized tissues. Thus, vascularized graftsare relatively tolerogenic (21, 25, 26), whereas skin and tissuegrafts are relatively immunogenic, probably because they releasecells or Ags from cells into the lymphatic system where they sen-sitize the host (27). Even in the case of autografts, direct thymicgrafts require a revascularization period after transplantation. The

FIGURE 7. a, Flow cytemetric analysis of lymphopoietic donor mac-rochimerism (percent of CD3-positive donor cells) in peripheral blood inrecipients of composite thymokidney (#12750 and #12827) and in recipi-ents of kidney and thymus separately (#12834 and #12858).b, Immuno-histochemical study of macrochimerism in a mesenteric lymph node in arecipient of composite thymokidney (#12750) on POD 30. Donor classI-positive cells (brown) are seen in T cell area of a mesenteric lymph node.

FIGURE 8. TNP-modified CTL re-sponses of a thymus-grafted animal(#11196) and a naive SLA-matched ani-mal. Percent specific lysis at E:T ratio of100:1 is shown.

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ischemic period in the first 1–3 wk leads to temporary loss of thethymic graft structure, which is reconstituted over the next 5–7 wk(45). Thus, during the period of revascularization, there is in-creased susceptibility to nonspecific graft loss and an increasedpotential for sensitization of the recipient. In contrast to directthymic grafts, transplantation of thymic tissue as a composite thy-mokidney graft avoids the necessity for revascularization in therecipient, and the thymic graft functions immediately.

Based upon our results, we would suggest the following poten-tial mechanisms through which the thymic grafting may inducetolerance in this model: 1) T cell progenitors may be positively andnegatively selected by donor thymic stroma and/or dendritic cellsby a mechanism similar to that of self-tolerance (2). Immunohis-tochemistry demonstrated that donor-type dendritic cells remainedfor .3 mo after the transplant. Negative selection of potentiallyautoreactive thymocytes occurs mainly in the thymus and isthought to be induced primarily by interaction with bone marrow-derived cells (2). Other reports have demonstrated that thymic ep-ithelial cells are capable of taking part in both positive and nega-tive selection of thymocytes (28, 29) and of inducing anergy (30).Thus, the long-term presence of donor stromal cells (donor epi-thelial cells, dendritic-like cells, and vascular endothelial cells) inthe donor thymic graft may play an important role in the inductionof tolerance by deletion, anergy, or a combination of the two.Immuhistochemistry also demonstrated that (1) recipient dendritic-like cells had migrated into the thymic graft within 2 wk. Themigrant recipient-type dendritic cells are cytokeratin2/class II1

cells, strongly suggesting that they are bone marrow-derived den-dritic cells and thus may delete self-reactive cells. Consistent withthis hypothesis, we evaluated T cell restriction in an animal(#11196) whose T cells had progressively decreased in numberduring the 3.7 years since thymectomy but were reconstituted afterthe composite thymokidney transplant. T cells were restricted byboth the recipient and donor MHC, indicating that newly devel-oped T cells were educated by both host and donor elements in thethymic graft.

2) All recipients in this study were not T cell depleted. Thus,mature T cells were present in recipients of composite thymokid-neys. However, mature peripheral T cells have also been shown tobecome unresponsive to donor Ag by recirculation to the thymus(31), and a similar mechanism could be operative for the thymicgraft. Re-entry of activated T cells into the thymus has been re-ported previously (31); thus, alloreactive T cells could enter thedonor thymic graft and could be educated. In addition, because therecipient and donor are class II- matched in this model, processedclass I Ags presented by class II Ags in the thymus would beexpected to be identical regardless of whether the APC were ofdonor or recipient origin. Tolerance at the level of CD4 helper cellsrecognizing class I peptides through the indirect pathway may bepossible in both cases. CyA can effectively inhibit both the CD4helper pathway and the direct CD8 helper pathway (32–34). Thus,T cell depletion was not required to induce rapid and stable toler-ance in this model.

3) Another possibility is that thymic emigrants from the thymicgraft, which may include regulatory cells, facilitate tolerance in-duction peripherally. Such peripheral tolerance could be mediatedby a changed cytokine milieu or by suppressive mechanisms. Sup-pression of this type has been reported previously in rodent mod-els. One group has identified CD4-positive cells as the regulatorycell population (35–37). However, direct thymic tissue transplantsdid not facilitate the induction of tolerance in our model and, there-fore, donor thymic emigrants may not be sufficient to inducetolerance.

Our strategy eventually may be clinically applicable for the in-duction of transplantation tolerance to xenografts, which is theultimate goal of these experiments. We have chosen to begin thestudies in an allogeneic system rather than in a discordant xeno-geneic system to assess the cellular immune response without thecomplications provided by natural Abs, which are a formidablebarrier to vascularized xenografts (38–41). The present data indi-cate that transplantation of a vascularized thymic graft is a poten-tial strategy to induce tolerance across a class I-mismatch barrier.Therefore, we are evaluating the effect of composite thymokidneygrafts on the induction of tolerance across a two-haplotype MHCmismatch barrier. Preliminary data demonstrate that vascularizedthymokidney grafts can induce tolerance across fully MHC-mis-matched barriers in thymectomized recipients after a T cell deple-tion regimen. Because xenogeneic T cell reactivity between humanand pig has been demonstrated to be at least as strong as that seenin allogeneic responses (42–44), the induction of T cell toleranceby this strategy may also prove to be of importance to the eventualsuccess of xenotransplantation.

AcknowlegementsWe thank Drs. David K. C. Cooper and Isabel McMorrow for their helpfulreview of the manuscript, Scott Arn for herd management and qualitycontrol typing, Joseph Ambroz for technical assistance, and Lisa A. Ber-nardo for help in manuscript preparation. We would also like to thankNovartis for generously providing CyA and Schering-Plough AnimalHealth for providing Flunixamine.

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