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The Journal of Immunology, 2001, 166: 1843-1854.
Copyright © 2001 by The American Association of Immunologists

Transplantation Tolerance and Autoimmunity After Xenogeneic Thymus Transplantation1

Guliang Xia, Jozef Goebels, Omer Rutgeerts, Michel Vandeputte and Mark Waer2

Laboratory for Experimental Transplantation, University of Leuven, Leuven, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Successful grafting of vascularized xenografts (Xgs) depends on the ability to reliably induce both T cell-independent and -dependent immune tolerance. After temporary NK cell depletion, B cell suppression, and pretransplant infusion of donor Ags, athymic rats simultaneously transplanted with hamster heart and thymus Xgs developed immunocompetent rat-derived T cells that tolerated the hamster Xgs but provoked multiple-organ autoimmunity. The autoimmune syndrome was probably due to an insufficient development of tolerance for some rat organs; for example, it led to thyroiditis in the recipient rat thyroid, but not in simultaneously transplanted donor hamster thyroid. Moreover, grafting a mixed hamster/rat thymic epithelial cell graft could prevent the autoimmune syndrome. These experiments indicate that host-type thymic epithelial cells may be essential for the establishment of complete self-tolerance and that mixed host/donor thymus grafts may induce T cell xenotolerance while maintaining self-tolerance in the recipient.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The thymus is well known to play critical roles not only in establishing and shaping a functional T cell repertoire, but also in inducing and maintaining self-tolerance as well as deletional transplantation (Tx)3 tolerance (1, 2). Either isogeneic or allogeneic thymus (allothymus) tissues have been shown to restore thymus-dependent immunity such as reactivity to mitogens or allogeneic Ags and the ability of IgG Ab production in congenitally athymic or neonatally thymectomized animals. Additionally, allothymus-reconstituted animals become tolerant to donor strain graft and resistant against third-party allograft (3).

In xenogeneic combinations, restoration of T cell functions and induction of donor tissue xenograft (Xg) tolerance has been documented in congenitally nude or neonatally thymectomized mice implanted with thymus Xgs, such as from concordant rat (4, 5, 6, 7) or from discordant rabbit donors (8). Human/murine thymopoiesis has been demonstrated in fetal or postnatal human thymus/liver tissue-grafted SCID mice; in this system, mouse- or human-derived T cells differentiating in the human thymus are tolerized for both human and mouse Ags in vitro (9, 10, 11). Also, fetal porcine thymus supports porcine or human thymopoiesis after grafting with porcine or human fetal liver tissue in SCID mice (12, 13) as well as murine lymphopoiesis in nude mice (14) and in adult thymectomized, T/NK cell-depleted mice (15). In addition, mouse T cells educated in a porcine thymus become tolerant to swine skin Xgs (16).

These experiments indicate that xenogeneic thymus (xenothymus) Tx is a useful and reliable approach to support a functional T cell repertoire and to induce T cell-specific xenotolerance, at least for nonprimarily vascularized Xgs, the rejection of which depends predominantly on T cells. However, for immediately vascularized Xgs to survive, T cell-specific tolerance is not sufficient, as various T cell-independent (T-I) immunities may lead to Xg rejection even before T cells are activated (17). This explains why, to date, no long-lasting xenotolerance for vascularized Xgs by thymus Tx has been demonstrated.

Previous studies by our group have demonstrated that T-I xenoreactivity, such as those mediated by IgM xenoantibodies (xAbs), NK cells, and macrophages (M{phi}s), is vigorous and sufficient to mediate hamster cardiac Xg rejection in T-deficient athymic rats (18). These immune barriers could be overcome by the induction of T-I xenotolerance using a tolerizing regimen (TR) including the infusion of xenoantigens (xAgs), temporary NK cell depletion, and short-term B cell suppression (19). In the present study we have explored whether combined T-I and T cell-dependent (T-D) Xg tolerance are induced using combined hamster thymus/heart Tx in similarly treated nude rats. We show that under these conditions, fetal or adult hamster thymus efficiently supports rat-derived T lymphopoiesis. Moreover, these T cells are immunocompetent and specifically tolerant for donor xAgs both in vitro and in vivo. However, hamster thymus-grafted rats develop multiple-organ autoimmune disease. Tx of a mixed hamster/rat thymus that maintains tolerance for hamster xAgs can prevent this autoimmunity. To our knowledge, this is the first demonstration that combined and long-lasting T-I and T-D Xg tolerance leading to a long-term survival of vascularized Xgs can be achieved without bone marrow Tx in animals with intact innate immunity and developing functional T cell repertoire after thymus Tx. These observations may be relevant for the induction of clinical Xg tolerance as well.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Six- to 8-wk-old congenitally athymic Rowett (RT1c) rnu/rnu or Wag/Rj (RT1u) rnu/rnu male nude rats (Harlan CPB, Zeist, The Netherlands) were used as recipients. They were bred in clean conditions and kept in laminar flow units. Inbred Au/Hö Han Rj hamsters (CERJ, Le Genest-St-Isle, France) and inbred WKAH (RT1k) rats were used as donors. Some adult donor hamsters and rats received 10.5 Gy of total body irradiation to deplete thymocytes 2 days before Tx.

Tolerizing regimen (TR)

The TR has been previously described in detail (19). It consisted of 1) a single i.v. transfusion of 1 ml of heparinized whole hamster blood on day -14; 2) a single i.v. injection of 100 µl of rabbit anti-asialo GM1 serum, a NK cell-depleting Abs purchased from Wako Pure Chemicals Industries (Osaka, Japan) that was diluted in 0.5 ml of PBS and administered on day -14; and 3) a 4-wk administration (day -14 to day 14, given by gavage) of 20 mg/kg/day of malononitriloamide x920715 (MNA715), an analogue derived from a novel immunosuppressant leflunomide (20).

Surgical procedures

Fetal thymic tissue implantation. Two lobes of fetal (2 wk gestation) hamster or rat thymus tissue were implanted under the right kidney capsule via a midline laparotomy incision. For mixed fetal hamster-rat thymus Tx, fetal WKAH rat thymi and fetal hamster thymi were cut into pieces. Then the majority of the thymocytes and bone marrow-derived cells were removed by putting the thymus pieces on a steel mesh and "squeezing" them using a rubber stamp. The remaining tissue, largely composed of thymic epithelial cells (as evidenced under the microscope), was implanted under the kidney capsule (i.e., one rat lobe and one hamster lobe per recipient). By doing so, hamster and rat thymus pieces were intensely mixed, which was subsequently confirmed by histology showing a complete mixture of areas composed of hamster or rat thymic epithelial cells (see below). To prevent the possible transfer of fetal thymocytes, rat and hamster donors (pregnant mothers) were lethally irradiated (9.5 Gy) 1 day before Tx.

Heterotopic heart grafting. Under diethyl ether anesthesia, cervical/abdominal hamster- or rat-into-rat cardiac Tx was performed using standard microsurgical techniques as described previously (19).

Vascularized adult thymus/heart composite grafting. A modified technique of a combined Tx of the donor thymus and heart was developed based upon the method described by Andrzejewski and Zielinski (21). Adult hamster or rat heart in continuity with two lobes of thymus was vascularized from the dorsal (descending) aorta and drained to the main pulmonary artery. After systemic heparinization, the donor hamster or rat main pulmonary artery (near its bifurcation) and dorsal aorta (below the left azygos vein) was divided and cut, and then the aorta was flushed with cold (4°C) heparinized (10 U/ml) PBS to protect the heart-thymus composite graft. Both sides of internal thoracic arteries and veins and both sides of carotid and subclavian arteries and veins were ligated near the chest wall so that both sides of the thymic arteries and veins were preserved. The left azygos vein was then ligated and cut, and the dorsal aorta was divided so as to provide an optimal anastomotic site. Finally, ligatures were placed around the supradiaphragmatic vena cava and the separate right and left pulmonary veins. The composite graft was then harvested. The donor dorsal aorta was anastomosed end-to-side to the recipient abdominal aorta, and the donor pulmonary artery was sutured to the recipient posterior (inferior) vena cava using running 10-0 nylon sutures. Graft beating was monitored by abdominal palpation twice daily. Rejection was diagnosed by cessation of palpable ventricular contractions and confirmed by direct visualization and subsequent histological examination.

FACScan analysis

Phenotypic analysis of T cell surface markers and identification of elicited IgM or IgG Abs directed against hamster or WKAH rat targets were measured by FACScan analysis. Unless otherwise mentioned, all mAbs were purchased from PharMingen (San Diego, CA).

Phenotype analysis. PBMC were prepared from aliquots of 100 µl of heparinized whole blood from hosts depleted of RBC by 0.83% NH4Cl solution and double labeled for 30 min at 4°C with the following mAbs to identify T cell subsets. 1) PE anti-rat CD3 (G4.18) together with either FITC anti-rat CD4 (OX-35) or FITC anti-rat CD8b (341) mAbs to identify CD3+CD4+ or CD3+CD8+ T cells, respectively. 2) PE anti-rat Thy1.1 (MRC OX-7, Serotec, Kidlington, Oxford, U.K.) together with FITC anti-rat CD4 (OX-35) or FITC anti-rat CD45RC (MRC OX-22, Serotec) were used for analysis of coexpression of CD4 or CD45RC molecules on Thy1+ cells. 3) CD4+ T cells were further divided into CD45RC+ and CD45RC- subpopulations by double staining with PE anti-rat CD4 (OX-38) and FITC anti-rat CD45RC. In some animals T cell activation markers such as CD25 (MCA730F, Serotec) or OX-40 (MCA273F, Serotec) expressed on circulating or spleen CD4+ (OX-38PE) T cells as well as the presence of donor hamster-derived lymphocytes (5311F, Intercell Technologies, Hopwell, NJ) were analyzed as well. Cells were washed, suspended in 1% paraformaldehyde/PBS, and analyzed by Becton Dickinson FACSort (San Jose, CA). Gated lymphocytes were analyzed for one- and two-color fluorescence labeling, and the results were expressed as the percentage of positive cells in PBMC. To calculate the recent thymic emigrants (RTE) that are characterized by the phenotype Thy1+CD4+CD45RC- (22, 23), gated CD4+ T cells were analyzed for the presence of anti-Thy1.1 and anti-CD45RC staining. False positive cells were excluded by isotype-matched irrelevant mAbs staining.

Serum Ab analysis. Aliquots of 100 µl of heparinized hamster or WKAH rat whole blood were depleted of RBC by 0.83% NH4Cl solution and incubated for 30 min at 4°C with 10 µl of serum from recipients. Subsequently, the cells were washed twice and stained with 25 µl of PE anti-rat IgM (G53-238) or 50 µl of FITC anti-rat IgG (STAR 17, Serotec). Results were expressed as the relative mean channel fluorescence, which was calculated as the mean fluorescence of stained cells divided by the mean fluorescence of cells incubated with naive nude serum and counterstained with PE anti-rat IgM or FITC anti-rat IgG mAbs.

Functional T cell assays

Proliferation assays. The proliferation of the T cells supported by allothymus or xenothymus was tested in a PHA stimulation assay and in a MLR using standard technique (13). For PHA stimulation tests, 1 x 105 PBMC were cultured with 1 µg/well of PHA (Sigma, St. Louis, MI). For allogeneic and/or xenogeneic MLR assays, 5 x 105 PBMC were stimulated with 5 x 105 irradiated (20 Gy) WKAH rat or hamster PBMC. For autologous MLR assays, 5 x 105 CD3+ splenocytes were stimulated with 5 x 105 irradiated (20 Gy) CD3- splenocytes (isolated by magnetic beads; see below) from sick animals or from control rats. The cells in quadruplicate wells in 200 µl of complete medium/well (RPMI 1640 supplemented with 10% FCS, 5 x 10-5 M 2-ME, 0.1 mg/ml streptomycin, and 100 U/ml penicillin) were incubated for 4 days at 37°C in a humidified atmosphere of 5% CO2 in flat-bottom microplates. During the last 16 h, 1 µCi of tritiated thymidine ([3H]; ICN Pharmaceuticals, Irvine, CA) was added. Subsequently, the cultures were harvested onto fiberglass filters, and a liquid scintillation analyzer measured the incorporation of [3H]thymidine (counts per minute).

Cell-mediated lympholysis (CML). CML was measured in a standard 4-h 51Cr release assay (24). Briefly, spleen cell suspensions (5 x 106/ml) were activated by gamma-irradiated (20 Gy) hamster or WKAH rat splenocytes (5 x 106/ml) for 5 days and used as effector cells. 51Cr (Na2CrO4, ICN, 200 µCi/1 x 106 cells)-labeled Con A (Sigma)-activated hamster or WKAH rat lymphoblasts were used as target cells. Effector cells were incubated in the wells of 96-well, U-bottom microplates in quadruplicate with 1 x 105 51Cr-labeled target cells (hamster and WKAH rat Con A blasts), giving two E:T cell ratios (50:1 and 25:1) in a total volume of 0.2 ml of complete medium. These plates were incubated at 37°C in humidified 5% CO2 in air for 4 h. There were 100 µl of supernatants harvested and counted in a Beckman gamma counter for 2 min. Spontaneous release was obtained from wells mixed with target cells and medium only. Maximal release was obtained from wells receiving 1% saponin. The percentage of cytotoxicity was calculated by the following formula: % lysis = 100 x [(experimental release - spontaneous release)/(maximal release - spontaneous release)]

Characterization of autoantibodies

Anti-DNA autoantibodies were measured by an ELISA (24), and organ-specific autoantibodies were demonstrated by indirect immunofluorescence staining using standard technique (19). For measuring anti-DNA autoantibodies, microplates were coated overnight at 4°C with aliquots of 100 µl of DNA from calf thymus (Sigma) diluted at 10 µg/ml and then washed with 0.05% Tween-20 in PBS (pH 9.6). After incubation with 3% BSA for 1 h at 37°C to block nonspecific binding, the wells were washed again. Aliquots of 100 µl of different dilutions (from 1/1,600 to 1/12,800) of serum were then incubated for 1 h at 37°C, the plates were washed, and alkaline phosphatase-conjugated rabbit anti-rat IgM or anti-rat IgG (Zymed, San Francisco, CA; diluted 1/1000) was added for 1 h at 37°C. The plates were washed, substrate solution (p-nitrophenyl phosphate, Sigma) was added, and absorbance (OD) at 405 nm was measured with an automated spectrophotometer (Bio-Tek, Winooski, VT). Results were expressed as the relative titer of OD, which was calculated as follows: relative titer = (OD of thymus-grafted rat sera - OD of negative control)/(OD of naive rat sera - OD of negative control).

For detecting organ-specific autoantibodies, cryostat sections (5 µm) of thyroid, salivary gland, and stomach of normal nude rats were mounted on slides coated with poly-L-lysine (Sigma) and dried overnight at room temperature. The sections were then fixed in 4°C acetone and sequentially incubated first with positive serum from wasting rats (diluted 1/20) and then with FITC goat anti-rat IgM or FITC goat anti-rat IgG (1/200 diluted; Cappel, West Chester, PA) mAbs. The frozen sections were mounted, then visualized and imaged using a Leica fluorescence microscopic system (Rockleigh, NJ).

Adoptive cell transfer experiments

Viable whole splenocytes from hamster thymus-grafted rats (2 mo post-Tx) were adoptively transferred into syngeneic naive nude rats or were first separated into CD3+ and CD3- subpopulations by magnetic cell sorting (25) and then transferred. Subsequently, animals were monitored for clinical symptoms of wasting, development of histologic lesions, and production of autoantibodies. Splenocytes from naive nude or euthymic rats were used as controls. For magnetic cell sorting, RBC and dead cells were first removed by Percoll (1.086) centrifugation, washed, and incubated for 20 min on ice with mouse anti-rat CD3 mAb (1F4, ProBio, Margate Kent, U.K.; 50 µl/106 cells) diluted 1/100 in special PBS (PBS supplemented with 0.5% BSA, 5 mM EDTA, and 0.01% sodium azide). The wells were washed, and nonspecific binding was blocked with 1/10 diluted normal rat serum for 10 min on ice. After washing twice, the cells were resuspended in special PBS at a ratio of 80 µl of a 107 cell suspension and 20 µl of rat anti-mouse IgM microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) and were incubated for 15 min on ice. CD3- cells were eluted from selection columns (Miltenyi Biotec) exposed to the magnetic field, and CD3+ T cells were eluted after removal from the magnetic field according to the manufacturer’s protocol. The purity of separation was >90% as evidenced by FACScan analysis.

Histology and immunohistochemistry

At the time of sacrifice, the following organs or tissues were harvested and fixed in 10% neutral buffered formalin or embedded in OCT medium (Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -80°C for immunohistochemical analysis: thymus and/or heart Xg, brain, thyroid, salivary gland, tongue, esophagus, stomach, liver, pancreas, intestine, colon, kidney, adrenal gland, prostate, seminal vesicles, testis, heart, lung, spleen, lymph nodes, skin, and ears. Tissue sections were embedded in paraffin, sectioned (5 µm), and stained with hematoxylin-eosin for light microscopy. Hamster Xgs were also stained with Masson’s Trichrome to show collagen and with acid orcein to visualize elastic, and rat kidneys were stained with periodic acid-Schiff for visualization of the glomerular basement membrane. The presence of rat-derived MHC class I+ and class II+ cells in thymus Xgs and of rat CD3+ T cells in rejected allografts was demonstrated by immunohistochemistry (13, 19). Briefly, cryostat sections (5 µm) were mounted on slides coated with poly-L-lysine and dried overnight at room temperature. The sections were fixed in acetone (4°C) for 15 min, washed in PBS, and then incubated with primary mAbs. For the demonstration of MHC class I+ and II+ cells, biotinylated mouse anti-rat MHC class I (OX-18) or anti-rat MHC class II (OX-6) mAbs were incubated overnight at 4°C, respectively. To reveal the presence of CD3+ cells, mouse anti-rat CD3 mAb (1F4) was first incubated overnight at 4°C, then washed and incubated with biotinylated goat anti-mouse Ig (Dako, Glostrup, Denmark) for 1 h. HRP-conjugated streptavidin (Dako) was added and visualized using the substrate diaminobenzidene (Sigma). Endogenous peroxidase activity was blocked by adding 0.3% H2O2, and nonspecific staining was blocked by incubation with 5% normal goat serum/PBS for 0.5 h before mAb incubation. Slides were counterstained with hematoxylin, mounted, and microscopically examined.

Statistical analysis

Data analysis was performed by two-sample Student’s t test, Wilcoxon test, and nonparametric Mann-Whitney U test, as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Survival of fetal thymus and/or adult heart Xgs

In accordance with our previous findings (18, 19), hamster cardiac Xgs were acutely rejected (3–4 days) in naive nude rats (group 1, Table IGo), whereas they were tolerated and survived indefinitely in TR-treated rats (group 2, Table IGo). When TR-treated rats were also given a fetal hamster thymus (two lobes) beneath the kidney capsule at the time of heart Tx (group 3), all heart Xgs survived continuously despite the generation of rat T cells by hamster thymus (see below). However, 2–3 mo later, all the animals developed an overt wasting syndrome (characterized by weight loss), leading to death in another 1–2 mo.


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  		Table I. Survival of fetal thymic tissue and/or adult heart grafts

 
To know whether the wasting syndrome was provoked by the thymus Xgs, the TR treatment, or a combination of both, another group (group 4) of animals received fetal hamster thymus alone. These xenothymic tissues were not rejected, as suggested by the generation of rat T cells (see below) and by autopsy. The latter observation was not a surprise, as it is generally known that the rejection of nonvascularized tissue Xgs (e.g., also pancreatic islets) strongly depends upon the presence of T cells, particularly CD4+ T cells (26). Again, all rats in group 4 developed a manifest wasting disease and had to be sacrificed for examination. Thus, the problem of the wasting syndrome was provoked by the presence of a thymus Xg. On the contrary, all untreated nude rats receiving fetal allothymus (WKAH rat) either in combination with cardiac grafts (group 5) or alone (group 6) became long-term survivors without any signs of wasting or rejection.

Survival of vascularized, adult composite thymus/heart Xgs

Next, a series of experiments was performed in nude rats to investigate the function of adult and immediately vascularized hamster thymus Xgs. For that purpose, adult hamster thymus/heart Xgs were grafted en bloc (see Materials and Methods). TR-treated rats receiving nonirradiated composite Xgs all died within 1 wk with functioning Xgs (group 7, Table IIGo). This mortality was due to an acute graft-vs-host disease (GVHD) mediated by mature thymocytes present in the adult thymus. This was supported by typical clinical signs of acute GVHD such as diarrhea; by histological examination demonstrating mononuclear cell infiltration into spleen, skin, and liver (data not shown); and by the clear presence of hamster lymphocytes using FACScan analysis (~10% of PBMC) in the peripheral circulation. The diagnosis of GVHD by donor thymocytes was further supported by the fact that depletion of thymocytes using pre-Tx donor irradiation successfully prevented it. These recipients of irradiated composite Xgs survived continuously even after withdrawal of immunosuppression (group 8). However, after ~ 2 mo the rats in group 8 also developed a wasting syndrome similar to the fetal thymus-grafted animals from groups 3 and 4 and had to be sacrificed for further examination. All untreated rats acutely rejected either nonirradiated (group 9, Table IIGo) or irradiated (group 10, Table IIGo) composite Xgs by day 3, confirming that, in contrast to fetal thymus tissue, nude rats could reject vascularized Xgs. All rats receiving irradiated allocomposite (WKAH rat) grafts indefinitely accepted their grafts without treatment (group 11, Table IIGo), whereas those receiving nonirradiated allocomposite grafts showed varying degrees of GVHD (data not shown).


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  		Table II. Survival of vascularized "composite" adult thymus/heart grafts

 
Histology and immunohistochemistry of thymus and/or heart Xgs

Consistent with our previous studies (18, 19), hamster hearts were acutely rejected in naive nude rats (group 1) by a mechanism involving IgM xAbs, NK cells, and M{phi}s, resulting in a typical picture of acute vascular rejection. In contrast, TR-treated rats (group 2) became tolerant for their hamster heart Xgs that were characterized by a normal histological picture without signs of acute or chronic rejection. In TR-treated, combined fetal thymus/adult heart-grafted rats (group 3), the fetal hamster thymi showed considerable growth and became secondarily vascularized beneath the kidney capsule. At ~3 mo post-Tx, the size of the thymus Xgs had increased 5- to 6-fold compared with the primary size at the time of Tx. Several small blood vessels originating from the kidney, usually six to eight vessels per thymus, were visualized under the Xg surface and concentrically entered into thymus. Microscopically, cortical and medullary compartments were well demarcated (Fig. 1Goa). In addition, thymus tissue were densely populated with rat-derived thymocytes, as shown by immunohistochemistry (see below), and the thymic stromal architecture was normal, showing the clear presence of epithelial cells, trabeculae, and the thymic corpuscles. In this group the simultaneously grafted adult cardiac Xgs showed normal histological structure (Fig. 1Gob). Thymi implanted under the kidney capsule of untreated rats (group 4) showed a similar macroscopic and histological appearance (Fig. 1Goc) and supported rat-derived T lymphocytes equally well.



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  		FIGURE 1. Histology of hamster thymus or heart Xgs. Three months post-Tx, fetal thymus Xg from a TR-treated nude rat showed normal thymus structure with the clear presence of epithelial corpuscles and trabeculae as well as normal thymocyte maturation under the rat kidney capsule (a). Tolerated heart Xg (3 mo post-Tx) from a combined fetal thymus/heart-xenografted rat (group 3) showed normal histological structure without infiltrate (b). Fetal hamster thymus grafted in naive nude rats showed a normal thymic structure removed at 3 mo post-Tx (c). Tolerated vascularized thymus from a composite-xenografted rat (group 8) showed normal meshwork structure and repopulation with rat thymocytes (d). Acutely rejected hamster heart (e) and thymus (f) from a vascularized composite Xg transplanted in an untreated rat (group 10) showed the typical picture of acute vascular rejection: edema, hemorrhage, tissue necrosis, and destruction.

 
In TR-treated rats, functioning composite Xgs from group 7 rats that died from GVHD were histologically normal without infiltration (data not shown). Tolerated hearts from xenocomposite of group 8 rats showed a normal histological structure resembling tolerated hearts from group 3 rats. In addition, tolerated thymi (initially irradiated) from the same group showed normal histological structure (Fig. 1God) and were repopulated with rat-derived thymocytes (see below). In untreated rats, both nonirradiated and irradiated composite Xgs were acutely rejected with the heart Xgs, showing a typical picture of acute vascular rejection: edema, hemorrhage, infiltration with polymorphonuclear and mononuclear cells, and tissue necrosis (Fig. 1Goe). The rejected thymus Xgs showed edema, hemorrhage, stromal architecture destruction, and cellular infiltrates (Fig. 1Gof).

Distribution of rat MHC class I and class II Ags was examined by immunohistochemistry using OX-18 and OX-6 mAbs, respectively. These mAbs are rat species specific (Fig. 2Go, a and b) and are not cross-reactive with hamster Ags (Fig. 2Go, c and d). In normal rat thymi, thymic epithelial cells and thymocytes from the deep cortex and medulla were readily stained with MHC class I Ags (Fig. 2Goa). The major MHC class II+ cells were epithelial cells in the cortex and dendritic cells and M{phi}s residing in the medulla (Fig. 2Gob). Tolerated fetal thymus from group 3 rats (3 mo after Tx) showed densely repopulating thymocytes that stained with mAbs for rat MHC class I in the deep cortex and medulla compartments (Fig. 2Goe). Furthermore, rat-derived MHC II + cells with the appearance of dendritic cells were clearly present and well distributed in the medulla (Fig. 2Gof). Hence, these staining patterns indicated that thymocytes and MHC II+ dendritic-like cells colonizing the hamster thymi were derived from rat progenitor cells. Similar observations were made in long term surviving vascularized hamster thymus from group 8 (data not shown) as well as by others in long term surviving human thymus Xgs in SCID mice (9).



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  		FIGURE 2. Immunoperoxidase staining for rat MHC class I and class II Ags. In normal rat thymus, thymic epithelial cells and thymocytes residing in deep cortex and medulla were MHC I+ (a), and thymic epithelial cells and medullary dendritic cells and M{phi}s were MHC II+ (b). These rat mAbs were not cross-reactive with normal hamster thymus (c and d). Distribution of rat MHC I and MHC II Ags in a tolerated fetal hamster thymus from group 3 harvested at 3 mo post-Tx revealed that rat thymocytes residing in the deep cortex and medulla were MHC I+ (e) and MHC II+ dendritic-like cells populated in the medulla (f).

 
T cell generation after xenogeneic or allothymus grafting

The capacity of the xenothymus to generate various lymphocyte subsets in the peripheral blood was sequentially analyzed and compared with the function of the allothymus. Rat-specific mAbs were used in all tests that detected lymphocytes originating from rat precursor cells. As shown in Fig. 3Goa, control nude rats lacked RTE. After Tx of a xenothymus (either fetal tissue or irradiated adult composite grafts), the percentage of RTE increased equally well as after allothymus (fetal tissue or irradiated adult composite grafts) Tx to reach a level at 1 mo after Tx comparable to the level in normal euthymic rats (Fig. 3Goa).



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  		FIGURE 3. Kinetics of rat T cell generation. Dynamic measurement of RTE (a), CD3+CD4+ T cells (b), and CD4+CD45RC+ T cells (c) after xenothymus (groups 3 and 8) or allothymus (groups 5 and 11) grafting compared with naive nude rats or normal euthymic rats.

 
As generally known (18), PBMC of naive nude rats showed very low percentages of CD3+CD4+ lymphocytes (Fig. 3Gob) or CD3+CD8+ lymphocytes (data not shown). After allothymus Tx the percentage of CD3+CD4+ T cells slowly increased to reach a subnormal level (compared with that in euthymic rats) at 6 mo post-Tx. Fetal xenothymus-grafted rats showed the slowest recovery compared with adult xenocomposite-grafted or allothymus-grafted groups (Fig. 3Gob). This was probably due to the smaller size of the fetal xenothymus that was implanted, because larger volumes of thymus Xgs (e.g., two thymi) resulted in faster generation of T cells (data not shown) (27). Very soon after thymus Tx, the total white blood cell count and the percentage of PBMC were similar to those in control euthymic rats in all groups. Hence, the relative percentages of lymphocytes compared with controls also reflect the ratios of absolute numbers. In agreement with other studies (24, 28), in all xenothymus-grafted rats the generation of CD3+CD8+ T cells (29) was slower than that of CD3+CD4+ T cells (data not shown).

Rat CD3+CD4+ T cells can be subdivided into two major subpopulations: CD4+CD45RC+ cells are naive mature or resting memory T cells, whereas CD4+CD45RC- cells are T cells that expand after Ag encounter (30). Although the precise function of these subsets is still unclear, CD4+CD45RC+ T cells secrete mainly Th1 cytokines, provoke lethal GVHD, and provide help for B cells during primary Ab response, whereas CD4+CD45RC- T cells rather belong to the Th2 lineage, help B cells during secondary Ab responses, and may suppress autoimmune reactions (31, 32). Before the onset of the wasting symptoms, the ratio between CD45RC+ and CD45RC- CD4 cells was similar and progressively increased in all thymus-grafted animals (Fig. 3Goc). After the appearance of the wasting symptoms the xenothymus-grafted animals showed a higher proportion of CD4+CD45RC- cells. This probably was a consequence of Ag-induced expansion of T cells (30, 31) and might be involved in the wasting syndrome (see below).

Although hamster lymphocytes were detected (~10% of PBMC) and caused lethal acute GVHD in nonirradiated xenocomposite-grafted and TR-treated rats (group 7), in all other xenothymus-grafted (fetal tissue or irradiated composite grafts) rats, there was no clear evidence using FACScan analysis for the presence of significant numbers of hamster lymphocytes in the peripheral circulation.

In vitro and in vivo reactivity of T cells generated by thymus grafts

As shown in Fig. 4Goa, PBMC from naive nude rats showed very low reactivity upon in vitro stimulation with allogeneic WKAH rat cells, xenogeneic hamster cells, or PHA mitogens. Allothymus (WKAH)-grafted rats developed significant reactivity for xenogeneic hamster cells or PHA mitogens and were tolerant for donor rat Ags. Also, xenothymus (Au/Hö hamster)-grafted rats proliferated vigorously after in vitro stimulation with allogeneic (WKAH rat) cells or PHA mitogens, whereas they were tolerant for donor hamster xAgs. Moreover, CD3+ T cells separated by magnetic beads (see Materials and Methods) from xenothymus-grafted rats showed no reactivity against host type stimulator cells (autologous MLR), but responded vigorously against third-party allogeneic (WKAH rat) or xenogeneic stimulator (Aura hamster) cells. Compared with euthymic control rats, lymphocyte reactivity in xenothymus- or allothymus-grafted rats was relatively weaker, but correlated with the percentage of CD3+ T cells that were present among the effector cells at the time when the tests were performed.



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  		FIGURE 4. In vitro and in vivo function of T cells supported by hamster thymus grafts. In vitro reactivity (stimulation index in PHA or MLR tests) and percentage of CD3+ T cells in PBMC of xenothymus generated T cells compared with naive nude, allothymus-grafted rats, and normal euthymic rats (a). T cells are specifically tolerant for donor hamster xAgs and reactive against allogeneic cells or PHA mitogen. After cardiac allografting, xenothymus-grafted rats produce high titers of T-dependent IgG allo-Abs as detected by FACScan analysis (b; white profile, preallotransplantation; black profile, after allograft rejection).

 
Next, the in vivo T cell immunity of xenothymus-grafted rats was investigated. For that purpose, allogeneic (WKAH rat) hearts were transplanted into fetal xenothymus-grafted nude rats (group 3) at 2 mo after thymus Tx. All these cardiac allografts (n = 3) were rejected after 16–17 days, whereas the primary hamster Xgs (thymus/heart) were unaffected. The rejection of allografts occurred later than in euthymic rats (7–9 days) probably due to the lower percentage of T cells in fetal xenothymus-grafted rats (Fig. 3Gob). The rejection was mediated by hamster thymus-supported T cells, as rejection was accompanied by the production of high titers of T-D rat IgG allo-Abs (Fig. 4Gob) and as rat CD3+ T cells were clearly demonstrated by immunohistochemistry in rejected cardiac allografts (data not shown). Similar results were obtained in allothymus-grafted rats that, however, rejected third-party allogeneic hearts somewhat more rapidly (-10 days) due to a relative higher percentage of T cells in these rats.

Development of T cell xenotolerance and autoimmunity in xenothymus-grafted rats

Rat T cells generated by hamster thymus were tolerant for hamster xAgs. This appeared from the observation that they showed specific in vitro nonresponsiveness for hamster xAgs in MLR (Fig. 4Goa) and by their specific inability to lyse hamster target cells in CML assays (data not shown). Moreover, transplanted hamster Xgs (thymus, heart, and thyroid; see below) in these rats were free from rejection. In addition, neither anti-hamster specific IgM nor IgG xAbs were identified in the serum or deposited within tolerated Xgs (data not shown).

At about 2 mo after thymus Tx (fetal tissue or vascularized adult composite grafts), all rats developed an overt wasting syndrome that usually became lethal 1–2 mo later. This disease was provoked by multiple organ-specific autoimmunity. Indeed, the primary structure of rat organs such as thyroid, salivary gland, and stomach was destroyed, and these organs were heavily infiltrated with mononuclear cells (Fig. 5Go, a–c). In addition, hamster thymus-grafted, but not allothymus-grafted, rats developed high titers of autoantibodies with reactivity against DNA (IgM titers of >1/6,400 and IgG titers of >1/12,800) as well as against some organs, e.g., anti-stomach (Fig. 5God). Furthermore, organ-specific autoantibodies (e.g., anti-stomach) were directed only against rat (Fig. 5God) and not against hamster stomach (Fig. 5Goe). Interestingly, in four rats fetal hamster thyroid was implanted together with hamster thymus beneath the kidney capsule. The thyroiditis was only found in the rat thyroids (Fig. 5Goa), not in the hamster thyroids (Fig. 5Gof).



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  		FIGURE 5. Histological examination of wasting nude rats after xenothymus Tx (2–3 mo). Thyroiditis (a), Sialoadenitis (b), Gastritis (c), and anti-rat stomach IgG autoantibodies (d) were present. Anti-stomach Abs were specifically reactive with rat stomach (d), but not with hamster stomach, by immunofluorescence assay (e). Thyroiditis did not occur in hamster thyroid grafts (f) when simultaneously implanted with the hamster thymus under the kidney capsule, while the rat thyroid developed thyroiditis (a).

 
T cells drove this autoimmune syndrome. At the time of initiation of the wasting syndrome, usually 2–3 mo after thymus Tx, the majority of CD3+CD4+ T cells lacked the CD45RC (Fig. 3Goc) and Thy-1 Ags (data not shown), indicating that they were activated (23, 30). In addition, expression of other activation markers, such as CD25 (IL-2R {alpha}-chain) or OX-40 (CD134, a member of the nerve growth factor receptor/TNF receptor superfamily) was found on ~30% of splenic CD4+ T cells (data not shown). This further pointed to an activation of CD3+CD4+ T cells in the animals with wasting disease (31). Finally, direct evidence of a role for T cells in the initiation and progression of autoimmunity appeared from adoptive transfer experiments. Two weeks after transfer of whole splenocytes from sick animals, syngeneic naive nude rats started to develop clinical signs of wasting syndrome and became very sick 2–3 wk later. Also, rats transferred with purified CD3+ splenocytes (isolated using magnetic cell sorting) from sick animals showed overt clinical signs of wasting syndrome ~1 mo after transfer and died about 1 mo later. Transfer of autoimmunity was confirmed histologically (e.g., presence of thyroiditis) and serologically (e.g., formation of anti-DNA Abs). Autoimmunity could not be transferred by CD3- splenocytes from wasting nude rats or by splenocytes or CD3+ cells from syngeneic euthymic rats.

Early thymic presence of rat-derived thymic epithelial cells and not of hemopoietic cells successfully prevents autoimmunity while maintaining T cell xenotolerance

Although immunohistochemistry of the thymus Xgs showed a clear presence of rat bone marrow-derived cells (see Fig. 2Gof), it could be hypothesized that this was insufficient or occurred too late to induce thymic tolerance for organ-specific peptides presented in the context of rat MHC. To test this hypothesis, immediately after Tx, TR-treated and hamster composite-grafted nude rats (n = 7) were injected intrathymically with 30 x 106 splenocytes from syngeneic nude rats (TR-treated) as a source of rat bone marrow-derived cells. This manipulation effectively increased the presence of MHC II+ cells in the thymus Xgs, as demonstrated by immunohistochemical staining (data not shown), but was unable to prevent autoimmunity. Actually, these rats still developed a lethal wasting syndrome (2–3 mo post-Tx), characterized by the production of anti-DNA autoantibodies, multiple organ autoimmune lesions, and predominant generation of CD45RC- CD4+ cells, and had to be sacrificed after 72 ± 16 days (Fig. 6Goa).



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  		FIGURE 6. Recipient and heart graft survival in nude rats receiving hamster, rat, or hamster/rat thymus grafts. In contrast to rats receiving hamster thymus grafts with or without intrathymic injection of extra rat APCs, rats receiving mixed hamster/rat thymus grafts survived indefinitely without any sign of autoimmunity (a). Hamster hearts survived indefinitely in mixed fetal rat/hamster thymus-grafted rats (n = 5), whereas they were rejected rapidly in three fetal rat thymus-grafted rats (b).

 
Another explanation for the organ-specific autoimmunity could be that xenothymus epithelium presented either species-specific peptides or organ-specific peptides, but only in association with hamster MHC class II Ags. If this hypothesis was true, the presence of rat thymic epithelial cells within the hamster thymus Xgs should prevent autoimmunity. To test this, mixed hamster/rat thymic epithelium grafts (see Materials and Methods) were grafted in five untreated nude rats. Due to the fact that firstly pre-Tx lethal irradiation of rat and hamster donors killed all or at least the majority of thymocytes, and next that thymic tissue was intensely squeezed to remove as much as possible of other types of cells such as dendritic cells, mixed thymic tissue grafts were predominantly composed of rat and hamster thymic epithelial cells and grew out as healthy thymus grafts (Fig. 8Goa). This was further confirmed by immunostaining for rat MHC II showing the presence of rat thymic epithelial cells and dendritic shape cells in various areas of mixed grafts (Fig. 8Gob), whereas in exclusive hamster thymus grafts, only the MHC II+ dendritic shape cells were found (Fig. 2Gof). In contrast to in rats given a hamster thymus graft (Fig. 6Goa), all mixed thymus-grafted rats were healthy and survived indefinitely (Fig. 6Goa). In none of these animals did autoimmune symptoms occur, as evidenced by the absence of clinical signs (weight loss, hunched back, etc.), by the absence of anti-DNA autoantibodies (titers were similar to allothymus-grafted rats), by the predominance of the CD45RC+ subset, and by the lack of histological evidence for thyroiditis, gastritis, or sialoadenitis. Functionally, mixed hamster/rat thymus grafts supported the generation of T cells characterized by the progressive increase in CD3+CD4+, CD3+CD8+ T cells and the CD45RC+CD4+ subset (Fig. 7Goa) equally well as exclusive rat thymus grafts (Fig. 7Gob). Moreover, T cells from the mixed thymus-grafted rats were tolerant to donor hamster xAgs both in vitro and in vivo. In vitro, specific nonresponsiveness was found against donor hamster (Au/Hö) or host rat (RH-rnu) stimulator cells and not against third-party hamster (Aura) or allogeneic rat (RA) cells in MLR (Fig. 7Goc). In vivo, five TR-treated nude rats were grafted with a mixed hamster/rat thymus in conjunction with a hamster heart. All these grafts survived equally well as in TR-treated nude rats grafted with a hamster thymus (Fig. 6Gob). This indicated that the tolerizing effect for hamster xAgs of the mixed hamster/rat thymus was as good as that of hamster thymus grafts. Moreover, the tolerizing effect for hamster of the mixed rat/hamster thymus was not due to the presence of rat thymic epithelial cells, as TR-treated nude rats receiving an exclusive rat thymus rejected concomitantly grafted hamster hearts (Fig. 6Gob). The hamster cardiac Xgs showed normal histology (Fig. 8Gof) without any sign of cellular or chronic (Fig. 8Go, g and h) rejection.



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  		FIGURE 8. Histology and immunohistochemistry of mixed thymus or heart Xgs. Histology of a mixed thymus graft removed at 3 mo post-Tx showed normal thymus structure and thymocyte maturation under the host kidney capsule (a). Immunoperoxidase staining for rat MHC II of the same graft (b) showed combined characteristics of a similar staining of fetal rat thymus grafts (c) and of fetal hamster thymus grafts transplanted under the rat kidney capsule (d) with the clear presence of rat MHC II+ dendritic-like cells and thymic epithelial cells. A higher magnification observation taken from b of rat thymic epithelial cells positively stained with rat MHC II is shown (e). Tolerated hamster cardiac Xg (3 mo post-Tx) from a TR-treated and mixed rat/hamster thymus-grafted rat showed normal myocytes without cellular infiltrate as demonstrated by hematoxylin-eosin staining (f), the absence of increased deposition of collagen as detected by Masson’s Trichrome staining (g), and the absence of intima proliferation as illustrated by Orcein staining (h).

 


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  		FIGURE 7. T cell phenotype and in vitro reactivity in nude rats receiving mixed hamster/rat thymus grafts. The generation of rat CD4 subsets in mixed rat/hamster thymus-grafted rats (a) was as good as that in allothymus-grafted rats (b). T cells supported by mixed rat/hamster thymus grafts were specifically tolerant for donor hamster cells and recipient rat cells, but were reactive against third-party hamster cells, third-party rat cells, and PHA mitogen (c).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Vigorous T-I and T-D immune mechanisms provoke rejection of vascularized Xgs (17, 18). In a previous study we were able to induce T-I xenotolerance for vascularized Xgs using a TR consisting of a temporary NK cell depletion, B cell immunosuppression together with pre-Tx infusion of xAgs (19). Here we have explored whether T-I Xg tolerance could be combined with the induction of T-D Xg tolerance using xenothymus Tx.

The induction of mixed bone marrow chimerism is probably one of the most profound ways to induce stable and long term Tx tolerance (33). In various models the induction of xenogeneic bone marrow chimerism over either a concordant (34, 35, 36) or discordant (37) barrier has been successfully achieved. For the present study we have chosen not to use a regimen involving chimerism, because in clinical application xenogeneic chimerism may still be associated with serious complications. Indeed, various species incompatibilities, such as between host-derived growth factors or bone marrow stroma and donor-derived hemopoietic stem cells, may prevent the establishment of long term and stable chimerism, although recently much progress has been achieved in this area (12, 37). Next, the risk for GVHD after bone marrow Tx remains a concern for clinician. Finally, although the availability of depleting mAbs seems to progressively obviate the need for lethal irradiation, some type of irradiation (e.g., thymic irradiation or a low dose of total body irradiation) is usually required for the induction of chimerism, which always implies a risk of tumorigenesis (33, 38). Hence, for the present study Tx of xenothymus was chosen as a tolerizing element for T cell compartment. This was based on the observations that the thymus is the principal and natural site where the T cell repertoire and self-tolerance is established and shaped (1, 39). Additionally, concordant (4, 5, 6, 7) and discordant (8, 16) thymus Tx have been demonstrated to support T lymphopoiesis as well as to induce T cell xenotolerance in the murine model.

TR-treated rats receiving both a hamster heart and a hamster thymus progressively developed high percentages of functional T cells that were able to reject third-party cardiac allografts, but were tolerant for hamster organs (heart, thymus, and thyroid). It is unlikely that the T cell xenotolerance was based on microchimerism (40), because hamster hemopoietic cells could not be demonstrated by FACScan analysis, and xenotolerance occurred equally well in the recipients receiving vascularized composite Xgs from lethally irradiated donors in which hamster hemopoietic precursors were destroyed.

Various problems may occur after xenothymus Tx that were only partly observed in the present study. First, mature T cells within the thymus Xgs may provoke GVHD in immune-incompetent recipients. This was the case when adult thymus Xgs were grafted in TR-treated rats (group 7), but could be prevented by using fetal or irradiated adult thymus Xgs. Another concern is that recipient T cells maturing in a donor xenothymus become restricted for donor MHC Ags. This may cause problems when in the peripheral immune compartment Ags are presented by host-type APCs expressing host-type MHC Ags (41, 42, 43). This was not the case in the hamster thymus-grafted rats, as after cardiac allografting high levels of rat IgG allo-Abs were produced. Hence, a good MHC interaction between rat T and B cells was preserved, which is in accordance with what has been found by other investigators (44, 45). Furthermore, xenothymus-supported T cells are fully immunocompetent, including resistance to parasitic infection (44).

The major concern of xenothymus Tx is whether host T cells develop enough self tolerance for host Ags after maturation within a xenothymus. With respect to rat MHC Ags, this was the case, as T cells from hamster thymus-grafted rats did not respond to host-type MHC Ags in an autologous MLR assay, whereas they reacted vigorously against allogeneic MHC Ags. The tolerance for rat MHC was probably related to the immigration of host-type bone marrow-derived cells within the xenothymus medulla, as demonstrated by our immunohistochemical observations. Thymic bone marrow-derived cells are well known to delete a large proportion of auto- or donor-reactive T cells, thus leading to T cell self-, allo-, or xenotolerance (46, 47, 48, 49).

An important finding of the present study that is relevant for an eventual clinical application of xenothymus Tx was that all hamster thymus-grafted rats developed a multiple-organ autoimmune syndrome, whereas allothymus-grafted rats did not. Although this problem may not necessarily occur in all species combinations, it was also found by others in mice grafted with thymus from various origins (7, 24, 45) and confirms the importance of a proper thymopoiesis for the maintenance of self tolerance (50, 51, 52, 53, 54). An explanation for this autoimmune syndrome could be that xenothymus leads to an imbalance between autoimmune effector and regulatory cells. Such an imbalance has been demonstrated in various models to initiate autoimmunity (32, 55, 56). However, we believe that it is unlikely to be the major reason in the present experiment. First, in the week before autoimmune symptoms (~2 mo), the ratio between CD45RC- and CD45RC+ CD4 lymphocyte subsets was similar in xenothymus- and allothymus-grafted rats. A predominance of the CD45RC+ subset was previously shown to induce autoimmunity, which could be suppressed by the CD45RC- subset in rats (31, 32). After the appearance of the autoimmune symptom, a predominance of the latter protective subset was found in our hamster thymus-grafted rats. This predominance of the CD45RC- subset is probably a consequence of autoantigen-induced activation of naive mature T cells with autoreactive potentiality, because naive T cells lose their CD45RC expression after Ag encounter (30, 31). Moreover, a general imbalance would also not explain why, for example, thyroiditis occurred in rat thyroids and not in hamster thyroids in our model. The latter finding suggests that there is insufficient tolerance for organ-specific peptides presented in the context of rat MHC Ags, whereas there is sufficient tolerance for similar peptides presented by hamster MHC Ags.

Ectopic or promiscuous expression of mRNA for numerous self-Ags such as insulin, thyroglobulin, and myelin proteolipid proteins has been demonstrated in the thymus. For insulin, there is evidence for expression at the protein level (57, 58, 59). Thus, thymus epithelium is now generally believed to be very important for the induction of tissue-specific tolerance (60, 61, 62, 63, 64, 65, 66, 67). In the context of xenothymus Tx, autoimmunity may occur as a consequence of either the species-specific nature of organ-specific peptides or of an insufficient or abnormal presentation of host self-Ags. The latter may be caused by an insufficient presence of host-type APCs in the xenothymus or by an exclusive presentation by donor-type thymic epithelial cell (68). The former possibility is unlikely, because firstly rat type MHC II+ cells were clearly demonstrated in the hamster thymus by immunohistochemistry, and secondly extra injection of rat splenocytes in the hamster thymus was unable to prevent autoimmunity. Therefore we favor the hypothesis that autoimmunity was due to the exclusive presentation of some organ-specific proteins by the xenothymus epithelium. It was recently reported that thymic epithelial cells, and not thymic bone marrow-derived cells, use an MHC II endogenous presentation pathway to induce tolerance to certain proteins (67, 69, 70). The fact that autoimmunity was prevented in the xenothymus-grafted rats when rat thymic epithelial cells were simultaneously grafted (mixed hamster/rat thymic epithelium) is compatible with the former hypothesis. The efficiency of the mixed donor/host thymus Tx depends on the presence within the same site of both host and donor thymic epithelial cells, as rats receiving donor and host thymus grafts under the capsules of different kidneys rejected their Xgs (data not shown). The latter observation is a further argument indicating that in the present model tolerance depends on an intrathymic selection event rather than on a post-thymic immunoregulatory mechanism.

For practical reasons the present study was undertaken in congenitally athymic rats. To be clinically more relevant, similar experiments need to be performed using T-sufficient rats. These experiments will probably be successful, as others have successfully achieved xenothymus Tx in adult thymectomized, T/NK cell-depleted recipients (16). The clinical pig-to-human situation involves the presence of preexisting natural anti-donor xAbs, which was not the case in the present concordant model. However, we have recently shown that the TR could also be successfully used in rats with high titers of preformed xAbs (71). The technical feasibility of simultaneous thymus and organ grafting has recently been demonstrated in a large animal model as well (72). Thus, we believe that concomitant xenografting of thymus and other vascularized organs may be a promising approach to induce Xg tolerance if some of the potential risks, such as autoimmunity, are anticipated and avoided by, for example, simultaneous Tx of donor/host type thymic epithelium.


   Acknowledgments
 
We are grateful to Prof. Dr. Brigstock, Ohio State University, for his critical review of this manuscript.


   Footnotes
 
1 This work was supported by the grants from the National Fund for Scientific Research and the Research Council of the University of Leuven. Back

2 Address correspondence and reprint requests to Prof. Mark Waer, Laboratory for Experimental Transplantation, University of Leuven, Campus Gasthuisberg O&N, Herestraat 49, B-3000 Leuven, Belgium. Back

3 Abbreviations used in this paper: Tx, transplantation; allothymus, allogeneic thymus; GVHD, graft-vs-host disease; M{phi}, macrophage; RTE, recent thymic emigrants; T-D, T cell-dependent; T-I, T cell-independent; TR, tolerizing regimen; xAb, xenoantibody; xAg, xenoantigen; xenothymus, xenogeneic thymus; Xg, xenograft; CML, cell-mediated lympholysis. Back

Received for publication May 25, 2000. Accepted for publication October 17, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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