S. Gojo

High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response

BACKGROUND In pig-to-primate organ transplantation, hyperacute rejection can be prevented, but the organ is rejected within days by acute vascular rejection, in which induced high-affinity anti-Gal alpha1-3Gal (alphaGal) IgG and possibly antibodies directed against new porcine (non-alphaGal) antigenic determinants are considered to play a major role. We have explored the role of an anti-CD40L monoclonal antibody in modifying the humoral response to porcine hematopoietic cells in baboons pretreated with a nonmyeloablative regimen. METHODS Porcine peripheral blood mobilized progenitor cells obtained by leukapheresis from both major histocompatibility complex-inbred miniature swine (n=7) and human decay-accelerating factor pigs (n=3) were transplanted into baboons. Group 1 baboons (n=3) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with ATG, complement depletion with cobra venom factor, short courses of cyclosporine, mycophenolate mofetil, porcine hematopoietic growth factors, and anti-alphaGal antibody depletion by immunoadsorption before transplantation of high doses (2-4 x 10(10)/cells/kg) of peripheral blood mobilized progenitor cells. In group 2 (n=5), cyclosporine was replaced by eight doses of anti-CD40L monoclonal antibodies over 14 days. The group 3 baboons (n=2) received the group 1 regimen plus 2 doses of anti-CD40L monoclonal antibodies (on days 0 and 2). RESULTS In group 1, sensitization to alphaGal (with increases in IgM and IgG of 3- to 6-fold and 100-fold, respectively) and the development of antibodies to new non-alphaGal porcine antigens occurred within 20 days. In group 2, no sensitization to alphaGal or non-alphaGal determinants was seen, but alphaGal-reactive antibodies did return to their pre- peripheral blood mobilized progenitor cells transplant levels. In group 3, attenuated sensitization to alphaGal antigens was seen after cessation of cyclosporine and mycophenolate mofetil therapy at 30 days (IgM 4-fold, IgG 8-30-fold), but no antibodies developed against new porcine determinants. In no baboon did anti-CD40L monoclonal antibodies prevent sensitization to its own murine antigens. CONCLUSIONS We believe these studies are the first to consistently demonstrate prevention of a secondary humoral response after cell or organ transplantation in a pig-to-primate model. The development of sensitization to the murine elements of the anti-CD40L monoclonal antibodies suggests that nonresponsiveness to cell membrane-bound antigen (e.g., alphaGal) is a specific phenomenon and not a general manifestation of immunological unresponsiveness. T cell costimulatory blockade may facilitate induction of mixed hematopoietic chimerism and, consequently, of tolerance to pig organs and tissues.

Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates.

Background. Efforts to achieve tolerance to transplanted pig organs in nonhuman primates by the induction of a state of mixed hematopoietic chimerism have been associated with disorders of coagulation and thrombosis. Activation of recipient vascular endothelium and platelets by porcine hematopoietic cells and/or activation of donor organ vascular endothelium and/or molecular differences between the species may play roles. Irradiation or drug therapy could possibly potentiate endothelial cell activation and/or injury. Methods. We have investigated parameters of coagulation and platelet activation in nonhuman primates after (1) a regimen aimed at inducing mixed hematopoietic chimerism and tolerance (TIR that included total body irradiation, T cell depletion, and splenectomy; (2) pig bone marrow or pig peripheral blood mobilized progenitor cell transplantation (PCTx); and/or (3) pig organ transplantation (POTx). Five experimental groups were studied. Baboons were the recipient subjects in all groups except Group 1. Gp 1 Cynomolgus monkeys (n=6) underwent TIR + allotransplantation of hematopoietic cells and a kidney or heart or TIR + concordant xenotransplantation (using baboons as donors) of cells and a kidney; Gp 2 Baboons (n=4) underwent TIR with or without (±) autologous hematopoietic cell infusion; Gp 3 (n=12) PCTx±TIR; Gp 4 (n=5) POTx±TIR; Gp 5 (n=4) TIR + PCTx + POTx. Platelet counts, with plasma prothrombin time, partial thromboplastin time, fibrinogen levels, fibrin split products and/or D-dimer were measured. Results. In the absence of a discordant (porcine) cellular or organ transplant (Groups 1 and 2), TIR resulted in transient thrombocytopenia only, in keeping with bone marrow depression from irradiation. PCTx alone (Group 3) was associated with the rapid development of a thrombotic thrombocytopenic (TTP)-like microangiopathic state, that persisted longer when PCTx was combined with TIR. POTx (±TIR) (Group 4) was associated with a gradual fall (over several days) in platelet counts and fibrinogen with disseminated intravascular coagulation (DIC); after graft excision, the DIC generally resolved. When TIR, PCTx and POTx were combined (Group 5), an initial TTP-like state was superseded by a consumptive picture of DIC within the first week, necessitating graft removal. Conclusions. Both PCTx and POTx lead to profound alterations in hemostasis and coagulation parameters that must be overcome if discordant xenotransplantation of hematopoietic cells and organs is to be fully successful. Disordered thromboregulation could exacerbate vascular damage and potentiate activation of coagulation pathways after exposure to xenogeneic cells or a vascularized xenograft.

TRANSFER OF SWINE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II GENES INTO AUTOLOGOUS BONE MARROW CELLS OF BABOONS FOR THE INDUCTION OF TOLERANCE ACROSS XENOGENEIC BARRIERS

BACKGROUND The present study examined the potential role of gene therapy in the induction of tolerance to anti-porcine major histocompatibility complex (SLA) class II-mediated responses after porcine renal or skin xenografts. METHODS Baboons were treated with a non-myeloablative or a myeloablative preparative regimen before bone marrow transplantation with autologous bone marrow cells retrovirally transduced to express both SLA class II DR and neomycin phosphotransferase (NeoR) genes, or the NeoR gene alone. Four months or more after bone marrow transplantation, the immunological response to a porcine kidney or skin xenograft was examined. Both the renal and skin xenografts were SLA DR-matched to the transgene, and recipients were conditioned by combinations of complement inhibitors, adsorption of natural antibodies, immunosuppressive therapy, and splenectomy. RESULTS Although the long-term presence of the SLA transgene was detected in the peripheral blood and/or bone marrow cells of all baboons, the transcription of the transgene was transient. Autopsy tissues were available from one animal and demonstrated expression of the SLA DR transgene in lymphohematopoietic tissues. After kidney and skin transplantation, xenografts were rejected after 8-22 days. Long-term follow-up of control animals demonstrated that high levels of induced IgG antibodies to new non-alphaGal epitopes developed after organ rejection. In contrast, induced non-alphaGal IgG antibody responses were minimal in the SLA DR-transduced baboons. CONCLUSIONS Transfer and expression of xenogeneic class II DR transgenes can be achieved in baboons. This therapy may prevent late T cell-dependent responses to porcine xenografts, which include induced non-alphaGal IgG antibody responses.

Plasma perfusion by apheresis through a Gal immunoaffinity column successfully depletes anti-Gal antibody: experience with 320 aphereses in baboons.

Abstract: Background: Anti‐Galα1–3Gal (Gal) antibodies (Gal Ab) contribute to the rejection of porcine organs transplanted into primates. Extracorporeal immunoadsorption (EIA) has been developed to eliminate Gal Ab from the circulation.

Porcine hematopoietic cell xenotransplantation in nonhuman primates is complicated by thrombotic microangiopathy.

Thrombotic microangiopathy (TM) is a serious complication of bone marrow transplantation (BMT) that resembles thrombotic thrombocytopenic purpura (TTP). In attempting to achieve hematopoietic cell chimerism in the pig-to-baboon model, we have observed TM following infusion of high doses (>1010 cells/kg) of porcine peripheral blood mobilized progenitor cells (PBPC) into baboons. We performed investigations to analyze the pathobiology of this TM and to test therapeutic interventions to ameliorate it. PBPC were obtained by leukapheresis of cytokine-stimulated swine. The initial observations were made in two baboons that underwent a non-myeloablative regimen (NMR) prior to PBPC transplantation (TX) (group 1). We then studied three experimental groups. Group 2 (n = 2) received NMR without PBPC TX. Group 3 (n = 2) received PBPC TX alone. Group 4 (n = 6) received NMR + PBPC TX combined with prostacyclin, low-dose heparin, methylprednisolone, and cyclosporine was replaced by anti-CD40L mAb in five cases. Baboons in groups 1 and 3 developed severe thrombocytopenia (<10 000/mm3), intravascular hemolysis with schistocytosis (>10/high powered field (hpf)), increase in plasma lactate dehydrogenase (LDH) (2500–9000 U/l), transient neurologic changes, renal insufficiency, and purpura. Autopsy on two baboons confirmed extensive platelet thrombi in the microcirculation, and, similar to clinical BMT-associated TM/TTP, no unusually large vWF multimers or changes in vWF protease activity were observed in the plasma of baboons with TM. In group 2, self-limited thrombocytopenia occurred for 10–15 days following NMR. Group 4 baboons developed thrombocytopenia (<20 000/mm3) rarely requiring platelet transfusion, minimal schistocytosis (<3/hpf), minor increase in LDH (<1000 U/l), with no clinical sequelae. We conclude that high-dose porcine PBPC infusion into baboons induces a microangiopathic state with vWF biochemical parameters resembling clinical BMT-associated TM/TTP and that administration of antithrombotic and anti-inflammatory agents can ameliorate this complication. Bone Marrow Transplantation (2001) 27, 1227–1236.

Gene therapy and transplantation.

Advances in molecular biology and in techniques of gene transfer have resulted in the development of practical approaches to human gene therapy. Many applications are of relevance to manipulation of the immune system and have potential in organ and cell transplantation. For example, gene therapy approaches may facilitate the induction of immunological tolerance to a donor organ or protect it locally against the hosts immune response. Based on a comprehensive review of the world literature, examples of current research efforts in both allogeneic and xenogeneic transplantation are presented and discussed.

Xenogeneic thymus transplantation in a pig-to-baboon model.

Background. We have tested whether fetal porcine thymic tissue transplantation can lead to tolerance across a discordant (pig-to-baboon) xenogeneic barrier. Methods. Six baboons underwent a conditioning regimen with thymectomy, splenectomy, and anti-monkey CD3 antibody conjugated to a diphtheria toxin binding site mutant (FN18-CRM9). Porcine fetal or neonatal thymic tissue was transplanted into three baboons. Three control baboons received either no transplanted pig tissue (n=1) or adult pig lymph node (n=2). Cellular responses and skin xenografts were used to test for tolerance. Results. Experimental baboons: After T-cell depletion and thymic transplantation, recovery of thymus-dependent naïve-type CD4 cells (CD4/CD45RAhigh) and in vitro xenogeneic hyporesponsiveness were observed. No sensitization of alpha-galactosyl antibody responses was observed. The thymic grafts survived up to 48 days. Porcine skin xenografts were performed in two of these animals with survival of 22 and 24 days. Control baboons: Only two of these animals were completely T-cell depleted, and both failed to recover thymus-dependent T cells (CD4/CD45RAhigh). In one animal, general in vitro hyporesponsiveness was observed, with subsequent death from infection. The second animal demonstrated delayed recovery of T cells and prolonged general hyporesponsiveness in vitro. Neither animal demonstrated prolongation of porcine skin grafts compared with allografts (both rejected by day 13). Conclusions. Porcine thymic tissue is able to induce xenogeneic hyporesponsiveness. More efficient thymic engraftment may allow this approach to induce xenograft tolerance.

Anti-Galalpha1-3Gal antibody levels in organ transplant recipients receiving immunosuppressive therapy.

The effect of long-term pharmacologic immunosuppression (PI) on anti-Galalpha1-3Gal (alphaGal) antibody (Ab) levels has not been determined previously in humans. In this study, we measured alpha Gal Ab levels by ELISA in 14 healthy volunteers (controls) and in 70 patients with grafts (kidney, heart, liver) who had received different combinations of PI (including cyclosporine, tacrolimus, azathioprine, mycophenolate mofetil, and steroids) for >3 months. There was great variation in Gal IgM (<80-fold) and IgG (<160-fold). There was no difference in Gal IgM or Gal IgG between any one group and any other. In kidney patients with either high (mean 68%) or low (mean 6%) panel-reactive alloantibodies, there was no difference in alpha Gal Ab level or serum cytotoxicity to pig cells. In vitro immunoadsorption of alphaGal Ab from the serum did not change panel-reactive alloantibody positivity. Therapy with OKT3, a mouse product that might stimulate alphaGal Ab production, led to no significant change in patient Ab levels. We conclude that long-term (>3 months) PI does not reduce Gal Ab levels sufficiently to be of clinical value in xenotransplantation.

Gene therapy--its potential in surgery.

Advances in techniques have resulted in practical applications for gene therapy, which is becoming applicable for the treatment of human disease. This review outlines the advantages and disadvantages of the techniques available. Examples of research efforts in the treatment of diseases of relevance to the surgeon (cardiovascular diseases, cancer, wound healing, fracture repair, and in organ transplantation) are presented.

Differential expression of Galalpha1,3Gal epitopes on fetal and adult porcine hematopoietic cells.

Galα1‐3Gal (Gal) is the major epitope on pig tissues bound by human natural antibodies. Xenogeneic hematopoietic cell transplantation is being investigated to induce immunological tolerance to xenografts. We have investigated the level of Gal expression on pig hematopoietic cells. Cells were collected from pig fetal liver and bone marrow (BM), and also from adult BM and peripheral blood, before and after treatment with pig‐specific hematopoietic growth factors. Fluorescent activated cell sorting (FACS) analysis was performed with the M86 monoclonal antibody (specific for Gal), lineage markers, and biotinylated stem cell factor (SCF) to detect c‐kit expression. In fetal pig BM and liver, there was no significant difference in Gal expression between monocytes/macrophages (myeloid cells) and lymphocytes. In adult hematopoietic cells from all sources, Gal‐positive subpopulations in T cells showed weak expression of Gal, whereas B cells demonstrated higher expression, and myeloid cells showed highest expression. Adult BM and mobilized peripheral blood progenitor cells contained small populations with very low or negligible expression of Gal. A very small population of c‐kit‐positive cells, indicating progenitor cells, were Gal‐negative. The small Gal‐negative population that exists in progenitor cells might explain why some pig colony forming units (CFU) can be resistant to human serum.