Henk Jan Schuurman

Heart transplantation in baboons using α1,3-galactosyltransferase gene-knockout pigs as donors: Initial experience

Hearts from α1,3-galactosyltransferase knockout pigs (GalT-KO, n = 8) were transplanted heterotopically into baboons using an anti-CD154 monoclonal antibody–based regimen. The elimination of the galactose-α1,3-galactose epitope prevented hyperacute rejection and extended survival of pig hearts in baboons for 2–6 months (median, 78 d); the predominant lesion associated with graft failure was a thrombotic microangiopathy, with resulting ischemic injury. There were no infectious complications directly related to the immunosuppressive regimen. The transplantation of hearts from GalT-KO pigs increased graft survival over previous studies.

Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates

Cell-based diabetes therapy requires an abundant cell source. Here, we report reversal of diabetes for more than 100 d in cynomolgus macaques after intraportal transplantation of cultured islets from genetically unmodified pigs without Gal-specific antibody manipulation. Immunotherapy with CD25-specific and CD154-specific monoclonal antibodies, FTY720 (or tacrolimus), everolimus and leflunomide suppressed indirect activation of T cells, elicitation of non-Gal pig-specific IgG antibody, intragraft expression of proinflammatory cytokines and invasion of infiltrating mononuclear cells into islets.

α1,3-galactosyltransferase gene-knockout pig heart transplantation in baboons with survival approaching 6 months

Background. The recent generation of α1,3-galactosyltransferase gene-knockout (GalT-KO) pigs has allowed investigation of the survival of GalT-KO pig organs in nonhuman primates. Methods. Heterotopic heart transplantation from GalT-KO pigs was carried out in baboons (n=8) using a human antihuman CD154 monoclonal antibody-based immunosuppressive regimen. Results. In six of the eight cases, graft survival extended to between approximately 2 and 6 months. All grafts developed thrombotic microangiopathy (TM). In particular, the clinical course of one baboon in which the graft functioned for 179 days is summarized. This baboon received aspirin (40 mg on alternate days) from day 4 in addition to heparin, which may have been a factor in the delay of onset and progression of TM and in prolonged graft survival. Maintenance therapy with anti-CD154 mAb, mycophenolate mofetil, and methylprednisolone was associated with persistently low numbers of CD3+CD4+ and CD3+CD8+ cells. Despite persisting depletion of these cells, no infectious complications occurred. Conclusions. It remains to be established whether TM is related to a very low level of natural preformed or T-cell-induced antibody deposition on the graft, inducing endothelial activation and injury, or to molecular incompatibilities in the coagulation mechanisms between pig and baboon, or to both. However, function of a pig organ in a baboon for a period approaching six months, which has not been reported previously, lends encouragement that the barriers to xenotransplantation will eventually be overcome.

Suppression of Natural and Elicited Antibodies in Pig-to-Baboon Heart Transplantation Using a Human Anti-Human CD154 mAb-Based Regimen

Natural and elicited antipig antibodies (Abs) lead to acute humoral xenograft rejection (AHXR). Ten baboons underwent heterotopic heart transplantation (Tx) from human decay‐accelerating factor (hDAF) pigs. Depletion of anti‐Galα1, 3Gal (Gal) Abs was achieved by the infusion of a Gal glycoconjugate from day – 1. Immunosuppression included induction of antithymocyte globulin, thymic irradiation, and cobra venom factor, and maintenance with a human antihuman CD154 mAb, mycophenolate mofetil, and methylprednisolone; heparin and prophylactic ganciclovir were also administered. Pig heart survival ranged from 4 to 139 (mean 37, median 27) days, with three functioning for >50 days. Graft failure (n = 8) was from classical AHXR [ 4], thrombotic microangiopathy [ 3], or intragraft thrombosis [ 1], with death (n = 2) from pneumonia [ 1], or possible drug toxicity (with features of thrombotic microangiopathy) [ 1]. Anti‐Gal Abs (in μg/mL) were depleted by Gal glycoconjugate before graft implantation from means of 41.3 to 6.3 (IgM) and 12.4–4.6 (IgG), respectively, and at graft excision were 6.3 and 1.7 μg/mL, respectively. No elicited Abs developed, and no cellular infiltration was seen. The treatment regimen was effective in maintaining low anti‐Gal Ab levels and in delaying or preventing AHXR. The combination of costimulatory blockade and heparin with Tx of a Gal‐negative pig organ may prolong graft survival further.

Thrombotic microangiopathy associated with humoral rejection of cardiac xenografts from α1,3-galactosyltransferase gene-knockout pigs in baboons

Heterotopic cardiac xenotransplantation from alpha1,3-galactosyltransferase gene-knockout (GalT-KO) swine to baboons was performed to characterize immunological reaction to the xenograft in the absence of anti-Gal antibody-mediated rejection. Eight baboons received heterotopic cardiac xenografts from GalT-KO porcine donors. All baboons were treated with chronic immunosuppressive therapy. Both histological and immunohistochemical studies were performed on biopsy and graftectomy samples. No hyperacute rejection was observed. Three baboons were euthanized or died 16 to 56 days after transplantation. The other five grafts ceased beating between days 59 and 179 (median, 78 days). All failing grafts exhibited thrombotic microangiopathy (TM) with platelet-rich fibrin thrombi in the microvasculature, myocardial ischemia and necrosis, and focal interstitial hemorrhage. TM developed in parallel with increases in immunoglobulin (IgM and IgG) and complement (C3, C4d, and C5b-9) deposition, as well as with subsequent increases in both TUNEL(+) endothelial cell death and procoagulant activation (increased expression of both tissue factor and von Willebrand factor and decreased expression of CD39). CD3(+) T-cell infiltration occurred in all grafts and weakly correlated with the development of TM. In conclusion, although the use of GalT-KO swine donors prevented hyperacute rejection and prolonged graft survival, slowly progressive humoral rejection--probably associated with non-Gal antibodies to the xenograft--and disordered thromboregulation represent major immunological barriers to long-term xenograft survival.

Species incompatibilities in the pig-to-macaque islet xenotransplant model affect transplant outcome: a comparison with allotransplantation

Graham ML, Bellin MD, Papas KK, Hering BJ, Schuurman H‐J. Species incompatibilities in the pig‐to‐macaque islet xenotransplant model affect transplant outcome: a comparison with allotransplantation. Xenotransplantation 2011; 18: 328–342.

Anti-non-Gal porcine endothelial cell antibodies in acute humoral xenograft rejection of hDAF-transgenic porcine hearts in cynomolgus monkeys.

Abstract:  Background:  Anti‐Galα1–3Gal (Gal) antibodies play a major role in hyperacute rejection and acute humoral xenograft rejection (AHXR) in porcine‐to‐nonhuman primate transplantation. The role of anti‐non‐Gal antibodies in AHXR is less well defined.

Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin.

Porcine islet xenotransplantation is considered a potential cell‐based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose‐stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3‐fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig‐to‐NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets.

Co-stimulation blockade targeting CD154 and CD28/B7 modulates the induced antibody response after a pig-to-baboon cardiac xenograft

Abstract:  Background:  The induced antibodies against Galα1,3Gal (Gal) and non‐Gal epitopes may contribute to delayed xenograft rejection (DXR). We asked whether blockade of the CD40/CD154 and CD28/B7 co‐stimulatory pathways modulates the baboon elicited antibody response to pig Gal and non‐Gal antigens.

Elicited Antibodies in Baboons Exposed to Tissues from 1,3-Galactosyltransferase Gene-Knockout Pigs

Background. This study investigates anti-nonGal antibodies (Abs) in baboons after α1,3-galactosyltransferase gene-knockout (GalT-KO) pig heart transplantation (Tx). Methods. Four baboons underwent pig heart Tx under chronic immunosuppression, which was discontinued after graftectomy. During follow-up, one baboon also received a pig splenocyte infusion. Hearts and splenocytes were from GalT-KO pigs (n=3) or pigs with low Gal expression (Gal-low, n=2), all of swine leukocyte antigen (SLA) dd haplotype. Several weeks after graftectomy, sera were tested by flow cytometry and cytotoxicity assay on porcine peripheral blood mononuclear cells (PBMC) for elicited anti-nonGal Abs. Sera were adsorbed on a Gal immunoaffinity matrix, and tested for SLA haplotype specificity using PBMC from SLA aa, cc, and dd haplotypes. Results. Before heart Tx, no baboon had anti-nonGal Abs demonstrable by binding or cytotoxicity to GalT-KO PBMC. All four baboons developed anti-nonGal Abs after Tx, demonstrable by flow cytometry, and three sera from baboons showed cytotoxicity to GalT-KO PBMC of SLAdd haplotype. After adsorption of anti-Gal Abs, the elicited anti-nonGal Abs showed similar binding to PBMCs from pigs of all three haplotypes (SLAdd, SLAaa, SLAcc). Conclusions. Anti-nonGal Abs developed after GalT-KO pig heart Tx into baboons. The most potent of these antibodies appeared to detect antigens shared by the three pig haplotypes tested. It remains unclear whether these antibodies are directed towards shared SLA determinants or other pig antigens, and whether antibodies with specificity for allelic SLA determinants are also present, but at lower titer.