Adoptive Transfer of Renal Allograft Tolerance in a Large Animal Model.

Our recent studies in an inbred swine model demonstrated that both peripheral and intra-graft regulatory cells were required for the adoptive transfer of tolerance to a second, naïve donor-matched kidney. Here, we have asked whether both peripheral and intra-graft regulatory elements are required for adoptive transfer of tolerance when only a long-term tolerant (LTT) kidney is transplanted. Nine highly-inbred swine underwent a tolerance-inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naïve kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor-matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance-inducing and/or tolerance-maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naïve donor-matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

High incidence of xenogenic bone marrow engraftment in pig-to-baboon intra-bone bone marrow transplantation.

Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3-13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.

Transgenic expression of human CD47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation.

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable chimerism levels were observed in the peripheral blood of these recipients. In contrast, transplantation of WT progenitor cells resulted in little or no bone marrow engraftment and no detectable peripheral chimerism. These results demonstrate a substantial protective effect of hCD47 expression on engraftment and persistence of porcine cells in this model, presumably by modulation of macrophage phagocytosis.

Skin graft tolerance across a discordant xenogeneic barrier.

Specific T-cell tolerance may be essential for successful xenotransplantation in humans. Grafting of thymectomized, T cell-depleted normal mice with xenogeneic fetal pig thymus and liver (FP THY/LIV) tissue results in the recovery of functional CD4 antigen-positive cells. We have tested T-cell tolerance by skin grafting. Donor-matched pig skin survived permanently (> 200 days), whereas allogeneic mouse skin was rapidly rejected. Nontolerant control mice rejected pig skin within 26 days. Both porcine and murine histocompatibility class IIhigh cells were detected in long-term thymus grafts, and T-cell repertoire analyses suggested that tolerance to both donors and recipients developed, at least in part, by intragraft clonal deletion. This study demonstrates the principle that tolerance, measured by the stringent criterion of skin grafting, can be induced across a widely disparate species barrier.

Results of gal-knockout porcine thymokidney xenografts.

Clinical transplantation for the treatment of end-stage organ disease is limited by a shortage of donor organs. Successful xenotransplantation could immediately overcome this limitation. The development of homozygous alpha1,3-galactosyltransferase knockout (GalT-KO) pigs removed hyperacute rejection as the major immunologic hurdle to xenotransplantation. Nevertheless, GalT-KO organs stimulate robust immunologic responses that are not prevented by immunosuppressive drugs. Murine studies show that recipient thymopoiesis in thymic xenografts induces xenotolerance. We transplanted life-supporting composite thymokidneys (composite thymus and kidneys) prepared in GalT-KO miniature swine to baboons in an attempt to induce tolerance in a preclinical xenotransplant model. Here, we report the results of seven xenogenic thymokidney transplants using a steroid-free immunosuppressive regimen that eliminated whole-body irradiation in all but one recipient. The regimen resulted in average recipient survival of over 50 days. This was associated with donor-specific unresponsiveness in vitro and early baboon thymopoiesis in the porcine thymus tissue of these grafts, suggesting the development of T-cell tolerance. The kidney grafts had no signs of cellular infiltration or deposition of IgG, and no grafts were lost due to rejection. These results show that xenogeneic thymus transplantation can support early primate thymopoiesis, which in turn may induce T-cell tolerance to solid organ xenografts.

Strain restricted typing sera (SRTS) for use in monitoring the genetic integrity of congenic strains.

A relatively simple procedure for serologic monitoring of the genetic integrity of congenic strains housed in a conventional colony is presented. Using a combination of 3 or 4 F1 immunizing cells, sera can be produced in each strain housed in the colony which will react in a complement-mediated cytotoxicity assay with peripheral lymphocytes from most if not all other strains in the colony. Routine screening of breeding stock with these strain restricted typing sera (SRTS) permits the sensitive detection of genetic contamination between the stocks maintained. These sera detect H-2, minor histocompatibility differences, and other cell surface differentiation antigens, and can also be used to identify the nature of a contaminant when isolated. In addition, when used within appropriate strain combinations, the sera can be useful in detecting antigenic determinants otherwise difficult to identify.

Patterns of T cell-accessory cell interaction in the generation of primary alloresponses in the pig.

Partially inbred, MHC-homozygous miniature swine provide a unique model for the study of organ transplantation and the induction of tolerance in large animals. Models of both vascularized solid organ transplantation and bone marrow transplantation have previously been established. The availability of monoclonal antibodies reactive with porcine leukocyte subset antigens now makes possible studies of the cellular immunology in this species, affording the opportunity to examine mechanisms of transplant tolerance and graft rejection in increasing detail. Using such antibodies and peripheral blood leukocytes from pigs of recombinant MHC haplotypes, we have examined porcine T cell-accessory cell interactions in vitro with attention to T cell subsets and the class of MHC alloantigen stimulation. Primary allospecific MLR and CML cultures were studied after depletion of accessory cells from responder and/or stimulator populations. Although class II MHC antigens were expressed on the majority of porcine T cells before and after depletion, these cells were insufficient for antigen presentation, since there was an absolute requirement for ACs in the generation of primary alloresponses. Proliferative and CTL alloresponses could be generated provided that ACs of either stimulator or responder type were present. Selective depletion of CD4+ T cells from the responder population demonstrated: (a) that the interaction mediated by self ACs was CD4-dependent; (b) that two pathways exist for interaction involving allogeneic ACs; and (c) that the interaction involving allogeneic class II is CD4-dependent, while that with allogeneic class I is not.

Highly disparate xenogeneic skin graft tolerance induction by fetal pig thymus in thymectomized mice: Conditioning requirements and the role of coimplantation of fetal pig liver.

BACKGROUND: Highly disparate xenogeneic pig skin graft tolerance and efficient repopulation of mouse CD4+ T cells are achieved in thymectomized (ATX) B6 mice that receive T cell and natural killer (NK) cell depletion by injection of a mixture of monoclonal antibodies (mAbs) (GK1.5, 2.43, 30-H12, and PK136) on days -6, -1, +7, and +14 and 3 Gy total body irradiation (TBI) followed by implantation of fetal pig thymus/liver (FP THY/LIV) grafts on day 0. The requirements for each treatment in this model to achieve pig skin graft tolerance have not previously been defined. Therefore, we performed a series of experiments to address the role of each treatment in achieving maximal skin graft tolerance. METHODS: Peripheral mouse CD4+ T-cell repopulation and pig skin graft survival were followed in this pig-to-mouse model in which recipient B6 mice were treated with modified regimens that omitted thymectomy, 3 Gy TBI, anti-Thy1.2, and anti-NK1.1 mAbs, injection of a mixture of mAbs on day +14, or coimplantation of FP LIV, respectively. RESULTS: Prolongation but not permanent survival of donor MHC-matched pig skin grafts was observed in euthymic B6 mice that received T and NK cell depletion, 3 Gy TBI, and 7 Gy thymic irradiation and FP THY/LIV in the mediastinum, suggesting that full xenogeneic tolerance was not achieved in euthymic mice. However, after grafting FP THY alone to ATX B6 mice treated either with the "standard" regimen, or with a conditioning regimen that omitted all components of the conditioning regimen except treatment with anti-CD4 and anti-CD8 mAbs, efficient peripheral repopulation of mouse CD4+ T cells and long-term donor MHC-matched pig skin graft acceptance were observed. CONCLUSIONS: Highly disparate xenogeneic pig skin graft tolerance can be achieved by grafting FP THY alone in anti-CD4 and anti-CD8 mAb-treated ATX B6 mice, but not in euthymic B6 mice. Additional treatment of ATX recipient mice with anti-Thy1.2 and NK1.1 mAbs and 3 Gy TBI is not essential for donor pig skin graft tolerance induction.

Mechanism of tolerance to class I-mismatched renal allografts in miniature swine: regulation of interleukin-2 receptor alpha-chain expression on CD8 peripheral blood lymphocytes of tolerant animals.

BACKGROUND: Donor-specific tolerance to renal allografts in miniature swine is uniformly induced across a two-haplotype class I plus minor histocompatibility antigen disparity by a 12-day course of cyclosporine. Recent studies have demonstrated that the thymus is essential for rapid and stable tolerance induction, because either prior thymectomy or a series of thymic biopsies induce a spontaneously reversible rejection crisis after the 12-day course of cyclosporine. The present study examined the peripheral cellular mechanisms of tolerance by analyzing cytotoxic effector pathways in peripheral blood lymphocytes (PBL) of tolerant animals. METHODS: The phenotype and cytotoxic T lymphocyte response of alloantigen-activated PBL cultures using cells from a series of tolerant animals with stable renal function (no thymic manipulation), or during a rejection crisis (induced by thymic biopsies), were studied. The in vitro findings were correlated with the in vivo clinical course of experimental animals. RESULTS: The data demonstrated that in vivo and in vitro tolerance was associated with a specific deficiency of interleukin-2 receptor (IL-2R) alpha-chain up-regulation on CD8 single-positive (SP) T cells expressing high levels of CD8 (CD8high) when PBL from tolerant animals are stimulated with donor class I alloantigen. Stimulation by third party class I alloantigen, or by donor antigen during a rejection crisis, produced efficient cytotoxic T lymphocyte responses and expression of IL-2Ralpha on CD8high SP cells. CONCLUSION: Antigen-specific regulation of the IL-2Ralpha expression on CD8high SP PBL is a principal event associated with and potentially involved in the mechanism of tolerance in this preclinical large animal model.

The induction of specific pig skin graft tolerance by grafting with neonatal pig thymus in thymectomized mice.

BACKGROUND: Xenogeneic donor-specific tolerance can be induced by transplanting fetal pig thymus and liver tissue (FP THY/LIV) to thymectomized (ATX), T/NK cell-depleted mice. By using neonatal pig tissue, we hoped to overcome two obstacles that arise with the use of fetal pig tissue: (1) the inability to keep fetal pigs alive after harvesting their thymic tissue, resulting in unavailability of their skin or other organs for grafting; and (2) the limited fetal thymic tissue yield, making application to large animals and humans more difficult. METHODS: Neonatal pig thymus tissue (NP THY) was grafted into ATX, T/NK cell-depleted, 3Gy whole body-irradiated, originally immunocompetent B6 mice to evaluate the ability of NP THY to reconstitute mouse CD4+ T cells and to induce xenogeneic tolerance to donor pig skin grafts. RESULTS: Repopulation of mouse CD4+ T cells in the peripheral tissues was observed in T/NK cell-depleted, ATX B6 mice that received NP THY with or without neonatal pig spleen (NP SPL), but not in those receiving NP SPL alone, indicating that pig thymus grafting was necessary and sufficient for mouse T cell recovery. Seven of nine NP THY/SPL-grafted ATX mice and two of six NP THY-grafted ATX mice that reconstituted >5% CD4+ cells in PBL accepted donor pig skin long-term without lymphocyte infiltration, whereas they rejected allogeneic BALB/c skin and third party pig skin grafts as rapidly as euthymic mice. CONCLUSIONS: NP THY can support the development of mouse CD4+ T cells that are functional and specifically tolerant to donor pig antigens in ATX, T/NK cell-depleted, 3 Gy whole body-irradiated, originally immunocompetent B6 mice. Additional grafting of NP SPL with NP THY improves the efficiency of tolerance induction in this model.

Tolerance to class I-disparate renal allografts in miniature swine. Maintenance of tolerance despite induction of specific antidonor CTL responses.

Miniature swine that become tolerant to renal allografts across an MHC class I barrier following a short course of cyclosporine are unresponsive to donor class I antigens in cell-mediated lymphocytotoxicity. However, skin grafts bearing donor class I plus third-party class II antigens are promptly rejected, and the animals then develop marked cell-mediated lymphocytoxic reactivity to donor class I antigens in vitro, but do not reject the kidney transplants. We show here that CTL generation is directed toward the same donor class I antigens as are expressed by the kidney donor, and is not the result of recognition in vitro of the tolerated class I antigen plus peptides of minor antigens shared between the skin graft donor and the stimulator/target cells. We also show that detection by CTLs of peptides expressed by skin but not by kidney is also not a sufficient explantation of the results, since the survival of skin grafts from the kidney donor is also prolonged, even after precursor CTL can be detected in vitro. The data are most consistent with suppression in vivo in tolerant animals of the helper pathways necessary for activation of precursor CTLs. Differences in patterns of cytokine expression by graft infiltrating cells may provide a mechanism for local suppression of help in this model. Finally, we have examined antibody production after sensitizing by skin grafts in long-term tolerant animals and have found that anti-donor class I antibodies are not produced, even though the same animals produce both anti-class II and anti-third-party class I antibodies.

The importance of nonimmune factors in reconstitution by discordant xenogeneic hematopoietic cells.

Bone marrow transplantation has been shown to induce donor-specific tolerance in rodent models. This approach could potentially be applied to xenotransplantation across discordant species barriers. To evaluate host factors resisting hematopoietic cell engraftment, we have developed two model systems utilizing the combination of swine into severe combined immunodeficient (SCID) mice. SCID mice lack functional B and T lymphocytes, and can therefore be used to evaluate nonimmune factors resisting marrow engraftment, and for adoptive transfer studies to test the role of immune cells and antibodies. First we transplanted swine bone marrow cells into SCID mice conditioned with whole-body irradiation (4 Gy). For nine weeks following the intravenous administration of 10(8) swine bone marrow cells, up to 3.8% of peripheral blood leukocytes were of swine origin, as determined by flow cytometry (FCM). These cells were all of the myeloid lineage. Swine IgG was also detectable in the serum for up to 14 weeks. The bone marrow of the reconstituted mice contained low percentages of swine myeloid cells, and swine myeloid progenitors could be detected for up to 20 weeks after bone marrow transplantation. In a second model, we grafted thymus and liver tissue from 45-69-day-old swine fetuses under the kidney capsule of 4 Gy-irradiated SCID mice. A suspension containing 10(8) swine fetal liver cells (FLC) was also administered i.p. Long-term repopulation with swine T cells was observed, with up to 1.5% swine T cells detected in the WBC, peritoneum, and spleen for at least 5.5 months postgrafting. These T cells expressed either CD4 or CD8, whereas up to 17.6% of cells in the thymic grafts expressed both CD4 and CD8. The i.p. FLC suspension was required for optimal long-term graft maintenance. Our studies show that (1) low level myeloid and B lymphocyte reconstitution can be achieved by transferring adult swine BMC to irradiated SCID recipients; (2) swine myeloid progenitors were detectable long-term in BMC of these mice, suggesting that stem cell engraftment was achieved; and (3) T cell reconstitution of SCID mice by swine progenitors requires cotransplantation of a swine stromal environment, as is provided by fetal swine thymus/liver grafts. We conclude that nonimmune factors such as those provided by species-specific stromal environments are important for reconstitution of some lineages by discordant hematopoietic stem cells.

Long-term acceptance of primarily vascularized renal allografts in miniature swine. Systemic tolerance versus graft adaptation.

We have previously demonstrated that tolerance to two-haplotype class I-mismatched renal allografts can be induced uniformly by a short course of cyclosporine. We report here that following transplant nephrectomy, 8 such long-term acceptor animals all accepted a second renal transplant MHC matched to the original donor without additional immunosuppression. These results indicate that the mechanism of tolerance to primarily vascularized renal allografts involves modification of the host's immune system by the first transplant. To assess the possibility that "graft adaptation" is also involved in the maintenance of tolerance, we retransplanted class I-disparate kidneys from tolerant animals into naive recipients MHC matched to the original recipient. Three of 4 such transplants were rejected acutely, while one animal demonstrated a markedly prolonged survival, but also eventually rejected. These results, therefore, demonstrate that: (1) graft adaptation is not required in order to maintain tolerance; (2) graft acceptance involves induction of systemic tolerance; and (3) graft adaptation may participate in kidney graft prolongation but is not sufficient to transfer tolerance to a secondary host.

Enhanced CD4 reconstitution by grafting neonatal porcine tissue in alternative locations is associated with donor-specific tolerance and suppression of preexisting xenoreactive T cells.

BACKGROUND: Donor-specific xenograft tolerance can be achieved by grafting fetal porcine thymus tissue to thymectomized (ATX) mice treated with natural killer (NK) and T-cell-depleting monoclonal antibodies plus 3 Gy of total body irradiation (TBI). Grafting of neonatal, instead of fetal, thymus, along with neonatal pig spleen, leads to a lower level of mouse CD4 cell reconstitution, with less reliable tolerance induction. For a number of reasons, it would be advantageous to use neonatal rather than fetal pigs as donors. We therefore investigated the possibility that grafting larger amounts of neonatal porcine thymus tissue to different sites could allow improved outcomes to be achieved. MATERIALS AND METHODS: Multiple or single fragments of neonatal porcine thymus tissue were grafted with a splenic fragment to different sites (mediastinum, mesentery, and kidney capsule) of ATX B6 mice treated with T- and NK-cell-depleting antibodies and 3Gy TBI. Mice also received an intraperitoneal injection containing 1 x 10(7) donor splenocytes. Donor-specific skin graft tolerance was evaluated, and CD4 reconstitution and mouse anti-donor xenoantibodies were followed by flow cytometry. RESULTS: Peripheral repopulation of CD4+ cells occurred by 7 weeks after transplantation in mice grafted with four fragments of neonatal porcine tissue in either the mediastinum or the mesentery, but not in mice grafted under both kidney capsules with the same amount of tissue. The level of CD4 reconstitution correlated with skin graft tolerance and an absence of induced anti-donor xenoantibodies. Seventy-five percent of mice with >20% of CD4+ cells among peripheral blood lymphocytes (PBL) by 13 weeks posttransplantation accepted donor porcine skin, while rejecting either non-donor neonatal porcine or mouse BALB/c skin allografts. In contrast, only 29% of grafted mice with <20% CD4+ cells in the peripheral blood at 13 weeks accepted donor porcine skin. Grafted mice with poor reconstitution showed either low or high levels of anti-pig xenoantibodies of the IgM, IgG1, and IgG2a isotypes. Grafted mice with >20% CD4+ cells all had low levels of anti-pig xenoantibodies of these isotypes and displayed mixed lymphocyte reaction (MLR) tolerance to donor pig major histocompatibility complex (MHC), with responsiveness to allogeneic mouse stimulators. CONCLUSION: Grafting neonatal porcine thymus into either the mediastinum or mesentery provides earlier and more efficient reconstitution of the CD4 compartment than does grafting under the kidney capsule. Good CD4 reconstitution was associated with optimal donor-specific skin graft tolerance and avoidance of the anti-donor xenoantibody responses observed in mice with poor CD4 reconstitution. These results also suggest that there is a suppressive component to the porcine xenograft tolerance induced with this approach.

Cardiac allograft vasculopathy is abrogated by anti-CD8 monoclonal antibody therapy.

BACKGROUND: Cardiac allograft vasculopathy, a diffuse and accelerated form of arteriosclerosis, is a major cause of graft loss or heart transplant recipient death after the first transplant year. This study examined the effects of depleting host CD8 + T lymphocytes on the development of cardiac allograft vasculopathy in miniature swine. METHODS: Cardiac allografts were heterotopically transplanted across a major histocompatibility complex class I barrier in partially inbred miniature swine and monitored for rejection by serial biopsies, electrocardiograms, and echocardiograms. Four control animals received cyclosporine on postoperative days 0 to 11. Another four miniswine were given 14.5 mg/kg of 76-2-11 (a mouse anti-swine CD8 monoclonal antibody) on postoperative day 0, in addition to a 12-day course of cyclosporine. Host CD8+ T cells and circulating 76-2-11 monoclonal antibodies were monitored by flow cytometry. RESULTS: As compared with cyclosporine-treated control animals, swine receiving 76-2-11 demonstrated near-complete depletion of peripheral CD8+ T cells by postoperative day 2, which persisted for 14 to 18 days. Mean allograft survival of the antibody-treated group and the control group was not statistically different (33 days versus 39 days, respectively) and both groups demonstrated severe interstitial rejection at necropsy. Control animals demonstrated florid intimal thickening of large and small arteries at necropsy. However, swine treated with 76-2-11 showed no intimal proliferation. CONCLUSIONS: Depletion of host CD8+ T cells prevents or delays the development of intimal proliferation in miniature swine. CD8+ lymphocytes play an important role in the early development of cardiac allograft vasculopathy in large animals.