Thymic tissue transplantation has been performed previously in adult mice to induce donor-specific tolerance across allogeneic and xenogeneic barriers. We have now attempted to extend this technique to a large animal preclinical model and describe here our initial studies of allogeneic thymic transplantation in miniature swine. Two miniature swine were thymectomized before thymic tissue transplantation, and two remained euthymic. Donor thymic tissue was harvested from SLA class I-mismatched juvenile pigs and placed into recipient sternocephalicus muscle, kidney capsule, and omentum. A 12-day course of cyclosporin A was started on the day of transplantation. Allogeneic thymic engraftment could only be achieved in euthymic and not in thymectomized miniature swine using this treatment regimen. Both nonthymectomized animals showed good graft development, with evidence of thymopoiesis, as indicated by positive CD1 and host-type SLA class I immunoperoxidase staining of immature graft-infiltrating cells. Both animals also demonstrated donor-specific T cell hyporesponsiveness, as measured by MLR and cell-mediated lympholysis. The thymic grafts continued to develop despite the appearance of high levels of anti-donor specific cytotoxic IgG Abs. Thus, thymic tissue transplanted across an SLA class I barrier can engraft and support host thymopoiesis in euthymic miniature swine. The presence of the host thymus was required for engraftment. These data support the potential of thymic transplantation as part of a regimen to induce donor-specific tolerance to xenogeneic organ grafts.

The thymus is of central importance in T cell development (1, 2). Both thymic epithelial cells and bone marrow-derived APCs participate in the process of selecting immature thymocytes. The thymic epithelium is believed to positively select thymocytes in the cortex of the thymus, so that only cells with a self MHC-restricted TCR are allowed to mature (reviewed in Refs. 3 and 4). The bone marrow-derived APCs, found mainly at the cortico-medullary junction, are largely responsible for negative selection, which leads to deletion of T cells with high affinity for self MHC (reviewed in 5). Recent studies both in vitro, using thymic epithelial cell cultures (6), and in vivo, using thymic transplantation models of mice (7, 8), suggest that the thymic epithelium may also be involved in the negative selection process. Thus, only thymocytes with a low to intermediate self-reactivity are selected for expansion and exportation to the periphery (6, 9, 10).

Successful transplantation of allogeneic or xenogeneic thymic tissue might induce donor-specific tolerance through the deletion of newly developing T cells by the same mechanism that is used in the thymus for deletion of T cells with high affinity for self. Thus, colonization of the donor thymus with bone marrow precursors would lead to negative selection to donor MHC. Migration of host APCs into the grafts would also assure negative selection to host MHC, leading to a specific state of tolerance to both self and donor-specific non-self.

Thymic tissue engraftment has been achieved in immunocompetent mice after thymectomy, followed by lethal whole body irradiation (11, 12), total lymphoid irradiation (13) or T cell and NK cell depletion with mAbs (8). In a xenogeneic pig-to-mouse model involving host thymectomy as well as T and NK cell depletion, the recovery of a tolerant recipient T cell repertoire was shown to be due in part to intrathymic deletion of donor- and host-reactive recipient thymocytes developing in the xenogeneic thymic graft (8, 14, 15). Of importance for potential clinical applications is the fact that both allo- and xenogeneic thymic grafts have been shown to support essentially normal T cell development with normal host-restricted immunocompetence (16, 17, 18).

In an attempt to transfer this technique to large animals, we have initiated a series of experiments in inbred Massachusetts General Hospital (MGH)4 miniature swine. Miniature swine provide the unique opportunity to study the effects of selective MHC matching on parameters of immunity in a reproducible fashion (19). Kidney transplantation across a class I barrier was shown to be successful in 100% of cases when high dose CyA was administered during the first 12 days after transplantation (20). We have begun these studies by using the same immunosuppressive regimen and MHC mismatch for thymic transplantation in miniature swine. We report here successful transplantation of class I-mismatched juvenile thymic tissue and the ability of such tissue to support host thymopoiesis and confer donor-specific hyporesponsiveness.

Two- to three-month-old SLAgg (class Icc/class IIdd) donors and 4- to 5-mo-old, class I-mismatched SLAdd (Idd/IIdd) recipients were selected from our herd of partially inbred miniature swine. The immunogenetic characteristics of this herd (21, 22) and of the intra-MHC recombinant haplotypes (23) have been described previously and are illustrated schematically in Fig. 1. Each experiment used one thymic tissue donor and two recipients. One of the two recipients in each experiment was thymectomized before thymic transplantation.

FIGURE 1.

SLA haplotypes of MGH miniature swine.

FIGURE 1.

SLA haplotypes of MGH miniature swine.

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All animals received preoperative sedation with i.m. xylazine (2 mg/kg), atropine (0.04 mg/kg), and Telazol (1.4 mg/kg; Elkins-Sinn, Cherry Hill, NJ). Isoflurane inhalation anesthesia was administered before endotracheal intubation and was continued throughout the entire operative procedure at a level permitting spontaneous respiration. Perioperatively, each animal received one prophylactic dose of cefazolin (40 mg/kg i.m.).

In the thymectomized group, a complete thymectomy was performed 3–4 wk before thymic transplantation using a combined midline cervicotomy and partial sternotomy as previously described (24). Briefly, the pretracheal muscles were retracted, and the trachea, internal jugular vein, and carotid artery were exposed from the mandibular area to the ventral pericardium. The entire thymus was then carefully dissected and removed. Adherent fatty tissue was also excised when necessary to ensure completeness.

Partial thymectomy was performed in the thymic donor on day 0 through a midline cervicotomy after exposing the major portion of the cervical thymus. Two pieces of thymus, ∼3 cm3 each, were harvested, placed in 0.9% saline solution, and immediately transferred to one euthymic and one thymectomized animal. The thymic tissue was minced into ∼10-mm3 fragments and implanted into three prepared sites in the recipients:

Sternocephalicus muscle.

Ten small pockets, each capable of holding five pieces of tissue, were formed in each sternocephalicus muscle using mosquito clamps. ∼1 cm3 of minced thymic tissue was placed into the pockets, and the sites were closed with 4-0 Prolene (Ethicon, Somerville, NJ), which also served to mark the implantation sites for future biopsies.

Omentum.

After midline laparotomy, a volume of ∼1 cm3 of minced thymic tissue was wrapped into an edge of the omentum and closed with 4-0 Prolene.

Kidney capsule.

One cubic centimeter of minced thymic tissue was spread out under both kidney capsules as described previously (25). Briefly, a 1-cm incision was made in the kidney capsule directly opposite the hilum. The capsule was then carefully detached from the underlying cortex using an curved hemostat, and thymic tissue was inserted beneath it.

All recipients received two central venous Silastic catheters (Dow Corning, Midland, MI) placed into each external jugular vein via direct cut down. The catheters were tunneled s.c., exiting dorsally. One catheter was used for CyA administration, and the other for obtaining blood samples.

Fresh split-thickness skin grafts (40 × 40 × 2.2 mm) were harvested from donors using a Zimmer dermatome and placed on graft beds, also prepared with a dermatome, on the lateral thorax as previously described (26). The day of rejection was defined as the time at which <10% of the skin graft showed signs of viability as judged by color, texture, and warmth to touch.

The only immunosuppressive treatment was a 12-day course of CyA, administered once per day to all animals beginning on day 0. The first dose on day 0 was 13 mg/kg body weight i.v. given intraoperatively. Animals were either maintained on i.v. solution or switched to an oral CyA solution (Neoral) on day 5, which was given by a gastrostomy tube inserted on the day of thymic transplantation. CyA trough levels were measured in whole blood by a mAb-based RIA. CyA dosage was adjusted daily to maintain a level between 400 and 800 ng/dl. The i.v. and oral (Neoral) preparations of CyA were provided by Novartis Pharmaceuticals (Hanover, NJ).

All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Publication 85-23, revised 1985) and were approved by the Massachusetts General Hospital animal research committee.

Freshly drawn, heparinized whole blood was diluted with HBSS (Life Technologies, Gaithersburg, MD), and mononuclear cells were obtained by gradient centrifugation using lymphocyte separation medium (Organon Teknika, Durham, NC). The mononuclear cells were washed once with HBSS before the contaminating red cells were lysed with ACK lysing buffer (BioWhittaker, Walkersville, MD). Cells were then washed in HBSS again and resuspended in tissue culture medium (see below). Cell suspensions were kept at 4°C until they were used in cellular assays.

MLR cultures, to test for proliferative response to alloantigen, have been described previously (28). Briefly, 4 × 105 responders and an equal number of irradiated (25 Gy) stimulators were incubated in 200 μl of standard MLR medium using flat-bottom 96-well plates (Costar, Cambridge, MA). After 5 days of incubation, 1 μCi of [3H]thymidine was added to each well, followed by an additional 5-h incubation. 3H incorporation was determined in triplicate samples by liquid scintillation.

CML assays were performed as described previously (27). Briefly, lymphocyte cultures containing 4 × 106 responder and 4 × 106 irradiated (25 Gy) stimulator PBL were incubated in 2 ml of medium for 6 days at 37°C in 7% CO2 and 100% humidity. These effector cells were harvested, and 5 × 106 cells were added to the first three wells of a 96-well round-bottom well before being serially diluted in triplicate (E:T cell ratio, 100:1, 50:1, 25:1, and 12.5:1). 51Cr (5 × 103; Amersham, Arlington Heights, IL)-labeled blasts generated from PHA (M-Form, Life Technologies) stimulation were used as specific targets and added to all wells. After 5.5 h of incubation at 37°C the supernatants were harvested with the Skatron collection system (Skatron, Sterling, VA), and 51Cr release was determined on a gamma counter (Micromedics, Huntsville, AL). Maximum lysis was obtained with a 5% solution of the nonionic detergent, Nonidet P-40 (BRL, Rockville, MD). Baseline levels were measured as the rate of spontaneous release of 51Cr from 5 × 103 targets. The results were expressed as the percentage of specific lysis: {[experimental release (cpm) − spontaneous release (cpm)]/[maximum release (cpm) − spontaneous release (cpm)]} × 100%.

Targets were matched to the stimulators, which included thymus and skin donor-matched PBL (SLAgg: class Icc/class IIdd), and skin donor-only-matched PBL (SLAhh: class Iaa/class IIdd) PBL. Targets SLA matched to the effectors were used as negative controls in all assays.

Animals were tested for the presence of anti-donor cytotoxic Abs using a two-stage, complement-dependent lymphocytotoxicity assay as described previously (26). The medium used consisted of medium 199 (Life Technologies) with 1% FCS added as a protein source. Targets that were of the same SLA haplotype as the donor thymus were used to test for the presence of cytotoxic Abs. Specific lysis was measured using the same formula as that used in the CML assays (see above). All targets were tested against a positive control (alloantiserum) and a baseline serum sample from the experimental animal to control for nonspecific complement cytotoxicity. To determine whether cytotoxic activity was due to IgM or IgG, sera were incubated with 0.2 M DTT (Sigma, St. Louis, MO) to eliminate IgM activity before the assay. The cytotoxicity index was calculated by multiplying the maximum percent lysis by the reciprocal dilution of half-maximum lysis.

Flow cytometry was used to follow the course of serum Abs sampled serially before and after thymic transplantation. Polyclonal, fluoresceinated, goat anti-swine IgG (γ-chain) or IgM (μ-chain) Abs (Kirkegraard & Perry Laboratories, Gaithersburg, MD) were used as second reagents after incubation for 30 min with the specific test sera. All steps were performed at 4°C using flow cytometry buffer (HBSS containing 0.5% BSA and 0.5% sodium azide). The total bound Abs were measured as the median fluorescence.

Macrochimerism was determined by one-color indirect Ab staining. Mouse anti-pig mAbs reactive with SLA class I (2.27.3a: IgG1), class Ic (16.7.E4.2: IgM), and class Id (2.12.3a; IgM) as previously described (29) were added in separate tubes and incubated for 30 min at 4°C. Non-cross-reacting anti-mouse class I mAbs (36-7-5, IgG2a and 12-2-2, IgM) were used as negative control Abs. All Abs were FITC conjugated. After washing twice, cells were resuspended in 0.5 ml of flow cytometry buffer. Analysis was performed using a Becton Dickinson FACScan II (San Jose, CA).

Sequential biopsies were performed on the day of transplantation; wk 2, 4, 8, 12, 16, 20, and 24; and every 6 wk thereafter, as well as postmortum, and staining was carried out using hematoxylin and eosin. Every scheduled biopsy of the sternocephalicus muscle was performed by completely removing one of the 20 grafts.

Frozen sections of sequential thymic graft biopsies were analyzed using the avidin-biotin-HRP complex technique reported previously (30). Donor and recipient cells were stained with murine anti-pig mAbs. An SLA class Ic-specific mAb (16.7.E4.2) was used to identify donor tissue, whereas an SLA class Id mAb (2.12.3a) served as a marker for graft-infiltrating cells. Immature thymocytes were stained with anti-CD1 mAb (76-7-4: IgG2a) (31), whereas mature thymocytes were stained with anti-CD3 mAb (2-6-15: IgG2a) (24). Briefly, 2- to 4-μm sections were incubated with 1% normal horse serum to inhibit nonspecific binding of horse IgG and with avidin to block endogenous biotin. After 20 min, the tissue was covered with optimally diluted primary Ab (mouse anti-pig mAb) and incubated for 60 min at room temperature. Sections were rinsed with PBS and incubated in a solution of biotin (1 mg/100 ml in PBS) with 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase. The biotinylated secondary Ab (horse anti-mouse) was added and incubated for 30–45 min. After a PBS wash, sections were incubated in an optimal dilution of avidin-biotin-peroxidase complex for 60 min, rinsed in PBS, and stained in a solution containing 3-amino-9-ethyl carbazole for 2–5 min. Staining was stopped by dipping slides into distilled water. Sections were then fixed in 4% paraformaldehyde and counterstained with Gill’s single-strength hematoxylin. Controls included omission of primary Ab or horse anti-mouse Ab.

To evaluate the optimal conditions required for thymic tissue engraftment and development, four animals received class I-mismatched minced thymic tissue placed into three distinct sites: sternocephalicus muscle, kidney capsule, and omentum. Two animals were thymectomized, and two were left euthymic before thymic transplantation to investigate the role of the recipient thymus in allogeneic thymic engraftment. CyA, given during the first 12 postoperative days, was the only immunosuppression used in these animals. The same immunosuppressive regimen has previously been shown to induce long term tolerance to SLA class I-mismatched kidney grafts in euthymic recipients, whereas untreated recipients uniformly rejected (20).

The animals in the first experiment (12311 Thx and 12364 EuThy) received a 12-day course of i.v. CyA. Their CyA trough levels were within the desired range between 400 and 800 ng/dl throughout this period. In the second experiment, the two animals (12578 Thx and 12579 EuThy) were switched to Neoral on day 5. Both animals had supratherapeutic trough levels and suffered from neurologic side effects (tremor and somnolence), but suffered no permanent sequelae.

Thymectomy before allogeneic thymic transplantation (12311 Thx and 12578 Thx) did not support engraftment in this model. In contrast, both euthymic animals showed thymic graft development with evidence of host thymopoiesis (12364 EuThy and 12579 EuThy). Initial graft biopsies performed on these animals in all three sites at 2 and 4 wk showed complete loss of thymic architecture (Fig. 2, a and b), with clusters of mature recipient-type T cells, as indicated by positive CD3 and class Id staining (data not shown). These cells were most likely infiltrating T cells rather than locally maturing T cells, although the latter cannot be ruled out. As judged by immunohistochemistry, stromal cells expressed mainly donor-type SLA class I (Fig. 3, d and f), whereas the vascular endothelial cells expressed either donor or host-type SLA class I (Fig. 3, c–f), indicating partial revascularization by the host. Immature, CD1-positive thymocytes were decreased at wk 2 (compared with the normal thymus) and were undetectable at wk 4. These immature T cells were probably remaining donor cells that disappeared over the first 4 wk. The thymic grafts were then progressively repopulated with CD1-positive T cells in both euthymic animals. The CD1 positive cells (Fig. 3,b) expressed class Id weakly (Fig. 3, c and e), consistent with thymopoiesis involving host T cell progenitors. The percentages of CD1- and class Id-positive host thymocytes were used as measurements of thymic graft development. These levels continuously increased compared with the levels of mature T cells observed in the thymic grafts in both animals until wk 16 (Table I). The immature T cells were distributed around the mature donor-type T cells, forming a thymic neocortex and medulla. Within the medulla, new Hassall’s corpuscles developed (Figs. 2,c and 3). By wk 12, the grafts could not be distinguished histologically from native thymus except by size. Macroscopically, the size of the i.m. grafts increased to ∼3–4 times that of the original implants. The omentum grafts also increased in size, but the kidney capsule grafts did not. Histology obtained on grafts from the omentum and kidney capsule revealed the same findings as those from the sternocephalicus muscle (data not shown). However, because the procedure for taking biopsies from the muscle was technically easier and less invasive, muscle grafts were biopsied preferentially at later time points. The histologic appearance of the thymic grafts was evaluated at 4-wk intervals, and with each biopsy performed, at least one graft was excised. Therefore, the total amount of thymic grafts continuously decreased. The euthymic animals were followed over a period of 12 mo after transplantation, until the majority of the grafts were removed. In one of the animals (12579 EuThy), the remaining grafts were structurally intact (Fig. 2 d). The remaining grafts of the other animal (12364 EuThy) may have been rejected after the recipient was sensitized with donor skin grafts (see below).

FIGURE 2.

Histologic appearance (H & E staining) of thymic grafts transplanted into the sternocephalicus muscle of a nonthymectomized animal (12579 EuThy). a, Marked mononuclear cell infiltrate without thymic structure was evident at wk 2 after transplantation. b, At wk 4 after thymic transplantation showing an area of central necrosis. c, Eight weeks after transplantation the areas of mature thymocytes are organized, and immature thymocytes are seen to be forming a neocortex (C) and medulla (M) with Hassall’s corpuscles (arrow). d, Thymic grafts then progressively repopulated with immature thymocytes (wk 51 is shown here).

FIGURE 2.

Histologic appearance (H & E staining) of thymic grafts transplanted into the sternocephalicus muscle of a nonthymectomized animal (12579 EuThy). a, Marked mononuclear cell infiltrate without thymic structure was evident at wk 2 after transplantation. b, At wk 4 after thymic transplantation showing an area of central necrosis. c, Eight weeks after transplantation the areas of mature thymocytes are organized, and immature thymocytes are seen to be forming a neocortex (C) and medulla (M) with Hassall’s corpuscles (arrow). d, Thymic grafts then progressively repopulated with immature thymocytes (wk 51 is shown here).

Close modal
FIGURE 3.

Immunoperoxidase staining of the allogeneic thymus showing evidence for thymopoiesis of host-type thymocytes 8 wk after transplantation (12364 EuThy). Magnification for a–d is ×160, and for e and f is ×320. Mainly mature thymocytes located in the medulla M stained positive for anti-CD3 (a), whereas the immature thymocytes of the cortex C stained positive for anti-CD1 (b). Antirecipient class I immunostaining was strongly positive for mature medullary T cells (c) and weakly positive on immature thymocytes in the thymic cortex (e). In contrast, thymocytes stained negative for anti-donor class I (d and f). Only endothelial cells that were located mainly at the cortico-medullary junction, the medulla, and the Hassall’s corpuscles (arrow) stained positive for donor class I.

FIGURE 3.

Immunoperoxidase staining of the allogeneic thymus showing evidence for thymopoiesis of host-type thymocytes 8 wk after transplantation (12364 EuThy). Magnification for a–d is ×160, and for e and f is ×320. Mainly mature thymocytes located in the medulla M stained positive for anti-CD3 (a), whereas the immature thymocytes of the cortex C stained positive for anti-CD1 (b). Antirecipient class I immunostaining was strongly positive for mature medullary T cells (c) and weakly positive on immature thymocytes in the thymic cortex (e). In contrast, thymocytes stained negative for anti-donor class I (d and f). Only endothelial cells that were located mainly at the cortico-medullary junction, the medulla, and the Hassall’s corpuscles (arrow) stained positive for donor class I.

Close modal
Table I.

Thymic graft development as measured by the relative percentage of immature host-type thymocytes (CD1 and SLA class Id positive) compared to all thymocytes (CD1 and CD3 positive)

WeekExpt. 1Expt. 2
POD% CD1+ recipient cellsPOD% CD1+ recipient cells
12311 Thx12364 EuThy12578 Thx12579 EuThy
16 0c 0c 18 0c 0c 
36 0c 0c 30 0c 0c 
56 0c 10c 57 0c 0c 
12 84a 0c 40c 78b 0c 20c 
16 106a 0c NDe 94b — 50c 
20 126a f 0c 121b — 20c 
24 156a — 20c 156b — 40d 
30  — ND 211b — 60c 
36 261a — 0c    
42  — ND    
48 331a — 0c    
WeekExpt. 1Expt. 2
POD% CD1+ recipient cellsPOD% CD1+ recipient cells
12311 Thx12364 EuThy12578 Thx12579 EuThy
16 0c 0c 18 0c 0c 
36 0c 0c 30 0c 0c 
56 0c 10c 57 0c 0c 
12 84a 0c 40c 78b 0c 20c 
16 106a 0c NDe 94b — 50c 
20 126a f 0c 121b — 20c 
24 156a — 20c 156b — 40d 
30  — ND 211b — 60c 
36 261a — 0c    
42  — ND    
48 331a — 0c    
a

Assay performed after recipient rejected donor skin graft.

b

Assay performed after recipient rejected donor and third-party class I disparate skin grafts.

c

Specimen obtained from sternocephalicus muscle.

d

Specimen obtained from kidney capsule.

e

ND, not determined.

f

—, Animal had been sacrificed.

In contrast to the animals that remained euthymic, both thymectomized animals showed a loss of thymic tissue with scarring and no evidence of thymic engraftment on any scheduled protocol biopsy. These results suggested that, as for class I-mismatched renal allografts transplanted with 12 days of cyclosporin (24), the host thymus may be crucial for engraftment of class I-mismatched thymic tissue.

MLR responses in naive animals were mainly directed against SLA class II, whereas the SLA class I proliferative responses were generally low (SI = 2–10). In contrast, CML responses in naive animals directed against SLA class I were always vigorous (32).

Rejection of the thymic grafts in the thymectomized animals led to a marked increase in anti-donor MLR (12311 Thx: SI = 35; 13578 Thx: SI = 43; measured at wk 8), consistent with sensitization (Fig. 4). In contrast, euthymic animals that accepted their thymic grafts continued to have low anti-donor responses, suggesting that sensitization did not occur.

FIGURE 4.

Mixed lymphocyte responses of all animals before and 8 wk after thymic transplantation. A marked increase in the anti-donor proliferative response (SLAgg) is seen only in the thymectomized animals that rejected the thymic grafts. In contrast, both euthymic animals remained hyporesponsive. Responses to SLA class I (SLAhh) and fully mismatched control stimulators (SLAYUC) are also shown. Results are reported as SI (stimulation index).

FIGURE 4.

Mixed lymphocyte responses of all animals before and 8 wk after thymic transplantation. A marked increase in the anti-donor proliferative response (SLAgg) is seen only in the thymectomized animals that rejected the thymic grafts. In contrast, both euthymic animals remained hyporesponsive. Responses to SLA class I (SLAhh) and fully mismatched control stimulators (SLAYUC) are also shown. Results are reported as SI (stimulation index).

Close modal

Acceptance of thymic grafts was also accompanied by donor-specific hypo- or nonresponsiveness in CML (Table II). Anti-third-party CML responses were generally normal, but anti-SLAhh responses were diminished, suggesting possible cross-tolerance to determinants shared by class I of SLAc and SLAa (Fig. 1). Stimulators from fully allogeneic outbred (Yucatan) animals produced vigorous responses in nearly all experiments. Representative data for one of the euthymic animals (12579 EuThy) at wk 0, 4, and 8, demonstrating the development of donor specific nonresponsiveness, are illustrated in Fig. 5.

Table II.

Sequential CML assays performed in both experimentsa

WeekFirst ExperimentSecond Experiment
POD12311 Thx12364 EuThyPOD12578 Thx12579 EuThy
Antidonor stimulatorAnti-third party stimulatorAntidonor stimulatorAnti-third party stimulatorAntidonor stimulatorAnti-third party stimulatorAntidonor stimulatorAnti-third party stimulator
SLAggSLAhhSLAyucSLAggSLAhhSLAyucSLAggSLAhhSLAyucSLAggSLAhhSLAyuc
21.4 5.6 ND 21.7 8.3 ND 25.2 14.4 1.0 29.3 19.3 5.0 
36 43.2 44.6 ND 30.0 24.2 ND 30 29.8 9.7 21.9 1.5 8.3 24.6 
56 18.4 32.3 ND 12.5 2.3 ND 57 28.3 31.4 28.7 0.9 30.7 32.5 
12 84b NDd ND ND 7.9 8.6 ND 78c 22.2 32.9 20.5 −3.0 1.0 20.4 
16 106b 35.0 36.2 36.8 0.8 6.8 28.6 94c — — — 19.4 43.5 24.1 
20 126b e — — 11.6 44.8 15.0 121c — — — 15.8 45.8 40.0 
24 156b — — — ND ND ND 156c — — — 11.5 47.9 26.2 
30  — — — ND ND ND 211c — — — 5.4 38.4 28.3 
36 261b — — — 21.7 41.3 38.4        
42  — — — ND ND ND        
48 331b — — — 42.2 50.2 44.3        
WeekFirst ExperimentSecond Experiment
POD12311 Thx12364 EuThyPOD12578 Thx12579 EuThy
Antidonor stimulatorAnti-third party stimulatorAntidonor stimulatorAnti-third party stimulatorAntidonor stimulatorAnti-third party stimulatorAntidonor stimulatorAnti-third party stimulator
SLAggSLAhhSLAyucSLAggSLAhhSLAyucSLAggSLAhhSLAyucSLAggSLAhhSLAyuc
21.4 5.6 ND 21.7 8.3 ND 25.2 14.4 1.0 29.3 19.3 5.0 
36 43.2 44.6 ND 30.0 24.2 ND 30 29.8 9.7 21.9 1.5 8.3 24.6 
56 18.4 32.3 ND 12.5 2.3 ND 57 28.3 31.4 28.7 0.9 30.7 32.5 
12 84b NDd ND ND 7.9 8.6 ND 78c 22.2 32.9 20.5 −3.0 1.0 20.4 
16 106b 35.0 36.2 36.8 0.8 6.8 28.6 94c — — — 19.4 43.5 24.1 
20 126b e — — 11.6 44.8 15.0 121c — — — 15.8 45.8 40.0 
24 156b — — — ND ND ND 156c — — — 11.5 47.9 26.2 
30  — — — ND ND ND 211c — — — 5.4 38.4 28.3 
36 261b — — — 21.7 41.3 38.4        
42  — — — ND ND ND        
48 331b — — — 42.2 50.2 44.3        
a

Each experiment included one euthymic and one thymectomized animal. Effectors were tested on targets that were matched to the stimulators. Data are expressed as percent specific lysis at an effector:target ratio of 100:1.

b

Assay performed after recipient rejected donor-specific skin graft.

c

Assay performed after recipient rejected donor-specific and third-party class I disparate skin grafts.

d

ND, not determined.

e

—, Animal had been sacrificed.

FIGURE 5.

Representative cell-mediated lympholysis responses of a euthymic animal (12579 EuThy) before and 4 and 8 wk after thymic transplantation are shown. The filled squares demonstrate the anti-donor (SLAgg) responses. Positive controls consisted of SLA class I (SLAhh) and fully mismatched (SLAYUC) targets. All targets, except those of the negative control (stimulator YUC, target SLAdd), were matched to the stimulator.

FIGURE 5.

Representative cell-mediated lympholysis responses of a euthymic animal (12579 EuThy) before and 4 and 8 wk after thymic transplantation are shown. The filled squares demonstrate the anti-donor (SLAgg) responses. Positive controls consisted of SLA class I (SLAhh) and fully mismatched (SLAYUC) targets. All targets, except those of the negative control (stimulator YUC, target SLAdd), were matched to the stimulator.

Close modal

The mechanism of the observed donor-specific hyporesponsiveness in CML was addressed by providing T cell help to the effectors through addition of IL-2 to the CML cultures. Alternatively, effectors were generated by culture with stimulators fully mismatched to the responder, but SLA class I matched to the thymic graft donor. In both experiments, anti-donor responses could not be restored (data not shown), suggesting that these T cells in the thymic graft recipients were either anergic or deleted.

Anti-donor cytotoxic Abs were detected 4 wk after thymic transplantation in both thymectomized and euthymic animals, as measured on donor-type PBL. Serum treatment with DTT did not diminish the cytotoxicity, suggesting that Abs of IgG class were responsible for at least part of the complement-mediated cytotoxicity. Representative data for one of the two engrafted animals (12579 EuThy) are shown in Fig. 6.

FIGURE 6.

Analysis of anti-thymic donor cytotoxic Abs performed on animal 12579 EuThy before (open symbols) and after donor skin transplantation (filled symbols). The 51Cr release was measured using chromium-labeled, thymic donor-matched PBL, in a two-stage complement-dependent lymphocytotoxicity assay. DTT was added in a second assay to eliminate IgM activity. Data are expressed as the percent specific lysis of donor-matched (SLAgg) PBL targets.

FIGURE 6.

Analysis of anti-thymic donor cytotoxic Abs performed on animal 12579 EuThy before (open symbols) and after donor skin transplantation (filled symbols). The 51Cr release was measured using chromium-labeled, thymic donor-matched PBL, in a two-stage complement-dependent lymphocytotoxicity assay. DTT was added in a second assay to eliminate IgM activity. Data are expressed as the percent specific lysis of donor-matched (SLAgg) PBL targets.

Close modal

Flow cytometry was performed to assess macrochimerism in a thymectomized and a euthymic animal (12311 Thx and 12364 EuThy). PBL, gated on lymphocytes, were stained for donor-type SLA class I. Samples were obtained from both animals on days 0, 4, 7, 10, 16, and 30 and again on day 126 in the euthymic pig only (12364 EuThy). In addition, the host thymus was analyzed in the same animal on days 0, 16, and 126. No SLA class Ic-positive cells could be detected at a detection level of 0.05% (data not shown).

In an attempt to determine whether tolerance to the thymic donor might have been induced, skin grafts obtained from the original thymic donor were transplanted onto each thymic graft recipient, 8 wk after thymic transplantation. Naive animals from our inbred herd of miniature swine generally reject class I-mismatched skin grafts within 8–10 days (33). Both animals that were thymectomized before thymic transplantation rejected the skin grafts between 9 and 10 days (12311 Thx and 12578 Thx, respectively). One of the euthymic animals (12364 EuThy) also rejected in 10 days, while the other (12579 EuThy) showed a prolonged skin survival of 18 days. After skin graft rejection, subsequent thymic graft development was impaired in both of these animals, as measured by the relative levels of immature CD1-positive thymocytes compared with the mature T cells in the grafts (Table I). In addition, both euthymic animals experienced an immediate increase in their levels of cytotoxic Abs (Fig. 6), and an increase several weeks later in their anti-donor CML responses (Table II). This apparent rejection process was seen only transiently in the animal with prolonged skin graft survival (12579 EuThy). The other animal (12364 EuThy) may have rejected its remaining thymic grafts between 28 and 40 wk after skin grafting (i.e., 36 and 48 wk after thymic transplantation).

We have previously demonstrated that fetal pig thymic tissue transplanted into T and NK cell-depleted thymectomized mice engrafts and supports host thymopoiesis. The developing mouse T cells were immunocompetent and demonstrated in vitro and in vivo tolerance to porcine xenoantigens (8, 16, 34, 35). Because of the potential of this methodology as a means of inducing tolerance clinically, it was deemed important to determine the feasibility of extending these findings to a large animal model. The present study was therefore undertaken to evaluate the requirements for allogeneic thymic engraftment in miniature swine before proceeding to a xenogeneic large animal system.

Class I-mismatched kidney grafts are uniformly accepted in euthymic pigs when transplantation is followed by a 12-day course of CyA. In contrast, such kidney grafts are rejected when transplanted without immunosuppression (20). Therefore, we adopted the same CyA treatment regimen and SLA disparity for development of a thymic transplantation model. We compared animals who underwent thymectomy before thymic transplantation to those who remained euthymic. Because previous studies in rodent models have demonstrated host thymectomy to be an absolute requirement for allogeneic or xenogeneic thymic tissue engraftment, it seemed possible that the same might be true in miniature swine (13, 35). In theory, the host thymus might be expected to continuously produce allo-reactive T cells and cause rejection of the thymic graft, while removal of the host thymus in conjunction with T cell depletion might allow the transplanted thymus to survive and sustain T cell development after engraftment.

Contrary to this expectation, not only was removal of the host thymus found to be unnecessary to permit engraftment of class I-mismatched thymic tissue, but such thymectomy was also found to be detrimental. Thus, after host thymectomy and class I-mismatched thymic transplantation followed by a 12-day course of CyA, sequential graft biopsies revealed that as early as 2 wk and continuing throughout the entire observation period, there was no evidence for viable thymic graft tissue. In contrast, the nonthymectomized animals successfully engrafted, and by 8 wk post-transplant the grafts had differentiated into tissue that fulfilled all the morphologic criteria of a functional thymus. The grafts were repopulated with immature, host-type thymocytes and showed normal thymic architecture, including a neocortex and medulla. In subsequent weeks, the relative number of immature thymocytes continuously increased, and Hassall’s corpuscles developed.

This requirement for the presence of the host thymus is probably peculiar to the use of our CyA treatment regimen for induction of tolerance across an MHC class I barrier (20). Under these circumstances, mature T cells that would otherwise react with the graft are apparently inactivated by a peripheral mechanism, currently under investigation in our renal transplantation studies. In those class I-mismatched renal transplantation studies, prior thymectomy was also found to be detrimental to the induction of tolerance (24). On the other hand, when T cell depletion is used to permit engraftment, it is likely that host thymectomy may not be detrimental and may even be required, as was the case in murine studies (8). We are now attempting to extend our thymic transplantation studies to more extensive histocompatibility barriers in our miniature swine, where, on the basis of our previous studies of renal transplantation, we anticipate that CyA alone will not be sufficient to induce long term graft acceptance (26). We therefore plan to use T cell depletion in these studies and will again examine the effects of thymectomy (G. W. Haller et al., work in progress).

There are additional reasons why thymectomy may have been detrimental to successful engraftment in these studies. These include recent evidence that the host thymus may be involved in the development of donor-specific regulatory cells (36, 37). In addition, in murine models, CyA treatment has been shown to lead to thymic medullary involution with destruction of the medullary dendritic cells. After CyA treatment is stopped, the thymus rapidly repopulates with dendritic cells (38, 39). It is therefore possible that in the present studies either donor dendritic cells or host cells presenting donor peptides may repopulate the thymus early after CyA treatment. This repopulation, which would of course require the presence of the host thymus, could be responsible for subsequent deletion of developing donor-reactive thymocytes.

In vitro cellular assays revealed that the thymic graft recipients had developed donor-specific hyporesponsiveness. Antidonor proliferative responses as well as cell-mediated cytotoxicity were markedly reduced or absent as early as 4 wk after transplantation. Surprisingly, however, the animals not only produced an anti-donor IgM response, but also demonstrated a class switch to IgG 4 wk after thymic transplantation. Because such class switching is thought to require T cell help, we speculate that T cell responsiveness may have been present transiently after cessation of CyA treatment on day 11. This T cell response may have disappeared or been suppressed by the time at which the MLR was first measured (4 wk after transplantation). However, the thymic grafts continued to develop, and endothelial cells remained positive for donor SLA class I despite the presence of donor-specific Abs. This finding suggests either a local suppressive phenomenon or the general absence of effector mechanism leading to destruction of Ab-coated cells in the thymus. Consistent with the latter possibility, failure of mAbs to deplete mature T cells in the thymus has previously been reported in murine studies (40).

Despite successful engraftment, we speculate that the immunosuppressive treatment selected for these experiments was insufficient to prevent sensitization. However, it is remarkable that these grafts continued to develop and remained viable as long as 1 yr after transplantation in the presence of alloantibodies. Even after sensitization with donor skin, which was originally performed with the intention of assessing donor-specific tolerance, one of the animals was able to recover donor-specific hyporesponsiveness in CML. These results demonstrate the potential of allogeneic thymus to induce tolerance in large animals and suggest that thymus may be resistant to both cellular and humoral rejection. These findings may be of particular importance in xenotransplantation, where preformed natural Abs are present (reviewed in 41).

We thank Drs. Kai Sonntag, Ignacio Rodriguez-Barbosa, and Shaun Kunisaki for their helpful review of the manuscript, and Novartis Pharmaceuticals Corp. (Hanover, NJ) for generously providing CyA.

1

This work was supported by National Institutes of Health Grants AI31046 and HL18646. G.W.H. and A.W. received partial support from the Deutsche Forschungsgemeinschaft.

4

Abbreviations used in this paper: CyA, cyclosporin A; CML, cell-mediated lympholysis; EuThy, euthymic pig; MGH, Massachusetts General Hospital; POD, postoperative day; SI, stimulation index; SLA, swine leukocyte Ag; Thx, thymectomized pig.

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