Pig-to-baboon heterotopic heart transplantation – exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade-based regimens
Hayato Iwase
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
These authors contributed equally to this work.Search for more papers by this authorBurcin Ekser
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Department of Surgery, Transplantation and Advanced Technologies, Vascular Surgery and Organ Transplant Unit, University Hospital of Catania, Catania, Italy
These authors contributed equally to this work.Search for more papers by this authorVikas Satyananda
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorJay Bhama
Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorHidetaka Hara
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorMohamed Ezzelarab
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorEdwin Klein
Division of Laboratory Animal Resources, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorRobert Wagner
Division of Laboratory Animal Resources, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorCassandra Long
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorJnanesh Thacker
Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorJiang Li
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorHao Zhou
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Center for Kidney Transplantation, Second Affiliated Hospital of the University of South China, Hengyang, Hunan, China
Search for more papers by this authorMaolin Jiang
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Center for Kidney Transplantation, Second Affiliated Hospital of the University of South China, Hengyang, Hunan, China
Search for more papers by this authorSantosh Nagaraju
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorHuidong Zhou
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Center for Kidney Transplantation, Second Affiliated Hospital of the University of South China, Hengyang, Hunan, China
Search for more papers by this authorMassimiliano Veroux
Department of Surgery, Transplantation and Advanced Technologies, Vascular Surgery and Organ Transplant Unit, University Hospital of Catania, Catania, Italy
Search for more papers by this authorPietro Bajona
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorMartin Wijkstrom
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorYi Wang
Center for Kidney Transplantation, Second Affiliated Hospital of the University of South China, Hengyang, Hunan, China
Search for more papers by this authorNikolai Klymiuk
Gene Center, Ludwig Maximilians Universitat Munchen (LMU), Munich, Germany
Search for more papers by this authorEckhard Wolf
Gene Center, Ludwig Maximilians Universitat Munchen (LMU), Munich, Germany
Search for more papers by this authorCorresponding Author
David K. C. Cooper
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Address reprint requests to David K. C. Cooper, Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Starzl Biomedical Science Tower, W1543, 200 Lothrop Street, Pittsburgh, PA 15261, USA
(E-mail: [email protected])
Search for more papers by this authorHayato Iwase
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
These authors contributed equally to this work.Search for more papers by this authorBurcin Ekser
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Department of Surgery, Transplantation and Advanced Technologies, Vascular Surgery and Organ Transplant Unit, University Hospital of Catania, Catania, Italy
These authors contributed equally to this work.Search for more papers by this authorVikas Satyananda
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorJay Bhama
Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorHidetaka Hara
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorMohamed Ezzelarab
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorEdwin Klein
Division of Laboratory Animal Resources, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorRobert Wagner
Division of Laboratory Animal Resources, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorCassandra Long
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorJnanesh Thacker
Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorJiang Li
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorHao Zhou
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Center for Kidney Transplantation, Second Affiliated Hospital of the University of South China, Hengyang, Hunan, China
Search for more papers by this authorMaolin Jiang
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Center for Kidney Transplantation, Second Affiliated Hospital of the University of South China, Hengyang, Hunan, China
Search for more papers by this authorSantosh Nagaraju
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorHuidong Zhou
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Center for Kidney Transplantation, Second Affiliated Hospital of the University of South China, Hengyang, Hunan, China
Search for more papers by this authorMassimiliano Veroux
Department of Surgery, Transplantation and Advanced Technologies, Vascular Surgery and Organ Transplant Unit, University Hospital of Catania, Catania, Italy
Search for more papers by this authorPietro Bajona
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorMartin Wijkstrom
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Search for more papers by this authorYi Wang
Center for Kidney Transplantation, Second Affiliated Hospital of the University of South China, Hengyang, Hunan, China
Search for more papers by this authorNikolai Klymiuk
Gene Center, Ludwig Maximilians Universitat Munchen (LMU), Munich, Germany
Search for more papers by this authorEckhard Wolf
Gene Center, Ludwig Maximilians Universitat Munchen (LMU), Munich, Germany
Search for more papers by this authorCorresponding Author
David K. C. Cooper
Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA
Address reprint requests to David K. C. Cooper, Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Starzl Biomedical Science Tower, W1543, 200 Lothrop Street, Pittsburgh, PA 15261, USA
(E-mail: [email protected])
Search for more papers by this authorAbstract
Background
Three costimulation blockade-based regimens have been explored after transplantation of hearts from pigs of varying genetic backgrounds to determine whether CTLA4-Ig (abatacept) or anti-CD40mAb+CTLA4-Ig (belatacept) can successfully replace anti-CD154mAb.
Methods
All pigs were on an α1,3-galactosyltransferase gene-knockout/CD46 transgenic (GTKO.CD46) background. Hearts transplanted into Group A baboons (n = 4) expressed additional CD55, and those into Group B (n = 3) expressed human thrombomodulin (TBM). Immunosuppression included anti-thymocyte globulin with anti-CD154mAb (Regimen 1: n = 2) or abatacept (Regimen 2: n = 2) or anti-CD40mAb+belatacept (Regimen 3: n = 2). Regimens 1 and 2 included induction anti-CD20mAb and continuous heparin. One further baboon in Group B (B16311) received a modified Regimen 1. Baboons were followed by clinical/laboratory monitoring of immune/coagulation parameters. At biopsy, graft failure, or euthanasia, the graft was examined by microscopy.
Results
Group A baboons survived 15 to 33 days, whereas Group B survived 52, 99, and 130 days, respectively. Thrombocytopenia and reduction in fibrinogen occurred within 21 days in Group A, suggesting thrombotic microangiopathy (TM), confirmed by histopathology. In Group B, with follow-up for >4 m, areas of myofiber degeneration and scarring were seen in two hearts at necropsy. A T-cell response was documented only in baboons receiving Regimen 2.
Conclusions
The combination of anti-CD40mAb+belatacept proved effective in preventing a T-cell response. The expression of TBM prevented thrombocytopenia and may possibly delay the development of TM and/or consumptive coagulopathy.
References
- 1Kolber-Simonds D, Lai L, Watt SR et al. Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proc Natl Acad Sci USA 2004; 101: 7335–7340.
- 2Phelps CJ, Koike C, Vaught TD et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science 2003; 299: 411–414.
- 3Kuwaki K, Tseng YL, Dor FJ et al. Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med 2005; 11: 29–31.
- 4Tseng YL, Kuwaki K, Dor FJ et al. alpha1,3-Galactosyltransferase gene-knockout pig heart transplantation in baboons with survival approaching 6 months. Transplantation 2005; 80: 1493–1500.
- 5Ezzelarab M, Garcia B, Azimzadeh A et al. The innate immune response and activation of coagulation in alpha1,3-galactosyltransferase gene-knockout xenograft recipients. Transplantation 2009; 87: 805–812.
- 6Houser SL, Kuwaki K, Knosalla C et al. Thrombotic microangiopathy and graft arteriopathy in pig hearts following transplantation into baboons. Xenotransplantation 2004; 11: 416–425.
- 7Shimizu A, Hisashi Y, Kuwaki K et al. Thrombotic microangiopathy associated with humoral rejection of cardiac xenografts from alpha1,3-galactosyltransferase gene-knockout pigs in baboons. Am J Pathol 2008; 172: 1471–1481.
- 8Mohiuddin MM, Corcoran PC, Singh AK et al. B-cell depletion extends the survival of GTKO.hCD46Tg pig heart xenografts in baboons for up to 8 months. Am J Transplant 2012; 12: 763–771.
- 9Lawson JH, Daniels LJ, Platt JL. The evaluation of thrombomodulin activity in porcine to human xenotransplantation. Transplant Proc 1997; 29: 884–885.
- 10Siegel JB, Grey ST, Lesnikoski BA et al. Xenogeneic endothelial cells activate human prothrombin. Transplantation 1997; 64: 888–896.
- 11Kopp CW, Grey ST, Siegel JB et al. Expression of human thrombomodulin cofactor activity in porcine endothelial cells. Transplantation 1998; 66: 244–251.
- 12Mohiuddin MM, Singh AK, Corcoran PC et al. One-year heterotopic cardiac xenograft survival in a pig to baboon model. Am J Transplant 2014; 14: 488–489.
- 13Mohiuddin M, Singh AK, Corcoran PC et al. Genetically engineered pigs and target-specific immunomodulation provide significant graft survival and hope for clinical cardiac xenotransplantation. JTCS 2014; 148: 1106–1113.
- 14Cozzi E, White DJ. The generation of transgenic pigs as potential organ donors for humans. Nat Med 1995; 1: 964–966.
- 15Petersen B, Ramackers W, Tiede A et al. Pigs transgenic for human thrombomodulin have elevated production of activated protein C. Xenotransplantation 2009; 16: 486–495.
- 16Miwa Y, Yamamoto K, Onishi A et al. Potential value of human thrombomodulin and DAF expression for coagulation control in pig-to-human xenotransplantation. Xenotransplantation 2010; 17: 26–37.
- 17Wuensch A, Baehr A, Bongoni AK et al. Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs. Transplantation 2014; 97: 138–147.
- 18Yazaki S, Iwamoto M, Onishi A et al. Production of cloned pigs expressing human thrombomodulin in endothelial cells. Xenotransplantation 2012; 19: 82–91.
- 19Ezzelarab MB, Ekser B, Echeverri G et al. Costimulation blockade in pig artery patch xenotransplantation – a simple model to monitor the adaptive immune response in nonhuman primates. Xenotransplantation 2012; 19: 221–232.
- 20Kawai T, Andrews D, Colvin RB, Sachs DH, Cosimi AB. Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand. Nat Med 2000; 6: 114.
- 21Knosalla C, Gollackner B, Cooper DK. Anti-CD154 monoclonal antibody and thromboembolism revisited. Transplantation 2002; 74: 416–417.
- 22Ayares D, Phelps C, Vaught TD et al. Multi-transgenic pigs for vascularized pig organ xenografts. Xenotransplantation 2011; 18: 269 (abstract #119).
- 23Klymiuk N, Wuensch A, Kurome M et al. GalT-KO/CD46/hTM triple-transgenic donor animals for pig-to-baboon heart transplantation. Xenotransplantation 2011; 18: 271 (Abstract #126).
- 24Loveland BE, Milland J, Kyriakou P et al. Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons. Xenotransplantation 2004; 11: 171–183.
- 25Cooper DKC, Ye Y, Niekrasz M. Heart transplantation in primates. In: DVPL Cramer, L Makowka, eds. Handbook of Animal Models in Transplantation Research. Boca Raton: CRC Press, 1994: 173–200.
- 26Iwase H, Ekser B, Satyananda V et al. Initial in vivo experience of pig artery patch transplantation in baboons using mutant MHC (CIITA-DN) pigs. Transplant Immunol. 2015: 99–108.
- 27Lowe M, Badell IR, Thompson P et al. A novel monoclonal antibody to CD40 prolongs islet allograft survival. Am J Transplant 2012; 12: 2079–2087, 52.
- 28Larsen CP, Pearson TC, Adams AB et al. Rational development of LEA29Y (belatacept), a high-affinity variant of CTLA4-Ig with potent immunosuppressive properties. Am J Transplant 2005; 5: 443–453.
- 29Ekser B, Bianchi J, Ball S et al. Comparison of hematologic, biochemical, and coagulation parameters in alpha1,3-galactosyltransferase gene-knockout pigs, wild-type pigs, and four primate species. Xenotransplantation 2012; 19: 342–354.
- 30Buhler L, Awwad M, Basker M et al. High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response. Transplantation 2000; 69: 2296–2304.
- 31Adams AB, Shirasugi N, Jones TR et al. Development of a chimeric anti-CD40 monoclonal antibody that synergizes with LEA29Y to prolong islet allograft survival. J Immunol 2005; 174: 542–550.
- 32Haanstra KG, Ringers J, Sick EA et al. Prevention of kidney allograft rejection using anti-CD40 and anti-CD86 in primates. Transplantation 2003; 75: 637–643.
- 33Kenyon NS, Chatzipetrou M, Masetti M et al. Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154. Proc Natl Acad Sci USA 1999; 96: 8132–8137.
- 34Kirk AD, Harlan DM, Armstrong NN et al. CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates. Proc Natl Acad Sci USA 1997; 94: 8789–8794.
- 35Pearson TC, Trambley J, Odom K et al. Anti-CD40 therapy extends renal allograft survival in rhesus macaques. Transplantation 2002; 74: 933–940.
- 36Tai HC, Campanile N, Ezzelarab M, Cooper DK, Phelps C. Measurement of anti-CD154 monoclonal antibody in primate sera by competitive inhibition ELISA. Xenotransplantation 2006; 13: 566–570.
- 37Thompson P, Cardona K, Russell M et al. CD40-specific costimulation blockade enhances neonatal porcine islet survival in nonhuman primates. Am J Transplant 2011; 11: 947–957.
- 38Ezzelarab M, Hara H, Busch J et al. Antibodies directed to pig non-Gal antigens in naive and sensitized baboons. Xenotransplantation 2006; 13: 400–407.
- 39Hara H, Ezzelarab M, Rood PP et al. Allosensitized humans are at no greater risk of humoral rejection of GT-KO pig organs than other humans. Xenotransplantation 2006; 13: 357–365.
- 40Hara H, Long C, Lin YJ et al. In vitro investigation of pig cells for resistance to human antibody-mediated rejection. Transpl Int 2008; 21: 1163–1174.
- 41Ekser B, Klein E, He J et al. Genetically-engineered pig-to-baboon liver xenotransplantation: histopathology of xenografts and native organs. PLoS ONE 2012; 7: e29720.
- 42Chen G, Qian H, Starzl T et al. Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys. Nat Med 2005; 11: 1295–1298.
- 43Ezzelarab M, Ezzelarab C, Wilhite T et al. Genetically-modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T-cell xenoresponses. Xenotransplantation 2011; 18: 183–195.
- 44Wilhite T, Ezzelarab C, Hara H et al. The effect of Gal expression on pig cells on the human T-cell xenoresponse. Xenotransplantation 2012; 19: 56–63.
- 45Ezzelarab C, Ayares D, Cooper DK, Ezzelarab MB. Human T-cell proliferation in response to thrombin-activated GTKO pig endothelial cells. Xenotransplantation 2012; 19: 311–316.
- 46Gorski A, Wasik M, Nowaczyk M, Korczak-Kowalska G. Immunomodulating activity of heparin. FASEB J 1991; 5: 2287–2291.
- 47Heinzelmann M, Bosshart H. Fondaparinux sodium lacks immunomodulatory effects of heparin. Am J Surg 2004; 187: 111–113.
- 48Hochart H, Jenkins PV, Preston RJ et al. Concentration-dependent roles for heparin in modifying lipopolysaccharide-induced activation of mononuclear cells in whole blood. Thromb Haemost 2008; 99: 570–575.
- 49Ranjbaran H, Wang Y, Manes TD et al. Heparin displaces interferon-gamma-inducible chemokines (IP-10, I-TAC, and Mig) sequestered in the vasculature and inhibits the transendothelial migration and arterial recruitment of T cells. Circulation 2006; 114: 1293–1300.
