Yasushi Fuchimoto

Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model

Bone marrow transplantation (BMT) has considerable potential for the treatment of malignancies, hemoglobinopathies, and autoimmune diseases, as well as the induction of transplantation allograft tolerance. Toxicities associated with standard preparative regimens for bone marrow transplantation, however, make this approach unacceptable for all but the most severe of these clinical situations. Here, we demonstrate that stable mixed hematopoietic cell chimerism and donor-specific tolerance can be established in miniature swine, using a relatively mild, non-myeloablative preparative regimen. We conditioned recipient swine with whole-body and thymic irradiation, and we depleted their T-cells by CD3 immunotoxin-treatment. Infusion of either bone marrow cells or cytokine-mobilized peripheral blood stem cells from leukocyte antigen-matched animals resulted in stable mixed chimerism, as detected by flow cytometry in the peripheral blood, thymus, and bone marrow, without any clinical evidence of graft-versus-host disease (GvHD). Long-term acceptance of donor skin and consistent rejection of third-party skin indicated that the recipients had developed donor-specific tolerance.

Mixed chimerism and tolerance without whole body irradiation in a large animal model

Mixed hematopoietic chimerism may provide a treatment for patients with nonmalignant hematologic diseases, and may tolerize patients to organ allografts without requiring chronic immunosuppression. However, the toxicity of the usual conditioning regimens has limited the clinical applicability of this approach. These regimens generally include some level of whole body irradiation (WBI), which is thought to facilitate engraftment either by making room for donor hematopoietic stem cells or by providing sufficient host immunosuppression to enable donor cells to engraft. Here, we have established mixed chimerism across both minor and major histocompatibility barriers in swine, by using high doses of peripheral blood stem cells in the absence of WBI. After mixed chimerism was established, swine leukocyte antigen-matched (SLA-matched) donor skin grafts were tolerated and maintained for a prolonged period, whereas third-party SLA-matched skin was rejected promptly. Donor-matched kidney allografts were also accepted without additional immunosuppression. Because of its low toxicity, this approach has potential for a wide range of clinical applications. Our data may indicate that niches for engrafting stem cells are filled by mass action and that WBI, which serves to empty some of these niches, can be omitted if the donor inoculum is sufficiently large and if adequate host T-cell depletion is achieved before transplant.

Porcine kidney and heart transplantation in baboons undergoing a tolerance induction regimen and antibody adsorption

BACKGROUNDnXenotransplantation would provide a solution to the current shortage of organs for transplantation. Our group has been successful in inducing tolerance in mice and monkey models of allogeneic transplantation. The present study attempts to extend the same tolerance-inducing regimen to a pig-to-baboon organ transplantation model.nnnMETHODSnNine baboons underwent a conditioning regimen (consisting of nonmyeloablative or myeloablative whole body and thymic irradiation, splenectomy, antithymocyte globulin, pharmacologic immunosuppression and porcine bone marrow transplantation [BMTx]), which has previously been demonstrated to induce donor-specific allograft tolerance in monkeys. In addition, immunoadsorption of anti-alphaGal antibody (Ab) was performed. Four of the nine baboons received pig kidney transplants (KTx), and one also underwent repeat transplantation with an SLA-matched kidney. Two received heterotopic pig heart transplants (HTx). Three baboons underwent conditioning without organ transplantation for long-term studies of natural Ab kinetics.nnnRESULTSnIn the three baboons that received the conditioning regimen without an organ transplant, immunoadsorption reduced Ab by approximately 90%, but recovery of Ab to pretreatment level or higher occurred within 7 days. In contrast, the level of Ab remained low after organ transplant. No Ab to pig antigens other than alphaGal was detected in any baboon before or after BMTx, KTx, or HTx. No graft succumbed to hyperacute rejection. KTx function began to deteriorate within 3-6 days, with oliguria and hematuria progressing to anuria, and the kidneys were excised after 3, 6, 9, 11, and 14 days, respectively. One HTx ceased functioning at 8 days; the second baboon died with a contracting HTx at 15 days. Features of coagulopathy and thrombocytopenia developed in all six transplanted baboons (high D-dimer, prolonged prothrombin time and partial thromboplastin time, and falling fibrinogen) resulting in serious bleeding complications in two baboons, one of which died on day 9. Donor organs showed progressive acute humoral rejection with deposits of IgM, IgG, and complement; a focal mononuclear cellular infiltrate was also observed. The ureter was the earliest structure of the KTx affected by rejection, with progression to necrosis.nnnCONCLUSIONSnThis conditioning regimen prevented hyperacute rejection but was ineffective in preventing the return of Ab, which was associated with the development of acute humoral rejection with features of coagulopathy. No baboon developed anti-pig Ab other than alphaGal Ab. Further modifications of the protocol directed toward suppression of production of Ab are required to successfully induce tolerance to pig organs in baboons.

Skin-specific alloantigens in miniature swine

BACKGROUNDnThe acceptance of skin allografts has historically been among the most challenging problems in the field of transplantation, attributed, at least in part, to the existence of antigens expressed by skin but not by other tissues. Many studies have suggested the existence of skin-specific antigens in rodents, but data in large-animal models are more limited.nnnMETHODSnWe have recently developed protocols for attaining stable mixed hematopoietic chimerism in miniature swine, using MHC-matched donors and recipients. We have now assessed tolerance to donor-derived skin and cardiac allografts in these chimeric animals.nnnRESULTSnSkin-graft rejection was seen in four of six animals receiving skin grafts taken from the respective hematopoietic donors. In the other two animals, donor-derived skin grafts survived indefinitely. No cardiac-allograft rejection was observed in mixed-chimeric animals that received heart transplants from their hematopoietic donors, even in animals that had already rejected skin allografts from the same donors. In all animals assessed, in vitro hyporesponsiveness to donor hematopoietic cells persisted.nnnCONCLUSIONnThese findings support the concept that skin expresses immunogenic alloantigens that either are not expressed or are not immunogenic in cardiac or hematopoietic tissue.

Mixed hematopoietic chimerism induces long-term tolerance to cardiac allografts in miniature swine

BACKGROUNDnTolerance to cardiac allografts has not been achieved in large animals using methods that are readily applicable to human recipients. We investigated the effects of mixed hematopoietic chimerism on cardiac allograft survival and chronic rejection in miniature swinennnMETHODSnRecipients were T-cell depleted using a porcine CD3 immunotoxin, and each received either of two nonmyeloablative preparative regimens previously demonstrated to permit the establishment of stable mixed hematopoietic chimerism across MHC-matched, minor antigen-mismatched histocompatibility barriers. Five to 12 months after the chimerism was induced, hearts from the original cell donors were heterotopically transplanted into the stable mixed chimeras.nnnRESULTSnCardiac allografts transplanted into untreated recipients across similar minor antigen barriers were rejected within 44 days (within 21, 28, 35, 39, 44 days among individual study subjects). In contrast, hearts transplanted into the mixed chimeras were all accepted long term ( > 153, > 225, > 286, > 362 days) without immunosuppressive drugs and developed minimal vasculopathy.nnnCONCLUSIONSnMixed hematopoietic chimerism, established in miniature swine using clinically relevant, non-myeloablative conditioning regimens, permits long-term cardiac allograft survival without chronic immunosuppressive therapy, significant vasculopathy, or graft-versus-host disease.

Variable relationship between chimerism and tolerance after hematopoietic cell transplantation without myelosuppressive conditioning

Background. We have previously described a mixed chimerism protocol that avoids myelosuppressive conditioning and permits hematopoietic cell transplantation across MHC barriers without the need for whole body irradiation in miniature swine. Here, we report our current experience including animals conditioned without thymic irradiation, and we attempt to define the relationship between long-term chimerism and stable tolerance in these animals. Methods. Recipient swine received in vivo T-cell depletion, with or without thymic irradiation on day −2. Cyclosporine was administered for 30 to 60 days beginning on day −1. A total of 1 to 2×1010/kg cytokine-mobilized donor hematopoietic cells were infused during 3 days. Chimerism was determined by flow cytometry. In vitro tolerance assays and donor-matched kidney transplantation were performed after cessation of cyclosporine. Results. Most recipients maintained stable chimerism (26 of 35) and were specifically tolerant to donor-matched cells in vitro regardless of whether they received thymic irradiation. Donor-matched kidney transplantations performed in chimeric animals without in vitro antidonor immune responses were accepted without immunosuppression. Some animals developed in vitro evidence of antidonor MHC responsiveness despite the persistence of donor cells in the peripheral blood. Donor-matched kidney transplantations performed in the face of these responses were rejected. Conclusions. These data indicate that this nonmyelosuppressive protocol can induce stable chimerism and robust tolerance even in animals conditioned without thymic irradiation. However, the data also demonstrate that macrochimerism does not always correlate with tolerance. Lack of in vitro antidonor immune responses in chimeric animals is an important predictor of renal allograft acceptance in this model.

Cytokine-mobilized peripheral blood progenitor cells for allogeneic reconstitution of miniature swine.

BACKGROUNDnBecause of the relative ease of acquisition, increased yield, and improved engraftment characteristics, mobilized peripheral blood progenitor (stem) cells (PBSCs) have recently become the preferred source for hematopoietic stem cell transplantation. In our laboratory, procurement of a megadose of PBSCs is necessary for on-going studies evaluating non-myelosuppressive transplant regimens for the induction of mixed chimerism and allograft tolerance. To exploit hematopoietic growth factor synergy, we have sought to combine growth factors with proven utility to improve PBSC mobilization and maximize our PBSC procurement through an automated collection procedure.nnnMETHODSnMobilization characteristics of PBSCs were determined in 2-5-month-old miniature swine. Animals received either swine recombinant stem cell factor (pSCF, 100 microg/kg) and swine recombinant interleukin 3 (pIL-3, 100 microg/kg), administered intramuscularly for 8 days, or pSCF, pIL-3, and human recombinant granulocyte-colony stimulating factor (hG-CSF), at 10 microg/kg. Leukapheresis was performed beginning on day 5 of cytokine treatment and continued daily for 3 days.nnnRESULTSnCollection of PBSCs from cytokine-mobilized animals via an automated leukapheresis procedure demonstrated a 10-fold increase in the number of total nucleated cells (TNC) (20-30 x 10(10) TNC) compared to bone marrow harvesting (2-3 x 10(10) total TNC). A more rapid rise in white blood cells (WBCs) was seen after administration of all three cytokines compared to pSCF and pIL-3 alone. An increase in colony-forming unit granulocyte-macrophage frequency measured daily from peripheral blood during cytokine treatment, was seen with the addition of hG-CSF to pSCF/pIL-3 correlating well with the rise in WBCs. Similarly, the addition of hG-CSF demonstrated a notable increase in the median progenitor cell yield from the 3-day leukapheresis procedure. Cytokine-mobilized PBSCs were capable of hematopoietic reconstitution. PBSCs mobilized with pSCF/pIL-3 were infused into an SLA-matched recipient conditioned with cyclophosphamide (50 mg/kg) and total body irradiation 1150 cGy. Neutrophil and platelet engraftment occurred on days 5 and 7, respectively, with minimal evidence of graft-versus-host disease. Complete donor chimerism has been demonstrated 331 days after transplant.nnnCONCLUSIONSnOur preliminary results show that in this well-defined miniature swine model, recombinant swine cytokine combinations (pSCF, pIL-3 with or without hG-CSF) successfully mobilize a high yield of progenitor cells for allogeneic transplantation. Furthermore, these cytokine-mobilized PBSCs demonstrate the potential to reconstitute hematopoiesis and provide long-term engraftment in miniature swine.

Peripheral regulation of graft-versus-host alloreactivity in mixed chimeric miniature swine.

Background. Despite the presence of circulating donor-derived T cells during the induction of mixed chimerism across MHC barriers in miniature swine, severe graft-versus-host disease was avoided in the majority of animals. In this study, we investigated the possible roles of recipient and donor lymphoid populations in the regulation of donor-anti-recipient alloreactivity. Methods. Mixed chimerism across a full MHC-mismatch barrier was established in miniature swine using a high-dose allogeneic peripheral blood stem cell protocol. Peripheral blood mononuclear cells from mixed chimeric swine were co-cultured with naïve donor-matched responders and naïve recipient-matched stimulators in mixed lymphocyte reactions. Results. Peripheral blood mononuclear cells from mixed chimeras inhibited donor-anti-recipient proliferation. This suppression was radioresistant to 25 Gy. Suppression of donor-anti-recipient alloreactivity was not observed in mixed lymphocyte co-cultures when donor-derived cells were added in the absence of recipient-derived cells. Conclusions. These results suggest an association between the presence of an active and relatively radioresistant cell population, demonstrable in vitro, and the regulation of graft-versus-host disease across MHC barriers in mixed chimeric miniature swine.

Neuropathy in miniature swine after administration of the mutant diphtheria toxin-based immunotoxin, pCD3-CRM9.

BACKGROUNDnEffective in vivo T-cell depletion is a critical component of many transplantation tolerance protocols. We have previously demonstrated T-cell depletion in miniature swine using a CRM9-based CD3-immunotoxin, pCD3-CRM9. CRM9 is a mutant form of diphtheria toxin (DT) that binds less efficiently than wild-type DT to the DT receptor (proHB-EGF) of primates. In this report, we describe and characterize the dose-dependent neurotoxicity associated with CRM9-based immunotoxin administration in swine.nnnMETHODSnMiniature swine were treated with varying doses of pCD3-CRM9 followed by daily monitoring for symptoms of neuropathy, including limb weakness, paresis, sluggishness, and/or respiratory distress. Animals demonstrating severe respiratory distress were euthanized and peripheral nerve, spinal cord, and skeletal muscle tissue samples were obtained at autopsy for microscopic examination. Unconjugated CRM9 was administered to one animal to define its toxicity independent of the effects of T-cell depletion.nnnRESULTSnExcellent T-cell depletion was obtained using doses of pCD3-CRM9 greater than 0.1 mg/kg. However, neurotoxicity was observed at these doses, as manifested by transient muscle weakness or paresis, which in some cases progressed to respiratory failure and death. Dorsal root ganglia samples revealed pathological changes typical of diphtheritic polyneuropathy. The animal receiving unconjugated CRM9 exhibited the same neurotoxic side effects as those receiving the pCD3-CRM9 conjugate.nnnCONCLUSIONSnAdministration of pCD3-CRM9 immunotoxin provides excellent T-cell depletion in miniature swine but is associated with significant dose-dependent neurotoxicity. A possible reason for CRM9-associated neurotoxicity in swine, but not primates, is suggested on the basis of a known amino acid difference in the exodomain of the DT receptor (proHB-EGF) of swine compared with that of primates.