J. S. Arn

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 xenotrans- plantation 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 en-graftment, 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 108 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

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.

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.

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.

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.

Utility of xenografts: Lack of correlation between PRA and natural antibodies to swine

Abstract: Among the patients that might potentially benefit from the availability of xenografts are those in kidney failure who demonstrate high levels of antibody reactivity to panels of typing lymphocytes. Such individuals with high PRA (panel reactive antibody) are unlikely to receive a renal allograft because they are highly sensitized to the vast majority of potential donors. In addition, all humans have demonstrable levels of natural antibodies reactive to distantly related species such as the pig. If there were a correlation between PRA and levels of natural antibodies, then such patients would also be at greater risk for hyperacute rejection of xenografts. We have therefore examined, in a blinded fashion, the porcine lymphocyte reactivity of sera from PRA positive donors. Subsets of the 105 sera tested were grouped by PRA level and analyzed for levels of natural antibodies detectable by a complement‐dependent cytotoxicity assay on porcine lymphocytes. There was no significant difference in the range of titers of natural antibodies between subsets. Thus, there was no demonstrable correlation between levels of PRA and levels of natural antibodies to porcine lymphocytes.

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.

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.

In vivo treatment with monoclonal antibodies directed against CD4 and CD8 antigens in miniature swine.

Two monoclonal antibodies (mAb) with specificities for mature T-cell subsets in miniature swine have been characterized previously. Antibody 74–12-4 recognizes the porcine CD4 accessory molecule and 76–2-11 is specific for the CD8 molecule. We have now examined the effects of in vivo administration of 74–12-4 and 76–2-11 on several parameters of transplantation immunity. No prolongation of class I disparate skin or kidney graft survival was observed in animals treated with either mAb alone or with a combination of both. In addition, in vivo treatment with these mAbs, in combination with subtherapeutic total body irradiation, failed to permit engraftment of allogeneic bone marrow. These therapeutic failures were thought likely to be a consequence of the fact that 74–12-4 coats, but does not deplete, CD4 cells in vivo. Because there are numerous anti-human mAbs that likewise fail to deplete in vivo, we have used 74–12-4 as a prototype to further manipulations aimed at achieving depletion. We attempted to eliminate 74–12-4-coated cells in two animals by subsequent administration of a hyperimmune pig anti-mouse Ig serum. In both such treated animals, administration of this serum produced surprisingly rapid clearance of 74–12-4 from the circulation and caused uncoating of CD4 cells, but no significant cell elimination was detected by flow cytometry. We have also prepared an anti-porcine-CD4 immunotoxin by conjugating 74–12-4 to the ribosome inhibitory plant toxin, pokeweed antiviral protein. This immunotoxin led to significant but not complete CD4 cell depletion from the peripheral blood in four of four treated animals. Further manipulations and possibly development of new anti-porcine CD4 mAbs may therefore be required to achieve complete depletion and successful mAb-mediated modification of transplantation responses.

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.