Induction of specific tolerance to class I-disparate renal allografts in miniature swine with cyclosporine.

Previous studies in miniature swine have suggested that the mechanism underlying the spontaneous development of tolerance in one third of one-haplotype class I disparate renal allografts (i.e., ag----ad) involves a relative T cell help deficit at the time of first exposure to antigen. If this hypothesis were correct, then one might expect the administration of an immunosuppressive agent capable of inhibiting lymphokine production during this period to lead to the induction of tolerance to class I MHC antigens in two-haplotype class I mismatched renal allografts (i.e., gg----dd), which are otherwise uniformly and acutely rejected. This hypothesis was tested in eight two-haplotype class I disparate, class II matched donor-recipient pairs, in which recipients were treated with cyclosporine 10 mg/kg, i.v. q.d. for 12 days. This protocol led to the induction of long-term (greater than 100 days) specific tolerance in 100% of recipients, as compared with control animals that rejected grafts in 13.7 +/- 0.9 days (P less than 0.0001). The specificity of tolerance was assessed both in vivo with subsequent skin grafts and in vitro by mixed lymphocyte response (MLR) and cell-mediated lymphocytotoxicity (CML). Survival of donor-specific skin grafts was prolonged compared with skin grafts bearing third-party class I antigens (19.5 +/- 2.0 versus 11.5 +/- 2.0 days, n = 4, P less than 0.05). Tolerant recipients had markedly diminished or absent anti-donor MLR and CML responses, but maintained normal reactivity to third party. Four of eight CsA-treated recipients showed detectable levels of anti-donor IgM, while none demonstrated the presence of anti-donor IgG, which was found in all rejecting controls.     

Successful induction of long-term specific tolerance to fully allogeneic renal allografts in miniature swine.

Major histocompatibility complex class II matching is of overwhelming importance for achieving tolerance to kidney transplants (KTX) in miniature swine. When class II antigens are matched, long-term specific tolerance across complete MHC class I antigen barriers can uniformly be induced by a 12-day perioperative course of cyclosporine. This same regimen is ineffective in fully MHC-mismatched combinations. We hypothesized that initial induction of tolerance to kidney donor class II antigens by bone marrow transplantation might allow tolerance to be induced to a subsequent fully allogeneic KTX in combination with CsA therapy. We report here the results of such fully allogeneic KTX performed in 4 recipients of prior single-haplotype class II-mismatched BMT. All animals received KTX from donors class II matched to the BMT donor and received a 12-day course of intravenous CsA (10 mg/kg/day). All four animals have maintained normal serum creatinine values (less than 2.0 mg/dl) for greater than 200 days posttransplant. Specific hyporesponsiveness to both BMT and KTX donor-type MHC antigens was found in mixed lymphocyte culture and cell-mediated lympholysis assays. Compared with third-party grafts, significantly prolonged survival of BMT donor-specific (P = 0.031) and KTX donor-specific (P = 0.031) skin grafts was observed. These results demonstrate that induction of tolerance to class II antigens by BMT allows a short course of CsA to induce specific tolerance to fully allogeneic renal allografts.     

Acceptance reaction: intragraft events associated with tolerance to renal allografts in miniature swine

Inbred miniature swine that are treated for 12 d with a high dose of cyclosporin A develop tolerance to MHC class II matched, class I-mismatched renal allografts. The aim of this study was to clarify the intrarenal allograft events associated with the development of tolerance in this protocol. Morphologic and immunologic studies were performed in serial biopsies from accepting grafts after 12 d of cyclosporin A treatment (n = 4) and were compared with those from untreated control rejecting grafts (n = 4). In accepting grafts with stable function, a transient interstitial infiltrate developed. The cellular infiltrate had many similarities to that in rejecting grafts; both had T cells and macrophages, similar proportions of T-cell subsets, and a similar frequency of in situ nick end labeling (TUNEL)+ apoptotic infiltrating cells. However, the cellular infiltrate in the acceptance reaction was distinguished by less T-cell activation (interleukin-2 receptor+), less proliferation (proliferating cell nuclear antigen+) of infiltrating cells, and less graft cell apoptosis in arteries, tubules, glomeruli, and peritubular capillaries. Thereafter, the infiltrate in the accepting grafts progressively resolved with decreased cell proliferation, activation, and apoptotic graft parenchymal cell injury, but the high frequency of apoptosis persisted in graft-infiltrating cells. In parallel to the intragraft events, donor-specific unresponsiveness developed as assessed by cell-mediated cytotoxicity by blood mononuclear cells in vitro. In conclusion, the acceptance reaction in transplanted grafts is characterized by progressive resolution of T-cell proliferation and activation and of cell-mediated graft injury, as well as prolonged T-cell apoptosis. These intragraft events suggest that both T-cell anergy and T-cell deletion occur in the graft during the development of tolerance. Some of the described immunopathologic findings (activation, proliferation, apoptosis) may be useful in distinguishing acceptance from rejection, as well as in predicting later graft acceptance in tolerance induction protocols.     

The failure of skin grafting to break tolerance to class I-disparate renal allografts in miniature swine despite inducing marked antidonor cellular immunity.

Long-term specific tolerance to one haplotype class I plus minor antigen disparate renal allografts develops without exogenous immunosuppression in approximately 35% of miniature swine (n = 128). Previous studies have suggested that this phenomenon is related to limited class I-specific helper T cell activity as evidenced by the failure of antibody class switching in vivo and the ability of exogenous interleukin 2 to elicit antidonor responses in vitro. To determine whether tolerance could be broken by inducing antidonor reactivity with donor antigen and a source of T cell help, multiple skin grafts bearing donor class I plus third-party class II antigens were placed on tolerant animals. Skin grafts were placed at least 3 months after the kidney transplant, at which time all recipients had normal renal function as measured by blood urea nitrogen and serum creatinine. First-set rejection of skin grafts by SLAad and SLAdd hosts occurred in 11.8 +/- 1.1 days (mean +/- SEM, n = 6) and in 9.3 +/- 0.9 days (n = 4), respectively. Coincident with skin rejection, most animals developed a transient rise in BUN to 62 +/- 11 mg/dl (n = 10) and a similar rise in Cr to 4.9 +/- 1.2 mg/dl (n = 10), with normal levels returning in all animals within two weeks. Subsequent skin grafts with the same disparity did not undergo second-set rejection and did not induce BUN or Cr elevations. Prior to skin grafting, animals showed no antidonor activity in mixed lymphocyte reaction or cell-mediated lymphocytotoxicity assays. After two skin grafts, all animals developed donor-specific CML and secondary MLR responses, and additional skin grafts amplified this cellular immunity. Development of marked antidonor immunity without a break in tolerance suggested that either graft adaptation or local suppression might be involved in maintaining tolerance to class I MHC antigens. In preliminary studies, an immunized SLAad animal and an immunized SLAdd animal were retransplanted with kidneys MHC matched to their first allografts. In both cases, the second graft was accepted permanently without immunosuppression, suggesting that graft adaptation is not necessary for the maintenance of tolerance to renal allografts in miniature swine.     

Transplantation in miniature swine. VI. Factors influencing survival of renal allografts.

Renal allografts were performed between and among animals from three herds of miniature swine that were selectively inbred to homozygosity at the major histocompatibility complex, MSLA. The results suggest several genetic factors which influence the survival of renal allografts in these animals. As expected, the major histocompatibility complex (MHC) was of dominant importance, and all MSLA-mismatched grafts were rejected promptly (12 +/- 3.7 days). Some MSLA-matched grafts were also rejected (30 +/- 15.0 days), indicating that non-MSLA loci also determine antigens which can lead to kidney rejection. Other MSLA-matched grafts were accepted indefinitely. At least one immune response gene that determined ability to reject kidneys across non-MSLA differences seemed to be segregating in our swine population. Animals that had accepted MSLA-matched renal grafts for extended periods demonstrated markedly prolonged survival of subsequent donor skin grafts compared to skin graft survival across the same non-MSLA difference in normal animals. This finding suggests that failure to reject kidneys across non-MSLA differences indicates systemic tolerance, and that there may be a relationship between the induction of such tolerance and the proposed immune response gene controlling rejection.     

Role of the thymus in transplantation tolerance in miniature swine. III. Surgical manipulation of the thymus interferes with stable induction of tolerance to class I-mismatched renal allografts

BACKGROUND: Previous studies have demonstrated that long-term tolerance of class I mismatched renal allografts in miniature swine is induced by a short course of cyclosporine (CyA), and that a total thymectomy 21 days before transplantation abrogates the induction of stable tolerance. We have now examined the effects of surgical manipulation of the thymus, with or without a reduction in the thymic volume, on the induction of tolerance. MATERIALS AND METHODS: Miniature swine receiving a transplant of a class I-mismatched renal allograft and 12 days of CyA underwent either (1) a partial thymectomy 21 days before kidney transplantation (day -21), (2) serial thymic biopsies (to evaluate the effect of surgical trauma and reduction in volume of the thymus) or serial incisions of the thymus thymus (to evaluate the effect of surgical trauma without changes in thymic volume), (3) a sham thymectomy on day -21, or serial sham thymic surgery on the same POD as the thymic biopsies and incisions (control animals). RESULTS: Control animals had a stable plasma creatinine, had donor-specific unresponsiveness in cell-mediated lympholysis (CML) assays, had absence of rejection in kidney biopsy specimens, and did not develop anti-donor class I immunoglobulin (Ig)G alloantibodies. Animals undergoing a partial thymectomy on day -21 or serial thymic biopsies showed severe renal dysfunction, histological evidence of rejection in kidney biopsy specimens and anti-donor reactivity in CML assays; all but one animal developed anti-donor class I IgG alloantibodies. Serial incisions of the thymus induced an increase in plasma creatinine and histological rejection in 1 of 3 animals and anti-donor cytotoxic T cells in vitro in all 3 animals. CONCLUSIONS: A partial thymectomy or serial thymic biopsies markedly interfere with the induction of tolerance to renal allografts. Serial thymic incisions also interfere with the induction of tolerance, but to a lesser degree. These studies may have implications for tolerance-inducing protocols that involve thymic manipulation.     

An MHC class II disparity raises the threshold for tolerance induction in pulmonary allografts in miniature swine

OBJECTIVES: The mechanisms and treatment of chronic rejection in pulmonary allotransplantation remain elusive. We have induced robust tolerance to class I-disparate lung allografts in miniature swine using an intensive 12-day course of tacrolimus. Here, we tested whether a tolerant state can be induced in swine receiving fully mismatched lung allografts. METHODS: Orthotopic left lung allografts were performed using MHC class I-disparate (group 1: n = 3) or fully disparate (group 2: n = 6) donors. The recipients received a 12-day postoperative course of tacrolimus (continuous intravenous infusion; target level = 35-50 ng/mL) as their only immunosuppression. RESULTS: All swine in group 1 maintained their grafts long term without developing any lesions of chronic rejection (>497, >432, >451 days). These recipients exhibited donor-specific hyporesponsiveness in cell-mediated lymphocytotoxity (CML) and mixed lymphocyte reaction (MLR) assays. In group 2, five of the six recipients maintained their grafts long term (sacrificed on postoperative days 515, 389, 429, 481, and 438 with viable grafts). Isolated lesions of obliterative bronchiolitis were occasionally seen on biopsy, and donor-specific hyporesponsiveness on assays was consistently observed. The remaining recipient rejected its graft on day 103 with histologic findings of obliterative bronchiolitis. CONCLUSIONS: We report long-term graft acceptance without chronic immunosuppression in five of six recipients across a full MHC disparity, albeit with some evidence of obliterative bronchiolitis. These data suggest that the class II disparity inherent in a fully mismatched transplant increases the requirement for tolerance induction.     

Role of the thymus in transplantation tolerance in miniature swine: II. Effect of steroids and age on the induction of tolerance to class I mismatched renal allografts

BACKGROUND: Recent studies in young (5-7 months) miniature swine have demonstrated that the thymus is involved in the rapid induction of stable tolerance to class I mismatched renal allografts after a 12-day course of Cyclosporine (CyA). Because both steroids and age are known to influence the structure and function of the thymus, we have now studied the effects of these two parameters on tolerance induction in this model. MATERIALS AND METHODS: In young swine, the administration of methylprednisolone (MP) during the standard tolerance-inducing regimen (a 12-day course of CyA) produced severe renal dysfunction and acute cellular rejection histologically. However, the renal allografts recovered and were accepted for >100 days with histological evidence of chronic rejection. To test the effect of age, two relatively old swine (55 and 71 months) received transplants of class I mismatched renal allografts and the standard 12-day course of CyA. One animal rejected the allograft acutely on postoperative day 22, and the second also rejected, but more slowly, with manifestations of chronic rejection. CONCLUSION: These findings suggest that both MP and old age interfere with the induction of stable tolerance in a fashion similar to the previously described effect of thymectomy. These results may have important implications for the mechanism of thymic-dependent tolerance, for the use of steroids in clinical protocols for the induction of allograft tolerance, and for the application of such protocols to adult patients.     

Regulatory mechanism of peripheral tolerance: in vitro evidence for dominant suppression of host responses during the maintenance phase of tolerance to renal allografts in miniature swine

BACKGROUND: Studies from our laboratory have demonstrated that a short course of cyclosporine leads to indefinite survival of renal allografts across an MHC class-I barrier in miniature swine. We have recently reported that a peripheral regulatory mechanism appears to be involved in the maintenance of this tolerance since peripheral blood lymphocytes (PBL), exposed to donor antigen in vitro specifically suppressed the generation of anti-donor cytotoxic activity by recipient-matched naive PBL. We have now further investigated the mechanism of this phenomenon to determine the level at which such regulation occurs, and investigated the phenotypes of the cells involved in maintaining dominant suppression. MATERIALS AND METHODS: PBL from long-term tolerant animals (>6 months after renal transplantation) were pre-stimulated in vitro with donor-type PBL. These cells were then incubated with recipient-matched naive responders and donor-type PBL stimulators in MLR assays. The proliferative activity of the cells was measured by [3H]-thymidine incorporation. Suppression was measured by inhibition of proliferation of naive cells in response to donor PBL when co-cultured with tolerant cells. Flow cytometry was used to study the phenotypes of cells that were present in cell cultures. RESULTS: Primed PBL from tolerant animals markedly suppressed the proliferative response of recipient-matched naive cells to donor-matched stimulators in vitro. No suppression of proliferation was observed in response to third party stimulators, indicating that the suppression was donor-specific. Primed PBL from naive animals stimulated with donor antigen and co-cultured with unprimed recipient-matched naive cells also demonstrated reduced proliferative responses. However, this decrease in proliferation appeared to be due to a 'burn-out' phenomenon, as assessed by kinetic studies, rather than due to true suppression. Expression of CD25 increased on a sub-population of T cells from tolerant animals following priming with donor antigen. These cell then markedly inhibited further activation of CD25 positive cells in co-cultures with naive responder cells, suggesting a possible mechanism of suppression. CONCLUSION: These results suggest that the mechanism of tolerance to class-I-mismatched renal allografts, involves a population of regulatory cells that are capable of suppressing proliferative anti-donor responses.     

Factors affecting patency of venous allografts in miniature swine

In immunologically defined National Institutes of Health miniswine, a segment of internal jugular vein was anastomosed to the carotid artery as an interposition graft. Patency of swine major histocompatibility complex matched, one haplotype mismatched, and complete mismatched veins was 9.8, 6.3, and 3.0 weeks respectively (p = 0.009). More than 90% of mismatched and 20% of matched allografts developed a positive crossmatch before occlusion (p = 0.006). The mixed lymphocyte response did not predict graft occlusion. Treatment of 10 swine with cyclosporine (10 mg/kg/day) did not significantly improve patency for one haplotype mismatched grafts. In haplotype mismatched veins, cryopreserved grafts occluded more rapidly than noncryopreserved grafts: mean 2.4 versus 6.3 weeks, respectively (p = 0.002). In all cryopreserved vein grafts, alloantibody appeared at or after graft occlusion rather than before occlusion as seen with fresh allografts (p = 0.046). The mean patency of cryopreserved versus fresh autografts was 3.3 and greater than 32 weeks, respectively (p = 0.004). In summary, these results indicate that (1) allograft patency is related to the degree of swine major histocompatibility complex match and development of cytotoxic alloantibodies; (2) moderate-dose cyclosporine does not prolong allograft patency nor suppress development of antibody; (3) cryopreservation may accelerate graft occlusion through nonimmunologic mechanisms.     

Tolerance to musculoskeletal allografts with transient lymphocyte chimerism in miniature swine

BACKGROUND: Although transplantation of musculoskeletal allografts in humans is technically feasible, the adverse effects of long-term immunosuppression subject the patient to high risks for correcting a non-life-threatening condition. Achieving immunologic tolerance to musculoskeletal allografts, without the need for chronic immunosuppression, could expand the clinical application of limb tissue allografting. Tolerance to musculoskeletal allografts has been accomplished previously in miniature swine in our laboratory. Although stable, mixed chimerism has been suggested as the mechanism underlying long-term tolerance in a rat limb model, the mechanism of this tolerance induction has not been established. This report explores the possible relationship between hematopoietic chimerism and tolerance to musculoskeletal allografts in swine. METHODS: Twelve miniature swine underwent vascularized musculoskeletal allograft transplantation from histocompatibility complex (MHC) matched, minor antigen-mismatched donors. Eight animals received a 12-day coprse of cyclosporine, one of which was excluded due to subtherapeutic levels. Four recipients were not immunosuppressed. Serial biopsies to assess graft viability and flow cytometry to assess chimerism were performed. Donor and third-party skin grafts were placed on recipients with surviving allografts greater than 100 days to validate tolerance. RESULTS: Both groups developed early peripheral chimerism, but this chimerism became undetectable by postoperative day 19 in the cyclosporine group and by day 13 in the control group. Animals receiving cyclosporine developed permanent tolerance to their allografts, whereas those not receiving cyclosporine rejected their allografts in 6-9 weeks. Animals demonstrating tolerance to their bone allografts also demonstrated prolonged donor skin graft survival. CONCLUSIONS: Induction of tolerance to musculoskeletal allografts can be achieved in the MHC matched swine. Although hematopoietic chimerism is present in the immediate postoperative period, persistent, long-term chimerism does not seem to be necessary for maintenance of such tolerance.     

Persistent chimerism despite antidonor MHC in vitro responses in miniature swine following allogeneic hematopoietic cell transplantation

BACKGROUND: T-cell chimerism predominates in miniature swine receiving hematopoietic-cell transplantation without myelosuppressive conditioning. Several chimeric recipients have become hyporesponsive to donor-major histocompatibility complex (MHC) in vitro and accepted donor-matched renal transplants without immunosuppression. However, some retained antidonor in vitro responses and subsequently rejected donor renal allografts despite the persistence of peripheral blood chimerism. In this study, we characterize the donor cells in both "tolerant" and "nontolerant" chimeric miniature swine. METHODS: Peripheral blood chimerism was determined by flow cytometry. In vitro antidonor responsiveness was determined by mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML). Donor cells were separated from chimeras by immunomagnetic bead separation and used as stimulators or targets in CML assays. Phenotypic analysis of donor cells in chimeras was performed using flow cytometry. RESULTS: Peripheral blood chimerism stabilized beyond 100 days and was made up almost entirely of T cells. PBMC from nontolerant chimeras could be stimulated in vitro to kill donor cells isolated from the mixed chimera itself. In contrast, PBMC from tolerant chimeras hyporesponsive to donor-type cells could not be stimulated in vitro to kill their own sorted donor cells. CONCLUSIONS: The in vivo persistence of donor T cells in mixed chimeric animals with in vitro antidonor responsiveness is not caused by an inability of these cells to be killed but rather by the poor stimulating capacity of these donor T cells. The nature of donor T cells that persist in the face of in vitro antidonor responses, has important implications for the induction of transplant tolerance by way of the generation of mixed chimerism.     

Detection of regulatory cells as an assay for allograft tolerance in miniature swine.

BACKGROUND: There is currently a great need for an in vitro assay to assess the presence of tolerance following allotransplantation to determine whether immunosuppressive medications can be discontinued. Our laboratory has recently developed an assay involving coculture inhibition of cell-mediated lympholysis that correlates with tolerance to allografts in swine leukocyte antigen (SLA) Class I-mismatched miniature swine. The potential for clinical application of this assay may depend on 2 important factors: (1) whether the assay can be used in the presence of immunosuppression; and (2) whether frozen-stored naive responder cells can be utilized. METHODS: Long-term tolerant MGH miniature swine that had accepted SLA Class I-mismatched kidney transplants after a 12-day course of cyclosporine or tacrolimus were studied. Two long-term tolerant and 2 naive control animals were treated with a clinically relevant dose of cyclosporine for 2 weeks (trough level 100 to 400 ng/ml) to simulate the ongoing "chronic" immunosuppression used in human recipients of allografts. Cells from tolerant or naive, recipient-matched animals were stimulated for 6 days with donor or third-party SLA. These primed cells were then cocultured with naive unstimulated recipient major histocompatibility complex (MHC)-matched responders and irradiated stimulators. Responder cells were tested both fresh and frozen. RESULTS: Suppression of cytotoxic responses of naive responder cells was observed in all coculture assays using cells from tolerant animals primed against donor antigen in vitro, but not in assays using similarly primed cells from naive animals. Responder cells from tolerant animals receiving immunosuppression had a suppressive activity similar to that from cells of the same animals not receiving immunosuppression. Similar suppression was also observed in coculture assays using either fresh or frozen naive responder cells. CONCLUSIONS: This coculture assay appears to correlate with the presence of tolerance under conditions applicable to the clinical setting. The assay appears to identify peripheral regulatory mechanisms of tolerance in allogeneic transplant recipients, and therefore may provide an approach for determining an appropriate timepoint at which to test withdrawal of immunosuppressive medications.     

Vascularized thymic lobe transplantation in miniature swine: I. Vascularized thymic lobe allografts support thymopoiesis

BACKGROUND: Vascularized thymokidney transplants have previously been shown to induce tolerance across major histocompatibility complex barriers. The ability to perform vascularized thymic lobe transplantation could permit such tolerance to be induced with any cotransplanted solid organ or tissue. For this reason, we have developed a technique for vascularized thymic lobe transplantation in miniature swine. METHODS: Thymic vessels (n=2) were anastomosed to the carotid artery and the external jugular vein of na?ve minor-mismatched recipients treated with a 12-day course of cyclosporine A (10 mg/kg/day). Graft survival and thymopoiesis were assessed by histology, immunohistochemistry, and fluorescence-activated cell sorting. Allele-specific antibodies 74-12-4 and pig allelic antigen (PAA) were used to distinguish donor and recipient cells. RESULTS: Allografts showed intact cortical and medullary structure posttransplantation, without evidence of rejection or ischemia. Recipient thymocytes repopulated the donor cortical thymus by POD30 and increased in the cortex and medulla by POD60. CONCLUSIONS: Our study demonstrates the technical feasibility of vascularized thymic lobe transplantation and the support of thymopoiesis by such transplants in a large animal model. This technique may offer a novel strategy to induce transplant tolerance across allogeneic and xenogeneic barriers, and to support long-term thymopoiesis in immunodeficient hosts.