, Volume 62, Issue 5, pp 811–821 | Cite as

Operational immune tolerance towards transplanted allogeneic pancreatic islets in mice and a non-human primate

  • Midhat H. AbdulredaEmail author
  • Dora M. Berman
  • Alexander Shishido
  • Christopher Martin
  • Maged Hossameldin
  • Ashley Tschiggfrie
  • Luis F. Hernandez
  • Ana Hernandez
  • Camillo Ricordi
  • Jean-Marie Parel
  • Ewa Jankowska-Gan
  • William J. Burlingham
  • Esdras A. Arrieta-Quintero
  • Victor L. Perez
  • Norma S. Kenyon
  • Per-Olof BerggrenEmail author



Patients with autoimmune type 1 diabetes transplanted with pancreatic islets to their liver experience significant improvement in quality of life through better control of blood sugar and enhanced awareness of hypoglycaemia. However, long-term survival and efficacy of the intrahepatic islet transplant are limited owing to liver-specific complications, such as immediate blood-mediated immune reaction, hypoxia, a highly enzymatic and inflammatory environment and locally elevated levels of drugs including immunosuppressive agents, all of which are injurious to islets. This has spurred a search for new islet transplant sites and for innovative ways to achieve long-term graft survival and efficacy without life-long systemic immunosuppression and its complications.


We used our previously established approach of islet transplant in the anterior chamber of the eye in allogeneic recipient mouse models and a baboon model of diabetes, which were treated transiently with anti-CD154/CD40L blocking antibody in the peri-transplant period. Survival of the intraocular islet allografts was assessed by direct visualisation in the eye and metabolic variables (blood glucose and C-peptide measurements). We evaluated longitudinally the cytokine profile in the local microenvironment of the intraocular islet allografts, represented in aqueous humour, under conditions of immune rejection vs tolerance. We also evaluated the recall response in the periphery of the baboon recipient using delayed-type hypersensitivity (DTH) assay, and in mice after repeat transplant in the kidney following initial transplant with allogeneic islets in the eye or kidney.


Results in mice showed >300 days immunosuppression-free survival of allogeneic islets transplanted in the eye or kidney. Notably, >70% of tolerant mice, initially transplanted in the eye, exhibited >400 days of graft survival after re-transplant in the kidney without immunosuppression compared with ~30% in mice that were initially transplanted in the kidney. Cytokine and DTH data provided evidence of T helper 2-driven local and peripheral immune regulatory mechanisms in support of operational immune tolerance towards the islet allografts in both models.


We are currently evaluating the safety and efficacy of intraocular islet transplantation in a phase 1 clinical trial. In this study, we demonstrate immunosuppression-free long-term survival of intraocular islet allografts in mice and in a baboon using transient peri-transplant immune intervention. These results highlight the potential for inducing islet transplant immune tolerance through the intraocular route. Therefore, the current findings are conceptually significant and may impact markedly on clinical islet transplantation in the treatment of diabetes.


Allogeneic rejection Anterior chamber of the eye Immune tolerance induction and maintenance Immunosuppression-free Intraocular transplantation Long-term graft survival Non-invasive longitudinal intravital imaging Pancreatic islet transplant Th2 cytokines 



Delayed-type hypersensitivity


Immediate blood-mediated immune reaction


Islet equivalents


Peripheral blood mononuclear cell


Postoperative day




T helper


Tetanus toxoid and diphtheria



The authors are grateful to W. Diaz, J. Geary and R. Rodriguez-Lopez (Diabetes Research Institute, University of Miami, USA) for their excellent care of non-human primates and associated procedures, and to A. Rabassa and E. Poumian-Ruiz (Diabetes Research Institute, University of Miami, USA) for assistance with islet isolation from the donor baboon. We also thank S. Dubovy and C. Maza (Bascom Palmer Eye Institute, University of Miami, USA) for help with sectioning and histological examination of the baboon eyes, and A. Mendez (Diabetes Research Institute, University of Miami, USA) and H. Salah-Uddin (Department of Psychiatry, University of Miami, USA) for help with Bio-Plex assay setup.

Contribution statement

MHA conceived the study, designed and conducted experiments, analysed and interpreted data and wrote the manuscript. DMB designed and conducted experiments, analysed and interpreted data and wrote the manuscript. AS, CM, MH, AT, LFH, AH and EAA-Q conducted experiments and collected data and proofread the manuscript. JMP planned experiments, interpreted data and proofread the manuscript. WJB and EJ-G performed trans vivo DTH assays, interpreted data and edited the manuscript. VLP designed experiments, performed intraocular islet transplantation and eye examinations in the baboon, interpreted data and edited the manuscript. CR, NSK and P-OB conceived the study, designed experiments, interpreted data and edited the manuscript. All authors approved the version of the manuscript to be published. MHA, DMB, and P-OB are the guarantors of this work.


This work was supported by funds from the Diabetes Research Institute Foundation (DRIF) and the Diabetes Wellness Foundation and by grants from the Stanley J. Glaser Foundation Research Award (UM SJG2016-2), the NIH/NIDDK/NIAID (K01DK097194, U01-AI-102456, R56AI130330, UC4DK116241), the Swedish Diabetes Association Fund, the Swedish Research Council, Novo Nordisk Foundation, the Family Erling-Persson Foundation, Strategic Research Program in Diabetes at Karolinska Institutet, the ERC-2013-AdG 338936-BetaImage, the Family Knut and Alice Wallenberg Foundation, Skandia Insurance Company Ltd, Diabetes and Wellness Foundation, the Bert von Kantzow Foundation and the Stichting af Jochnick Foundation.

Duality of interest

P-OB is cofounder and CEO of Biocrine, an unlisted biotech company that is using the anterior chamber of the eye technique as a research tool. MHA is consultant for the same company. All other authors declare that there is no duality of interest associated with their contribution to this manuscript.

Supplementary material

125_2019_4814_MOESM1_ESM.pdf (529 kb)
ESM (PDF 529 kb)


  1. 1.
    Hering BJ, Clarke WR, Bridges ND et al (2016) Phase 3 trial of transplantation of human islets in type 1 diabetes complicated by severe hypoglycemia. Diabetes Care 39(7):1230–1240. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Tharavanij T, Betancourt A, Messinger S et al (2008) Improved long-term health-related quality of life after islet transplantation. Transplantation 86(9):1161–1167. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Shapiro AM (2012) Islet transplantation in type 1 diabetes: ongoing challenges, refined procedures, and long-term outcome. Rev Diabet Stud 9(4):385–406. CrossRefPubMedGoogle Scholar
  4. 4.
    Berman DM, Molano RD, Fotino C et al (2016) Bioengineering the endocrine pancreas: intraomental islet transplantation within a biologic resorbable scaffold. Diabetes 65(5):1350–1361. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Wolf-van Buerck L, Schuster M, Baehr A et al (2015) Engraftment and reversal of diabetes after intramuscular transplantation of neonatal porcine islet-like clusters. Xenotransplantation 22(6):443–450. CrossRefPubMedGoogle Scholar
  6. 6.
    Maffi P, Balzano G, Ponzoni M et al (2013) Autologous pancreatic islet transplantation in human bone marrow. Diabetes 62(10):3523–3531. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Korsgren O, Nilsson B (2009) Improving islet transplantation: a road map for a widespread application for the cure of persons with type I diabetes. Curr Opin Organ Transplant 14(6):683–687. CrossRefPubMedGoogle Scholar
  8. 8.
    Perez VL, Caicedo A, Berman DM et al (2011) The anterior chamber of the eye as a clinical transplantation site for the treatment of diabetes: a study in a baboon model of diabetes. Diabetologia 54(5):1121–1126. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Shishido ACA, Rodriguez-Diaz R, Berggren P-O, Abdulreda MH (2016) Clinical intraocular islet transplantation is not a number issue. CellR4 4:e2120Google Scholar
  10. 10.
    Szot GL, Yadav M, Lang J et al (2015) Tolerance induction and reversal of diabetes in mice transplanted with human embryonic stem cell-derived pancreatic endoderm. Cell Stem Cell 16(2):148–157. CrossRefPubMedGoogle Scholar
  11. 11.
    Lee K, Nguyen V, Lee KM, Kang SM, Tang Q (2014) Attenuation of donor-reactive T cells allows effective control of allograft rejection using regulatory T cell therapy. Am J Transplant 14(1):27–38. CrossRefPubMedGoogle Scholar
  12. 12.
    Speier S, Nyqvist D, Cabrera O et al (2008) Noninvasive in vivo imaging of pancreatic islet cell biology. Nat Med 14(5):574–578. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Abdulreda MH, Faleo G, Molano RD et al (2011) High-resolution, noninvasive longitudinal live imaging of immune responses. Proc Natl Acad Sci U S A 108(31):12863–12868. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Abdulreda MH, Rodriguez-Diaz R, Caicedo A, Berggren PO (2016) Liraglutide compromises pancreatic beta cell function in a humanized mouse model. Cell Metab 23(3):541–546. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Carlsson PO, Palm F (2002) Oxygen tension in isolated transplanted rat islets and in islets of rat whole-pancreas transplants. Transpl Int 15(11):581–585. CrossRefPubMedGoogle Scholar
  16. 16.
    McLaren JW, Dinslage S, Dillon JP, Roberts JE, Brubaker RF (1998) Measuring oxygen tension in the anterior chamber of rabbits. Invest Ophthalmol Vis Sci 39(10):1899–1909PubMedGoogle Scholar
  17. 17.
    Sharifipour F, Yazdani S, Pakravan M, Idani E (2013) Aqueous oxygen tension in glaucomatous and nonglaucomatous eyes. J Glaucoma 22(8):608–613. CrossRefPubMedGoogle Scholar
  18. 18.
    Nyqvist D, Speier S, Rodriguez-Diaz R et al (2011) Donor islet endothelial cells in pancreatic islet revascularization. Diabetes 60(10):2571–2577. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Miska J, Abdulreda MH, Devarajan P et al (2014) Real-time immune cell interactions in target tissue during autoimmune-induced damage and graft tolerance. J Exp Med 211(3):441–456. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Berman DM, Cabrera O, Kenyon NM et al (2007) Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model. Transplantation 84(3):308–315. CrossRefPubMedGoogle Scholar
  21. 21.
    Kenyon NS, Fernandez LA, Lehmann R et al (1999) Long-term survival and function of intrahepatic islet allografts in baboons treated with humanized anti-CD154. Diabetes 48(7):1473–1481. CrossRefPubMedGoogle Scholar
  22. 22.
    Pileggi A, Molano RD, Berney T et al (2001) Heme oxygenase-1 induction in islet cells results in protection from apoptosis and improved in vivo function after transplantation. Diabetes 50(9):1983–1991. CrossRefPubMedGoogle Scholar
  23. 23.
    Abdulreda MH, Caicedo A, Berggren P-O (2013) Transplantation into the anterior chamber of the eye for longitudinal, non-invasive in vivo imaging with single-cell resolution in real-time. J Vis Exp:e50466Google Scholar
  24. 24.
    Han D, Berman DM, Willman M et al (2010) Choice of immunosuppression influences cytomegalovirus DNAemia in cynomolgus monkey (Macaca fascicularis) islet allograft recipients. Cell Transplant 19(12):1547–1561. CrossRefPubMedGoogle Scholar
  25. 25.
    Burlingham WJ, Jankowska-Gan E, VanBuskirk A, Orosz CG, Lee JH, Kusaka S (2000) Loss of tolerance to a maternal kidney transplant is selective for HLA class II: evidence from trans-vivo DTH and alloantibody analysis. Hum Immunol 61(12):1395–1402. CrossRefPubMedGoogle Scholar
  26. 26.
    Jankowska-Gan E, Hegde S, Burlingham WJ (2013) Trans-vivo delayed type hypersensitivity assay for antigen specific regulation. J Vis Exp:e4454Google Scholar
  27. 27.
    Cure P, Pileggi A, Froud T et al (2008) Improved metabolic control and quality of life in seven patients with type 1 diabetes following islet after kidney transplantation. Transplantation 85(6):801–812. CrossRefPubMedGoogle Scholar
  28. 28.
    Abdulreda MH, Berggren PO (2013) Islet inflammation in plain sight. Diabetes Obes Metab 15(Suppl 3):105–116. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Rodriguez-Diaz R, Speier S, Molano RD et al (2012) Noninvasive in vivo model demonstrating the effects of autonomic innervation on pancreatic islet function. Proc Natl Acad Sci U S A 109(52):21456–21461. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Faustman DL, Steinman RM, Gebel HM, Hauptfeld V, Davie JM, Lacy PE (1984) Prevention of rejection of murine islet allografts by pretreatment with anti-dendritic cell antibody. Proc Natl Acad Sci U S A 81(12):3864–3868. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Terasaka R, Lacy PE, Hauptfeld V, Bucy RP, Davie JM (1986) The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts. Diabetes 35(1):83–88. CrossRefPubMedGoogle Scholar
  32. 32.
    Giovannoni L, Muller YD, Lacotte S et al (2015) Enhancement of islet engraftment and achievement of long-term islet allograft survival by toll-like receptor 4 blockade. Transplantation 99(1):29–35. CrossRefPubMedGoogle Scholar
  33. 33.
    Koulmanda M, Qipo A, Fan Z et al (2012) Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term triple therapy. Am J Transplant 12(5):1296–1302. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Almaca J, Molina J, Arrojo EDR et al (2014) Young capillary vessels rejuvenate aged pancreatic islets. Proc Natl Acad Sci U S A 111(49):17612–17617. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Chmelova H, Cohrs CM, Chouinard JA et al (2015) Distinct roles of β-cell mass and function during type 1 diabetes onset and remission. Diabetes 64(6):2148–2160. CrossRefPubMedGoogle Scholar
  36. 36.
    Ul-Haq Z, Naz S, Mesaik MA (2016) Interleukin-4 receptor signaling and its binding mechanism: a therapeutic insight from inhibitors tool box. Cytokine Growth Factor Rev 32:3–15. CrossRefPubMedGoogle Scholar
  37. 37.
    Paul WE (2015) History of interleukin-4. Cytokine 75(1):3–7. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Roth F, De La Fuente AC, Vella JL, Zoso A, Inverardi L, Serafini P (2012) Aptamer-mediated blockade of IL4Rα triggers apoptosis of MDSCs and limits tumor progression. Cancer Res 72(6):1373–1383. CrossRefPubMedGoogle Scholar
  39. 39.
    Xu Q, Lee J, Jankowska-Gan E et al (2007) Human CD4+CD25low adaptive T regulatory cells suppress delayed-type hypersensitivity during transplant tolerance. J Immunol 178(6):3983–3995. CrossRefPubMedGoogle Scholar
  40. 40.
    Torrealba JR, Katayama M, Fechner JH Jr et al (2004) Metastable tolerance to rhesus monkey renal transplants is correlated with allograft TGF-β1+CD4+ T regulatory cell infiltrates. J Immunol 172(9):5753–5764. CrossRefPubMedGoogle Scholar
  41. 41.
    Billingham RE, Brent L, Medawar PB (1953) Actively acquired tolerance of foreign cells. Nature 172(4379):603–606. CrossRefPubMedGoogle Scholar
  42. 42.
    Gonzalez-Nieto L, Domingues A, Ricciardi M et al (2016) Analysis of simian immunodeficiency virus-specific CD8+ T cells in Rhesus macaques by peptide-MHC-I tetramer staining. J Vis Exp:e54881Google Scholar
  43. 43.
    Nadazdin O, Boskovic S, Murakami T et al (2011) Host alloreactive memory T cells influence tolerance to kidney allografts in nonhuman primates. Sci Transl Med 3:86ra51CrossRefGoogle Scholar
  44. 44.
    Tomita Y, Satomi M, Bracamonte-Baran W et al (2016) Kinetics of alloantigen-specific regulatory CD4 T cell development and tissue distribution after donor-specific transfusion and costimulatory blockade. Transplant Direct 2(5):e73. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Burlingham WJ, Jankowska-Gan E, Kempton S, Haynes L, Kaufman DB (2015) Patterns of immune regulation in rhesus macaque and human families. Transplant Direct 1:e20CrossRefGoogle Scholar
  46. 46.
    Kawai T, Andrews D, Colvin RB, Sachs DH, Cosimi AB (2000) Thromboembolic complications after treatment with monoclonal antibody against CD40 ligand. Nat Med 6(2):114. CrossRefPubMedGoogle Scholar
  47. 47.
    Boumpas DT, Furie R, Manzi S et al (2003) A short course of BG9588 (anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis Rheum 48(3):719–727. CrossRefPubMedGoogle Scholar
  48. 48.
    Pinelli DF, Ford ML (2015) Novel insights into anti-CD40/CD154 immunotherapy in transplant tolerance. Immunotherapy 7(4):399–410. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Kim SC, Wakwe W, Higginbotham LB et al (2017) Fc-silent anti-CD154 domain antibody effectively prevents nonhuman primate renal allograft rejection. Am J Transplant 17(5):1182–1192. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Midhat H. Abdulreda
    • 1
    • 2
    • 3
    • 4
    Email author
  • Dora M. Berman
    • 1
    • 2
  • Alexander Shishido
    • 1
  • Christopher Martin
    • 1
  • Maged Hossameldin
    • 1
  • Ashley Tschiggfrie
    • 1
  • Luis F. Hernandez
    • 1
  • Ana Hernandez
    • 1
  • Camillo Ricordi
    • 1
    • 2
    • 5
  • Jean-Marie Parel
    • 4
  • Ewa Jankowska-Gan
    • 6
  • William J. Burlingham
    • 6
  • Esdras A. Arrieta-Quintero
    • 4
  • Victor L. Perez
    • 4
    • 7
  • Norma S. Kenyon
    • 1
    • 2
    • 3
  • Per-Olof Berggren
    • 1
    • 2
    • 8
    Email author
  1. 1.Diabetes Research Institute and Cell Transplant CenterUniversity of Miami Miller School of MedicineMiamiUSA
  2. 2.Department of SurgeryUniversity of Miami Miller School of MedicineMiamiUSA
  3. 3.Department of Microbiology and ImmunologyUniversity of Miami Miller School of MedicineMiamiUSA
  4. 4.Bascom Palmer Eye InstituteUniversity of Miami Miller School of MedicineMiamiUSA
  5. 5.Diabetes Research Institute FederationHollywoodUSA
  6. 6.Department of Surgery, School of Medicine and Public HealthUniversity of WisconsinMadisonUSA
  7. 7.Duke OphthalmologyDuke UniversityDurhamUSA
  8. 8.The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska InstitutetKarolinska University Hospital L1StockholmSweden

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